The Role of Pseudomonas aeruginosa DsbA-1 in Bacterial Pathogenesis: Current Research and Future Prospects

Abstract

Disulfide bond isomerase proteins (Dsbs) have been extensively characterized in gram-negative bacteria. Recently research efforts is being placed on their biology in gram-positive species. Modern “omics” technologies, allowed assessment of the contribution of the Dsbs to bacterial pathogenesis. The author cloned and characterized the dsbA 1 protein from Pseudomonas aeruginosa in the late 1990s. The global proteome analysis demonstrated that the dsbA gene is under the direct regulatory control of the extracytoplasmic function (ECF) sigma factor AlgT(U) or sigma-22. This is unique to P. aeruginosa. Disruption of dsbA gene results in pleiotropic phenotype: defect in assembly of cysteine disulfide bond containing proteins-as shown in many others. Recently, omics-based approaches identified expression changes in dsbA gene under different physiological states of bacterial pathogens-primarily in free-living, biofilm state, or under infectious disease conditions. Involvement of dsbA function in biofilm formation was shown using dsbA gene disruption mutants. This chapter documents past and current findings and concludes with future trends in research on Dsbs including peptidomimetics.

1. Introduction

This chapter examines the biology of disulfide bond isomerase (Dsb) proteins and how these proteins are involved in assembly of virulence determinants in the bacterial cell [1]. We first examine the function of these proteins within the cell. DsbAs are proteins that facilitate the formation of the disulfide bond. This bond exists between two cysteine amino acid residues. The “R” group in this amino acid contains thiol or “SH” group at its end. The thiol group needs to be oxidized and DsbA or DsbA-like proteins aid in this. Slow oxidation of the thiol group is possible (Figure 1). The disulfide bond can be intrachain-within one polypeptide or alternatively between two polypeptide chains (intermolecular). Several kinds of proteins have disulfide bonds. The DsbA protein is localized in the periplasmic space in gram-negative bacteria. It is involved in oxidation reactions with the thiol groups in proteins. It is regenerated by a cognate DsbB protein localized in the inner membrane of the gram-negative bacterial cell [3]. In gram-positive bacteria, the role of DsbA and Dsbs is less well understood. This is likely due to the lack of known and tractable model organisms and/or the lack of genetic tools for analysis [4]. Peculiar characteristics of gram-positives will be discussed later in the text [5]. Scientists study DsbA function by disrupting the coding sequence of the dsbA gene. Typically this is done by insertion of a nonpolar cassette insertion that encodes a Gentamycin drug resistance marker [6]. The lack of DsbA function causes a slew of phenotypic changes in the mutant [6]. These changes are well characterized and typically similar in gram-negative bacteria [7]. Interestingly you can complement a dsbA disruption in P. aeruginosa by complementing with a cloned E. coli dsbA gene. Inspite of the dsbA mutation, the strain will survive and typically exhibits growth characteristics similar to the wild-type parent strain [6]. This property makes it easy to study the dsbA mutation. Similar growth rates between the mutant and wild-type strains indicate that the effects of dsbA mutation do not suppress growth of bacteria. Growth differences could further complicate analysis. Due to similar growth rates its likely phenotypic differences are due to dsbA disruption. This allows direct comparison of wild type to the mutant data set [6]. These observations are supported by complementation studies: wherein a cloned dsbA gene restores the defective phenotypes in the mutant [6]. This is almost true for at least all gram-negative bacteria I have come across and worked with including Sinorhizobium meliloti, Haemophilus influenzae, and the gram-positive bacteria Staphylococcus aureus [unpublished data].

Figure 1.

SH group and disulfide bond in protein. Panel A: The generation of the reactive intermediate the “thiolate” ion. This reactive center can further react to form a disulfide bond. The disulfide bond stabilizes protein structure. Most proteins are held by chaperones and maintained till their ultimate death and destruction [2]. In Panel B you can see both intermolecular disulfide bonds (between heavy and light chain) and intramolecular (In the heavy chain).

