Microarray analysis of B. pseudomallei CPS III expression following intraperitoneal inoculation in the hamster model of melioidosis.
\r\n\tWith this goal in mind, together with the US Prof. John M. Ballato and the InechOpen publishing house since 2011 we have published in 2011, 2013, 2015 and 2017 4 books of our serial “Optoelectronics” and the book “Excitons”, edited in 2018 by Prof. Sergei L. Pyshkin. Publishing the new book “Luminescence” we are pleased to note the growing number of countries participating in this undertaking as well as for a long time fruitfully cooperating scientists from the United States and the Republic of Moldova.
\r\n\tSpecialists from all over the world have published in edited by us books their works in the field of research of the luminescent properties of various materials suitable for use in optoelectronic devices, the development of new structures and the results of their application in practice.
Polysaccharide capsules are structures found on the cell surface of a broad range of bacterial species. The polysaccharide capsule often constitutes the outermost layer of the cell, and therefore is often involved in mediating direct interactions between the bacteria and its environment. It is due to these interactions that polysaccharide capsules have been implicated as important virulence factors for many bacterial pathogens.
Bacterial extracellular polysaccharides (EPS) may be classified as either capsular polysaccharides (CPS), where the polysaccharide is intimately associated with the cell surface, or as slime polysaccharides, where the polysaccharide is loosely associated with the cell [1]. Differentiation between these forms is difficult since CPS may be released from the cell, giving the appearance of a slime polysaccharide [1]. In turn, distinguishing between CPS and other cell surface polysaccharides, such as O-antigenic moieties of lipopolysaccharide (LPS), may also be difficult, since CPS may be found associated with LPS [1].
Capsular polysaccharides are highly hydrated molecules that are over 95% water [2]. They are often linked to the cell surface of the bacterium via covalent attachments to either phospholipid or lipid-A molecules, although some CPS may be associated with the cell in the absence of a membrane anchor [1, 3]. Capsular polysaccharides can be either homo- or heteropolymers composed of repeating monosaccharides joined by glycosidic linkages [4]. The multiple hydroxyl groups present within each monosaccharide may be involved in the formation of the glycosidic bond, therefore, any two monosaccharides may be joined in a number of configurations, which leads to large structural diversity among CPS types. In the case of human pathogens, a large number of different capsule serotypes have been identified, and certain CPS or K-antigens have been associated with specific infections [4]. For example, the Escherichia coli K1 antigen, a homopolymer of α2,8-linked N-acetylneuraminic acid (NeuNAc), is the major cause of neonatal meningitis [5]. While bacterial species may demonstrate great structural diversity in synthesizing capsules, chemically identical capsular polysaccharides may also be synthesized by different bacterial species. The Neisseria meningitidis group B capsular polysaccharide is identical to the K1 polymer of E. coli, and the E. coli K18, K22, and K100 antigens have the same constituents and structure as the Haemophilus influenza serotype b capsule [6, 7]. The conservation of CPS types between bacterial species raises interesting questions regarding the evolution of capsules and the transmission and acquisition of capsule biosynthesis genes [4].
The genetic loci necessary for the production of bacterial capsules are primarily clustered at a single chromosomal locus, which allows for the coordinate regulation of a large number of genes that may be involved in both the biosynthesis and export of capsular polysaccharides [4, 1]. In most bacterial species, the capsule gene clusters demonstrate conserved genetic organization. The capsules of E. coli have been classified into four groups based on genetic organization and biosynthetic criteria and capsules of other bacteria may resemble these prototypes. Group 1 capsules include the E. coli K30 capsule and the capsules of Klebsiella sp. and Erwinia sp. [8]. Group 2 capsules include the E. coli K1 and K5 capsules, as well as the capsules produced by Neisseria sp. and Haemophilus sp. Group 3 capsules include the E. coli K10 capsule. Group 4 capsules include the capsules of E. coli K40 and 0111. Both group 2 and group 3 capsule gene clusters are organized into 3 regions. Regions 1 and 3 are involved in the export and modification of the capsular polysaccharides and are conserved between members of the group, while region 2 contains the genes responsible for the biosynthesis of the capsule and is usually serotype specific [4, 8]. Generally these regions are organized into one transcriptional unit and the regions within the capsule locus are divergently transcribed [4, 8]. In addition, some genes within a region may be translationally coupled, such as the kpsU and kpsC genes in group 2 E. coli capsules, and the kpsM and kpsT genes in group 3 E. coli capsules, which allows for balanced expression of two proteins [4].
The A+T composition of capsule gene clusters is often significantly higher than the rest of the chromosome, suggesting a common ancestry of capsule genes in gram-negative bacteria [9]. It is likely that these A+T rich regions have been horizontally transferred between bacterial species. In addition, the A+T ratio of region 2 DNA of group 2 E. coli capsule gene clusters compared to regions 1 and 3 confirms that capsule diversity has been achieved in part through the acquisition of different region 2 sequences [4].
The production of a polysaccharide capsule is widespread in pathogenic bacteria. A number of functions have been assigned to bacterial capsules including: prevention of dessication, adherence, resistance to non-specific host immunity, resistance to specific host immunity, and mediating the diffusion of molecules through to the cell surface [4, 1].
Capsules may form a hydrated gel around the surface of the bacterial cell, which may protect the bacteria from the harmful effects of dessication [10]. This may increase the survival of encapsulated bacteria outside of the host, promoting the transmission of pathogenic bacteria from one host to another [4]. Mucoid isolates of E. coli, Acinetobacter\n\t\t\t\tcalcoaceticus, and Erwinia stewartii are more resistant to drying than isogenic nonmucoid strains [11]. Studies with E. coli have shown that the expression of genes encoding for the colanic acid capsule is increased by dessication [11]. In addition, alginate production by Pseudomonas aeruginosa is triggered by high osmolarity, which may be a consequence of dessication [12].
Capsular polysaccharides may promote adherence of bacteria to both surfaces and other bacterial cells, which may facilitate colonization of a particular niche and may lead to the formation of biofilms [13]. Cell-surface polysaccharides have been shown to mediate the attachment of bacterial cells to one another, leading to biofilm formation and persistence of the organisms during colonization [1, 14].
Capsular polysaccharides are one of the components responsible for resistance to the non-specific immunity of the host. The presence of a capsule is thought to confer resistance to non-specific host defense mechanisms such as complement and complement-mediated opsonophagocytosis [4]. Bacterial capsules may resist complement-mediated killing by providing a permeability barrier to complement components, which masks the underlying cell surface structures that activate complement [15]. The capsule may also act in concert with O-antigens to confer resistance to complement-mediated killing [16]. As a result, a combination of cell surface structures is responsible for conferring resistance to killing by the complement cascade [4]. Finally, capsules are responsible for resistance to complement-mediated opsonophagocytosis. This resistance may be due to steric effects, which results in the capsule acting as a barrier between the C3b deposited on the bacterial surface and the C3b receptors present on phagocytes [4]. Alternatively, the resistance to opsonophagocytosis may be due to the net negative charge of the polysaccharide capsule [17].
Capsules may also confer resistance to the specific immune response of the host. Although most capsular polysaccharides can elicit an immune response, some capsules are poorly immunogenic [4]. Examples of such capsules include those containing NeuNAc, such as the E. coli K1 capsule or the capsule of Neisseria meningitidis serogroup B, and the E. coli K5 capsule, which is similar to desulfoheparin [18, 19]. Because these capsules are structurally similar to polysaccharides encountered on host tissue, these capsules are poorly immunogenic, and elicit a poor antibody response in the host [20].
Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative, facultatively anaerobic, motile bacillus that is commonly found in the soil and stagnant waters in a number of regions around the world, particularly in areas that fall between 20o north and 20o south of the equator [21, 22]. Infection by B. pseudomallei is often due to either direct inoculation into wounds and skin abrasions or to inhalation of contaminated material [22, 23, 24,]. This would explain the prevalence of the disease among rice farmers as well as helicopter pilots in the Vietnam War who developed melioidosis due to inhalation of contaminated dust [24]. Melioidosis may present as an acute pneumonia or an acute septicemia, which is the most severe form of the disease. The disease may also manifest as a chronic infection involving long-lasting suppurative abscesses in numerous sites in the body. Infection with B. pseudomallei may even result in a subclinical infection and remain undetected for a number of years. Both the chronic and subclinical forms generally remain undiagnosed until activated by a traumatic event or a decrease in immunocompetence [25]. B. pseudomallei is inherently resistant to a number of antibiotics, and even with aggressive antibiotic therapy, the mortality rate remains high, and the incidence of relapse is common [26, 27].
At the time our studies were initiated some cell-associated antigens had been identified and characterized in B. pseudomallei. Cell-associated antigens include exopolysaccharide (EPS) and lipopolysaccharide (LPS) [28, 29, 30]. The EPS produced by B. pseudomallei was determined to be an unbranched polymer of repeating tetrasaccharide units with the structure -3)-2-O-acetyl-β-D-Galp-(1-4)-α-D-Galp-(1-3)-β-D-Galp-(1-5)-β-D-KDOp-(2- [31, 32]. The role of EPS in virulence was not known, but sera from patients with melioidosis had been shown to contain antibodies against EPS [30]. Two other EPS structures were also identified; a branched 1,4-linked glucan polymer ((CP-1a) and a triple-branched heptasaccharide repeating unit composed of rhamnose, mannose, galactose, glucose, and glucuronic acid (CP-2) [33]. The genes involved in the synthesis of these capsules, and the role of these capsules in virulence had not been identified. The LPS of B. pseudomallei was structurally characterized and reported to contain two types of O-polysaccharide moieties termed type I O-PS and type II O-PS [34, 35]. Type II O-PS was found to be an unbranched heteropolymer with repeating D-glucose and L-talose residues with the structure -3)-β-D-glucopyranose-(1-3)-6-deoxy-α-L-talopyranose-(1-. Type II O-PS had been shown to be involved in serum resistance [36]. Type II O-PS mutants also demonstrated reduced virulence in three animal models of B. pseudomallei infection [36]. Type I O-PS was determined to be an unbranched homopolymer with the structure -3)-2-O-acetyl-6-deoxy-β-D-manno-heptopyranose-(1-, however, the role for this polysaccharide in infection had not been defined, nor the genes responsible for its biosynthesis been identified.
B. thailandensis is a nonpathogenic soil organism originally isolated in Thailand [37]. Based on biochemical, immunological, and genetic data, B. pseudomallei and B. thailandensis are closely related species. However, these two organisms differ in a number of ways and have been classified into two different species [38]. The rRNA sequence of B. thailandensis differs from that of B. pseudomallei by 15 nucleotides, and there are significant differences in genomic macrorestriction patterns between these organisms [39]. The biochemical profiles of these two species differ in that B. thailandensis can utilize L-arabinose whereas B. pseudomallei does not [38, 40]. The most distinct difference between these two species, however, is their relative virulence. The 50% lethal dose (LD50) for B. pseudomallei in the Syrian hamster model of acute melioidosis is <10 organisms, whereas the LD50 for B. thailandensis is approximately 106 organisms [38]. It has also been shown that the two species can be differentiated based on their propensity to cause disease in humans. Environmental strains isolated in Thailand that are able to assimilate L-arabinose are not associated with human infection, whereas clinical isolates are not able to utilize L-arabinose [41].
