Polysaccharide Export Research Articles

Ozone-Resistant Bacteria, an Inconvenient Hazard in Water Reclamation: Resistance Mechanism, Propagating Capacity, and Potential Risks.

Resistant bacteria have always been of research interest worldwide. In the urban water system, the increased disinfectant usage gives more chances for undesirable disinfection-resistant bacteria. As the strongest oxidative disinfectant in large-scale water treatment, ozone might select ozone-resistant bacteria (ORB), which, however, have rarely been reported and are inexplicit for their resistant mechanisms and physiological characteristics. In this study, six strains of ORB were screened from a water reclamation plant in Beijing. Three of them (O7, CR19, and O4) were more resistant to ozone than all previously reported ORB or even spores. The ozone consumption capacity of extracellular polymeric substances and cell walls was proved to be the main sources of bacterial ozone resistance, rather than intracellular antioxidant enzymes. The transcriptome results elucidated that strong ORB possessed a combined antioxidant mechanism consisting of the enhanced transcription of protein synthesis, protein export, and polysaccharide export genes (LptF, LptB, NodJ, LivK, LviG, MetQ, MetN, and GltU). This study confirmed the existence of ORB in urban water systems and brought doubts to the idea of a traditional control strategy against chlorine-resistant bacteria. A salient "trade-off" effect between the ozone resistance and propagation ability indicated the weakness and potential control approaches of ORB.

A computational model for lipid-anchored polysaccharide export by the outer membrane protein GfcD

Many bacteria are protected by different types of polysaccharide capsules, structures formed of long repetitive glycan chains that are sometimes free and sometimes anchored to the outer membrane via lipid tails. One type, called group 4 capsule, results from the expression of the gfcABCDE-etp-etk operon in Escherichia coli. Of the proteins encoded in this operon, GfcE is thought to provide the export pore for free polysaccharide chains, but none of the proteins has been implicated in the export of chains carrying a lipid anchor. For this function, GfcD has been a focus of attention as the only outer membrane β-barrel encoded in the operon. AlphaFold predicts two β-barrel domains in GfcD, a canonical N-terminal one of 12 strands and an unusual C-terminal one of 13 strands, which features a large lateral aperture between strands β1 and β13. This immediately suggests a lateral exit gate for hydrophobic molecules into the membrane, analogous to the one proposed for the lipopolysaccharide export pore LptD. Here, we report an unsteered molecular dynamics study of GfcD embedded in the bacterial outer membrane, with the common polysaccharide anchor, lipid A, inserted in the pore of the C-terminal barrel. Our results show that the lateral aperture does not collapse during simulations and membrane lipids nevertheless do not penetrate the barrel but the lipid chains of the lipid A molecule readily exit into the membrane.

Development of a novel multi-epitope vaccine for brucellosis prevention

Brucellosis, a zoonotic disease caused by Brucella, presents a significant threat to both animal and human health. In animals, the disease can lead to infertility, miscarriage, and high fever, while in humans, symptoms may include recurrent fever, fatigue, sweating, hepatosplenomegaly, and joint and muscle pain following infection. Treatment often involves long-term antibiotic therapy, placing a substantial psychological and financial burden on patients. While vaccination is crucial for prevention, current animal vaccines have drawbacks such as residual virulence, and a safe and effective human vaccine is lacking. Hence, the development of a vaccine for brucellosis is imperative. In this study, we utilized bioinformatics methods to design a multi-epitope vaccine targeting Brucella. Targeting Heme transporter BhuA and polysaccharide export protein, we identified antigenic epitopes, including six cytotoxic T lymphocyte (CTL) dominant epitopes, six helper T lymphocyte (HTL) dominant epitopes, one conformation B cell dominant epitope, and three linear B cell dominant epitopes. By linking these epitopes with appropriate linkers and incorporating a Toll-like receptor (TLR) agonist (human beta-defensin-2) and an auxiliary peptide (Pan HLA-DR epitopes), we constructed the multi-epitope vaccine (MEV). The MEV demonstrated high antigenicity, non-toxicity, non-allergenicity, non-human homology, stability, and solubility. Molecular docking analysis and molecular dynamics simulations confirmed the interaction and stability of the MEV with receptors (MHCI, MHCII, TLR4). Codon optimization and in silico cloning validated the translation efficiency and successful expression of MEV in Escherichia coli. Immunological simulations further demonstrated the efficacy of MEV in inducing robust immune responses. In conclusion, our findings suggest that the engineered MEVs have the potential to stimulate both humoral and cellular immune responses, offering valuable insights for the future development of safe and efficient Brucella vaccines.

