Examples of different structures of the bacterial O antigen.
One of the most complex topics within bacterial anatomy and physiology is that of exopolysaccharides. These molecules have various structures and functions and also provide different types of advantages to their producing microorganisms, including surface variability, resistance to innate and acquired immunity mechanisms, the ability to adhere to different surface and cell types and resistance to antibiotic activity.
These bacterial systems are closely linked to the different genera and species that contain them. The organisation and expression of the genes that code for these external structures, genetic expression mechanisms and biosynthesis routes are extremely heterogeneous.
Although numerous classic microbial physiology and biochemical studies have focused on analysis of the external structures of microorganisms, not until recently has the study of exopolysaccharides become important, due to the role of exopolysaccharides in bacterial pathogenicity and the ecology of microbial populations and their possible role in the colonisation, residence and adaptation mechanisms in various ecosystems. Among the most important exopolysaccharides are those described below.
2. The glycocalyx
Although it can not be considered a bacterial structure, the glycocalyx is a heterogeneous set of exopolymers that have diverse biochemical compositions . The exopolymers are located immediately adjacent to the microorganism wall and are present as hydrophobic gels that are weakly associated with the external bacterial structures.
The production and presence of these exopolymers provide bacteria with a high degree of surface hydrophobicity that enables interaction between cellular and inert surfaces and subsequent bacterial colonisation through the development of microcolonies and biofilms.
Furthermore, the presence of certain biopolymers is related to the ability to resist antibiotic action by capturing these compounds through periplasmic glucans. An example of this is resistance to tobramycin, which is captured by cyclic-β (1,3)-glucan .
In contrast to the glycocalyx, a bacterial capsule is a well-defined external structure with a characteristic composition for each bacterial genus and species that provides a number of advantages, which are primarily related virulence, to the producing microorganism.
One of the classic examples of the importance of the capsule is provided by
It has been documented that the capsule participates in bacterial adhesion mechanisms and that its synthesis is stimulates by low stress conditions, such as the presence of serum, low Fe++ concentration and high CO2 tensions. Although in certain microorganisms, the presence of a capsule is discreet, in others, such as
Because of the production and exportation of bacterial exopolymers, the strains increase their degree of surface hydrophobicity, which facilitates interaction, adsorption and residence on a wide range of surfaces that, in principle, hinder bacterial colonisation. Regarding the production of biofilms, it has been documented that each bacterial genus and species responds to different signals from the environment and the host, as is the case of induction by tobramycin and the response capacity of the
In the relationship between
The physiology of bacteria that are found in biofilms is heterogeneous and depends on the specific site that the microorganism occupies in the microcolony. Nutrient gradients occur from the surface of the biofilm to the most internal parts, thereby influencing the bacterial physiology and consequently modifying the speed of growth, the generation time, the susceptibility to antibiotics (due to factors such as the presence of a diffusion barrier to antibiotics), the antigenic variability of individuals, the susceptibility to opsonisation and phagocytosis and even the alginate functions as a negative immunomodulator for the host .
Another example of the formation of biofilms is that produced by bacteria of the genus
5. Gram-negative lipopolysaccharide (LPS): structure and function
LPS is essential in the structure and function of the external membrane of gram-negative bacterial cell walls. LPS intervenes in the transportation of hydrophobic molecules to the interior of bacterial cells and are an essential factor in host-microorganism interactions.
LPS is an amphipathic glycoconjugate that constitutes 10% to 15% of the total molecules in the external membrane and represents 75% of the total of bacterial surface . There are three different LPS domains: a) Lipid A, which is the domain that is anchored to the membrane and the hydrophobic and endotoxic portions of the structure; b) The core oligosaccharide, which is the domain that connects lipid A to antigen O and is divided into the inner core and the outer core. The inner core is joined to lipid A and consists of unusual monosaccharides, including 2-keto-3-deoxy-octanoate (Kdo) and L-glycero-D-mannoheptose. The outer core is joined to the O antigen and is made of common sugars such as hexoses and hexosamines ; and c) The O polysaccharide, which is the hydrophilic and immunodominant domain of LPS and is an oligosaccharide of repeated units that is projected from the core toward the exterior of the bacterial surface.