To identify the genetic determinants that confer enhanced virulence in B. pseudomallei, a method combining subtractive hybridization, insertional mutagenesis, and animal virulence studies was developed [42]. Subtractive hybridization was carried out between the virulent B. pseudomallei and the weakly virulent B. thailandensis in order to isolate DNA sequences encoding for virulence determinants unique to B. pseudomallei. The genomic DNA sample from B. pseudomallei containing the sequences of interest was known as the tester DNA, and genomic DNA from B. thailandensis, the reference sample, was called the driver DNA. Tester and driver DNAs were digested and subjected to two rounds of hybridization. The remaining unhybridized sequences were considered tester-specific sequences. To enrich for tester-specific sequences, excess driver DNA was added in the hybridizations. The tester-specific sequences were then amplified by PCR and cloned into the plasmids pPCR or pZErO-2.1. Screening of the subtraction library revealed a number of DNA sequences unique to B. pseudomallei. Fifteen distinct plasmid inserts from the library were sequenced. The DNA inserts ranged from 100 to 800 bp in length and were found to contain an average G+C content of approximately 44 to 52%, which is considerably lower than the 68% G+C content of the B. pseudomallei chromosome. The DNA sequences were analyzed using the NCBI BLASTX program. One of the plasmid inserts, pDD1015, was found to share limited homology with WbpX, a glycosyltransferase, from Pseudomonas aeruginosa [43].
The 373-bp DNA insert from pDD1015 was cloned into a mobilizable suicide vector, pSKM11 [44]. The resulting plasmid, pSR1015, was mobilized into wildtype B. pseudomallei 1026b to create the mutant strain SR1015. Since the insert from pDD1015 was found to demonstrate homology to a glycosyltransferase from P. aeruginosa, it was postulated that it might encode a protein involved in carbohydrate synthesis. Since three carbohydrate structures had been previously purified and characterized, antibodies to each of these polysaccharides were available. To define the phenotype of SR1015, an ELISA was performed with the EPS-specific monoclonal antibody 3015, and B. pseudomallei 1026b and SR1015 were both found to contain EPS [45]. SR1015 was also shown to contain type II O-PS and to be serum resistant. Immunogold electron microscopy studies using rabbit polyclonal sera specific for a type I O-PS–flagellin conjugate was performed on the parent strain, 1026b, and SR1015 (Figure 1). B. pseudomallei 1026b reacted with antibodies to both flagellin and type I O-PS, as was evident by the distribution of gold particles around the bacterial surface and extending out along the flagella (Figure 1A). Unlike B. pseudomallei 1026b, SR1015 reacted only with the antibodies to flagellin, as the gold particles were found associated only with the flagella (Figure 1B). B. thailandensis, the negative control, did not react with the antibodies either to flagellin or to type I O-PS (Figure 1C). B. stabilis LMG7000 was also shown to react to the antibodies to type I O-PS, indicating this organism may produce a similar capsule (Figure 1D). Western blot analysis of proteinase K-digested whole cells from B. pseudomallei 1026b, B. thailandensis E264, and B. pseudomallei SR1015 using rabbit polyclonal sera raised to O-PS–flagellin protein conjugate confirmed the lack of type I O-PS in SR1015. Type I and type II O-PS were stained in B. pseudomallei 1026b, while only type II O-PS was stained in the lanes corresponding to B. pseudomallei SR1015 and B. thailandensis. These results indicated that we had identified and insertionally inactivated a gene involved in the synthesis of the type I O-PS of B. pseudomallei [42]. SR1015 was tested for virulence in the Syrian hamster model of acute septicemic melioidosis. The LD50 for SR1015 after 48 h was 3.5 x 105 CFU, while the LD50 of the parent strain, 1026b, was <10 CFU. The LD50 for SR1015 was similar to that for the weakly virulent B. thailandensis (6.8 x 105 CFU) [42]. This demonstrated that SR1015 is severely attenuated for virulence in this animal model of melioidosis and that type I O-PS is a major virulence determinant of B. pseudomallei. We later determined that the type I O-PS was a capsular polysaccharide (CPS I), not an O-PS moiety, which will be discussed below.
Immunogold electron microscopy of B. pseudomallei 1026b (A) and SR1015 (B), B. thailandensis E264 (C), and B. stabilis LMG7000 (D). Bacteria were reacted with polyclonal rabbit antiserum directed against an O-PS–flagellin protein conjugate absorbed with B. thailandensis E264 to remove the antibodies directed against type II O-PS, washed, and reacted with a goat anti-rabbit IgG-gold (5 nm) conjugate. Original magnification x330,000.
Two methods were used to clone the genes involved in the production and export of type I O-PS. The DNA flanking the insertion of pSR1015 was cloned from SR1015 and sequenced. We also used transposon mutagenesis to clone the genes involved in production of the polysaccharide; this was done to obtain any unlinked genes that may be involved in polysaccharide production. Approximately 1,300 transposon mutants were screened for loss of type I O-PS by ELISA. Six mutants were identified, and the DNA flanking the transposon insertion was cloned and sequenced. The Tn5-OT182 mutants SLR5, SLR8, SLR13, SLR18, and SLR19 mapped to the same region of the chromosome. Sequence analysis of the cloned fragments revealed the presence of 26 potential open reading frames involved in the synthesis and export of type I O-PS [42]. The open reading frames that predicted proteins involved in polysaccharide biosynthesis were found to demonstrate homology to proteins involved in the synthesis of a polysaccharide structure composed primarily of mannose. The other reading frames in the locus predicted proteins involved in the transport of capsular polysaccharides in a variety of bacteria, particularly those that produce group 2 and group 3 capsular polysaccharides [8]. The genes responsible for the production of type I O-PS were found to be similar to other loci encoding for capsular polysaccharides in that they are divergently transcribed [4]. The gene cluster involved in the production of this polysaccharide is also similar to group 3 capsule gene clusters in that there are no genes encoding KpsF and KpsU, which are present in group 2 capsule gene clusters [8]. However, the organization of the B.\n\t\t\t\t\tpseudomallei type I O-PS gene cluster differs in that it does not contain two export regions flanking a single biosynthetic region as seen in other group 3 capsule polysaccharide clusters [46]. The biosynthetic genes identified are not organized into one continuous transcriptional unit; instead, wcbB, manC, and wcbP are separated from the rest of the biosynthetic genes. The overall G+C content of this region is about 58%, lower than the G+C content of the rest of the chromosome (68%). The low G+C content in these clusters suggests that polysaccharide genes have a common origin and may have been transferred horizontally between species [9]. The genes involved in the production of this polysaccharide were named according to the bacterial polysaccharide gene nomenclature scheme [47]. The gene products associated with this cluster are shown in Figure 4. Mutations constructed in a number of these genes confirmed their role in the production of this polysaccharide [42].
The polysaccharide with the structure -3)-2-O-acetyl-6-deoxy-β-D-manno-heptopyranose-(1- was originally isolated and characterized as an O-PS component of LPS in B. pseudomallei and was designated type I O-PS [35]. However, our results suggested that this polysaccharide was a capsule rather than an O-PS moiety. The genes involved in the production of this capsule demonstrated strong homology to the genes involved in the production of capsular polysaccharides in many organisms, including N. meningitidis, H. influenzae, and E. coli. In addition, the export genes associated with this cluster are not associated with the previously characterized O-PS gene cluster [36]. Western blot analysis of proteinase K cell extracts and silver staining demonstrated that this polysaccharide has a high molecular mass (200 kDa) and lacks the banding pattern seen with O-PS moieties. Studies by our laboratory indicated that mutants in the production of the core oligosaccharide of the LPS are still capable of producing this polysaccharide [48]. Based on the above criteria and the genetic similarity to group 3 capsules, we proposed that this polysaccharide is a group 3 capsule and designated this capsule CPS I. This conclusion was further supported by Isshiki et al who separated this polysaccharide from a smooth lipopolysaccharide preparation of B. pseudomallei [49].
The role of CPS I in the pathogenicity of B. pseudomallei was investigated by performing further animal studies, serum bactericidal assays, complement protein C3b deposition assays, and radio-labelled phagocytic assays [50]. These experiments were facilitated by constructing a deletion strain harbouring a mutation in one of the CPS I genes and by complementation of this strain. An in-frame deletion was constructed in wcbB, a gene which encodes a glycosyltransferase, resulting in the capsule-minus strain SZ210. To confirm the role of wcbB in the biosynthesis of capsule, SZ210 was complemented by the introduction of a wild-type copy of the wcbB gene cloned into the mobilizable broad-host-range plasmid pBHR1 (MoBiTec). Western blot analysis of proteinase K-digested whole cells was performed using mouse monoclonal antibody directed to B. pseudomallei capsule to assess capsule production by these strains. Similar to the capsule minus strain SR1015 and B. thailandensis E264, which is known to lack this capsule, SZ210 was found to be negative for CPS I production, as indicated by the absence of a 200 kDa band that is present for wild-type 1026b. Complementation of SZ210 by providing the wild type wcbB gene in trans restored capsule production. Whole-cell extracts from the complemented strain SZ210(pSZ219) reacted to the capsule antibody producing the 200-kDa band corresponding to the B. pseudomallei capsule.
To establish a correlation between CPS I production and clinical infection a number of strains of B. pseudomallei isolated from a variety of clinical specimens were tested for capsule production by western blot analysis with polyclonal rabbit antisera to B. pseudomallei CPS I. Out of the 55 clinical strains tested for capsule production, 52 were found to produce this capsule. Three strains, 420a, 415c, and 375a were found to be negative for capsule production, similar to B. thailandensis E264. However, one of the capsule genes, wzt2, was successfully amplified from these three strains and following inoculation in the animal model, all three of these strains were found to produce capsule by western blot analysis. This indicated that CPS I production may be regulated in some strains and its expression may be induced in vivo. Therefore all of the 55 clinical strains of B. pseudomallei tested were found to produce capsule, establishing a 100% correlation between CPS I production and clinical infection [51].
Syrian golden hamsters were inoculated intraperitoneally with 101 to 105 cells of either wild type B. pseudomallei 1026b, capsule mutants SR1015 and SZ210, or the complemented strain SZ210(pSZ219). One group of animals inoculated with SR1015 also received 100 µg of purified B. pseudomallei capsule. After 48 h, the LD50 values were calculated, and the blood of the infected animals was diluted and plated for bacterial quantitation. The addition of purified capsule significantly increased the virulence of the capsule mutant strain SR1015. The LD50 value was calculated to be 34 CFU, similar to the LD50 value of wild-type B. pseudomallei 1026b (<10 CFU). In contrast, the LD50 value for SR1015 without the addition of purified capsule was calculated to be 3.5 x 105 CFU, 10,000- fold higher than when capsule was added to the inoculum. In addition, purified capsule enhanced the survival of SR1015 in the blood. Bacteria could not be detected in the blood of hamsters inoculated with SR1015 alone. However, the number of SR1015 CFU recovered from the blood of infected animals was 9.0 x 102 CFU/ml when capsule was added to the inoculum, an almost-1,000-fold increase. This number was comparable to the number of wild-type B. pseudomallei 1026b bacteria recovered from the blood. The addition of capsule was not toxic to the hamsters, as hamsters inoculated with 100 µg of purified capsule alone survived for the duration of the experiment without any ill effects. The LD50 value for the capsule mutant strain SZ210 containing an in frame deletion of the wcbB gene was calculated to be 9.6 x 104 CFU, and the number of bacteria in the blood was determined to be 10 CFU/ml. Complementation of this strain restored virulence in the animal model, resulting in an LD50 value of 12 CFU, comparable to that of wild type B. pseudomallei 1026b. Furthermore, the number of bacteria in the blood of animals infected with the complemented strain, SZ210(pSZ219), was determined to be 4.9 x 105 CFU/ml, similar to the number of bacteria recovered from animals infected with 1026b [50].