Exploring Roles of the Polysaccharide Capsule in Pathogenesis of Hypervirulent Acinetobacter baumannii Clinical Isolate Lac-4.

The frequently multidrug-resistant bacterial pathogen Acinetobacter baumannii is a leading cause of nosocomial infections, including ventilator-associated pneumonia, such that the World Health Organization and US Centers for Disease Control and Prevention have declared it a top priority candidate for novel drug development. Nearly all clinical A. baumannii strains express a thick surface polysaccharide capsule that protects against desiccation, host defenses, and disinfectants. In this study, we investigated the contribution of the polysaccharide capsule to virulence caused by the A. baumannii clinical isolate Ab Lac-4, which is rare in its ability to cause pneumonia and disseminated sepsis in healthy mice. We assessed the role of the capsule in wildtype Lac-4 (WT) by generating a premature stop codon in wza, which codes for the polysaccharide export protein. The wza# mutant was hypersensitive to killing by complement, whole blood, and healthy human neutrophils compared to WT and a revertant mutant (wza-Rev). Furthermore, the wza# mutant was highly attenuated in murine sepsis and unable to disseminate from the lungs during pneumonia. This study reinforces the capsule as a key contributor to Ab Lac-4 hypervirulence.

Genomic Comparative Analysis of Two Multi-Drug Resistance (MDR) Acinetobacter baumannii Clinical Strains Assigned to International Clonal Lineage II Recovered Pre- and Post-COVID-19 Pandemic.

After the emergence of COVID-19, numerous cases of A. baumannii/SARS-CoV-2 co-infection were reported. Whether the co-infecting A. baumannii strains have distinctive characteristics remains unknown. A. baumannii AMA_NO was isolated in 2021 from a patient with COVID-19. AMA166 was isolated from a mini-BAL used on a patient with pneumonia in 2016. Both genomes were similar, but they possessed 337 (AMA_NO) and 93 (AMA166) unique genes that were associated with biofilm formation, flagellar assembly, antibiotic resistance, secretion systems, and other functions. The antibiotic resistance genes were found within mobile genetic elements. While both strains harbored the carbapenemase-coding gene blaOXA-23, only the strain AMA_NO carried blaNDM-1. Representative functions coded for by virulence genes are the synthesis of the outer core of lipooligosaccharide (OCL5), biosynthesis and export of the capsular polysaccharide (KL2 cluster), high-efficiency iron uptake systems (acinetobactin and baumannoferrin), adherence, and quorum sensing. A comparative phylogenetic analysis including 239 additional sequence type (ST) 2 representative genomes showed high similarity to A. baumannii ABBL141. Since the degree of similarity that was observed between A. baumannii AMA_NO and AMA166 is higher than that found among other ST2 strains, we propose that they derive from a unique background based on core-genome phylogeny and comparative genome analysis. Acquisition or shedding of specific genes could increase the ability of A. baumannii to infect patients with COVID-19.

Bacterial Outer Membrane Polysaccharide Export (OPX) Proteins Occupy Three Structural Classes with Selective β-Barrel Porin Requirements for Polymer Secretion.

ABSTRACTSecretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous, as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes implicating outer membrane (OM) polysaccharide export (OPX) proteins in cell-surface polymer translocation. In the social predatory bacterium Myxococcus xanthus, the exopolysaccharide (EPS) pathway WzaX, major spore coat (MASC) pathway WzaS, and biosurfactant polysaccharide (BPS) pathway WzaB were herein found to be truncated OPX homologues of Escherichia coli Wza lacking OM-spanning α-helices. Comparative genomics across all bacteria (>91,000 OPX proteins identified and analyzed), complemented with cryo-electron tomography cell-envelope analyses, revealed such “truncated” WzaX/S/B architecture to be the most common among three defined OPX-protein structural classes independent of periplasm thickness. Fold recognition and deep learning revealed the conserved M. xanthus proteins MXAN_7418/3226/1916 (encoded beside wzaX/S/B, respectively) to be integral OM β-barrels, with structural homology to the poly-N-acetyl-d-glucosamine synthase-dependent pathway porin PgaA. Such bacterial porins were identified near numerous genes for all three OPX protein classes. Interior MXAN_7418/3226/1916 β-barrel electrostatics were found to match properties of their associated polymers. With MXAN_3226 essential for MASC export, and MXAN_7418 herein shown to mediate EPS translocation, we have designated this new secretion machinery component “Wzp” (i.e., Wz porin), with the final step of M. xanthus EPS/MASC/BPS secretion across the OM now proposed to be mediated by WzpX/S/B (i.e., MXAN_7418/3226/1916). Importantly, these data support a novel and widespread secretion paradigm for polysaccharide biosynthesis pathways in which those containing OPX components that cannot span the OM instead utilize β-barrel porins to mediate polysaccharide transport across the OM.IMPORTANCE Diverse bacteria assemble and secrete polysaccharides that alter their physiologies through modulation of motility, biofilm formation, and host immune system evasion. Most such pathways require outer membrane (OM) polysaccharide export (OPX) proteins for sugar-polymer transport to the cell surface. In the prototypic Escherichia coli Group-1-capsule biosynthesis system, eight copies of this canonical OPX protein cross the OM with an α-helix, forming a polysaccharide-export pore. Herein, we instead reveal that most OPX proteins across all bacteria lack this α-helix, raising questions as to the manner by which most secreted polysaccharides actually exit cells. In the model developmental bacterium Myxococcus xanthus, we show this process to depend on OPX-coupled OM-spanning β-barrel porins, with similar porins encoded near numerous OPX genes in diverse bacteria. Knowledge of the terminal polysaccharide secretion step will enable development of antimicrobial compounds targeted to blocking polymer export from outside the cell, thus bypassing any requirements for antimicrobial compound uptake by the cell.

Evidence for a Widespread Third System for Bacterial Polysaccharide Export across the Outer Membrane Comprising a Composite OPX/β-Barrel Translocon.

ABSTRACTIn Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM) polysaccharide export (OPX) type translocon exports the polysaccharide across the OM. The paradigm OPX protein Wza of Escherichia coli is an octamer in which the eight C-terminal domains form an α-helical OM pore and the eight copies of the three N-terminal domains (D1 to D3) form a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18-stranded β-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS (exopolysaccharide) is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments, phylogenomics, and computational structural biology, we identify and characterize EpsX as an OM 18-stranded β-barrel protein important for EPS synthesis and identify AlgE, a β-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, consists only of the periplasmic D1 and D2 domains and completely lacks the domain for spanning the OM (herein termed a D1D2OPX protein). In vivo, EpsX and EpsY mutually stabilize each other and interact in in vivo pulldown experiments supporting their direct interaction. Based on these observations, we propose that EpsY and EpsX make up and represent a third type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning β-barrel translocon together with the periplasmic D1D2OPX protein EpsY. Based on computational genomics, similar composite systems are widespread in Gram-negative bacteria.