The O antigen has a polysaccharide chain that varies in length with up to 40 repeated units of dideoxyhexoses. At least 20 different sugar molecules may compose the O antigen, including molecules that are rarely found in nature, such as abequose, colitose, paratose and tyvelose. These components are strain-specific. The O antigen displays a large degree of inter-species and intra-species variation, which is related to the nature, order and union of the different sugars (Figure 1) .
The O antigen is the immunodominant part of LPS and therefore is the easiest target for the humoral response of the host. For this reason, the O antigen is the basis for the serological classification of gram-negative bacteria. The O antigen is recognised by the innate immune response and participates in complement activation and in the inhibition of the formulation of the complex that attacks the membrane [6,7].
6. Biosynthesis of LPS
LPS is the primary component in the surface of gram-negative bacteria. The synthesis of LPS structures, which consist of lipid A, the core and antigen O, begins in the cytoplasm, where these structures are assembled. The structures are translocated to compartments such as the periplasm until the final destination is reached, which is the surface of the external membrane. The synthesis process has been widely studied in
The formation of lipid A is carried out in the internal face of the cytoplasmic membrane, and nine enzymes participate: LpxA, LpxC, LpxD, LpxH, LpxB, LpxK, KtdA, LpxL and LpxM. The biosynthesis of LPS begins with the formation of uridine diphosphate-diacyl-
Notably, the acyltransferases – LpxA, LpxD, LpxL and LpxM – selectively catalyse the different substrates and employ different acyl donors. For the first steps of the synthesis pathway of lipid A, the enzymes LpxA, LpxB and LpxD are required, with 3R-hydroxyacyl -Acyl Carrier Protein (3R-hydroxyacyl-ACP) serving as a donor. This compound is dehydrated by FabZ to form trans-2-acyl-ACP, which is also used as a donor of fatty acids in the biosynthesis of phospholipids. The synthesis of other LPSs in bacteria, such as
6.1. The core oligosaccharides
The assembly of lipid A from the core oligosaccharides (Kdo2 – lipid A) is the next step in the synthesis of LPS. This step is performed on the cytoplasmic surface of the internal membrane by glycosyltransferases, which are associated with the membrane and with nucleotide sugars as donors.
The core oligosaccharides normally contains 10 to 15 monosaccharides and may be divided into two structural regions, which are the inner core and the outer core, which are ultimately connected to lipid A and antigen O, respectively, in the final structure of LPS. The inner core contains residues of Kdo and Hep (L-glycero-D-manno-heptose). Kdo is the most conserved component in the nuclear region of the LPS. In contrast, the outer core is more variable, depend on the strain. However, the vertebral column of the oligosaccharide is typically composed of six units, and upon joining with other units, the column forms structures. The sugars commonly found in the core oligosaccharides are D-glucose, D-galactose, Kdo and Hep [9,10].
6.2. The O antigen
The majority of O antigens are heteropolymers, although a portion of O antigens may be composed of a single monosaccharide. The synthesis of the O antigen is performed in the same location as the core oligosaccharides, and this synthesis also uses nucleotide sugars as donors. In the majority of bacteria, a cluster of genes known as
The following hypotheses have been proposed regarding the assembly and transfer process of antigen O: a) a pathway dependent on Wzy, which is the prototype system; b) a pathway dependent on ABC transporters, which are typically used by linear polysaccharide structures; c) a pathway dependent on synthase, which involves glycosyltransferases capable of synthesising within a single polypeptide and is an uncommon pathway and finally d) seroconversion reactions, in which the addition of acetyl residues or glucose residues modifies antigen O. Within the prototype pathway dependent on Wzy, in bacteria such as
6.3. LPS and its transportation to the external membrane
When an LPS is formed, it must pass through the periplasmic space to reach the external membrane [9,10], and this process is facilitated by protein LptA (periplasmic), LptB (cytosolic), LptC, LptF, LptG (internal membrane) and LptD and LptE (external membrane). Several of these proteins act in complexes. For example, in the case of the transporter ABC, LptBFG and LptA and LptC translocate the LPS to the internal side of the external membrane such that the proteins LptD and LptE place it on the surface of the membrane. It has been observed an absence of LptA or LptB or both causes the accumulation of LPS in the periplasm [11–17].
