Structural diversity among Acinetobacter baumannii K-antigens and its implication in the in silico serotyping.

Acinetobacter baumannii is an emerging opportunistic pathogen. It exhibits multi-, extreme-, and pan-drug resistance against several classes of antibiotics. Capsular polysaccharide (CPS or K-antigen) is one of the major virulence factors which aids A. baumannii in evading the host immune system. K-antigens of A. baumannii exploit the Wzx/Wzy-dependent pathway that involves 13 different proteins for its assembly and transport onto the outer membrane. A total of 64 (out of 237 K-locus(KL) types) known K-antigen sugar repeating structures are discussed here and are classified into seven groups based on their initial sugars, QuiNAc4NAc, GalNAc, GlcNAc, Gal, QuiNAc/FucNAc, FucNAc, and GlcNAc along with Leg5Ac7Ac/Leg5Ac7R. Thus, the corresponding seven initializing glycosyltransferases (ItrA1, ItrA2, ItrA3, ItrA4, ItrB1, ItrB3, and ItrA3 along with ItrB2) exhibit serotype specificity. The modeled 3D-structural repository of the 64 K-antigens can be accessed at https://project.iith.ac.in/ABSD/k_antigen.html. The topology of K-antigens further reveals the presence of 2-6 and 0-4 sugar monomers in the main and side chains, respectively. The presence of negatively (predominant) or neutrally charged K-antigens is observed in A. baumannii. Such diversity in the K-antigen sugar composition provides the K-typing specificity (viz., 18-69% in terms of reliability) for Wza, Wzb, Wzc, Wzx, and Wzy proteins involved in the Wzx/Wzy-dependent pathway. Interestingly, the degree of uniqueness of these proteins among different K-types is estimated to be 76.79%, considering the 237 reference sequences. This article summarizes the A. baumannii K-antigen structural diversity and creation of a K-antigen digital repository and provides a systematic analysis of the K-antigen assembly and transportation marker proteins.

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI.

The Pss-I region of Rhizobium leguminosarum bv. trifolii TA1 comprises more than 20 genes coding for glycosyltransferases, modifying enzymes, and polymerization/export proteins, altogether determining the biosynthesis of symbiotically relevant exopolysaccharides. In this study, the role of homologous PssG and PssI glycosyltransferases in exopolysaccharide subunit synthesis were analyzed. It was shown that the glycosyltransferase-encoding genes of the Pss-I region were part of a single large transcriptional unit with potential downstream promoters activated in specific conditions. The ΔpssG and ΔpssI mutants produced significantly lower amounts of the exopolysaccharide, while the double deletion mutant ΔpssIΔpssG produced no exopolysaccharide. Complementation of double mutation with individual genes restored exopolysaccharide synthesis, but only to the level similar to that observed for the single ΔpssI or ΔpssG mutants, indicating that PssG and PssI serve complementary functions in the process. PssG and PssI interacted with each other in vivo and in vitro. Moreover, PssI displayed an expanded in vivo interaction network comprising other GTs involved in subunit assembly and polymerization/export proteins. PssG and PssI proteins were shown to interact with the inner membrane through amphipathic helices at their C-termini, and PssG also required other proteins involved in exopolysaccharide synthesis to localize in the membrane protein fraction.

Repeat-Unit Elongations To Produce Bacterial Complex Long Polysaccharide Chains, an O-Antigen Perspective.