To further demonstrate the role of the capsule in infection by B. pseudomallei, an experiment was designed to investigate differences in tissue distribution between the capsule mutant strain and the wild type in infected hamsters. Animals were inoculated with 102 CFU of either wild-type B.\n\t\t\t\t\tpseudomallei 1026b or the capsule mutant SR1015. At different time points, the animals were sacrificed, and the numbers of bacteria in the blood, liver, lungs, and spleen of each animal were determined. As seen in Figure 2, the numbers of B. pseudomallei 1026b and SR1015 bacteria were nearly undetectable at 12 h (Figure 2A). By 24 h, the numbers of 1026b bacteria recovered from the blood, lung, liver, and spleen increased, while SR1015 was detected only in the spleen (Figure 2B). By 48 h, very high numbers of 1026b bacteria were recovered from all of the organs taken, representing a dramatic increase compared to the inoculum (Figure 2C). In contrast, all of the organs taken from hamsters infected with SR1015 contained fewer bacteria (Figure 2C). Of particular interest was the fact that the number of SR1015 bacteria recovered from the blood at 48 h was lower than in the inoculum, suggesting that the capsule mutant was cleared from the blood more effectively than the wild type. The number of SR1015 bacteria recovered from the spleen was higher than the number of SR1015 bacteria in the blood, suggesting that SR1015 was being cleared from the blood and sequestered in the spleen. The difference in virulence between the two strains can be attributed to capsule production, since the CPS I mutant strain was found to have a growth rate similar to that of the wild-type strain 1026b [50].
Differences in tissue distribution between B. pseudomallei strains 1026b and SR1015 in the Syrian hamster model of acute melioidosis. Female Syrian hamsters (three per group) were inoculated intraperitoneally with 102 CFU of either strain, and at 12, 24, and 48 h, two groups of animals were sacrificed and bacterial quantitation of the tissues was determined. The data represent the average number of bacteria found in each tissue and the standard deviation for a given time point.
To define the role of the capsule for persistence in the blood, serum bactericidal assays were performed with the addition of purified capsule to determine if capsule had an effect on the survival of serum-sensitive strains of B. pseudomallei. For these experiments, we utilized a double mutant that we constructed in the laboratory, SLR5, which lacks both capsule and O-polysaccharide, since the capsule mutant SR1015 was previously found to be serum resistant [42]. The survival of SLR5 was extremely poor when incubated in the presence of 30% normal human serum (NHS). However, the addition of purified capsule increased the survival of SLR5 in NHS. The addition of 50 µg of capsule to the reaction increased the numbers of SLR5 to 5.9 x 101 CFU/ml, and the addition of 100 µg of capsule increased the survival of SLR5 by nearly 1,000-fold to 1.9 x 103 CFU/ml. Furthermore, pre-incubation of 30% NHS with 100 µg of capsule (PI-CPS) before the addition of bacteria increased the survival of SLR5 100,000-fold to 4.4 x 106 CFU/ml. This was similar to the survival of SLR5 when incubated with serum that was heat-inactivated (HI-NHS). These effects were found to be specific to capsule, since the addition of 50 or 100 µg of purified B. pseudomallei O-PS or preincubation of the serum with O-PS did not increase the survival of serum-sensitive SLR5 [50].
Since capsule mutants of B. pseudomallei are serum resistant in that they are not susceptible to lysis by the membrane attack complex (MAC) because they still produce O-PS, we postulated that the ability of the capsule to enhance survival in the blood could be due to its ability to inhibit C3b deposition and opsonization. To investigate the effect of capsule on C3b deposition, the amount of C3b deposited on the surfaces of wild-type B. pseudomallei 1026b and the capsule mutant, SR1015, in the presence of serum was determined by Western blot analysis using a mouse monoclonal antibody specific to human complement factor C3b. The deposition of C3b was found to be more pronounced in the capsule mutant SR1015 than in the wild type in both 10 and 30% NHS. Similar results were observed with the capsule mutant SZ210, a strain containing an in-frame deletion of the wcbB gene. More C3b was detected when SZ210 was incubated in both 10 and 30% NHS than with 1026b. Optical densitometry measurements were performed in order to quantitate the difference in C3b deposition between the strains. The average amount of C3b deposited on the surfaces of SR1015 and SZ210 bacteria was 3.5-fold higher than for 1026b in 10% NHS and 2.5-fold higher in 30% NHS. In addition, there was a shift in the molecular mass of C3b, which normally runs at 185 kDa, indicating a covalent attachment of the molecule to the bacterial surface. The nature of this attachment was not investigated; however, C3b is thought to covalently attach to the bacterial surface through an ester or amide linkage [50, 52].
Immunofluorescence microscopy analysis was also performed to demonstrate the difference in C3b deposition between the capsule mutant and the wild type. The same experiment described above was performed, and samples were reacted with the mouse monoclonal antibody to human complement factor C3b, except that the samples were reacted with a secondary antibody conjugated to Cy3 and stained with DAPI for visualization of bacterial cells. As shown in Figure 3, the B. pseudomallei capsule mutant SR1015 demonstrated more reactivity to the antibody to human C3b in the presence of serum than the wild-type 1026b. This is evident from the red fluorescence that corresponds to the C3b bound to the bacterial surface surrounding the blue DAPI-stained cells seen when the capsule mutant was incubated in the presence of 10% NHS (Figure 3D to F). In contrast, the amount of red fluorescence surrounding the DAPI-stained wild-type cells was minimal in the presence of 10% NHS (Figure 3A to C). There was a dramatic difference in the amount of C3b deposited on the surface of the capsule mutant compared to the wild type, which was detectable after only 15 min of incubation of the bacteria with human serum (Figure 3B and E). By 60 min, there was some C3b deposition on wild-type B. pseudomallei; however, there was still more C3b deposited on the surface of the capsule mutant (Figure 3C and F) [50]. This experiment was not performed with 30% NHS due to excessive clumping of the samples during the fixation process, which resulted in inconsistent and poor staining of the cells. Western blot analysis was also performed to determine the amount of complement factor C3b deposition on the surface of B. thailandensis E264, a related nonpathogenic organism. The amount of C3b deposition in B. thailandensis E264 was more pronounced than with B. pseudomallei 1026b and was similar to the amount of C3b deposited on the surface of the capsule mutant, B. pseudomallei SR1015, in the presence of human serum. The amount of C3b deposition that occurred on the surface of B. thailandensis was expected, since the organism is known to lack this capsule [42, 50].
Immunofluorescence microscopy analysis of decreased complement factor C3b deposition in 10% normal human serum by B. pseudomallei capsule. B. pseudomallei 1026b and SR1015 were incubated in 10% normal human serum (NHS), reacted with a mouse monoclonal antibody to human complement factor C3b, reacted with a rabbit anti-mouse IgG conjugated to Cy3 (Jackson Laboratories), and stained with DAPI for visualization of whole bacterial cells (Sigma). (A) B. pseudomallei 1026b incubated in PBS; (B) 1026b incubated in 10% NHS for 15 min; (C) 1026b incubated in 10% NHS for 60 min; (D) B. pseudomallei SR1015 incubated in PBS; (E) SR1015 incubated in 10% NHS for 15 min; (F) SR1015 incubated in 10% NHS for 60 min. The blue fluorescence indicates the DAPI stained bacteria, and the red fluorescence indicates the binding of complement factor C3b to the bacterial surface.
The capsule mutant SR1015 was phagocytosed more significantly by PMNL than the wild-type strain. The proportion of wild-type B. pseudomallei 1026b phagocytosed in the presence of 10% NHS was 35.9%, while the proportion of the capsule mutant SR1015 phagocytosed was 51.7% (P < 0.001). When each strain was incubated in the presence of 30% NHS, 59.3% of the wild-type strain 1026b was phagocytosed by the PMNL after 30 min compared to 82.3% for the capsule mutant (P < 0.001) [50].
The lux reporter strain B. pseudomallei SZ211 was constructed by cloning an internal fragment of the wcbB gene into pGSV3-lux, a suicide vector containing the lux operon from Photorhabdus luminescens [53]. Absorbance (OD540) and luminescence (in relative light units) measurements were taken every 2 h. Capsule expression was higher in the presence of M9 plus 1% glucose plus 30% normal human serum (NHS) and M9 plus 1% glucose plus 30% heat-inactivated serum (HI-NHS) than in M9 plus 1% glucose alone. The increase in light production of SZ211 in the presence of serum supports the requirement for capsule for survival in serum. The strain B. pseudomallei SZ213 was constructed by cloning an internal region of the wbiA gene, which encodes an O-acetyltransferase required for O-acetylation of the O-PS component of B. pseudomallei LPS [36]. Light production of this strain was measured under the same conditions to determine whether LPS expression was induced in the presence of serum. Similar to the capsule, LPS expression was elevated in the presence of both 30% NHS and 30% HI-NHS. The levels of expression of both capsule and LPS were not significantly different in NHS and HI-NHS, suggesting that the environment of the serum may be required for induction of gene expression rather than complement [50].
Sequence analysis of the completed genome of B. pseudomallei revealed four operons with the predicted function of capsular polysaccharide biosynthesis and export [54]. One of these operons, with the gene identifiers BPSL2786-2810, corresponds to the previously characterized mannoheptose capsule designated CPS I [42, 50]. Three other operons were identified. These three capsule operons in the genome of B. pseudomallei were further analyzed using the BLAST program and Artemis. The operons are illustrated in Figure 4. The operon consisting of the genes BPSS0417-0429, was designated CPS II (Figure 4B). Another operon, BPSS1825-1835, was designated CPS III and the predicted homologues were investigated further (Figure 4C). A fourth operon, CPS IV, was found to contain genes that may be involved in the synthesis of a capsule with the gene identifiers BPSL2769-2785 (Figure 4D) [54, 55].
Organization of the chromosomal regions containing the genes comprising the B. pseudomallei capsule operons. The direction of transcription is represented by arrows, and the gene names demonstrating the highest degree of homology to the B.\n\t\t\t\t\t\tpseudomallei open reading frames are indicated. The relative sizes of each locus are indicated. (A) B. pseudomallei capsule cluster I (CPS I). (B) B. pseudomallei capsule cluster II (CPS II). (C) B.\n\t\t\t\t\t\tpseudomallei capsule cluster III (CPS III). (D) B. pseudomallei capsule cluster IV (CPS IV).
Comparative analysis of the genomes of three Burkholderia species, B. pseudomallei, B. mallei and B. thailandensis, was performed to determine whether all of the predicted B. pseudomallei capsule operons were present in B. mallei and B. thailandensis as well. CPS II, III, and IV were found to be present in B. pseudomallei and B. thailandensis, but not B. mallei. This is in contrast to CPS I, which is present in B. pseudomallei and B. mallei, but not B. thailandensis [42, 50, 54, 55, 56, 57]. CPS II was found to be identical between B. pseudomallei and B. thailandensis, but B. thailandensis was found to contain two flanking hypothetical genes not present in B. pseudomallei. The CPS II genes were found to be deleted entirely from B. mallei. A large chromosomal region ranging from open reading frames BPSS0404 to BPSS0491 including CPS II was shown to be deleted in B. mallei compared to B. pseudomallei and replaced with a large chromosomal region containing open reading frames BMAA0555 (IS407A orfB) to BMAA1784, a unique hypothetical protein not found in B. pseudomallei. The two genomes align with the presence of the alkyl hydroperoxidase reductase genes ahpC and ahpF, but these are organized in the opposite orientation in B. mallei compared to B. pseudomallei. The entire CPS III operon and flanking genes were shown to be the same in both B. pseudomallei and B. thailandensis. In contrast, the majority of the CPS III cluster was deleted from B. mallei with the exception of the wcaJ and manC genes, as well as the two flanking hypothetical genes on one side, and another hypothetical gene on the other side of the deleted region. The deletion of the CPS III genes in B. mallei was found to be replaced with the IS407A orfA and orfB genes. The entire CPS IV region was found to be replaced in B. mallei and flanked by two IS407A elements. The open reading frame BPSL2785, which encodes a hypothetical protein, is present in B. mallei (BMA2284.1) ATCC 23344 as well as a number of other B. mallei strains, but is organized in the opposite orientation. The genomes of B. pseudomallei, B. thailandensis, and B. mallei all diverge upstream of the CPS IV region, but all three organisms were found to align at the location of the ompA and hypothetical genes [55].