2. Pseudomonas aeruginosa: an opportunistic pathogen

P. aeruginosa has a large genome size (6.3 Mb). It is a gram-negative rod-shaped bacterium that is often classified as an opportunistic pathogen. It is also a member of the ESKAPE pathogen group [8]. This opportunistic pathogen typically does not cause lethal disease in normal healthy human individuals. However, in people with comorbidities and other lifestyle disorders and accidents (burns surgical wounds and in-dwelling devices) P. aeruginosa can cause severe protracted illness and is a common cause of nosocomial or hospital-acquired infections [8]. It is a designated superbug. Thus, understanding its pathogenesis is key. Its genome (sequenced in the year 2000, Pathogenesis Corporation) is relatively more complex than Escherichia coli with some fascinating distinctions [9]. During the years 1996 through 2000, the author was involved in delineating the stress response regulon of AlgU/T or Sigma-22. Sigma-22 is a member of a conserved subfamily of Sigma 70 sigma factors. This subfamily of sigma factors is united through: the conservation of its protein structure; types of genes that is group regulates; the unique mechanism of activation in response to extracytoplasmic stress and potential consensus elements in the promoters of its induced genes. It’s appropriately named the Extracytoplasmic function (ECF) subfamily of sigma factors—the fourth member of the Sigma 70 group of sigma factors [10]. For a long time, scientists have considered colonization of a host species to involve a stress response in the invading bacterial pathogen. The pathogen adapts to the stress and then colonizes a niche within the host. Different OMICS based technologies have been developed. Scientists are using these technologies to study the stress response pathogenic bacteria generate in response to altered host species environment they encounter. There are 19 ECF sigma factors in P. aeruginosa. The extent of the regulons controlled by ECF sigma factors in P. aeruginosa is under study. The author used a reverse genetic approach proteomics-based approach to identify targets for the well-characterized sigma factor AlgT/U or Sigma-22. AlgT was first identified as a major virulence determinant due to its involvement in activation of the alginate biosynthesis operon. Alginate is a mucous exopolysaccharide that is a major virulence determinant in CF disease pathogenesis involving P. aeruginosa. Its regulation was characterized by Prof. Dennis E. Ohman a Ph.D. Mentor of the author, to whom the author is highly obliged [6].

3. Alginate: the Mucous exopolysaccharide

Alginate is a mucous polysaccharide that is made of individual sugar units that are assembled in the cell and extruded out as a polymer. It is an important virulence determinant of P. aeruginosa [11, 12]. Especially in the context of persistent Cystic fibrosis infections, where opportunistic P. aeruginosa infections can and do turn lethal. It is now believed that bacterial and viral co-infections are the main reason for the greater extent of morbidity of the disease in CF patients. Novel methods are being developed to fight these infections. These will be discussed in the summary section of the chapter.

Alginate is a polymer composed of P. aeruginosa alginate is composed of D-mannuronic acid residues interspersed with L-guluronic acid residues. The hydroxyl groups of the D-mannuronic acid residues may be O-acetylated at the C2′ and/or C3′ positions. The polymer is unbranched and has a long chain. The alginate biosynthesis operon is about 18 Kb long and is regulated by the ECF sigma factor AlgT. The acetylation function is carried out by the proteins AlgJ and AlgX, where AlgX might be involved in sequestering the nascent alginate polymer in the periplasm. The coating of the exopolymer alginate may provide a survival advantage to cells. It reduces the intake of antibiotics and phagocytosis. Alginate also prevents desiccation and provides protection from oxidizing agents [13]. Consequently, alginate-producing isolates of P. aeruginosa may have a survival advantage in particular in CF patients.

The lung function decreases upon the appearance of the mucoid (Alginate + phenotype) in P. aeruginosa in CF lung. This also correlates with a decrease of some other bacterial species like S. aureus and H. influenzae. The mucoid phenotype is a manifestation of the production of alginate. So, we need to focus on what alginate does. Alginate can scavenge hypochlorite produced by phagocytic cells. P. aeruginosa alginate also reduces the chemotaxis of polymorphonuclear leukocytes (PMN) into CF lungs. Alginate innately can inhibit the activation of the complement system. P. aeruginosa cells and synthesizing alginate are inherently resistant to phagocytosis by both PMN and macrophages in comparison to nonmucoid strains. Additionally, binding of the bacteria to host factors such as respiratory mucins or growth in a biofilm can aid in the colonization of CF lung environment and stabilization of infection (Figure 2) [16].

Figure 2.

Colony morphology of Pseudomonas aeruginosa on plates- mucoid phenotype. Mucoid (A) and nonmucoid (B) Colony variants of Pseudomonas aeruginosa on sheep blood agar plates. The mucoid phenotype is due to excess production of the polysaccharide Alginate [14, 15].