The O-antigen, a long polysaccharide that constitutes the distal part of the outer membrane-anchored lipopolysaccharide, is one of the critical components in the protective outer membrane of Gram-negative bacteria. Most species produce one of the structurally diverse O-antigens, with nearly all the polysaccharide components having complex structures made by the Wzx/Wzy pathway. This pathway produces repeat-units of mostly 3-8 sugars on the cytosolic face of the cytoplasmic membrane that is translocated by Wzx flippase to the periplasmic face and polymerized by Wzy polymerase to give long-chain polysaccharides. The Wzy polymerase is a highly diverse integral membrane protein typically containing 10-14 transmembrane segments. Biochemical evidence confirmed that Wzy polymerase is the sole driver of polymerization, and recent progress also began to demystify its interacting partner, Wzz, shedding some light to speculate how the proteins may operate together during polysaccharide biogenesis. However, our knowledge of how the highly variable Wzy proteins work as part of the O-antigen processing machinery remains poor. Here, we discuss the progress to the current understanding of repeat-unit polymerization and propose an updated model to explain the formation of additional short chain O-antigen polymers found in the lipopolysaccharide of diverse Gram-negative species and their importance in the biosynthetic process.

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

Secretion 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 ß-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 ß-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 ß-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 ß-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.

Bacterial Glycocalyx Integrity Impacts Tolerance of Myxococcus xanthus to Antibiotics and Oxidative-Stress Agents.

The presence of an exopolysaccharide (EPS) layer surrounding bacterial cells, termed a "glycocalyx", confers protection against toxic molecules. However, the effect of glycocalyx integrity on the tolerance to such agents is poorly understood. Using a modified disc-diffusion assay, we tested the susceptibility to a panel of antibiotics and oxidative stress-inducing compounds of various mutant strains of the social predatory Gram-negative soil bacterium Myxococcus xanthus; the selected mutants were those that manifest different physical states of their respective EPS glycocalyces. While the overall presence of an EPS layer was indeed beneficial for tolerance, the integrity of this layer was also found to affect the susceptibility of the bacterium to killing; however, this finding was not universal, and instead was dependent on the specific compound tested. Thus, the integrity of the cell-surface EPS glycocalyx plays an important role in the tolerance of M. xanthus to harmful compounds.

Topology of the Shigella flexneri Enterobacterial Common Antigen polymerase WzyE.

Enterobacteriales have evolved a specialized outer membrane polysaccharide [Enterobacterial Common Antigen (ECA)] which allows them to persist in various environmental niches. Biosynthesis of ECA initiates on the cytoplasmic leaflet of the inner membrane (IM) where glycosyltransferases assemble ECA repeat units (RUs). Complete RUs are then translocated across the IM and assembled into polymers by ECA-specific homologues of the Wzy-dependent pathway. Consisting of the membrane proteins Wzx, Wzy and Wzz, the Wzy-dependent pathway is the most common polysaccharide biosynthetic pathway in Gram-negative bacteria where it is most notably involved in LPS O antigen (Oag) biosynthesis. As such, the majority of research directed towards these proteins has been orientated towards Oag biosynthetic homologues with little directed towards ECA homologues. Belonging to the Shape, Elongation, Division and Sporulation (SEDS) protein family, Wzy proteins are polymerases, and are characterized as possessing little or no peptide homology among homologues as well as being polytopic membrane proteins with functionally relevant residues within periplasmic loops, as defined by C-terminal reporter fusion topology mapping. Here, we present the first the first major study into the ECA polymerase WzyE. Multiple sequence alignments and topology mapping showed that WzyE is unlike WzyB proteins involved with Oag biosynthesis WzyE displays high peptide conservation across Enterobacteriales. In silico structures and reporter mapping allowed us to identify possible functionally conserved residues with WzyESF's periplasmic loops, which we showed were crucial for its function. This work provides novel insight into Wzy proteins and suggests that WzyE is an optimal model to investigate Wzy proteins and the Wzy-dependent pathway.

Interdependence of Shigella flexneri O Antigen and Enterobacterial Common Antigen Biosynthetic Pathways.