In order to assess the role of CPS III in virulence a mutant in the CPS III operon was tested for virulence compared to wild type B. pseudomallei in the Syrian hamster model of melioidosis. Syrian golden hamsters were inoculated intraperitoneally with 101 to 103 cells of either wild-type B.\n\t\t\t\t\tpseudomallei 1026b or the capsule mutant SZ1829. After 48 h, the LD50 values were determined. SZ1829 had LD50 values of <10 CFU, identical to that of wild type B. pseudomallei, indicating that this capsule is not required for virulence. In addition, the bacterial load in the blood of the infected hamsters was similar to that of wild-type and significantly higher than that of the non-pathogenic B. thailandensis E264 and the CPS I mutant, B. pseudomallei SR1015, both of which are incapable of establishing bacteremia [37, 50]. This indicates that CPS III does not contribute to persistence in the blood. Similar results were obtained for CPS II and CPS IV mutants, but we went on to further characterize CPS III.
A lux reporter strain was constructed in the CPS III operon by cloning an internal fragment of one of the genes into pGSV3-lux, a suicide vector containing a promoterless lux operon from Photorhabdus luminescens [53]. Regulation of this capsule in an environment similar to that encountered in the host was determined by growing the lux reporter strain, SZ1829, in the presence of M9 plus 1% glucose versus M9 plus 1% glucose plus 30% normal human serum (NHS). Absorbance (OD540) and luminescence (in relative light units) measurements were taken every hour. The expression of CPS III (SZ1829) was higher in M9 plus 1% glucose alone compared to M9 plus 1% glucose plus 30% NHS. The expression of SZ1829 was 3-4 fold lower in 30% NHS. This was in contrast to the expression of CPS I (SZ211) (see section 3.5), which was significantly more highly expressed in 30% NHS at a level of 3-4 fold compared to growth in M9 plus 1% glucose alone [55]. Although CPS III demonstrated higher expression initially in 30% NHS, this may have been due to the fact that the addition of NHS caused precipitation in the media which affected the optical density of the cultures.
The expression of the lux operon in reporter strains SZ211 (CPS I-) and SZ1829 (CPS III-) was also measured in water to determine whether CPS III was induced in this environment. Overnight cultures of SZ211 and SZ1829 were inoculated into sterile water and incubated at 37oC without shaking. Capsule expression was determined as described above, but the luminescence/absorbance calculations for water were compared to the values for these strains when grown in LB. CPS III was found to be induced in water compared to LB. The expression of SZ1829 was found to be significant with an increase of 2-3 fold over the course of the experiment. The expression of CPS I was found to be greater than 4 fold higher in LB compared to water [55].
Microarray analysis of capsule expression was performed using a low-density DNA microarray. RNA was isolated from the livers and lungs of hamsters infected with B. pseudomallei and from B. pseudomallei grown in LB. The results of the microarray experiment are shown in Table 1 [55]. The level of gene expression, or fold change, is represented as the ratio of gene expression in the hamster compared to growth in LB. As shown in Table 1, CPS III genes were not found to be significantly expressed in vivo since most of the fold changes were determined to be less than 2-fold. Many of the genes had negative fold change values, indicating that these genes are suppressed in the host environment. The highest fold change result was 2.337866 for BPSS1827, a predicted glucose-6-phosphate isomerase, which is still much lower than the fold changes observed for CPS I genes, which were significantly higher [58].
Gene ID | Predicted function | Fold Change (in vivo vs. in vitro) |
BPSS1825 | Glycosyltransferase | 1.757155 |
BPSS1826 | Glycosyltransferase | 0.093565 |
BPSS1827 | Glucose-6-phosphate isomerase | 2.337866 |
BPSS1828 | Glycosyltransferase | -0.66607 |
BPSS1829 | Glycosyltransferase | -0.18061 |
BPSS1830 | Capsule export, tyrosine-protein kinase | -0.30655 |
BPSS 1831 | Capsule export, outer membrane protein | 0.725849 |
BPSS1832 | Transport, tyrosine-protein phosphatase | 1.52665 |
BPSS1833 | UDP-glucose-6-dehydrogenase | -0.71411 |
BPSS1834 | Sugar transferase | -0.07963 |
BPSS 1835 | Mannose-1-phosphate guanyltransferase | -0.75075 |
Microarray analysis of B. pseudomallei CPS III expression following intraperitoneal inoculation in the hamster model of melioidosis.
Glycosyl composition analysis was performed on the purified capsule by combined gas chromatography/mass spectrometry (GC/MS). GC/MS results indicated that CPS III is composed of galactose, glucose, mannose, xylose, and rhamnose residues, with the highest proportion of carbohydrate being galactose and glucose. Glycosyl linkage analysis was also performed [55]. The predominant glycosyl residue detected was a terminally-linked heptopyranosyl (t-Hep) at a percentage of 23.2. Other residues detected were a terminally-linked and a 4-linked glucopyranosyl (t-Glcp) (4-Glcp) at percentages of 14.6 and 10.8, respectively [55].
Although significant advances have been made in the field, melioidosis continues to be a public health concern in many regions of the world [59]. Completion of the sequencing of the B. pseudomallei genome has revealed potential virulence determinants and comparative genomics between the genomes of B. pseudomallei, B. thailandensis, and B. mallei species has contributed to a better understanding of the organism. Further studies are ongoing to define the pathogenesis of B. pseudomallei and to identify effective vaccine candidates and diagnostic targets [54, 59].
To obtain virulence determinants unique to B. pseudomallei, we used subtractive hybridization between this organism and a related nonpathogenic organism, B. thailandensis. Analysis of the subtractive hybridization library revealed that B. pseudomallei contains a number of DNA sequences that are not found in B. thailandensis. One of the subtraction clones, pDD1015, demonstrated weak homology to a glycosyltransferase, WbpX, from P. aeruginosa [43]. The insert from pDD1015 was cloned into a mobilizable suicide vector for insertional inactivation of the glycosyltransferase gene in wild-type B. pseudomallei. The resulting strain, SR1015, was markedly less virulent than the parent strain in an animal model. We determined that SR1015 harbored a mutation in a glycosyltransferase gene involved in the production of a capsular polysaccharide which we subsequently designated as CPS I. We then identified the operon involved in the biosynthesis and transport of this capsular polysaccharide (CPS I) [42]. The genes identified encode for proteins that are similar to proteins involved in the biosynthesis and export of capsular polysaccharides, particularly those involved in the production of group 3 capsular polysaccharides. Group 3 capsules include the E. coli K10 capsule and may also include the H. influenzae group b capsule and the capsule produced by N. meningitidis serogroup B [8]. Group 3 capsules are always coexpressed with O serogroups, are not thermoregulated, are transported by an ABC-2 exporter system, and do not contain the kpsU and kpsF genes, and usually the gene clusters map near the serA locus [8]. Thus far, no serA locus that is associated with the type I O-PS cluster was identified, but this polysaccharide is coexpressed with O antigen and lacks the kpsU and kpsF genes, and genes encoding for a putative ABC-2 transporter have been identified. The genes involved in the production of group 3 capsules are organized into regions and are divergently transcribed. Regions 1 and 3 are generally conserved and contain genes involved in export of the polysaccharide. These regions flank region 2, which contains the biosynthetic genes and is not conserved between serotypes [4]. The genetic organization of the CPS I is also similar to that of other capsule gene clusters in that the genes are organized into more than one transcriptional unit and appear to be divergently transcribed [42].
The polysaccharide with the structure -3)-2-O-acetyl-6-deoxy-β-D-manno-heptopyranose-(1- was originally isolated and characterized as an O-PS component of LPS in B. pseudomallei and was designated type I O-PS [35]. However, our results suggested that this polysaccharide is a capsule rather than an O-PS moiety. The genes involved in the production of this capsule demonstrated strong homology to the genes involved in the production of capsular polysaccharides in many organisms, including N. meningitidis, H. influenzae, and E. coli. In addition, the export genes associated with this cluster are not associated with the previously characterized O-PS gene cluster [36]. Western blot analysis of proteinase K cell extracts and silver staining showed that this polysaccharide has a high molecular mass (200 kDa) and lacks the banding pattern seen with O-PS moieties. This conclusion was further supported by another group of researchers that demonstrated this polysaccharide is a capsule rather than an O-PS component of LPS because it lacks a lipid A moiety and was not capable of macrophage activation [49]. Studies by our laboratory have indicated that mutants in the production of the core oligosaccharide of the LPS are still capable of producing this polysaccharide [48]. Based on the above criteria and the genetic similarity to group 3 capsules, we proposed that this polysaccharide is a capsule.
Virulence genes of a number of pathogenic bacteria are located on pathogenicity islands (PAIs), regions on the bacterial chromosome that are present in the genome of pathogenic strains but rarely present in those of nonpathogenic strains. The PAIs may range in size from about 30 kb to 200 kb and often differ in G+C content from the remaining bacterial genome; the PAIs are often associated with the carriage of many virulence genes. These genetic units are often flanked by direct repeats and may be associated with tRNA genes or insertion sequence (IS) elements at their boundaries. They may also be associated with the presence of mobility genes, such as IS elements, integrases, transposases, and origins of plasmid replication. These DNA regions are considered to be unstable in that they may be subject to deletion with high frequency or undergo duplications and amplifications [7]. A number of PAIs have been described for both gram-positive and gram-negative bacteria, and the application of subtraction hybridization has been used to successfully identify such genetic elements [7]. The subtractive hybridization that was carried out between B. pseudomallei and B. thailandensis led to the identification of a number of sequences that were found to be A-T rich compared to the rest of the B. pseudomallei chromosome. This, combined with the fact that insertional mutagenesis of the glycosyltransferase gene identified by this method resulted in an avirulent strain, suggests that we may have identified DNA sequences from a putative PAI and that the capsular polysaccharide gene cluster may be located on this island. It is possible that B. pseudomallei, B. mallei, and B. stabilis acquired DNA encoding for capsule as well as other potential, yet unidentified virulence factors by horizontal transfer recently in evolution. B. pseudomallei and B. mallei are known to contain IS elements that are present in B. cepacia but not in B. thailandensis [56, 60].
Capsule production has been correlated with virulence in many bacteria, particularly those causing serious invasive infections of humans [61]. Our studies demonstrated that CPS I is critical for the virulence of B. pseudomallei [42, 50]. A number of functions have been suggested for polysaccharide capsules: prevention of desiccation for transmission and survival, adherence for colonization, resistance to complement-mediated phagocytosis and complement-mediated killing, and resistance to specific host immunity due to a poor antibody response to the capsule [4].
To establish a correlation between capsule production and clinical infection a number of B. pseudomallei strains isolated from clinical specimens were tested for CPS I production. All 55 strains tested were found to produce CPS I by western blot analysis [51]. In addition 10 strains of B. thailandensis were tested and found negative for CPS I production, confirming the importance of CPS I in virulence as well as clinical infection.
CPS I production by B. pseudomallei was shown to contribute to the persistence of the organism in the blood of the host. All CPS I mutants tested in the animal model could not be isolated from the blood following infection. The addition of purified capsule was shown to increase the virulence of the CPS I mutant strains SR1015 and SZ210 in the animal model. Differences in tissue distribution between wild type B. pseudomallei and SR1015 in infected hamsters indicated that SR1015 was cleared from the blood because the numbers of SR1015 in the blood of infected hamsters was 10,000-fold lower than that of wild type 1026b and lower than the initial inoculum of 100 cfu/ml [50].