Structural basis for the recognition of the bacterial tyrosine kinase Wzc by its cognate tyrosine phosphatase Wzb

Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster. This process is driven by the opposing effects of intermolecular autophosphorylation, and dephosphorylation catalyzed by tyrosine phosphatases. In the absence of structural insight into the interactions between a BY-kinase and its phosphatase partner in atomic detail, the precise mechanism of this regulatory process has remained poorly defined. To address this gap in knowledge, we have determined the structure of the transiently assembled complex between the catalytic core of the Escherichia coli (K-12) BY-kinase Wzc and its counteracting low-molecular weight protein tyrosine phosphatase (LMW-PTP) Wzb using solution NMR techniques. Unambiguous distance restraints from paramagnetic relaxation effects were supplemented with ambiguous interaction restraints from static spectral perturbations and transient chemical shift changes inferred from relaxation dispersion measurements and used in a computational docking protocol for structure determination. This structurepresents an atomic picture of the mode of interaction between an LMW-PTP and its BY-kinase substrate, and provides mechanistic insight into the phosphorylation-coupled assembly/disassembly process proposed to drive BY-kinase function.

Prochloraz alone or in combination with nano-CuO promotes the conjugative transfer of antibiotic resistance genes between Escherichia coli in pure water

Conjugative plasmid transfer is a major contributor to the spread of antibiotic resistance genes (ARGs). However, the role of conventional fungicides on conjugative plasmid transfer has been neglected. Based on the condition that the increasing use of the combination of nano- and conventional fungicides will lead to combined contamination, the effects of a conventional fungicide prochloraz alone or in combination with nano-CuO on the conjugation of plasmid RP4 between Escherichia coli in phosphate-buffered saline were investigated in this study. The results demonstrated that 50 µg/L prochloraz alone significantly increased the conjugative transfer by 1.82 folds. The combination of 100 µg/L nano-CuO and prochloraz at 5, 50, and 500 µg/L significantly increased the conjugation by 2.56, 3.61, and 2.13 folds, respectively. The promotion of conjugative transfer of ARGs mediated by fungicides is mainly attributed to (i) the increased cell membrane permeability, (ii) the increased cell adhesion via enhancing the synthesis of polysaccharides in extracellular polymeric substances, and (iii) the up-regulation of the genes relevant to conjugation, oxidative stress, SOS response, outer membrane, polysaccharide export, intercellular adhesion, and ATP synthesis. Our findings provide evidence for the contribution of fungicides to ARGs transfer, which is significant to control the risk of ARGs dissemination.

Impaired cell-cell communication in the multicellular cyanobacterium Anabaena affects carbon uptake, photosynthesis, and the cell wall.

SummaryCell-cell communication is an essential attribute of multicellular organisms. The effects of perturbed communication were studied in septal protein mutants of the heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 model organism. Strains bearing sepJ and sepJ/fraC/fraD deletions showed differences in growth, pigment absorption spectra, and spatial patterns of expression of the hetR gene encoding a heterocyst differentiation master regulator. Global changes in gene expression resulting from deletion of those genes were mapped by RNA sequencing analysis of wild-type and mutant strains, both under nitrogen-replete and nitrogen-poor conditions. The effects of sepJ and fraC/fraD deletions were non-additive, and perturbed cell-cell communication led to significant changes in global gene expression. Most significant effects, related to carbon metabolism, included increased expression of genes encoding carbon uptake systems and components of the photosynthetic apparatus, as well as decreased expression of genes encoding cell wall components related to heterocyst differentiation and to polysaccharide export.

Comparative Methylome Analysis of Campylobacter jejuni Strain YH002 Reveals a Putative Novel Motif and Diverse Epigenetic Regulations of Virulence Genes.

Campylobacter jejuni is a major cause of foodborne gastroenteritis worldwide inflicting palpable socioeconomic costs. The ability of this pathogen to successfully infect its hosts is determined not only by the presence of specific virulence genes but also by the pathogen’s capacity to appropriately regulate those virulence genes. Therefore, DNA methylation can play a critical role in both aspects of this process because it serves as both a means to protect the integrity of the cellular DNA from invasion and as a mechanism to control transcriptional regulation within the cell. In the present study we report the comparative methylome data of C. jejuni YH002, a multidrug resistant strain isolated from retail beef liver. Investigation into the methylome identified a putative novel motif (CGCGA) of a type II restriction-modification (RM) system. Comparison of methylomes of the strain to well-studied C. jejuni strains highlighted non-uniform methylation patterns among the strains though the existence of the typical type I and type IV RM systems were also observed. Additional investigations into the existence of DNA methylation sites within gene promoters, which may ultimately result in altered levels of transcription, revealed several virulence genes putatively regulated using this mode of action. Of those identified, a flagella gene (flhB), a RNA polymerase sigma factor (rpoN), a capsular polysaccharide export protein (kpsD), and a multidrug efflux pump were highly notable.