Outer membrane (OM) polysaccharides allow bacteria to resist harsh environmental conditions and antimicrobial agents, traffic to and persist in pathogenic niches, and evade immune responses. Shigella flexneri has two OM polysaccharide populations, being enterobacterial common antigen (ECA) and lipopolysaccharide (LPS) O antigen (Oag); both are polymerized into chains by separate homologs of the Wzy-dependent pathway. The two polysaccharide pathways, along with peptidoglycan (PG) biosynthesis, compete for the universal biosynthetic membrane anchor, undecaprenyl phosphate (Und-P), as the finite pool of available Und-P is critical in all three cell wall biosynthetic pathways. Interactions between the two OM polysaccharide pathways have been proposed in the past where, through the use of mutants in both pathways, various perturbations have been observed. Here, we show for the first time that mutations in one of the two OM polysaccharide pathways can affect each other, dependent on where the mutation lies along the pathway, while the second pathway remains genetically intact. We then expand on this and show that the mutations also affect PG biosynthesis pathways and provide data which supports that the classical mutant phenotypes of cell wall mutants are due to a lack of available Und-P. Our work here provides another layer in understanding the complex intricacies of the cell wall biosynthetic pathways and demonstrates their interdependence on Und-P, the universal biosynthetic membrane anchor. IMPORTANCE Bacterial outer membrane polysaccharides play key roles in a range of bacterial activities from homeostasis to virulence. Two such OM polysaccharide populations are ECA and LPS Oag, which are synthesized by separate homologs of the Wzy-dependent pathway. Both ECA and LPS Oag biosynthesis join with PG biosynthesis to form the cell wall biosynthetic pathways, which all are interdependent on the availability of Und-P for proper function. Our data show the direct effects of cell wall pathway mutations affecting all related pathways when they themselves remain genetically unchanged. This work furthers our understanding of the complexities and interdependence of the three cell wall pathways.

Identification of a Region in Shigella flexneri WzyB Disrupting the Interaction with WzzpHS2.

Shigella flexneri can synthesize polysaccharide chains having complex sugars and a regulated number of repeating units. S. flexneri lipopolysaccharide O antigen (Oag) is synthesized by the Wzy-dependent pathway, which is the most common pathway used in bacteria for polysaccharide synthesis. The inner membrane protein WzyB polymerizes the Oag repeat units into chains, while the polysaccharide copolymerases WzzB and WzzpHS2 determine the average number of repeat units or "the modal length," termed short type and very long type. Our data show for the first time a direct interaction between WzyB and WzzpHS2, with and without the use of the chemical cross-linker dithiobis (succinimidyl propionate) (DSP). Additionally, mutations generated via random and site-directed mutagenesis identify a region of WzyB that caused diminished function and significantly decreased very long Oag chain polymerization, and that affected the aforementioned interaction. These results provide insight into the mechanisms underlying the regulation of Oag biosynthesis. IMPORTANCE Complex polysaccharide chains are synthesized by bacteria, usually at a regulated number of repeating units, which has broad implications for bacterial pathogenesis. One example is the O antigen (Oag) component of lipopolysaccharide that is predominantly synthesized by the Wzy-dependent pathway. Our findings show for the first time a direct physical interaction between WzyB and WzzpHS2. Additionally, a set of Wzy mutant constructs were generated, revealing a proposed active site/switch region involved in the activity of WzyB and the physical interaction with WzzpHS2. Combined, these findings further understanding of the Wzy-dependent pathway. The identification of a novel interaction with the polysaccharide copolymerase WzzpHS2 and the region of WzyB that is involved in this aforementioned interaction and its impact on WzyB Oag synthesis activity have significant implication for the prevention/treatment of bacterial diseases and discovery of novel biotechnologies.

Physiological and genomic analysis of newly-isolated polysaccharide synthesizing cyanobacterium Chroococcus sp. FPU101 and chemical analysis of the exopolysaccharide.

