Klebsiella LPS O1-antigen prevents complement-mediated killing by inhibiting C9 polymerization.

The Gram-negative bacterium Klebsiella pneumoniae is an important human pathogen. Its treatment has been complicated by the emergence of multi-drug resistant strains. The human complement system is an important part of our innate immune response that can directly kill Gram-negative bacteria by assembling membrane attack complex (MAC) pores into the bacterial outer membrane. To resist this attack, Gram-negative bacteria can modify their lipopolysaccharide (LPS). Especially the decoration of the LPS outer core with the O-antigen polysaccharide has been linked to increased bacterial survival in serum, but not studied in detail. In this study, we characterized various clinical Klebsiella pneumoniae isolates and show that expression of the LPS O1-antigen correlates with resistance to complement-mediated killing. Mechanistic data reveal that the O1-antigen does not inhibit C3b deposition and C5 conversion. In contrast, we see more efficient formation of C5a, and deposition of C6 and C9 when an O-antigen is present. Further downstream analyses revealed that the O1-antigen prevents correct insertion and polymerization of the final MAC component C9 into the bacterial membrane. Altogether, we show that the LPS O1-antigen is a key determining factor for complement resistance by K. pneumoniae and provide insights into the molecular basis of O1-mediated MAC evasion.

Molecular serotyping of diarrheagenic Escherichia coli with a MeltArray assay reveals distinct correlation between serotype and pathotype.

Diarrheagenic Escherichia coli serotypes are associated with various clinical syndromes, yet the precise correlation between serotype and pathotype remains unclear. A major barrier to such studies is the reliance on antisera-based serotyping, which is culture-dependent, low-throughput, and cost-ineffective. We have established a highly multiplex PCR-based serotyping assay, termed the MeltArray E. coli serotyping (EST) assay, capable of identifying 163 O-antigen-encoding genes and 53 H-antigen-encoding genes of E. coli. The assay successfully identified serotypes directly from both simulated and real fecal samples, as demonstrated through spike-in validation experiments and a retrospective study. In a multi-province study involving 637 E. coli strains, it revealed that the five major diarrheagenic pathotypes have distinct serotype compositions. Notably, it differentiated 257 Shigella isolates into four major Shigella species, distinguishing them from enteroinvasive E. coli based on their distinct serotype profiles. The assay's universality was further corroborated by in silico analysis of whole-genome sequences from the EnteroBase. We conclude that the MeltArray EST assay represents a paradigm-shifting tool for molecular serotyping of E. coli, with potential routine applications for comprehensive serotype analysis, disease diagnosis, and outbreak detection.

Development of a Shigella conjugate vaccine targeting Shigella flexneri 6 that is immunogenic and provides protection against virulent challenge.

Immunity protective against shigella infection targets the bacterial O-specific polysaccharide (OSP) component of lipopolysaccharide. A multivalent shigella vaccine would ideally target the most common global Shigella species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. We previously reported development of shigella conjugate vaccines (SCVs) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using a platform squaric acid chemistry conjugation approach and carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. Here we report development of a SCV targeting S. flexneri 6 (SCV-Sf6) using the same platform approach. We demonstrated that SCV-Sf6 was recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG and IgM responses, as well as rTTHc-specific IgG responses. Immune responses were increased when administered with aluminum phosphate adjuvant. Vaccination induced bactericidal antibody responses against S. flexneri 6, and vaccinated animals were protected against lethal challenge with virulent S. flexneri 6. Our results assist in the development of a multivalent vaccine protective against shigellosis.

Structure, biosynthesis and regulation of the T1 antigen, a phase-variable surface polysaccharide conserved in many Salmonella serovars.

The bacterial genus Salmonella includes diverse isolates with multiple variations in the structure of the main polysaccharide component (O antigen) of membrane lipopolysaccharides. In addition, some isolates produce a transient (T) antigen, such as the T1 polysaccharide identified in the 1960s in an isolate of Salmonella enterica Paratyphi B. The structure and biosynthesis of the T1 antigen have remained enigmatic. Here, we use biophysical, biochemical and genetic methods to show that the T1 antigen is a complex linear glycan containing tandem homopolymeric domains of galactofuranose and ribofuranose, linked to lipid A-core, like a typical O antigen. T1 is a phase-variable antigen, regulated by recombinational inversion of the promoter upstream of the T1 genetic locus through a mechanism not observed for other bacterial O antigens. The T1 locus is conserved across many Salmonella isolates, but is mutated or absent in most typhoidal serovars and in serovar Enteritidis.

Synergy between Group 2 capsules and lipopolysaccharide underpins serum resistance in extra-intestinal pathogenic Escherichia coli.