In the majority of bacteria, the genes that code for the enzymes involved in the biosynthesis of the O antigen are found in clusters. However, in the case of
7. Regulation of the expression of LPS
The biosynthesis of LPS is performed through two separate pathways. One pathway involves the formation of lipid A and the core, and the other pathway involves the formation of the O antigen. In the synthesis of LPS, a large number of genes participate, many of which are part of clusters located in different regions of the bacterial chromosome and, in some organisms, in plasmids [22,23].
7.1. Regulation of the biosynthesis of lipid A
Lipid A and the core oligosaccharide are formed in a continuous process, which is separate from the synthesis of the O antigen. In the majority of
The genes involved in the first steps of the biosynthesis of lipid A in
The proteins involved in the biosynthetic pathways of UDP-GlcNAc, UDP-Glc and UDP-Gal are coded in constitutive genes.
L-glycero-D-manno-heptose is added to its derivative ADP, which is synthesised from sedoheptulose 7-phosphate in four steps. The genes
Kdo is transferred from CMP-Kdo and synthesised from arabinose-5P and PEP by a three-stage pathway. Two of the genes in this process, namely,
PhoP-PhoQ is a two-component system that regulates virulence through adaptation to limited magnesium environments and regulates numerous cellular activities in gram-negative bacteria. This regulon consists of an external membrane sensor, PhoQ, and a cytoplasmic regulator, PhoP, and is activated by the acidic pH and by certain antimicrobial peptides (APs). PhoP-PhoQ is repressed by millimolar concentrations of magnesium and calcium. PhoQ senses the concentration of magnesium and of APs throughout a periplasmic domain, which undergoes a conformational change when it is joined to these compounds and results in autophosphorylation. The activation of the PhoP-PhoQ system may allow for the activation or repression of 40 genes .
The regulon PrmAB of
7.2. Regulation of the biosynthesis of the core
Genes in the
The central operon
7.3. Regulation of the biosynthesis of the O polysaccharide
The genes involved in the biosynthesis of the O antigen are generally found in the chromosome in the cluster of the O antigen or
The genes that code for the proteins that participate in the synthesis of the O antigen form three main groups: a) proteins involved in the biosynthesis of the precursors of nucleotide sugars of the O antigen; b) protein glycosyltransferases, which sequentially transfer various precursor sugars to form an oligosaccharide of a lipid carrier, undecaprenyl phosphate (UndP), which is located in the cytoplasmic face of the internal membrane and c) genes for the processing of the O antigen, which are involved in the translocation through the membrane and polymerisation (Figure 4) [32,33]. A fraction of O antigens includes acetyl-O groups, and others include residues; therefore, in the corresponding clusters, the transferases for them are coded. The differences among the many forms of the O antigen are due to the genetic variation in the cluster of the O antigen. The genes for the initial steps, which are also involved in conserved functions, do not duplicate in the cluster of the O antigen .
The genes of the biosynthetic pathway of the three precursors of the nucleotides of sugar are grouped within the genetic cluster of the O antigen.
The transferases coded by
The majority of the O antigen operons are constitutively expressed and are preceded by a sequence of 39 bp, known as JUMPStart (Figure 5B).This sequence includes two elements known as
8. Variability of the LPS
In bacterial pathogens, the most variable structures are those that are expressed on the cell surface. Intra-species genetic individuality has inheritable traits that may be observed in a sample of individuals. The genetic variability is derived from the combination of a certain number of genes, which each exists as a family of alleles that differ in structure and function. An example is the family located in the biosynthetic locus of the O antigen, and an example of this phenomenon at the population level is the hypervariability in the structure of this biomolecule . Among the components of the surface of the gram-negative bacteria, LPS is the primary constituents of the external membrane, which is a heterogeneous surface that is significantly involved in the process of the microorganism adapting to its environment.
LPS primarily consists of conserved segments, such as lipid A and the core, and secondarily consists of a hypervariable segment, which is the O antigen. The conserved domains of the LPS are shared regions among bacterial species, which intervene in the development and in the survival of the bacteria. The O antigen may display modifications such alterations in the length of the oligosaccharide chain and changes in the surface composition and in the chemical configuration, due to the addition of glycosyl or fucosyl groups or even non-hydrocarbonated substitutes, such as acetyl or methyl groups, which could affect the cellular structure .