A unicellular cyanobacterium that produces a large amount of exopolysaccharide (EPS) was isolated. The isolate, named Chroococcus sp. FPU101, grew between 20 and 30°C and at light intensities between 10 and 80 µmol m-2 s-1. Purified EPS from Chroococcus sp. FPU101 had a molecular size of 5.9 × 103 kDa and contained galactose, rhamnose, fucose, xylose, mannose, glucose, galacturonic acid, and glucuronic acid at a molar ratio of 17.2:15.9:14.1:11.0:9.6:9.5:13.0:9.7. The EPS content significantly increased when the NaCl concentration in the medium was increased from 1.7 to 100 mM. However, high NaCl concentrations did not significantly affect the molecular size or chemical composition of the EPS. The genes wza, wzb, wzc, wzx, wzy, and wzz that are involved in EPS synthesis were conserved in the genome of Chroococcus sp. FPU101, which was sequenced in this study. These results suggest that the Wzy-dependent pathway is potentially involved in EPS production in this organism.

Screening for texturing Leuconostoc and genomics behind polysaccharide production.

Synthesis of polysaccharides by Leuconostoc can result in improved texture of fermented products. A total of 249 Leuconostoc strains were screened for homo-polysaccharide production and for texturing capabilities in milk. A total of six Ln. mesenteroides strains with superior texturing properties had the genetic blueprint for both homo- (HoPS) and hetero-polysaccharide (HePS) synthesis. Only one strain produced texture in milk without added sucrose, suggesting HePS synthesis via the Wzy dependent pathway. In milk acidification experiments with added sucrose, all six strains depleted the sucrose and released fructose. Thus, they can be used for both texture and possibly also for sweetness enhancement.

Sugar-Coated Killer: Serotype 3 Pneumococcal Disease.

Capsular polysaccharide (CPS), which surrounds the bacteria, is one of the most significant and multifaceted contributors to Streptococcus pneumoniae virulence. Capsule prevents entrapment in mucus during colonization, traps water to protect against desiccation, can serve as an energy reserve, and protects the bacterium against complement-mediated opsonization and immune cell phagocytosis. To date, 100 biochemically and serologically distinct capsule types have been identified for S. pneumoniae; 20 to 30 of which have well-defined propensity to cause opportunistic human infection. Among these, serotype 3 is perhaps the most problematic as serotype 3 infections are characterized as having severe clinical manifestations including empyema, bacteremia, cardiotoxicity, and meningitis; consequently, with a fatality rate of 30%-47%. Moreover, serotype 3 resists antibody-mediated clearance despite its inclusion in the current 13-valent conjugate vaccine formulation. This review covers the role of capsule in pneumococcal pathogenesis and the importance of serotype 3 on human disease. We discuss how serotype 3 capsule synthesis and presentation on the bacterial surface is distinct from other serotypes, the biochemical and physiological properties of this capsule type that facilitate its ability to cause disease, and why existing vaccines are unable to confer protection. We conclude with discussion of the clonal properties of serotype 3 and how these have changed since introduction of the 13-valent vaccine in 2000.

A Diverse Repertoire of Exopolysaccharide Biosynthesis Gene Clusters in Lactobacillus Revealed by Comparative Analysis in 106 Sequenced Genomes.

Production of exopolysaccharides (EPS) is one of the unique features of Lactobacillus genus. EPS not only have many physiological roles such as in stress tolerance, quorum sensing and biofilm formation, but also have numerous applications in the food and pharmaceutical industries. In this study, we identified and compared EPS biosynthesis gene clusters in 106 sequenced Lactobacillus genomes representing 27 species. Of the 146 identified clusters, only 41 showed the typical generic organization of genes as reported earlier. Hierarchical clustering showed highly varied nature of the clusters in terms of the gene composition; nonetheless, habitat-wise grouping was observed for the gene clusters from host-adapted and nomadic strains. Of the core genes required for EPS biosynthesis, epsA, B, C, D and E showed higher conservation, whereas gt, wzx and wzy showed high variability in terms of the number and composition of the protein families. Analysis of the distribution pattern of the protein families indicated a higher proportion of mutually exclusive families in clusters from host-adapted and nomadic strains, whereas those from the free-living group had very few unique families. Taken together, this analysis highlights high variability in the EPS gene clusters amongst Lactobacillus with some of their properties correlated to the habitats.