Revelation of Function and Inhibition of Wza Through Single-Channel Studies.

Antibacterial resistance (AR) is causing more and more bacterial infections that cannot be cured by using the antibacterial drugs that are currently available. It is predicted that 10 million people will die every year by 2050 from infections caused by antibacterial resistant strains, surpassing the predicted numbers of deaths caused by cancer. AR is therefore a global challenge and novel antibacterial strategies are in high demand. To this end, the work on exploring the pore properties of a bacterial sugar transporter, WzaK30, has led to the discovery of the first inhibitor against bacterial capsular polysaccharides export.Recently, single-molecule recapitulation of capsular polysaccharide (CPS) export and pore formation properties of Wza barrel peptides have also revealed the possibility of a next-generation of Wza strategies. These strategies are based upon the first examination and understanding of the pore properties of wild-type (WT) and mutant WzaK30 in single-molecule electrical channel recording. The initially reported experimental procedures have been further developed to enable efficient studies of other Wza homologs that are more common in bacterial pathogens causing significant bacterial infections. Therefore, this chapter presents the most recent protocols and logistics behind the research on Wza channel activity, antibacterials, and strategies. The disciplines covered here include computation, molecular biology, biochemistry, electrophysiology, microbiology, and biophysics.

Articles of Significant Interest in This Issue

Exopolysaccharide (EPS), a secreted polysaccharide, has important functions in the social life cycle of Myxococcus xanthus. Most insights into the function of EPS have been obtained from analyses of regulatory mutants with altered EPS accumulation. Pérez-Burgos et al. (e00335-20) show that the EPS biosynthetic machinery makes up a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis and export. Mutants of this pathway have not only reduced EPS synthesis but also reduced type IV pilus-dependent motility and a conditional defect in development. Surprisingly, the defect in type IV pilus-dependent motility could be traced down to a defect in type IV pilus extension.

Identification of a hormogonium polysaccharide-specific gene set conserved in filamentous cyanobacteria.

Cyanobacteria comprise a phylum defined by the capacity for oxygenic photosynthesis. Members of this phylum are frequently motile as well. Strains that display gliding or twitching motility across semisolid surfaces are powered by a conserved type IV pilus system (T4P). Among the filamentous, heterocyst-forming cyanobacteria, motility is usually confined to specialized filaments known as hormogonia, and requires the deposition of an associated hormogonium polysaccharide (HPS). The genes involved in assembly and export of HPS are largely undefined, and it has been hypothesized that HPS exits the outer membrane via an atypical T4P-driven mechanism. Here, several novel hps loci, primarily encoding glycosyl transferases, are identified. Mutational analysis demonstrates that the majority of these genes are essential for both motility and production of HPS. Notably, most mutant strains accumulate wild-type cellular levels of the major pilin PilA, but not extracellular PilA, indicating dysregulation of the T4P motors, and, therefore, a regulatory interaction between HPS assembly and T4P activity. A co-occurrence analysis of Hps orthologs among cyanobacteria identified an extended set of putative Hps proteins comprising most components of a Wzx/Wzy-type polysaccharide synthesis and export system. This implies that HPS may be secreted through a more canonical pathway, rather than a T4P-mediated mechanism.

Identification of the Wzx flippase, Wzy polymerase and sugar-modifying enzymes for spore coat polysaccharide biosynthesis in Myxococcus xanthus.