4. Elastase the well characterized secreted protease

Numerous proteases are secreted by P. aeruginosa. They are concentrated outside the cell (extracellular). They operate as virulence factors. The LasB and LasA elastases are the most important. Alkaline protease (AprA), type IV protease (PIV), P. aeruginosa small protease (PASP), large ExoProtease A (LepA), P. aeruginosa aminopeptidase (PAAP), and MucD are the other proteases that P. aeruginosa produces [14]. The T2SS transports the LasA and LasB elastases extracellular environment [17]. The Quorum sensing (QS) system regulates the expression of their genes (lasA & lasB) [14]. Elastase or pseudolysin are other names for LasB elastase. The host protein Elastin is broken down by LasB a zinc-dependent metalloprotease [17]. It is the most prevalent extracellular enzyme, making it a focus of diligent research. In addition to its elastolytic activity, it also has other substrates, including immunoglobulins, surfactant proteins (SP-A and SP-D), cytokines (TNF-, IFN-, IL-6, or IL-2), and IL-2. Biofilm development is also impacted by LasB [13, 16]. We concentrate particularly on elastase because it is the most abundant enzyme. In addition, the author characterized its secretion in a dsbA mutant. The disulfide bonds exist in Elastase and the type II secretion system components [14]. By using a western blot and assessing the protein’s activity using a common elastase activity assay, we were able to demonstrate decreased levels of the Elastase protein and reduced elastolytic activity in our DsbA characterization (Figure 3) [6]. The serine protease LasA or staphylolysin is produced by the lasA gene. A zinc-dependent metalloendopeptidase called alkaline protease, also known as aeruginolysin, is released through the T1SS and is made by the aprA gene [14]. Its primary protein targets are endothelium’s fibronectin and laminin complement proteins (C1q, C2, and C3) and cytokines (IFN-c, TNF-a, and IL-6). Destruction of these immune system proteins by bacterial proteases is a way for the bacteria to suppress both the innate and adaptive immune response [14].

Figure 3.

Multiple sequence alignment dsbA proteins & catalytic site. Top Panel: Graphical representation of an inhibitor designed to fit into the hydrophobic pocket/groove of DsbA. The region around the active site is highly conserved. It is believed that the sequence around the catalytic domain CPHC motif determines the reactivity of the Cysteine residues. You can see strong conservation around the CPHC motif in the MSA in Panel 2. Sequences 1 through 4 are Vibrio Cholera, Haemophilus, E. coli, and Pseudomonas DsbA proteins.

A dsbA mutation can also impair certain lipolytic enzymes [14]. Fat-burning enzymes are secreted by the T2SS. These lipases break down lipids to produce free fatty acids and glycerol as by-products [14]. P. aeruginosa produces a large amount of LipA, which must first be activated by the chaperon lif [18, 19]. It is encoded in the lipA/lipH operon, along with its cognate foldase LipH, which is also necessary for the production of LipC [19].

5. The regulation of ECF sigma factor AlgU in P. aeruginosa

The ECF sigma factor family is a subset of the sigma 70 families of sigma factors. ECF stands for extracytoplasmic factor. These sigma factors are typically sequestered in an inner membrane complex (A complex of proteins encoded by genes in an operon encoding the ECF sigma factor) with the sigma factor residing in the cytoplasmic side of the bacterial cell. In response to different extra cytoplasmic signals or stress signals, the inner membrane complex is cleaved through selective proteolysis of different components and the sigma factor is released for gene expression. This type of regulation is well characterized for E. coli and now for P. aeruginosa as well (Figure 4). The operon encoding the ECF sigma factor typically has a similar organization with conservation shown in the gene arrangement, mode of activation, expression, protein structure (Sigma factor), and consensus elements in the promoters recognized by ECF sigma factors. There is also a likelihood of complementarity of function. For instance, the E. coli sigma factor RpoE can complement an AlgT/U mutation. This complementarity of function may exist for other bacterial species.

Figure 4.

ECF Sigma factor activation promoter consensus sequence. (a) ECF sigma factor activation and promoter specificity. (b)Truncate MucA22 cannot bind MucB so its attacked by AlgW and degraded releasing Sigma 22 or AlgT. ECf signals are discussed in Text. MucP is another protease indicated in proteolysis. (c) Promoter Sequence Specificity: Different residues and spacer element account for recognition sequence of a consensus promoter.