8.1. Intra-species variability in the composition of the O antigen
The heterogeneity in the expression of LPS may provide a medium to discriminate among the bacterial species. This heterogeneity is responsible for the well-known ladder profile that can be detected in silver-stained SDS-PAGE gels (Figure 6). This method is used to determine the number and repeated units of oligosaccharides that constitute the O antigen, which has been useful in epidemiological studies . The smooth strains contain the entire LPS, whereas the semi-rough strains have a subunit of the O antigen, and the rough strains lose the subunits of the O antigen.In Table 1, examples are presented of various sequences from different genera and species, which mark the variability of intra-species and inter-species LPS that impedes the immune response control of bacterial infections, among other issues.
An increase in temperature during microbial growth causes changes in the concentration of carbohydrates in LPS, which modifies their composition . In a study on
The different profiles from the isolates obtained from the sub-cultures of
Several isolates of
The isolates of
8.2. Heterogeneity of populations and phase variation
Phase variation is used by various bacterial species to generate diversity within a population. Phase variation is a process of change in the expression of the epitopes of the cellular surface of the bacteria . Bacterial cells may phenotypically vary even within a clone population, which allows them to adapt to their environment or even to evade the immune response of the host. Phase variation is a phenomenon that generates phenotypic heterogeneity within a population by means of gene regulation, which changes genes from a state of expression in which they are “turned on” to a state of non-expression in which they are “turned off”. The state of expression is inheritable, reversible and affects the same phenotype.
Antigenic variation is referred to as the expression of an alternative form of an antigen of the cellular surface, such as polysaccharides, lipoproteins and type IV pili, which at the molecular level, share characteristics with the phase variation mechanisms. During this adaptation process, the bacteria display reversible phenotypic changes as a result of genetic changes or epigenetic alterations in a specific locus. The mechanisms which allow for phase variation are: genetics (Slipped-strand mispairing, recombination) and epigenetic (DNA methylation) [47,48].
Numerous studies have been performed to reveal the genetic basis of the variation of the O antigen. In certain cases, the variability in the expression of the genes is regulated by elements in
One of the mechanisms that regulate phase variation at the molecular level is the slipping of one of the DNA strands, which causes a mispairing between the daughter strand and the parent strand during the replication of the DNA. This process is known as slipped-strand mispairing (SSM). The genomic sequences susceptible to SSM are those which contain short repetitions, microsatellites or a variable number of in tandem repetitions, which may cause a change in the expression of genes at the level of the transcription processes or translation according to the location of the repeated sequence in relation to the promoter and the codifying sequence . At a transcriptional SSM, this may lead to the activation or deactivation of the promoter region of the target gene, as occurs in
Within the genome of
One group of genes that generate phase variation are those that code for enzymes that intervene in the biosynthesis of LPS, which may cause variants of the gene product in the same bacterial population. The LPS of the majority of
The genes that code for the FucTs have elements in
The α 1,2-fucosyltransferase (FutC) catalyses the addition of fucose in the conversion process of LeX to LeY. Sanabria-Valentín et al., through
Phage recombination: seroconversion
The O antigen is a determinant of the virulence necessary for the pathogenicity of
The temperate phages of
The bacteriophage codes for an acetyltransferase and produces a conversion to the 3b serotype. The lysogenisation of the SfV bacteriophage produces modifications of the type V O antigen, which involves the addition of a glycosyl group through a bond of α 1,3 to the rhamnose II of the repeated tetrasaccharide unit. Similar to other phages that intervene in the glycosylation process, the genes involved in the conversion of serotypes are located immediate downstream from the
It has been suggested that GtrB catalyses the transfer of glucose from UDP-glucose to bactoprenol phosphate to form UndP-β-glucose in the cytoplasm. This molecule is subsequently translocated by GtrA in the periplasm before the glucosyl residue is joined by Gtr(type) for the growth of the O antigen unit .