The rod-shaped cells of Myxococcus xanthus, a Gram-negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesised by the ExoA-I proteins, some of which are part of a Wzx/Wzy-dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterise two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores. Our data support that MXAN_3260, renamed ExoM and MXAN_3026, renamed ExoJ, are the Wzx flippase and Wzy polymerase, respectively, responsible for translocation and polymerisation of the repeat unit of the spore coat polysaccharide. Moreover, we provide evidence that three glycosyltransferases (MXAN_3027/ExoK, MXAN_3262/ExoO and MXAN_3263/ExoP) and a polysaccharide deacetylase (MXAN_3259/ExoL) are important for formation of the intact spore coat, while ExoE is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for initiating repeat unit synthesis, likely by transferring N-acetylgalactosamine-1-P to undecaprenyl-phosphate. Together, our data generate a more complete model of the Exo pathway for spore coat polysaccharide biosynthesis and export.

Wza gene knockout decreases Acinetobacter baumannii virulence and affects Wzy-dependent capsular polysaccharide synthesis

ABSTRACT To investigate the virulence of capsular polysaccharide export protein (Wza) in carbapenem-resistant Acinetobacter baumannii and its effect on capsule formation. wza gene knockout and complementation strains were constructed, and changes in bacterial virulence were observed using in vitro adhesion, antiserum complement killing, anti-oxidation experiments, and infections in Galleria mellonella and mice. The effect of wza knockout on the genes wzb and wzc and wzi were assessed by RT-PCR. We successfully constructed wza knockout and complementation strains. Compared with wild-type (WT) strains, wza knockout strains displayed lower adhesion to A549 cells (p = 0.044), lower antiserum complement killing ability (p = 0.001), and lower mortality of G. mellonella (p = 0.010) and mice (p = 0.033). Expression levels of wzb, wzc and wzi were decreased in wza knockout strains. The antioxidant capacity of Wza knockout bacteria was only slightly decreased. Complementation of the wza gene returned the adhesion ability, antiserum complement killing ability, and mortality of G. mellonella and mice to WT levels. Expression of wzb, wzc and wzi was also returned to WT levels following wza complementation. The results clearly demonstrate that Wza is toxic. Wza affects the expression of other proteins of the Wzy capsule polysaccharide synthesis pathway, which affects the assembly, export, and extracellular fixation of capsular polysaccharide, resulting in synergistic effects that decrease bacterial virulence.

Extracellular Vesicles as Vehicles for the Delivery of Biologically Active Fungal Molecules.

Extracellular vesicles (EVs) are membranous structures surrounded by a lipid bilayer required for the export of fungal proteins, lipids, toxins, nucleic acids, pigments, and polysaccharides. Proteomic studies of the content of fungal EVs revealed the presence of molecules involved in cell metabolism, signal transduction, and virulence, among others. EVs are evolutionarily conserved in all three domains of life and play important roles in cell-cell communication. Recently, the bidirectional transport of EVs was characterized through the demonstration that EVs can be released and captured by fungal cells. In fungi, EVs participate in immunomodulation through the delivery of virulence factors, antigens and allergens, but further studies are necessary to investigate their potential roles as carriers of diagnostic biomarkers and in drug delivery or antifungal resistance transmission. In this review, we discuss the roles of fungal EVs and their cargo in cell-cell communication, host-pathogen interactions, and environmental perception. The functions of EVs as vehicles for transporting fungal proteins and virulence factors are also addressed, as well as their use as biomarkers for the diagnosis of diseases and possible participation in antifungal responses.

The gfc operon is involved in the formation of the O antigen capsule in Aeromonas hydrophila and contributes to virulence in channel catfish

A hypervirulent A. hydrophila (vAh) pathotype has been identified as the etiologic agent involved in disease outbreaks in farmed carp and catfish species in China and the Southeastern United States, respectively. To assess the role of the LPS O-antigen in vAh virulence, the O-antigen ligase (waaL) and O-antigen polymerase (wzy) genes were deleted. While neither waaL nor wzy were found to be required for vAh virulence, a waaL mutant was found to have a polar effect on an adjacent group 4 capsule (gfc) genetic operon that was predicted to play a role in capsule assembly. Mutations in the gfc operon attenuated vAh virulence in channel catfish, particularly the mutant lacking gfcD that is predicted to encode an outer membrane protein required for capsular polysaccharide export. Furthermore, the vAh gfcD mutant was found to lack significant biofilm-forming capacity or buoyancy compared to wild-type vAh and induced an adaptive immune response that protected catfish from vAh challenge. This study indicates the importance of the capsular polysaccharide assembly process in the pathogenesis of this highly virulent A. hydrophila pathotype.