CPS I production was shown to be responsible for persistence in the blood by evasion of the complement cascade and the mechanism for this was determined to be through the reduction of C3b deposition and opsonophagocytosis. The addition of purified CPS I to serum bactericidal assays showed that the capsule contributes to increased resistance of serum sensitive strains lacking the O-polysaccharide moiety (O-PS) of LPS to the bactericidal effects of normal human serum. However, CPS I mutants themselves were not found to be serum sensitive because they still produced O-PS, which was previously shown to be responsible for serum resistance, because it prevents lysis by the MAC complex [36]. This led us to postulate that CPS I was affecting the complement cascade through some other mechanism and it was found that this mechanism was through the reduction of C3b deposition and opsonization [50]. Both Western blot analysis and immunofluorescence microscopy experiments using a mouse monoclonal antibody to human C3b demonstrated the inhibition of C3b deposition by CPS I. In both experiments C3b deposition was more pronounced on the surface of the CPS I mutant compared to wild type. Also evident was that some C3b deposition occurred in the wild type, but this was expected since bacterial capsules are known to allow the diffusion of some C3b to the bacterial surface and B. pseudomallei is capable of activating the alternative pathway of complement culminating in the formation of the MAC complex [36, 62]. The accumulation of C3b affects the amplification step of the complement cascade and therefore, the less C3b deposited the less C5a is generated for phagocyte recruitment [63]. This explains the increased clearance of CPS I mutants from the blood. This conclusion was supported by the fact that B. thailandensis, the non-pathogenic organism which lacks CPS I, has been shown to be serum resistant, but is not capable of establishing a bacteremia in the Syrian hamster model of acute melioidosis [36, 37, 42]. Effective opsonization of invading bacteria results in enhanced phagocytosis and clearance of organisms form the blood of an infected host [52]. Quantitative radiolabelled phagocytic assays were also performed to establish a correlation between opsonization of the bacteria and phagocytosis by polymorphonuclear leukocytes. In the presence of serum, the CPS I mutant was more readily phagocytosed than wild type [50].
The expression of CPS I in the presence of normal human serum was found to be significantly elevated, also confirming that this capsule contributes to survival in the host. The presence of CPS I enables B.\n\t\t\t\tpseudomallei to survive in the blood through the inhibition of complement factor C3b deposition and phagocytosis [50]. The presence of this capsule facilitates survival as well as spreading to other organs, which can explain the overwhelming septicemia that is common in culture-positive melioidosis patients [64]. Therefore CPS I production is critical to the virulence of B. pseudomallei and further research will enhance the development of preventative strategies for melioidosis since this polysaccharide is one of the components of a B. pseudomallei subunit vaccine [28, 65].
Sequence analysis of the genome of B. pseudomallei revealed the presence of four operons possibly involved in polysaccharide capsule biosynthesis. One of these operons, (CPS I), corresponded to the previously identified and characterized mannoheptose capsule that was shown to be responsible for virulence and comprises one of the currently proposed melioidosis and glanders subunit conjugate vaccine [28, 66, 42, 50, 67]. The CPS I capsule cluster is present in the genome of B. mallei as well, but the complete cluster is not found in the genome of B. thailandensis [56, 57, 68]. This correlates with previous studies that have shown that this capsule is produced by B. mallei, but not by B. thailandensis [36, 37, 42, 56].
Three other putative capsule operons were identified by sequence analysis and all of these operons were found to be present in B. pseudomallei and B. thailandensis, but not B. mallei. Since these capsules are found in B. thailandensis and B. pseudomallei, they may be required for either survival in the host or in the environment; however, further studies are required to determine the roles of CPS II and CPS IV.
CPS III, located on chromosome 2, was found to contain 11 genes involved in the biosynthesis of a polysaccharide and was shown to be present in the genomes of B. pseudomallei and B. thailandensis, but not B. mallei. A mutation in the CPS III cluster did not affect production of CPS I and so it can be concluded that this operon encodes for gene products responsible for the biosynthesis of a separate capsule. CPS III was not found to contribute to the pathogenesis of B. pseudomallei. This capsule was not shown to be highly expressed in vivo by microarray analysis and was not required for virulence in the animal model. The CPS III mutant, SZ1829, which contains a mutation in the BPSS1829 gene as a result of insertional inactivation, was found to be as virulent in the animal model as wild type B. pseudomallei. The expression of this capsule was shown to be elevated when incubated in water, but suppressed in the presence of normal human serum [55]. The presence of the CPS III cluster in B. pseudomallei and B. thailandensis, both of which can survive for long periods in the environment compared to B. mallei, the increased expression of this capsule in water, and the low level of expression of this capsule in vivo, suggests that this capsule may contribute to the survival of B. pseudomallei in the environment [69].
Previous studies have demonstrated that B. pseudomallei produces three other capsular polysaccharides in addition to CPS I and these have been structurally characterized. One is an acidic polysaccharide with the structure, -3)-2-O-acetyl-β-D-Galp-(1-4)-α-D-Galp-(1-3)-β-D-Galp-(1-5)-β-D-KDOp-(2-, which is recognized by patient sera [32]. The other two are: a branched 1,4-linked glucan polymer ((CP-1a) and a triple-branched heptasaccharide repeating unit composed of rhamnose, mannose, galatose, glucose, and glucoronic acid (CP-2) [49]. Combined GC/MS analysis of CPS III revealed that the composition of this capsule demonstrates some similarity to the composition of the previously described capsule CP-2 composed of rhamnose, mannose, galactose, glucose, and glucoronic acid; however, the proportions of carbohydrate residues were not similar, and the CPS III capsule was also found to contain xylose and not glucoronic acid. In addition, CPS III was determined to be composed primarily of heptose [55]. Therefore it is evident that the capsule identified in this study is not one of the previously described capsule structures. Some of the previously characterized capsules produced by B. pseudomallei have been shown to be produced under unique conditions [32, 49]. Strain variation, differences in expression of the capsules, and discrepancies between purification strategies may also explain why a number of capsules have been shown to be produced by this organism. Nevertheless, the genes BPSS 1825-1835 appear to be involved in the biosynthesis of a capsule with this composition. Further analysis by 2D NMR would be required to definitively establish a connection between CPS III and one of the other published structures.
Studies by another laboratory have also focused on the presence of these capsule clusters in B. pseudomallei. Sarkar-Tyson et al. identified two polysaccharide clusters, one of which corresponds to the CPS III presented in this paper, but the authors identified this cluster as type IV O-PS (2007). The type IV O-PS was found to be involved in virulence in a mouse model [70]. However, a mutant in this polysaccharide did not demonstrate any difference in hydrophobicity compared to wild-type, indicating that this polysaccharide does not contribute to making the cell surface more hydrophobic, which is an advantageous characteristic for some pathogenic bacteria. The differences in virulence compared to the current work can be attributed to the use of different animal models; however, all other data seem to indicate that this capsule is not required for virulence.
A study was recently published which outlines the identification of another capsule produced by B. pseudomallei [71]. This capsule was determined to be composed of 1,3-linked α-D-mannose residues. This capsular polysaccharide was also found to be produced by B. mallei. The genes involved in the synthesis of this polysaccharide have not yet been identified and work is also underway to determine the role for this novel capsule in the pathogenesis of melioidosis and glanders.
B. pseudomallei is an environmental saprophyte often found in soil and stagnant water and incidence of the disease is high in rice farmers in Southeast Asia [22, 69]. This organism harbors a large genome which explains its ability to survive for long periods of time in the environment as well exist as a significant pathogen in both humans and animals. The presence of multiple polysaccharide clusters in the genome and the production of multiple capsule structures under differing conditions may contribute to the ability of this organism to adapt to a variety of conditions. As demonstrated in this study, capsule expression is dependent on the particular environment, which indicates that B. pseudomallei produces these capsules to promote a survival advantage either in the host or in the environment. Further studies aimed at characterizing the capsules of B. pseudomallei will be beneficial to understand the pathogenesis of this organism and to advance further vaccine development.
The author would like to thank Dr. Donald Woods and all colleagues in the Burkholderia pseudomallei research community for their support and discussions over the years. The work described in this chapter was funded by the following sources: Department of Defense contract DAMD 17-98-C-8003, the Medical Research Council of Canada, the Canadian Bacterial Diseases Network of Centers of Excellence, Canadian Institutes for Health Research MOP 36343, and a Research Incentive Grant from Athabasca University. Carbohydrate analysis was conducted by the Complex Carbohydrate Research Center at the University of Georgia and this was supported in part by the Department of Energy-funded (DE-FG09-93ER-20097) Center for Plant and Microbial Complex Carbohydrates. Microarray analysis was performed at the Southern Alberta Microarray Facility.
Stem cells are undifferentiated cells possessing greater capacity of self-renewal and multilineage differentiation potential. This makes them unique candidates for curing a diverse variety of human degenerative diseases. Based on their potency, stem cells are classified into three broad groups: embryonic stem cells (ESCs), fetal stem cells (FSCs), and adult stem cells (ASC). ESCs are pluripotent stem cells isolated from inner cell mass of blastocysts of human embryos. The uniqueness of ESCs lies in the fact that they are capable of differentiation into all three primary germ layers, i.e., ectoderm, endoderm, and mesoderm. However, due to high tendency of teratoma formation and ethical issues regarding the destruction of human embryos, the clinical applications of ESCs are restricted. Alternatively, fetal and adult tissue-derived stem cells are gaining popularity with little ethical concerns. Fetal stem cells can be isolated from extraembryonic tissues like cord blood, amniotic fluid, Wharton’s jelly, the placenta, and amniotic membrane [1, 2, 3]. However, adult stem cells (ASCs) are multipotent cells and are usually harvested from the bone marrow, adipose tissue, dental pulp, etc. All together, these cells possess clonogenic and self-renewing potential and plasticity to differentiate and often transdifferentiate into different tissue types.
Isolated stem cells from both adult and fetal tissues are multipotent and are recognized as MSCs. Notably, despite similar morphology and phenotypic properties, these tissue-specific MSCs have subtle differences in their regenerative potential due to the impact of stem cell niche on cell fate, known as stem cell niche theory, genetic variability, and/or epigenetic alterations [4]. Several studies have been carried out to show that there are differences in regenerative capacity of MSCs populations of the same passage number that have been isolated from different pockets of the body [5]. A recent comprehensive report also supports the hypothesis that tissue-specific MSCs express certain source-specific markers [6]. Dominic et al. established criteria to define MSCs on the basis of the following characteristics: (1) plastic-adherent cells; (2) expression of surface markers, CD73, CD90, CD105, and HLA-ABC, but negative expression from hematopoietic lineage-specific markers, CD34, CD45, CD14, CD11b, CD19, or HLA-DR; and (3) potential to differentiate into trilineage, i.e., osteoblast, adipocytes, and chondrocytes [7].
Due to their enhanced regenerative potential, the use of MSCs has become an emerging strategy for the treatment of injured or degenerated tissues. It was observed that in in vivo scenario, MSCs showed profound immunomodulatory effect [8]. The most important characteristic of MSCs is its immunomodulatory property which augments and modulates both adaptive and innate immune responses as it initiates the wound healing paradigm.
MSCs are also known for their immune-privileged property due to their low immunogenicity. Human MSCs show low levels of human leukocyte antigen (HLA) class I, and they do not express HLA-DR which is necessary to escape immune surveillance. The presence of HLA class I is important as low levels of HLA class I protect cells from the natural killer (NK) cell-mediated cytotoxicity. On the contrary, cells which do not express HLA class I are targeted and destroyed easily. Another essential characteristic is that they home and migrate to the site of damage where there is secretion of inflammatory chemokines. These events are mediated by several chemokine receptors which aid in their migration and homing potential to the sites of inflammation [9]. Owing to the immune tolerance property of MSCs, they possess several clinical advantages due to which these cells are also referred as “universal donors” [10, 11]. However, as true with any other cell-based therapy, the evaluation of safety and efficacy of these MSCs in allogeneic strategies for clinical use is of utmost importance (Figure 1).