Typically the AlgT/U sigma factor is sequestered in the cytoplasmic side in association with MucA, which is stabilized by MucB-via interaction with MucA periplasmic domain or C terminus end. In the mucA22 allele, the encoded MucA is prematurely truncated so the C terminal part that interacts with MucB is not made. The MucB interacting domain is missing. Consequently, the MucA22-AlgT interaction is destabilized as well. This is subject to proteolytic activity from AlgW. Further degradation of the cytoplasmic component of MucA is mediated by MucP and ClpXP as well. This process is called regulated inner membrane proteolysis or RIP for short. Once MucA has degraded, the AlgT/U molecule is free to reach the RNA polymerase so that it can be expressing genes that are part of its regulon. The extracytoplasmic stimuli that contribute to MucB and MucA degradation are misfolded proteins in the OM or periplasmic space, osmotic stress, antibiotics, and H_zO₂. RIP of MucA leads to activation of σ 22 and expression of the σ 22 regulon. RIP of MucA is accomplished by the proteases AlgW, MucP, ClpXP, and Prc (Figure 4) [14, 15, 19].

6. The hypothesis testing: stress response regulon of AlgT/U

As AlgT/U belongs to a highly conserved subfamily of Sigma 70-like proteins called the ECF (Extracytoplasmic Function family of sigma factors) we would anticipate that its regulon contains genes that have been seen to be regulated by other ECF sigma factors in different bacterial species. For instance, we have RpoE in E. coli. It may contain genes within its regulon that exist in P. aeruginosa and consequently could be part of AlgU/T regulon or any of the other 19 ECF sigma factors found in P. aeruginosa. Since AlgT is important for infection, which involves a stress response. Induction of AlgT on infection may likely turn on unique genes that could be important for virulence. Even though molecular genetics for P. aeruginosa was well developed with several genetic tools, the genome for P. aeruginosa PAO1 was near completion with large sequence contigs available (In 2000!!). Dr. Ohman’s group developed several tools to assess AlgT gene function in different strain backgrounds including the mucA22 allele background (where AlgT is fully active with a copious amount of alginate production.). We resorted to trying a reverse genetic approach to identify targets from AlgT/U. We compared whole cell lysates of PAO1—the prototypic P. aeruginosa strain with PDO300 that carries the mucA22 allele in PAO1 strain. Due to the mucA22 mutation, we should see the enhanced expression of AlgT-driven genes (ADG). Enhanced expression of ADGs should yield some protein spots that exhibit a higher steady-state level (higher staining protein spots, stained with Coomassie blue, that binds protein in a most linear fashion) of protein in the mucA22 strain PDO300 than PAO1. A visual and densitometric comparison identified several spots that exhibited increased expression in PDO300 than PAO1. This observation told us that the proteomic strategy was working. We also identified proteins involved in alginate biosynthesis to be elevated in the PDO300 strain (mucA22 allele, alginate overproducing). We chose to use Coomassie over the silver stain as all literature suggested that Coomassie gave more consistent results over decreased sensitivity to silver stain and better results with Edman degradation protocols. Consequently, we consistently identified about 900 proteins that represent about 1/3 of the entire expressed genome of P. aeruginosa (assuming 3000 genes are expressed at any given time). This was the first proteome study of AlgT regulon at that time. Subsequently, other comparisons might have been done. We reliably identified DsbA, Porin F, AlgD, and AlgA in our proteome studies to be elevated in mucA22 allele-bearing strain PDO300. This correlates perfectly with the genetic studies back then. Another unidentified protein about 14 KdA size, is YgaU (E. coli) ortholog called LysM in P. aeruginosa is also elevated in PDO300 (mucA22) ([20], unpublished data). We constructed transcriptional lacZ reporter fusions and introduced them into PAO1 and PDO300 strain backgrounds and showed elevated transcription in PDO300 over PAO1. Again, supporting the genetic studies and proteomic results. We also discovered some proteins that exhibited increased accumulation in the non-mucoid state N2-succinylornithine 5-aminotransferase or AruC is one such example [Data not shown 5, [21]]. Porin F, one of the most abundant porins, showed enhanced accumulation in mucoid scenario, but its promoter fusion showed no effective enhancement under mucoid conditions (AlgT+) [6]. Recently sequencing of PA14 was completed [22]. PA14 is more virulent than PAO1. More proteomic work and characterization of dsbA can be done in this strain [23].