Physiological properties and genetic analysis related to exopolysaccharide (EPS) production in the fresh-water unicellular cyanobacterium Aphanothece sacrum (Suizenji Nori).

The clonal strains, phycoerythrin(PE)-rich- and PE-poor strains, of the unicellular, fresh water cyanobacterium Aphanothece sacrum (Suringar) Okada (Suizenji Nori, in Japanese) were isolated from traditional open-air aquafarms in Japan. A. sacrum appeared to be oligotrophic on the basis of its growth characteristics. The optimum temperature for growth was around 20°C. Maximum growth and biomass increase at 20°C was obtained under light intensities between 40 to 80 µmol m-2 s-1 (fluorescent lamps, 12 h light/12 h dark cycles) and between 40 to 120 µmol m-2 s-1 for PE-rich and PE-poor strains, respectively, of A. sacrum . Purified exopolysaccharide (EPS) of A. sacrum has a molecular weight of ca. 104 kDa with five major monosaccharides (glucose, xylose, rhamnose, galactose and mannose; ≥85 mol%). We also deciphered the whole genome sequence of the two strains of A. sacrum. The putative genes involved in the polymerization, chain length control, and export of EPS would contribute to understand the biosynthetic process of their extremely high molecular weight EPS. The putative genes encoding Wzx-Wzy-Wzz- and Wza-Wzb-Wzc were conserved in the A. sacrum strains FPU1 and FPU3. This result suggests that the Wzy-dependent pathway participates in the EPS production of A. sacrum.

Synthesis of Rhizobial Exopolysaccharides and Their Importance for Symbiosis with Legume Plants.

Rhizobia dwell and multiply in the soil and represent a unique group of bacteria able to enter into a symbiotic interaction with plants from the Fabaceae family and fix atmospheric nitrogen inside de novo created plant organs, called nodules. One of the key determinants of the successful interaction between these bacteria and plants are exopolysaccharides, which represent species-specific homo- and heteropolymers of different carbohydrate units frequently decorated by non-carbohydrate substituents. Exopolysaccharides are typically built from repeat units assembled by the Wzx/Wzy-dependent pathway, where individual subunits are synthesized in conjunction with the lipid anchor undecaprenylphosphate (und-PP), due to the activity of glycosyltransferases. Complete oligosaccharide repeat units are transferred to the periplasmic space by the activity of the Wzx flippase, and, while still being anchored in the membrane, they are joined by the polymerase Wzy. Here we have focused on the genetic control over the process of exopolysaccharides (EPS) biosynthesis in rhizobia, with emphasis put on the recent advancements in understanding the mode of action of the key proteins operating in the pathway. A role played by exopolysaccharide in Rhizobium-legume symbiosis, including recent data confirming the signaling function of EPS, is also discussed.

Challenges and perspectives in combinatorial assembly of novel exopolysaccharide biosynthesis pathways.

Because of their rheological properties various microbial polysaccharides are applied as thickeners and viscosifiers both in food and non-food industries. A broad variety of microorganisms secrete structurally diverse exopolysaccharides (EPS) that contribute to their surface attachment, protection against abiotic or biotic stress factors, and nutrient gathering. Theoretically, a massive number of EPS structures are possible through variations in monosaccharide sequences, condensation linkages and non-sugar decorations. Given the already-high diversity of EPS structures, taken together with the principal of combinatorial biosynthetic pathways, microbial polysaccharides are an attractive class of macromolecules with which to generate novel structures via synthetic biology approaches. However, previous manipulations primarily focused on increasing polysaccharide yield, with structural modifications restricted to removal of side chains or non-sugar decorations. This article outlines the biosynthetic pathways of the bacterial heteroexopolysaccharides xanthan and succinoglycan, which are used as thickening and stabilizing agents in food and non-food industries. Challenges and perspectives of combining synthetic biology approaches with directed evolution to overcome obstacles in assembly of novel EPS biosynthesis pathways are discussed.