A diagrammatic representation of cellular characteristics, mode of action, and their therapeutic potential of mesenchymal stem cells with current status of clinical trial.
The initial reports of immunoregulatory properties were started with bone marrow-derived MSCs (BM-MSCs) [12]. Later, other sources of MSCs such as adipose tissue-derived MSCs (AD-MSCs) and Wharton’s jelly-derived MSCs (WJ-MSCs) were also explored for their immunomodulatory properties [1].
However, several challenges need to be overcome prior to the clinical applications of MSCs. Hence, a thorough insight of the various biological properties of MSCs will elucidate the mechanisms of MSC-based transplantation for immunomodulation.
A key factor of survival in multicellular organisms is the maintenance and balance of homeostatic state. In the absence of inflammation, phagocytic cell is recruited to remove the apoptotic cells, whereas during acute injury, it is accompanied by inflammation, and the cell components that are released from necrotic cells result in microvascular damage due to increased vasopermeability and infiltration of macrophages and neutrophils [13]. During the process of phagocytosis of necrotic cells, there is secretion of pro-inflammatory mediators such as interleukin-1 (IL-1), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α), various chemokines which further initiate downstream signaling pathways [14]. Adaptive immune response actively participates in the repair of damaged tissues in close association with CD4+, CD8+, T, and B cells [13]. Recently, MSCs have been recognized to be actively involved in damaged tissue repair processes. As a functional unit for development and regeneration in various tissues, they hold utmost importance in maintaining proper functioning of tissues [15, 16]. In their undifferentiated and self-renewable state, the balance among interaction and protection of MSCs appears to be achieved by maintaining the stem cells in a specialized microenvironment called “niche.” This niche provides accommodation to different molecules to brace and coordinate stem cell activities pertaining to growth, proliferation, differentiation, and functionality. In particular, cell-cell interaction in the niche provides structural support, regulates adhesive interaction, and activates signals by secretion of certain molecules that can control stem cell functions. Stem cell immunoregulatory responses occur due to its close association with vasculature which provides metabolic cues and a conduit due to which inflammatory cytokines and immune cells, as well as humoral factors, can be delivered to the niche. In addition, the niche also provides biochemical and biomechanical parameters such as temperature, shear force, and chemical signals, which also influence stem cell behavior and fate in response to the external environment. In the process of tissue repair, MSCs are able to affect the inflamed microenvironment by secreting a cascade of various adhesion molecules, growth factors, and pro- and anti-inflammatory cytokines [17]. Since MSCs display notable immunomodulatory properties and they are able to dodge the immune system recognition mechanisms, they can potentially modulate the defense mechanisms of the host. In inflammatory condition, MSCs located in immediate location or originating from the bone marrow region start migrating to the site of injury. At the site, these MSCs associate themselves closely with numerous types of immune cells in order to initiate regeneration process of damaged tissue, which is typically accompanied with cytokine storm. The combinational sensitization of MSCs by IFN-γ and TNF-α induces the release of chemokines where they participate in chemotaxis and are able to inhibit proliferation of inflammatory effector cells. Several molecules participate in the activation, homing, and functionality of MSCs [18] (Figure 2). The commencement of homing process is led by selectins present on the endothelium. Specifically, for bone marrow homing, the expression of hematopoietic cell E-/L-selectin ligand (HCELL) is very important which is a functional glycoform of CD44 present on the migrating cells, while MSCs do not express HCELL but express CD44. The subsequent molecules participated in the activation of MSCs are mainly chemokine receptors [19, 20].
Immunoregulatory action of MSCs at chronic inflammation (left panel) versus acute inflammation (right panel). MSCs home to the injury site due to local cytokine storm secreted by activated immune cells. Activation and migration of MSCs lead to secretion of multiple immunomodulatory and growth factors. Depending on the cytokine signal (acute versus chronic inflammation), MSCs initiate the immunoregulatory response and repair the injury site or are unable to inhibit the persisting chronic inflammatory signals resulting in cellular fibrosis.
Chemokines are defined as positively charged short peptides (7–13 kDa). Broadly, four families of chemokines have been recognized: CCL family with adjacent cysteine residues, CXCL family with cysteine residues separated by a single amino acid, CXCL family with two instead of four cysteines, and CX3CL family with cysteines separated by three amino acids. For MSCs to home to injury site, these cells bear chemokine receptors and are identified at site of injury due to production of the chemokines. Hence a thorough understanding of the functioning of chemokine receptor profile is important for optimizing the process of both internal and external homing processes of MSCs to wound site. The site of injury produces abundant chemokines which may provide signal to MSCs or may function as chemoattractants [21]. During the process of regeneration or repair, the injury site tightly regulates the process of chemokine expression profile or expression pattern of each chemokine, which plays a unique role in directed migration of cells toward the site of injury. The subsequent expression of chemokines to attract specific immune cell types is conducted by immune response of injury.
Integrins are key players associated with the balance activation-dependent arrest of MSCs in the second last step of homing. They are known for cell-cell mediated matrix and adhesion, and they belong to the largest family of receptors. Mammals contain 18α and 18β subunits of integrin that combine to form at least 24 different heterodimers, each of which corresponds to a specific set of cell surface, extracellular matrix (ECM) or soluble protein ligands. They are multifaceted receptors, transmitting bidirectional signals across the cell membrane, which is crucial for building a suitable interaction between the exterior and interior of the cell. Numerous cell processes like morphology, migration, proliferation, differentiation, and apoptosis are unexpected on this recurrent discussion. According to few reports, it was suggested that MSC homing will be affected if integrin-β1 is inhibited. Also, interaction of vascular cell adhesion molecule-1-very late antigen-4 (VCAM1-VL4) is involved functionally in MSC homing [22].
During the last stage of transmigration across the endothelial cell layer and below the basement membrane, various lytic enzymes are essential to cleave components of the basement membrane, such as the matrix metalloproteinases (MMPs) [23]. Specifically, gelatinases, MMP-2 and MMP-9, preferentially degrade collagen and gelatin, two of the major components of the basement membrane which facilitate MSC migration. The MSCs reportedly help migrate MMP-2 and tissue inhibitor of metalloproteinases 3 (TIMP-3) [23].
The initial reports pointing to MSC homing were toward investigating the origin of BM-MSCs after allogeneic bone marrow transplantation. Those studies also concluded that the hematopoietic cell population was provided by the donor, but the stem cells were provided by the recipient [24]. Therefore, a number of trials at both the preclinical and clinical levels have been carried out, and MSCs are seen to help migrate in a variety of tissues. Initial studies in animal models also confirmed that the presence of MSCs transplanted to donor was present in the bone marrow, thymus, spleen, and liver [12, 24, 25].
To elucidate the dynamics of MSCs migration, a systemic infusion of MSCs was studied by using varied techniques, i.e., after infusion of MSCs, they were first trapped in the lungs, and eventually, the cells disappear from the lungs and are distributed to other organs. Other aspects of MSCs homing was also studied by few groups under which they studied the factors such as early cell passage, irradiation and younger animals and observed that they influence the short-term bone marrow homing and condition which in result increases the homing [26, 27, 28, 29, 30].
Once MSCs are activated and recruited to the site of injury, there is onset of T-cell activation because of the presence of various pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-1β, etc. IFN-γ is a critical player in providing stimulatory signals for activation and expansion of T cells and its subsets, such as it begins to suppress the T cell proliferation, differentiation, and inhibition of various biological functions. Other than IFN-γ, TNF-α and IL-1β also activate MSCs, either in synergy or alone. After stimulation with pro-inflammatory cytokines, MSCs also release other significant immunomodulatory factors. These stimulated cells modulate many immune effecters in vitro as well as in an animal model [31, 32].
To participate in tissue repair, MSCs must be in close association with several stromal and immune cells. The mode of action of MSC tissue repair is complex wherein MSC-derived immunoregulatory factors play a critical role.
MSCs are reported to release an array of growth factors and immunomodulatory molecules (Table 1).
Secretome profile of hMSCs and their functions.
To investigate how the inflammatory microenvironment modulated secretion of anti-inflammatory factors at the sites of tissue damage and it was concluded that MSC-mediated immunosuppression occurs in the microenvironment surrounding the MSCs: the inflammatory factors produced during the immune response act to turn on the immunosuppressive capacity of MSCs. For example, during pregnancy, the developed immunological tolerance along with the fetus development highlights the key role of fetus-derived MSCs. These pleotropic cells inhibit a group of cells involved in innate and adaptive immunity such as B cells, dendritic cells (DC), macrophages, and various effector cells such as NK, CD4+ T, CD8+ T, regulatory T (Treg), and NKT cells [33, 34]. The contribution of MSC-derived molecules toward the immunoregulation has been discussed (Figure 3).
Representative image shows the immune response of MSCs by secretion of IM factors (left panel). Immunoregulatory function of MSCs on different cell types of the innate and adaptive immune cells. (Right panel) paracrine effect of MSCs through secretion of exosomes and their fusion with the target cell membrane and release of the biological active content for immunomodulatory effect.
Indoleamine-2,3-dioxygenase is a mammalian cytosolic enzyme responsible for catalyzing the initial step in tryptophan catabolism via the kynurenine degradation pathways. IDO is comprised of two alpha helical domains with a heme group located between them and is an essential amino acid which catalyzes the rate-limiting step in the degradation of tryptophan, with the kynurenine pathway [35]. Any reduction in the concentration of local tryptophan or its metabolite results in immunomodulatory effect by IDO expressing cells.
The studies carried out with placental cells showed that they are capable of preventing maternal T-cell destruction of the fetus during pregnancy, which happens due to the expression of IDO in placental cells. During pregnancy, the fetus expresses paternal antigens that do not provoke rejection by the mother like other semi-allogeneic grafts [36, 37]. Dendritic cells can also express IDO and thus induce a tolerogenic response. Su et al. suggest that MSCs do not have the innate ability to express IDO but gain this ability following stimulation by the pro-inflammatory cytokines IFN-γ and TNF-α in combination with IL-1β [38]. Recently, the role of IDO in MSC-mediated immunoregulation has been demonstrated in the suppression of various immune cell populations, including T cells and NK cells [39, 40].
HLA-G is a major histocompatibility complex class I antigen encoded by a gene on chromosome 6p21. It differs from classical HLA class I molecules by its restricted tissue distribution and limited polymorphism in the coding region. HLA-G can be expressed as seven distinct protein isoforms, each encoded by a specific, alternatively spliced transcript. Four isoforms are membrane-bound proteins (HLA-G1, HLA-G2, HLA-G3, and HLA-G4), and the other three isoforms are soluble proteins (HLA-G5, HLA-G6, and HLA-G7) [41]. It exerts its immunomodulatory functions by interacting with multiple receptors such as LILRB1(ILT2/CD85j), LILRB2 (ILT4/CD85d), and KIR2DL4 (CD158d) which are differentially expressed by immune cells. Besides these receptors, HLA-G can also bind to CD8 without T-cell receptor (TCR) interaction, provoking NK cells and activated CD8 + T cell-induced apoptosis as well as FASL upregulation and secretion [42, 43]. HLA-G plays a fundamental role in maternal tolerance and transplantation. HLA-G expression by MSCs can be positively modulated by IL-10 and leukemia inhibitory factor (LIF). Other molecules such as glucocorticoid and interferon-β (IFN-β) are found to regulate HLA-G expression in immune cells. HLA-G has been investigated for allogeneic solid organ transplantation and has been well associated with reduced number of immune rejection cases in kidney and liver allogeneic transplantations [44, 45].