7. Characterization of dsbA Gene through Cassette mutagenesis

Our proteomic studies show that a DsbA-like protein is induced under mucoid conditions. We then cloned the gene inserted a nonpolar gentamycin cassette in it and introduced it into the bacterial cell to create a dsbA gene on the chromosome that carries the nonpolar gentamycin cassette insertion [6]. We also created a complementing clone that carries the dsbA gene in trans. We compared PAO1 wild type to its two variants: PAO1 with dsbA::Gm cassette insertion (PAO1 dsbA insertion mutant) and PAO1 dsbA insertion mutant with a complementing dsbA gene in trans. Since dsbA disruption causes a pleiotropic phenotype [18]. We examined these strains for deficits in virulence factor production and activity (elastase, alginate, twitching motility, alkaline phosphatase, protease activity {Skim milk agar} and sensitivity to DTT). There are two dsbAs in P. aeruginosa and we characterized pDsbA 1. So far, no function has been ascribed to pDsbA-2. There are two dsbB-1 and dsbB-2, both of which can be used to regenerate DsbA-1. Thus in our analysis, we showed convincingly that a dsbA mutation is deleterious to the production, and secretion of several virulence factors, such as the metalloprotease Elastase. Elastase clearly showed a deficiency in secretion and activity (measured extracellularly). The twitching motility phenotype was reduced [6]. We did not look at individual components of the pilus but the pilA gene encodes cysteines and the PilA protein is involved in surface and host cell adhesion, colonization, biofilm maturation, virulence, and twitching, a form of surface-associated motility facilitated by cycles of extension, adhesion, and retraction of T4P fibers [19]. It is also involved in type two secretion and calcium signaling. There is a conserved C terminal disulfide loop (DSL) that when mutated by replacing the “C” residue abrogates assembly of Type 4 pilus or T4P entirely, other substitutions in and around the DSL also affect pilus assembly as measured by twitching motility and structure-based studies and surface assembly of the Type 4 pilus [24]. Suffice it to say, we conclusively showed that we had characterized the right gene, in particular, because two proteins PDsbA-1 and PDsbA-2 encoded by the corresponding genes exist. We only characterized pDsbA-1 because firstly only that gene exhibited enhanced protein accumulation under mucoid scenario. Second, it was at that time beyond the scope of the Author’s PhD. work. We then focussed on the transcription of the dsbA gene: We showed through promoter fusions that dsbA gene expression was enhanced three-fold under mucoid conditions. We then focussed on the promoter region of the dsbA gene. The Pseudomonas genome was partially sequenced and from the various contigs, we could PCR amplify the promoter region to generate transcriptional fusions to lacZ gene in an integrative vector pMS7. Single copy gene fusions were created in PDO300 (mucA22 allele, Mucoid phenotype, AlgT+ scenario) and PAO1 (the prototypic P. aeruginosa strain which is nonmucoid and AlgT). The genomic organization of the single-copy lacZ fusions was verified by genomic PCR. Subsequently, the author characterized DsbA-like proteins in H. influenzae (por periplasmic oxidoreductase) and in the plant symbiont S. meliloti (TlpA: thioredoxin-like protein A.) Por also exhibited high sensitivity to DTT. We discuss more about Por and TlpA in the next sections [25, 26]. The disruption studies i.e., dsbA disruption by cassette mutagenesis, exhibits loss of virulence factor maturation as shown in other bacterial species (Table 1).

Gene name	Bacterial Species	Phenotype	Virulence defect	Comments
dsbA	E. coli 0157	Reduced production of virulence factors	Reduced colonization of C. elegans gut and biofilm formation	Reduced virulence phenotype; At 9 days >50% better survival of C. elegans mutant strain.
dsbA	Pseudomonas aeruginosa	Reduced Twitching motility, pilA expression reduced	Reduced slow and fast killing in the C. elegans assay.	DsbA alters several virulence factors in the bacterium.
dsbA	Erwina carotovora	Less secreted enzymes, production of quorum sensing signal	Motility is highly reduced in dsbA mutant. Rotted tissue in potato tuber assay was much reduced in the dsbA mutant.	DsbA is responsible for so many traits.; Secretome and other things.; Secretion of quorum sensing signals.
sdbA	Streptococcus pyogenes	Catalyst for the enzyme SpeA	SpeA is oxidized by SdbA and maintains biological function	In the crystal structure of SpeA, Cys87 and Cys98 are linked by disulfide bond

Table 1.

DsbA orthologs and their role in different bacteria.