Epigenetic mechanisms: DNA methylation
The term epigenetic is defined as “inheritable changes in genetic expression that occur without alterations in the DNA nucleotide sequence”. Thus, an epigenetic mechanism may be understood as a complex system to use the genetic information selectively by activating and deactivating various functional genes. Epigenetic modifications may imply methylation of cytosine residue in the DNA. DNA methylation has been observed in various bacterial species. In bacteria, methylation is part of a defence mechanism to reduce the amount of horizontal genetic transference among species. DNA methylation constitutes an epigenetic marker that identifies the template strand during the replication of the DNA. Generally, the methylation of the regulatory elements of genes, such as promoters, enhancers, insulators and repressors, suppresses this function .
The modifications of the O antigen that may affect the serotype are related to those that contain the operon that code for the glycosyltransferases (
Within a clone population of
Through studies based on the analysis of gene expression, the presence of mutations, the level of DNA methylation and the
Understanding the variation of the LPS structure is important because the composition and the length of the O antigen chain may be an indicator of the virulence, and this characteristic often differs within a single bacterial strain .
9. The importance of the variability of the O antigen of LPS
The modifications that are present in the O antigen and that cause its variability play an important role in infections by gram-negative bacteria, given that the modifications may influence adherence, colonisation and the ability to evade the host’s defence mechanisms.
9.1. The role of the variation of LPS in the immune response
LPS activates not only the innate immune response but also the adaptive response. The first contact that LPS has with the immune system is with lipid A, which is recognised by the receptors involved in the innate immune response, while the structure of the O antigen participates in the adaptive response (synthesis of antibodies). LPS is a potent stimulator of the cells of the immune system, given that it induces the production of pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β) and acute phase proteins.
Although the variability is greater in the O antigen within LPS, there is also variability within lipid A. This variability is due to the length of the chains and the saturation of its acyl groups. Because these groups are strong immunostimulants, both the changes in the number of their chains and the presence of phosphorylations within the structure of the lipid A may influence its biological activity .
Lipid A is the structure of LPS that is recognised by the TLR4 receptors, which are part of the so-called Toll Like Receptors (TLRs), which are expressed by cells of the innate immune system and are stimulated by pathogen-associated molecular patterns (PAMPs). The stimulation of the LPS in certain cells of the monocyte-macrophage lineage, lymphoid cells and even cells that are not part of the immune system, such as epithelial cells, endothelial cells and vascular cells, occurs with the participation of other molecules, such as LPS binding protein (LBP), CD14 and MD-2. The transduction signals of TLR4 are divided into MyD88-dependent and MyD88-independent (also called TRI-dependent) groups. These signals may be regulated at various levels. For example, the RP105 and SIGIRR (Single immunoglobulin IL-IR related molecule) molecules inhibit the start of the signalling cascade .
Recognition through TLR4 is crucial for the control of infection, but changes in the signalling pathways may cause sepsis or evasion of the pathogen. The importance of signalling via TLR4-MD2 in response to gram-negative pathogens make this pathway an alternative to search for therapeutic targets not only for infectious diseases but also for other diseases with inflammatory aetiology, such as cancer, atherosclerosis, asthma and autoimmune conditions.
Antagonists of TLR4-MD2 have been identified, and several of these are based on the lipid A structures and other inhibitor molecules [63–65]. The intention is to use this type of antagonist therapy to treat septic shock. Additionally, several TLR4 antagonists primarily those that activate the TRAF (TNF receptor- associated factor) or TRIM (Tripartite motif) pathways have been proposed as adjuvants.
However, certain pathogens have the ability to modify the structure of lipid A and its detection by the host. For example, some isolates of
The large variability that the O antigen displays allows for the existence of various clones within a single species, which offers a selective advantage in the niche occupied by this clone and is precisely the interaction between the O antigen and the immune system that permits this advantage.
Many pathogens have the capability of varying the antigens that are attached to their surface and therefore can vary their antigenic composition. This variation is typically mediated by the regulation of the expression of genes. By varying their antigenicity, the pathogens have a greater ability to evade the immune response of the host, and this variability makes it more difficult to design vaccines for these pathogens .