Prostaglandins are small molecule derivatives of arachidonic acid (AA), produced by cyclooxygenase (COX, constitutively active cyclooxygenase COX1 and inducible COX2) and PG synthases. It can be produced by all cell types of the body, with epithelia, fibroblast, and infiltrating inflammatory cells representing the major sources of PGE-2 in the course of an immune response. The receptors of PGE2 (EP1–EP4) are present on multiple cell types, reflecting the ubiquitous function of PGE-2. It is relatively stable in vitro although its decay is accelerated by albumin [46]. In contrast, PGE-2 has a very rapid throughput rate in in vivo conditions and is quickly eliminated from tissues and circulation. This property of PGE-2 is most likely to contribute toward immune pathology and constitutes a potential target for immunomodulation. It is worth noting that the effect of PGE-2 in MSC-mediated immunoregulation in most cases is exerted in combination with other immunosuppressive molecules. With human MSCs, PGE-2 has been found to act with IDO to alter T-cell proliferation, during proliferation, cytotoxicity, and cytokine production by NK cells [47].
Nitric oxide synthases are family of enzymes catalyzing the production of nitric oxide from L-arginine. The enzymes convert arginine into citrulline and produce NO in the process. NO activity is independent of the level of calcium in the cell. However, its activity as other NO isoforms is dependent upon the binding of calmodulin (CaM). NO in high concentration is known to inhibit immune responses through mechanisms that remain largely unidentified. In addition, upon induction cytokines such as TNF-α and IFN-γ, alone or in combination, stimulate NO. This has a significant impact on both primary and secondary immune responses. For example, NO targets dendritic cells (DCs) that have a crucial role in making powerful immune response. It was found to prevent maturation of rat lung DCs by inhibiting granulocyte-macrophage colony-stimulating factors. Similarly, NO inhibits TNF-α and prevents DC maturation in humans [48]. MSCs produce large amounts of chemokines and adhesion molecules; immune cells accumulate in close proximity to the MSCs, where the high concentration of secreted NO can suppress the immune cells [49, 50].
IL-10 is produced by both myeloid and lymphoid cells. While it is good immune suppressor, it has some immune stimulatory effects. IL-10 is recognized by its effect on T cells, macrophages, and monocytes which ultimately prohibit inflammatory responses. Thus, it regulates growth and differentiation of B cells, T cells, NK cells, and other cells of the immune system hence influencing inflammatory responses. IL-10 has the capability to inhibit the production of IL-2, TNF-α, IL-12, and IFN-γ. Furthermore, it will downregulate HLA class I. Although IL-10 has been implicated in MSC-mediated immunosuppression, direct IL-10 production by MSCs has not been demonstrated so far. Instead, contact of antigen-presenting cells such as dendritic cells or monocytes with MSCs has been found to induce IL-10 production [51, 52, 53].
In addition to the above molecules, several additional mediators are produced by MSCs or other adult stem/progenitor cells upon inflammatory stimulation, such as the inhibitory surface protein programmed death ligand 1 (PD-L1) [54], heme oxygenase-1 (HO-1) [55], leukemia inhibitory factor (LIF), galectins [56], and TGF-β [57]. However, their modes of action and underlying molecular mechanisms that drive MSC-mediated immunosuppression require further investigation.
The antigen-specific immune system allows the development of immunological memory. It comprises of CD4+ T helper and CD8+ cytotoxic T lymphocytes that deliver a customized antigen-specific immune response following antigen processing and presentation by antigen-presenting cells (APCs). T helper cells comprise a subpopulation of cells called Tregs, which are specialized in suppression of T cell-mediated immune response [58]. The innate immune system plays an important role in the activation and subsequent course of adaptive immune response [59]. In addition, MSCs are able to suppress in vitro T-cell proliferation induced by cellular or non-specific mitogenic stimuli through the secretion of various soluble factors that include (transforming growth factor-beta 1) TGF-β, HGF, PGE-2, IDO, HLA-G5, and NO. The effect of these suppressive factors is upregulated by pre-sensitization of MSCs with TNF-α and IFN-γ. It is also known that MSCs polarize T cells toward a regulatory phenotype that serves as an important mechanism by which MSCs dampen inflammation [60, 61]. Tregs comprise a subpopulation of T helper cells, which are specialized in suppression of T cell-mediated immune response and characteristically express the forkhead box P3 (Fox P3) transcription factor. These are two main subsets of Tregs including a population of Fox P3+ natural Tregs which are thymus derived and specific for self-antigen and induced or adaptive Tregs that are derived from mature CD4 + CD35-FoxP3 precursors in the periphery following inflammatory stimuli. The in vitro co-culturing of MSCs with PBMNCs induced the differentiation of CD4+ T cells into CD25 + FoxP3+ expressing regulatory T cells [40, 62]. The possible reason of abovementioned mechanism is due to cell-cell contact of MSCs with helper T cells and secretion of PGE-2 and TGF-β. All together, these studies indicate that MSCs are able to maintain the balance between inflammatory effector T cells and anti-inflammatory Tregs.
B cells are also a major cell type involved in adaptive immune response, known for antigen presentation and antibody production. The balance between the different B-cell subsets has been identified as an important factor for optimal graft outcomes. To support the beneficial effect of B-cell depletion at the time of transplantation to impair T cell-mediated allo-response, the CD8 and CD4 T-cell memory is impaired when the antigen-presenting function of B cells is absent [63]. The exposure of enriched B-cell population to irradiated third party PBMNCs led to an increase in immunoglobulin (Ig) production that was abrogated by the addition of MSCs. There are diverse results among the studies to analyze the effect of MSCs upon exposure of isolated pure B cells [64]. These effects have been shown to be cell-cell contact independent or indirect through inhibition of pDC-induced B-cell maturation. On exposure, MSCs increased the viability of B cells and mediated the arrest of cell cycle at G0/G1 and inhibition of their differentiation into plasma cells and subsequent Ig formation, whereas it was observed that pre-treatment of MSCs with IFN-γ was necessary for their suppressive effect on B cells [66]. The activated B cells and memory B-cell subsets when exposed to MSCs were seen to increase their survival and proliferation [65]. The studies carried out by Schu et al. [66] showed that when allogeneic MSCs were injected into rats, a strong humoral response was elicited as compared to injection with syngeneic cells in an immunocompetent host.
To support whether the allogeneic MSCs exert a humoral response in the recipient to prove the notion, they performed the experiment in which a rat was injected with allogeneic MSCs; on the other hand, contradictory reports were also published stating after transplantation none of them developed anti-MSC antibodies [66]. These studies indicate some disparity of humoral response directed against the injected MSCs, and possible reasons may be the source of MSCs, number of injected cells and frequency of injections, route of administration, or concurrent immunosuppression used.
Natural killer cells or NK cells are a type of cytotoxic lymphocytes critical to the innate immune system, evolve as progenitors in the bone marrow, and circulate as mature cells in the blood. They provide rapid responses to viral-infected cells, acting 3 days after infection, and respond to tumor formation. They play a major role in the mechanisms of rejection of graft and are central to the regulation of cytotoxicity in response to human leukocyte antigen molecule. With increasing trends in therapeutic usage of MSCs for treatment of GvHD, it is important to investigate the underlying effects of interaction of MSC and NK cells. They function in the manner that they get activated and inhibited on cell surface because of receptors transmitting the signal into the cell. Usually, NK cell possesses regulatory functions and can secrete cytokines and chemokines which modulate the host’s immune response. IL-12 is the most important pro-inflammatory factor which responds to penetrating pathogens and acts through its high affinity receptors. It is released from accessory cells like monocytes, macrophages, and dendritic cells (DCs). Also, the most important cytokine released by NK cells is IFN-γ which is produced upon stimulation of IL-12. The NK cell-derived IFN-y reinforces the expression of IL-12 and DCs via feedback mechanism. BM-MSCs directly interfere with the proliferation, cytokine production, and in some cases cytotoxicity of NK cells. MSC-NK interactions are complex and largely dependent on the microenvironment and activation status of the NK cells. Mainly, MSCs suppress the production of IL-2, IL-15, and INF-γ but not the cytotoxicity of freshly isolated NK cells. In addition, when activated NK cells come into contact with the MSCs, it interferes with NK-mediated cytotoxicity which is primarily mediated by cell-cell contact and secretion of IDO, PGE-2, TGF-β1, and HLA-G5. Other reports mentioned that when licensed MSCs were exposed to IFN-γ, they are protected from NK-mediated cell killing, potentially due to their upregulated cell surface expression of HLA-I and downregulation of ULBP-3. This alongside an increased production of both IDO and PGE-2 offers multiple mechanisms for dampening NK responsiveness to the MSCs [59, 67, 68].
The potent antigen-presenting cells (APCs) and dendritic cells (DCs) play a pivotal role in initiating immune response. The life span of DCs can be divided into two major phases, an immature stage and a mature stage. These phases can be differentiated further on the basis of molecules expressed (CD80, CD86, OX62, HLA-II, and CD11b/c) on their surface. DCs can be immunostimulatory or immunosuppressive, depending upon their maturation stage and specific DC subset. Immature DCs (iDCs) express low levels of HLA-II but no co-stimulatory molecules. The interaction of MSCs with DCs leads to the inhibition of maturation of monocytes and CD34+ precursor cells. Moreover, the direct activation of DCs leads to the release of PGE-2, IL-6, TSG-6, MCSF, and jagged-2 mediated signaling. Tolerogenic phenotype occurs when DC secretome of pro-inflammatory cytokines (TNF-α and IL-12) shifts toward anti-inflammatory IL-10 in which further downstream induces Th2 and Treg responses [69, 70, 71].
Advances in stem cell technology have opened interesting perspectives within the realm of regenerative medicine. As reported, MSCs participate in repair and regenerative processes via different mechanisms like homing and transdifferentiation and immunomodulation, which depends on paracrine mechanisms [72, 73]. The initial studies using MSCs were based on local engrafting of MSCs and differentiating into multiple tissue types. However, with the in-depth study of different mechanisms of MSC action, it has been reported that <1% MSCs are able to survive transiently after systemic administration [74]. This suggests that paracrine mechanisms through secretion of various molecules called secretome might be the possible mechanism for MSC regenerative potential. This has attracted significant attention for the potential use of MSC secretome in tissue repair and regeneration.
The secretome released by MSCs includes various biologically active growth factors and cytokines which aid in immunomodulatory properties of MSCs [75]. However, we cannot neglect the fact that the secretome released in the milieu of ECM of the cells is an easy target for denaturation due to the presence of proteases and other enzymes in the microenvironmental niche. Therefore, these growth factors and cytokines have been shown to be packed into small vesicles called exosomes which are secreted by MSCs in the extracellular milieu of cell along with the secretome [76]. They function through encapsulation of biological active molecules such as miRNA, proteins, and immunomodulatory molecules and protect them from degradation.
Exosomes are lipid membrane-bound extracellular vesicles which possess a diameter of 30–120 nm and a density of 1.09–1.18 g/mL and are secreted by all cell types. These exosomes carry cellular components like proteins and nucleic acids and aid in cell-cell communication. The exosome was first discovered in 1984 by Johnstone in sheep reticulocytes [77]. It was initially believed that exosomes remove unwanted proteins from cells. Later on, it was demonstrated that many other cell types also secrete exosomes including immune cells, cancer cells, stem cells, and many more [78].
Exosomes are endosomal in origin, formed within multivesicular endosomes (MVEs). These vesicles are being released when membranes of MVEs fuse with that of the cellular plasma membrane. These exosomes express various surface markers like CD63, CD81, and CD9. They carry surface molecules that are present on the parent cell which aids in identification of exosomes and their parent cell source as well [77].