Structural and Functional Variation in Outer Membrane Polysaccharide Export (OPX) Proteins from the Two Major Capsule Assembly Pathways Present in Escherichia coli.

Capsular polysaccharides (CPSs) are virulence factors for many important pathogens. In Escherichia coli, CPSs are synthesized via two distinct pathways, but both require proteins from the outer membrane polysaccharide export (OPX) family to complete CPS export from the periplasm to the cell surface. In this study, we compare the properties of the OPX proteins from the prototypical group 1 (Wzy-dependent) and group 2 (ABC transporter-dependent) pathways in E. coli K30 (Wza) and E. coli K2 (KpsD), respectively. In addition, we compare an OPX from Salmonella enterica serovar Typhi (VexA), which shares structural properties with Wza, while operating in an ABC transporter-dependent pathway. These proteins differ in distribution in the cell envelope and formation of stable multimers, but these properties do not align with acylation or the interfacing biosynthetic pathway. In E. coli K2, murein lipoprotein (Lpp) plays a role in peptidoglycan association of KpsD, and loss of this interaction correlates with impaired group 2 capsule production. VexA also depends on Lpp for peptidoglycan association, but CPS production is unaffected in an lpp mutant. In contrast, Wza and group 1 capsule production is unaffected by the absence of Lpp. These results point to complex structure-function relationships between different OPX proteins.IMPORTANCE Capsules are protective layers of polysaccharides that surround the cell surface of many bacteria, including that of Escherichia coli isolates and Salmonella enterica serovar Typhi. Capsular polysaccharides (CPSs) are often essential for virulence because they facilitate evasion of host immune responses. The attenuation of unencapsulated mutants in animal models and the involvement of protein families with conserved features make the CPS export pathway a novel candidate for therapeutic strategies. However, appropriate "antivirulence" strategies require a fundamental understanding of the underpinning cellular processes. Investigating export proteins that are conserved across different biosynthesis strategies will give important insight into how CPS is transported to the cell surface.

Proteins in the periplasmic space and outer membrane vesicles of Rhizobium etli CE3 grown in minimal medium are largely distinct and change with growth phase.

Rhizobium etli CE3 grown in succinate-ammonium minimal medium (MM) excreted outer membrane vesicles (OMVs) with diameters of 40 to 100 nm. Proteins from the OMVs and the periplasmic space were isolated from 6 and 24 h cultures and identified by proteome analysis. A total of 770 proteins were identified: 73.8 and 21.3 % of these occurred only in the periplasm and OMVs, respectively, and only 4.9 % were found in both locations. The majority of proteins found in either location were present only at 6 or 24 h: in the periplasm and OMVs, only 24 and 9 % of proteins, respectively, were present at both sampling times, indicating a time-dependent differential sorting of proteins into the two compartments. The OMVs contained proteins with physiologically varied roles, including Rhizobium adhering proteins (Rap), polysaccharidases, polysaccharide export proteins, auto-aggregation and adherence proteins, glycosyl transferases, peptidoglycan binding and cross-linking enzymes, potential cell wall-modifying enzymes, porins, multidrug efflux RND family proteins, ABC transporter proteins and heat shock proteins. As expected, proteins with known periplasmic localizations (phosphatases, phosphodiesterases, pyrophosphatases) were found only in the periplasm, along with numerous proteins involved in amino acid and carbohydrate metabolism and transport. Nearly one-quarter of the proteins present in the OMVs were also found in our previous analysis of the R. etli total exproteome of MM-grown cells, indicating that these nanoparticles are an important mechanism for protein excretion in this species.