The O antigen is considered to be highly immunogenic and induces the production of antibodies that may activate the complement pathway, either through the classic pathway or an alternate pathway, which leads to cellular death or phagocytosis. Certain modifications in the oligosaccharide chain of the O antigen may alter the interaction of the complement pathway. Several O antigens of pathogens are similar to host molecules and facilitate invasion through mimicking in the host; for example, O antigens of the LPS of
The mimicking property may also serve to evade the immune system, as is the case of
The expression of Lewis antigens and their fucosylation have biological effects in the pathogenesis of this bacterium. The O antigen of
9.2. Changes in the LPS related to resistance
The hydrophobic antibiotics that reach the interior of the cells due to the permeability of the external membrane are aminoglycosides, macrolides, rifamycins, novobiocin, fusidic acid and cationic peptides. The tetracycline and the quinolones use pathways that are mediated by lipids and porins. The central region of the LPS is important because it provides a barrier against hydrophobic antibiotics and other components; isolates that express a long LPS have intrinsic resistance to these factors .
The polymyxins, which include polymyxin B and colistin (polymyxin E), belong to a group of natural antimicrobials that are found in eukaryotic cells; this group is known as the cationic antimicrobial peptides. The polymyxins are active against gram-negative pathogens, such as
The LPS has a negative charge and provides integrity and stability to the external membrane of the bacteria. Polymyxin has a positive charge, displacing the Mg+2 or Ca+2 and bonding to LPS, which as a consequence, destabilises and destroys the internal and external membrane .
Gram-negative bacteria may develop resistance to colistin and polymyxin B. The most important mechanisms involve modifications in the external membrane through changes of the LPS. The modification of the LPS occurs with the addition of 4-amino-4-deoxy-L-arabinose (Lara4N) to a phosphate group in lipid A. This addition causes an decreases in the negative charge of the lipid A, which decreases the affinity of the positively charged polymyxins [ 68–70].
The biosynthesis of LAra4N is mediated by the regulatory systems PmrA/PmrB and PhoP/PhoQ . One of the primary roles of the activation of PmrAB is the modification of the LPS. These modifications include additions of Ara4N and pEtN to the lipid A and of pEtN to the core of LPS. The modifications mask phosphate groups with positive charges, thereby affecting the electrostatic interaction with certain cationic compounds. The biosynthesis of LAra4N depends on the genes of the operon of resistance to polymyxin, which is known as
In the bacterial pathogens, the most variable structures are those expressed in the cell surface. LPS is one of the principal antigenic structures of cell surface of gram-negative bacteria. A great variability in LPS has been demonstrated and principally in O antigen of gram-negative bacteria. This variability is present not only in the longitude of the oligosaccharide chains but also in the composition and structure of LPS.
Many of the functions of O antigen are associated to the longitude of the chain and to the variability of its structural features. This variability could affect the function, physical and chemical properties as well as the target site of LPS and determines the changes in the virulence of the microorganism that favor its adaptation to fluctuating enviroment which in many occasions are hostile to the microorganism and permit its evasion of the immune response of the host. The variation in O antigen structure has demonstrated that its composition and the longitude of its chain could be biological markers of virulence and this characteristic could differ within the same bacterial strain. The variability of LPS could derive from adaptations that involve associated changes to the synthesis of this molecule. The antigenic variability could occur by means of genetic and epigenetic mechanisms. The lost or gain of genes associated to variability of LPS is due to the events of genetic material interchange produced by lateral transference of genes which leads to strain selection with new characteristics and the evolution of the bacteria by modification of this structure.
One of the most important aspects of LPS function is its participation as immunogenic molecule and its role in bacterial classification based on O antigen and its variability. In general, it is seen that the modifications of O antigen play an important role in the process of infection including the adherence, the colonization, and the ability to evade defensive mechanisms of the host especially the innate resistance.
The study of the events of variation of LPS and its effects on pathogenecity and virulence represents a field of study of great interest to understand bacterial physiology and its mechanisms of adaptation and evolution.
The immunogenicity and variability of O antigen confer to gram-negative bacteria an important characteristic for its serological typification. The 0-antigen is subject to an intense selection on the part of immune system, which could be the principal factor for the different forms in which it is presented. For this reason, the variability of O antigen has been an area of intense research.