Moreover, the exosomes secreted by stem cells carry various proteins (growth factors and cytokines) and nucleic acids (mRNA and miRNA) that can influence their mode of action [79]. The content carried by these vesicles depends on the type of cell (including source of cells) and its state of activation. Once released, these vesicles have local as well as remote effect by interacting with the neighboring cells or by circulating in the body fluids (bloodstream, saliva, serum, etc.).
The exosomes were studied for their multifaceted application in antigen presentation, and vastly studied immune cell was dendritic cells. The clinical studies have been conducted to evaluate the dendritic cell derived exosomes for their therapeutic potential. However, compared to preclinical studies, only a few clinical trials have been conducted using exosomes. Reported studies were conducted where dendritic cell-derived exosomes were evaluated for their safety, tolerability, and efficacy in cancer patients. Exosomes carry parental cell surface marker expression. In this regard, DC derived exosomes are HLA-II positive as a result they can only be used in patient specific studies [80, 81]. In contrast, MSCs are immunologically naïve as they express only HLA class I molecules and lack HLA class II, CD40, CD80 and CD86 expression on their cell surface. Also, they are capable of immune escape and fail to induce an immune response by the transplanted host. Similarly, exosomes secreted by them are also immunologically naïve [82]. Considering all of the above properties, several recent studies have focused their research on evaluation of stem cell-derived exosomes in the area of immunomodulation with fewer reports.
In a recent study, exosomes derived from MSCs were specifically identified to mimic the effect of MSCs, and this paved the way to cell-free therapeutic approach using exosomes instead of the cell itself [83]. The first report using MSC exosomes were in cardiovascular diseases where Lai et al. [85] identified exosomes as the cardioprotective components in MSC paracrine secretion [84]. This was followed by several other studies where exosomes isolated from tissue-specific MSCs were studied for their therapeutic potential in various diseases.
Initial studies were performed on bone marrow-derived exosomes for evaluating their regenerative potential in cardiovascular diseases [85], acute kidney injury [86], bone defects, etc. [87]. By 2013, only researchers started exploring the regenerative potential of exosomes derived from adipose tissue and Wharton’s jelly sources. These studies have explored the various mechanisms by which these exosomes mimic MSCs. The content of these exosomes was evaluated by using various techniques like RNA sequencing, mass spectrometry, etc., to identify different molecules and their target effect.
Conforti et al. reported the effect of MSC-derived vesicles on B-cell proliferation which was further confirmed by Di Trapani’s group in 2016 [88, 89] . They observed that exosomes had higher levels of miRNAs compared to MSCs and induce inflammatory priming via increasing levels of miR-155 and miR-146. These are two miRNAs involved in the activation and inhibition of inflammatory reactions. Similar studies were reported where MSC-derived exosomes were shown to increase the ratio between regulatory and effector T cells along with the increase in cytokine such as IL-10 [90]. Similarly, Chen et al. [91] has also reported immunomodulatory effects of MSC-derived exosomes toward peripheral blood mononuclear cells (PBMNCs) focusing specifically on T cells. It was observed that there was significant inhibition of pro-inflammatory cytokines, IL-1β and TNF-α, but enhancement of the expression of anti-inflammatory cytokine, TGF-β1. This cytokine profile in their study mimics the immunomodulatory effect of MSCs [91]. Zhang et al. showed that these exosomes may polarize monocytes toward M2-like phenotype, which in turn induces CD4+ T-cell differentiation into regulatory T cells [92].
Blazquez et al. demonstrated AD-MSC-derived exosomes as a therapeutic agent for the treatment of inflammation-related diseases. They showed that exosomes exerted an inhibitory effect on the differentiation of activated T cells, reduced T-cell proliferation, and IFN-γ secretion in an in vitro stimulated T-cell model [93]. Favaro et al. has shown the effect of BM-MSC-derived exosomes on PBMNCs isolated from type I diabetic patients. These exosomes were able to inhibit the IFN-γ production and significantly increased the production of immunomodulatory mediators such as PGE-2, TGF-β, IL-10, and IL-6 [94]. The in vitro studies were complemented by the in vivo studies which confirmed the immunosuppressive effect of exosomes in mouse allogeneic skin grafting models [95]. Bai et al 2017 have subcutaneously administered exosomes isolated from human embryonic stem cell-derived MSCs and showed that there was delayed occurrence of GvHD for 2 days, concomitant with increasing Treg polarization. In continuation, the author has also demonstrated that exosomes released from WJ-MSCs can effectively ameliorate experimental autoimmune uveoretinitis (EAU) in rats by inhibiting the migration of inflammatory cells [95]. Moreover, there is only single case study on humans in which MSC exosomes have been tested in the treatment of resistant grade IV acute GvHD patient which experienced improvement in symptoms for 5 months. There were no side effects reported, and the decrease of pro-inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ was observed. The anti-inflammatory molecules IL-10, TGF-β1, and HLA-G contained in the exosome preparations were believed to contribute to the immunosuppressive effect of MSC-Exo [96].
Although there are limited studies available using MSC-derived exosomes, future advancements into research and gain in in-depth knowledge of immunomodulatory properties of these MSC exosomes could be seen. These nano-vesicles can be developed as cell-free therapies. The use of these exosomes as cell-free therapies provides following key advantages:
Less tumorigenicity.
Easy storage without application of potentially toxic cryopreservative agents.
Mass production through tailor-made cell lines.
Potential to be used as ready-to-go biologic product.
Off-the-shelf secretome therapies.
The time and cost of expansion and maintenance could be greatly reduced.
The biological product obtained could be modified to desired cell-specific effects.
Despite these advantages of MSC-derived exosomes, there has been a lack of manufacturing process that is required to generate exosomes with clinically relevant quantities. Therefore, there is an urgent need for technological advancements. Nevertheless, regulatory requirements will be necessary to establish the safety and efficacy profile of these exosome products.
MSCs delivered alone or with a biomaterial have been used in a variety of regenerative medicine strategies. In vivo evidence supports the hypothesis that MSCs have immunosuppressive properties that include prevention of graft versus host disease (GvHD), decreased graft rejection, prevention of experimental acute encephalomyelitis, prolonged skin graft survival, etc. [8]. However, in recent years, consistent reports on its immunomodulatory properties have opened up newer avenues for studying MSCs, other than regenerative medicine. As of May 2018, there were over 843 MSC-related clinical trials registered on the NIH Clinical Trial Database (
Apart from GvHD condition, multiple sclerosis (MS), joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA), inflammatory bowel diseases (IBD) and inflammatory airway, and pulmonary diseases are few examples of inflammatory diseases in which preclinical studies have established strong therapeutic effect of MSCs [101]. In multiple sclerosis, reports showed that MSC treatment increases accumulation of Th2 cytokines-IL-4 and IL-5 and generation of Treg in vivo both of which help reduce EAE symptomatology. The possible molecular mechanism by which MSCs polarize CD4 T cells in EAE is via IDO. Indeed, both small and large animal studies demonstrate that MSCs decrease inflammation in joint diseases and facilitate cartilage repair [102].
The critical part of IBD is the uncontrolled immune response to intestinal microbes, and it is progressively fatal without curative treatment, making MSCs an attractive therapeutic option for these chronic inflammatory diseases. In several experimental models of IBD, MSCs given by intraperitoneal or intravenous routes showed prevention of DSS-induced injury of the intestines. It was observed that MSCs can specifically reduce Th1 and Th17 responses as well as serum level of pro-inflammatory cytokines (IL-1b, IL-6, IL-17, TNF-α, and IFN-γ) while enhancing the numbers of Tregs and splenic (myeloid-derived suppressor cells) MDSCs. A very recent trial using allogeneic placenta-derived MSC-like cells (which were not registered) also showed favorable immune responses. Thus, MSC therapy for IBD—especially CD fistula formation—appears to be safe and a viable option [103, 104].
Pulmonary diseases like chronic obstructive lung disease (COPD) are driven by alveolar macrophages, cytotoxic T cells, and neutrophils leading to progressive limitations in airflow with small airway fibrosis and alveolar destruction. In in vivo studies documented post-MSC infusion showed downregulation of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 and upregulation of VEGF and TGF-β [105, 106]. In addition, MSCs or MSC-conditioned medium improved tissue damage and survival. This involves MSC-derived factors with microvesicles such as exosomes, which are considered as carriers.
MSCs are excellent candidates for therapeutic use as cellular therapies can potentially revolutionize the current pharmaceutical landscape. Emerging data suggests that MSCs have an immunomodulatory function, but thorough understanding of the mechanisms underlying the complex molecular interplay between MSCs and inflammatory responses will be crucial for exploiting MSC-based therapies in therapeutic applications. One important aspect is to delineate functional differences in tissue-specific MSCs isolated from different sources; current ISCT standardization does not include immune-related functional tests or more detailed molecular validation. Based on the evidence of several clinical trials, the safety of this therapy appears clear; however, the efficacy of such cell therapy is largely uncertain. The overwhelming positive results seen in preclinical animal studies have not yet been translated into clinic. In brief, there is still much to learn, explore, and optimize with regard to the interactions of MSCs in human pathological conditions. In the near future, based on current development and results, MSCs are expected to hold tremendous potential to achieve clinical relevance in regenerative therapy.
The authors would like to thank Mr. Manish Prajapati, for his expert help in designing the graphics for this book chapter. Authors would also like to thank Dr. Rituparna Chaudhari and Dr. Swati Midha for their critical evaluation of this book chapter.
The authors declare that they have no competing interest.
ESC | embryonic stem cell |
ASC | adult stem cell |
MSC | mesenchymal stem cell |
ISCT | International Society for Cellular Therapy |
HLA | human leukocyte antigen |
NK Cells | natural killer cells |
BM-MSCs | bone marrow-derived MSCs |
AD-MSCs | adipose tissue-derived MSCs |
WJ-MSCs | Wharton’s jelly-derived MSCs |
INF-γ | interferon gamma |
TNF-α | tumor necrosis factor-α |
IL-1 | interleukin-1 |
HCELL | hematopoietic cell E-/L-selectin ligand |
ECM | extracellular matrix |
VCAM1 | vascular cell adhesion molecule-1 |
VL4 | very late antigen-4 |
MMP | matrix metalloproteinases |
TIMP-3 | tissue inhibitor of metalloproteinases 3 |
NKT | natural killer T cell |
DC | dendritic cells |
Treg | regulatory T cells |
IDO | indoleamine-2,3-dioxygenase |
HLA-G | human leukocyte antigen-G |
LILRB1 | leukocyte immunoglobulin-like receptor B1 |
LILRB2 | leukocyte immunoglobulin-like receptor B2 |
KIR2DL4 | killer cell immunoglobulin like receptor |
TCR | T-cell receptor |
IFN-beta | interferon-B |
PGE-2 | prostaglandin E2 |
AA | arachidonic acid |
COX | cyclooxygenase |
iNOs | inducible nitric oxide synthase |
CaM | calmodulin |
PD-L1 | protein programmed death ligand 1 |
HO-1 | heme oxygenase-1 |
LIF | leukemia inhibitory factor |
APCs | antigen-presenting cells |
TGF-β1 | transforming growth factor-beta 1 |
HGF | hepatocyte growth factor |
FoxP3 | forkhead box P3 |
PBMNCs | peripheral blood mononuclear cells |
Ig | immunoglobulin |
GvHD | graft versus host disease |
ULBP-3 | UL16-binding protein 3 (ULBP3) |
MVEs | multivesicular endosomes |
EAU | autoimmune uveoretinitis |
HSCs | hematopoietic stem cells |
MS | multiple sclerosis |
OA | osteoarthritis |
RA | rheumatoid arthritis |
IBD | inflammatory bowel diseases |
EAE | experimental autoimmune encephalomyelitis |
COPD | chronic obstructive lung disease |
VEGF | vascular endothelial growth factor |
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