A short list of 4 DsbA orthologs from different bacterial species all having a common theme- defective in the production of virulence factors. This invariably results in a reduction in virulence of the associated pathogen harboring a mutation in the respective DsbA.

8. A close look at the dsbA gene/operon: primer extensions

Since the transcriptional fusion data suggested a clear induction of dsbA gene under mucoid conditions, we chose to examine the putative promoter region around the dsbA open reading frame in greater detail. We used a simple primer extension analysis procedure on whole cell mRNA isolated from PAO1, PDO300 and PDO300 with dsbAin trans [27]. As the data clearly shows in Figure 5, right panel, appearance of a novel transcript (T1) under mucoid conditions. It also appears to be driven by promoter elements that are the consensus for a ECF sigma factor (left panel, Figure 5) [28]. This data is also replicated in the Clinical isolate FRD1 and its derivative FRD440 [29]. Now we have a very substantial study to show enhanced expression of the dsbA gene, accumulation of the protein DsbA (Proteomic data) under mucoid conditions (AlgT+ scenario).

Figure 5.

Analysis of dsbA promoter by primer extension. Top Left panel A Arrangement of the transcript initiation site. Top Right Panel Primer extension reaction Lane Order No RNA PAO1, PDO300, FRD1 & FRD440. Three initiation sites with T1 appearing in mucoid conditions. Bottom Panel arrangement of orf’s schematic diagram. Top Left Panel in B we see the alignment of the promoter sequence with the consensus −35 and −10 regions with a 15/17 nucleotide spacer region.

Considering this study, there have been numerous characterizations of DsbA and DsbA-like proteins in several bacterial systems [6]. We have a wealth of data suggesting the importance of these protein systems (including DsbB) in virulence determination and maturation of virulence factors [30].

9. Novel regulation of the dsbA gene in P. aeruginosa: comparison with E. coli and others

Interestingly our extensive work with dsbA discovered this novel mechanism of regulation. So far my literature searches have identified only one-gram-positive organism where this is replicated: direct control of expression of dsbA gene by the ECF sigma factor. In our case, we have proteomic gene expression studies {promoter fusion studies in various strains} and primer extension studies that show direct evidence of AlgT/U directed transcription of the dsbA gene [5 and Primer extension data]. This is clearly distinct from E. coli where ECF factor RpoE does not directly enhance dsbA {EC} gene expression. In E. coli the CpxA/R two-component regulator directs transcription of the dsbA gene along with other genes that encode proteins involved in folding and assembly of proteins. The first characterized genes in the CpxA/R regulon were Peptidyl prolyl isomerase, DegP protease, DsbA, among others [31, 32]. The regulon is much larger now. SigL is an ECF sigma factor identified in Mycobacterium tuberculosis, disruption of the sigL gene causes a loss in virulence indicated by increased survival time in the BALB/c mice. Mpt53, annotated as a DsbE-like protein, is a gene in the SigL regulon. It has been characterized biochemically and structurally [33]. It contains a thioredoxin active site and is a strong oxidant, in contrast to the weak reducing activity of E. coli DsbE. These biochemical data suggest a possible role for Mpt53 as an extracellular oxidant that may be required for proper folding of reduced unfolded secreted proteins, a function more similar to that of E. coli DsbA [33]. It seems the P. aeruginosa DsbA-1 protein’s gene is regulated in a manner similar to Mpt53: Directly via an ECF sigma factor [33].

10. DsbA in biofilms: gene expression and disruption studies

DsbA has been implicated in Biofilm formation as well [12]. Inés Reigada et al a dual mix species biofilm of P. aeruginosa and S. aureus, proteins were assessed in the surfaceome and exoproteome compartments were determined for both bacterial species and compared between monospecies biofilms and dual-species biofilms [12]. DsbA levels were elevated in P. aeruginosa surfaceome. Alginate is probably not critical for biofilm formation [34]. So far our tests with the PAO1 dsbA::Gm strain suggest the dsbA disruption augments the biofilm kinetics. We see slightly faster biofilm formation in the dsbA mutant [Preliminary findings]. Quite similarly a dsbA mutant in Pseudomonas putida also shows enhanced exopolysaccharide production and increased biofilm formation on plastic [35, 36]. However, disruption studies in other organisms show a reduction in biofilm formation [30]. I believe that in P. aeruginosa dsbA contribution is essential along with alginate in biofilm mode. Especially in Cystic fibrosis [34], where the presence of alginate and excess of DsbA (due to induction) may be helpful in the production of other virulence determinants [15, 18]. Regulation of biofilm through the biofilm master regulator CsgD and fimbrial subunit CsgA. Deletion of either of these proteins causes a lack of enhancement of biofilm formation seen due to disruption of dsbA in P. aeruginosa [37]. Now with the advent of microarrays and such technologies, we can assess dsbA expression in biofilm and sessile bacterial populations as well. Mark A Webber’s Group showed, using the transposon mutagenesis approach, a strong temporal contribution to biofilm fitness for some genes, including somewhere expression changed between being beneficial or detrimental depending on the stage at which they are expressed, including dksA and dsbA. These studies were carried out on E. coli [38]. DsbA is involved in early biofilm for E. coli [38].

11. Future potential of DsbA research and avenues

Interestingly DsbA and DsbA-like proteins are not limited to gram-positive and negative bacteria like P. aeruginosa and E. coli alone. But in Eukaryotes specific advances have been made. Briefly, in eukaryotes, protein disulfide isomerases primarily facilitate the formation of protein disulfide bonds between cysteine residues. This process takes place in the endoplasmic reticulum lumen environment. Biology of PDI’s or protein disulfide isomerase-like molecules from five different species of malaria parasites—Plasmodium berghei, Plasmodium yoeli, Plasmodium knowlesi, and Plasmodium falciparum—are potential targets for research. Based on their chromosomal positions, P. falciparum four investigated protein disulfide isomerases have been given the names PfPDI-8, PfPDI-9, PfPDI-11, and PfPDI-14. The prototype PDI molecule with two thioredoxin domains (including CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL, is PfPDI-8. Recently, Fiona Angrisano et al demonstrate that a plasmodium protein (PDI-Trans/PBANKA_0820300) is a PDI that is male specifc, surface-expressed, and is essential for fertilization/transmission. It exhibits disulfide isomerase activity that is up-regulated postgamete activation. Using bacitracin-a PDI inhibitor they show a reduction in male gamete activation and fusion. This PDI activity can be inhibited by an antibody directed against the extracellular domain [39, 40].

With respect to DsbA and bacterial pathogenesis lot of research has been done. First and foremost many downstream targets of DsbA have been identified and the contribution of DsbA to pathogenesis has been assessed by demonstrating that disruption of the gene for DsbA has a deleterious effect on pathogenesis due to reduction in the expression of virulence factors most notably as functional virulence molecules at the correct locale in the cell [41, 42].

The focus areas are likely to be the following:

1. Design of broad-spectrum mimetics that can bind to the hydrophobic domain of the DsbA catalytic site [31]. This has been shown to disrupt the DsbA function. Assessment of in vivo function of these mimetics.

2. Identification of downstream targets both in biofilm and planktonic versions of a given bacterial species. Special focus on inner membrane and outer membrane proteins. Potential proteins involved in Quorum sensing, etc. [42].

3. Assessment of thiol status of proteins and correlation with protein amounts, so a thiol to protein quantity determination as an assessment to a protein’s ability to be functional. Since a protein that is likely to have disulfide bonds will likely be more stable, so a protein that loses its disulfide bonds becomes less stable and consequently it steady-state levels drop and it is also less likely to be functional. Thus a correlation of thiol status of given major virulence factors or complexes to their amount is a key finding for a targeted attack on novel virulence determinants in newly emerging pathogenic bacterial species. Some assessments are being done for in vivo thiol status [43].

4. A combination of metabolomic and proteomic and genomic approaches to obtain a systemic understanding of DsbA and downstream protein targets in bacterial pathogenesis [42]. Scientists have developed metabolomic profiles for bacteria that might reside in the CF lung: For P. aeruginosa it was Cyanide production [44]. Later studies showed that beyond identifying the presence of a specific molecule, groups of P. aeruginosa-colonized CF patients could be differentiated from noncolonized CF patients based on overall volatile breath profiles [45].

Finally, increased understanding is likely to generate avenues for clinical trials incorporating targeted therapy on DsbA and DsbA targets at least in biofilms. Multiple strategies are used to limit biofilm-based infections on implants [46, 47]. Research on DsbA mimetics and other targets could yield new treatment modalities [46, 48, 49].

12. Summary

In summary, we have examined my old work and how we demonstrated the direct involvement of AlgT with dsbA gene expression. This is unique to P. aeruginosa. So far literature searches have not identified a similar regulatory mechanism in any other gram-negative bacterium. We have recently seen a similar ECF sigma factor directed control on a protein disulfide isomerase gene in a gram-positive bacterium. Our hypothesis as to why the DsbA gene is upregulated in a mucoid scenario is the fact that I think the alginate exopolysaccharide creates a difficult capsule to allow other virulence factors to go out of the cell. I have shown using western blot (data not shown) that Elastase secretion on a per cell basis (normalized for OD_600nm is lower in cells producing alginate versus cells producing no alginate. Intracellular accumulation of elastase was identical in both alginate-producing and nonproducing strains. To support this hypothesis we should examine situations or bacterial systems here such induction of dsbA is seen routinely. We can then evaluate dsbA function towards pathogenesis by looking at steady state levels of virulence determinants and components of virulence factor secretion machinery. In addition, since following dsbA gene induction and concomitant accumulation of dsbA protein we could see a better disulfide bond status—so maybe compare the protein levels to thiol status in proteins [2, 43, 50, 51]. In addition, the ECF response to induce dsbA gene and subsequently DsbA protein levels could be a molecular switch to up the thiol status of the Pseudomonas bacterial cell in response to harsh conditions from host immune response. This was seen with acidophilic iron-oxidizing bacterium Leptospirillum ferriphilum where exposure to ROS upped the activity of intracellular DsbA-like proteins [52]. Along with metabolomic profiling of compounds can be done. It is clear from various studies that disulfide bonds are crucial for protein stability and that DsbA facilitating disulfide bonds could be a crucial target for future mimetics that may be targeted to the hydrophobic domain of its enzymatic region [48]. Alternatively, novel downstream targets that affect pathogenesis, even more, could be targeted for drug design or mimetics. Very briefly I discovered more disulfide isomerase-like proteins por in H. influenzae and TlpA in S. meliloti. A TlpA mutant caused a complete loss in nodule forming capacity in the mutant strain. That could be recovered in the complementation clone (Data not shown). With Por (Por: Periplasmic oxidoreductase). I characterized the periplasmic fraction and in a small pilot, experiment identified a novel target RibH a component of the Riboflavin synthase (data not shown. See Figure 3 for MSA alignments of DsbA orthologs from different species of bacteria).

13. Conclusion

Briefly, this chapter describes the involvement of DsbA in the pathogenesis of P. aeruginosa. We describe dsbA gene characterization with gentamycin cassette mutagenesis. The effect of disruption of the dsbA gene on virulence factor production is provided. More recently on biofilm formation as well. Since P. aeruginosa is an important pathogen we are constantly developing strategies to identify ways to eliminate it. A series of novel spirothiazolopyridine derivatives were designed and prepared with strong antimicrobial and antifungal properties [53]. Such compounds are likely to enhance our ability to stay abreast of AMR and novel pathogens. In addition, novel diagnosis methods being developed should be used to assess microbial contamination through innovative methods of diagnoses [54]. This collection of studies of Dsb proteins from different bacteria clearly shows their strong involvement in bacterial pathogenesis. We propose a more defined focus on the studies of these proteins and the development of methods to suppress their function. We also show a direct interaction of AlgT with the dsbA promoter a so far unique regulation not seen in gram-negative bacteria. Since Dsb proteins might be involved in biofilm we suggest a focus on their role in biofilm. We also suggest study of phenazine compounds like pyocyanin due to their role in bacterial pathogenesis [55]. It would be fascinating to see the involvement of DsbA in phenazine compound secretion and their redox status.

Acknowledgments

I would like to thank my Ph.D. Mentor Dr. Dennis E. Ohman, Chair Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA, USA. He gave all the care and encouragement for the development of this stress response regulon hypothesis and the use of proteomics to identify components of this pathway. A similar proteomic strategy was used by other members of the Dennis Ohman Laboratory who contributed to my work through helpful discussions. I am thankful to Dr.(s) Sang Jin Suh, Laura Suh, Lynn Wood, Sumita Jain, Kerain Grande and Joanne Lee Johnston. Further thanks to members of my family, parents and brother who supported me through tough times of my Ph.D. research work. A special thanks to close friends like Dr. Gobardhan Das and Dr. Arshad Jilani. A special thanks to join me on this book chapter n to Dr. Sunil Kumar contributed to the section on Eukaryotic PDIs. His insight into their biology is appreciated.