Escherichia coli (E. coli) is a major cause of urinary tract infections, bacteraemia, and sepsis. CFT073 is a prototypic, urosepsis isolate of sequence type (ST) 73. This laboratory, among others, has shown that strain CFT073 is resistant to serum, with capsule and other extracellular polysaccharides imparting resistance. The interplay of such polysaccharides remains under-explored. This study has shown that CFT073 mutants deficient in lipopolysaccharide (LPS) O-antigen and capsule display exquisite serum sensitivity. Additionally, O-antigen and LPS outer core mutants displayed significantly decreased surface K2 capsule, coupled with increased unbound K2 capsule being detected in the supernatant. The R1 core and O6 antigen are involved in the tethering of K2 capsule to the CFT073 cell surface, highlighting the importance of the R1 core in serum resistance. The dependence of capsule on LPS was shown to be post-transcriptional and related to changes in cell surface hydrophobicity. Furthermore, immunofluorescence microscopy suggested that the surface pattern of capsule is altered in such LPS core mutants, which display a punctate capsule pattern. Finally, targeting LPS biosynthesis using sub-inhibitory concentrations of a WaaG inhibitor resulted in increased serum sensitivity and decreased capsule in CFT073. Interestingly, the dependency of capsule on LPS has been observed previously in other Enterobacteria, indicating that the synergy between these polysaccharides is not just strain, serotype or species-specific but may be conserved across several pathogenic Gram-negative species. Therefore, using WaaG inhibitor derivatives to target LPS is a promising therapeutic strategy to reduce morbidity and mortality by reducing or eliminating surface capsule.

Eliminating Lipopolysaccharide (LPS) Using Lactic Acid Bacteria (LAB).

Gram-negative bacteria such as Escherichia coli, among intestinal bacteria, have lipopolysaccharide (LPS), which induces inflammation of human intestines. However, lactic acid bacteria (LAB) can improve human intraintestinal conditions. One reason is that ingestion of LAB prevents bacterial diarrhea. This chapter describes a method of LPS elimination using lactic acid bacteria (LAB). First, the LPS concentration is assayed using an LPS assay kit with the limulus cascade reaction made by limulus amebocyte lysate. Some LABs, four bacillus strains and one coccus strain, have LPS-elimination activity. Particularly, the coccus strain Pediococcus pentosaceus eliminates LPS to 43%. The cells fractionate and eliminate four fractions: the extracellular fraction, cell membrane fraction, cytoplasm fraction, and cell wall fraction. Only the cell wall digesting fraction eliminates LPS to 45%. Results confirm that the LAB eliminates all LPS having O-antigen under a low-sugar medium condition at temperatures of 15-30 °C. This method can be used for assay of LPS elimination by LABs exactly and easily for the probiotics field.

Multiplexed real-time PCR for the detection and differentiation of Klebsiella pneumoniae O-antigen serotypes.

Klebsiella pneumoniae has emerged as a global health threat due to its role in the spread of antimicrobial resistance and because it is a frequent cause of hospital-acquired infections and neonatal sepsis. Capsular and lipopolysaccharide (LPS) O-antigen polysaccharide surface antigens are major immunogens that are useful for strain classification and are candidates for vaccine development. We have developed real-time PCR reagents for molecular serotyping, subtyping, and quantitation of the most prevalent LPS O-antigen types (i.e., O1, O2, O3, and O5) of Klebsiella pneumoniae. We describe two applications for this O-typing assay: for screening culture isolates and for direct typing of Klebsiella pneumoniae present in stool samples. We find 100% concordance between the results of the O-typing assay and whole-genome sequencing of 81 culture isolates, and >90% agreement in O-typing performed directly on specimens of human stool, with disagreement arising primarily from a lack of sensitivity of the culture-based comparator method. Additionally, we find evidence for mixed O-type populations at varying levels of abundance in direct tests of stool from a hospitalized patient population. Taken together, these results demonstrate that this novel O-typing assay can be a useful tool for K. pneumoniae epidemiologic and vaccine studies.IMPORTANCEKlebsiella pneumoniae is an important opportunistic pathogen. The gastrointestinal (GI) tract is the primary reservoir of K. pneumoniae in humans, and GI carriage is believed to be a prerequisite for invasive infection. Knowledge about the dynamics and duration of GI carriage has been hampered by the lack of tools suitable for detection and strain discrimination. Real-time PCR is particularly suited to the higher-throughput workflows used in population-based studies, which are needed to improve our understanding of carriage dynamics and the factors influencing K. pneumoniae colonization.

Synthesis of Salmonella enteritidis Antigenic Tetrasaccharide Repeating Unit by Employing Cationic Gold(I)-Catalyzed Glycosylation Involving Glycosyl N-1,1-Dimethylpropargyl Carbamate Donors.

Synthesis of an antigenic tetrasaccharide repeating unit of the O-polysaccharide of Salmonella enteritidis lipopolysaccharide has been accomplished. Those four monosaccharides were assembled stereoselectively by employing our recently developed cationic gold(I)-catalyzed glycosylation methodology involving various glycosyl N-1,1-dimethylpropargyl carbamate donors. The newly formed α-anomeric stereochemical configuration was controlled by the axial C2-OBz of the glycosyl donors via anchimeric assistance.

Chemical Synthesis and Antigenicity Evaluation of an Aminoglycoside Trisaccharide Repeating Unit of Pseudomonas aeruginosa Serotype O5 O-Antigen Containing a Rare Dimeric-ManpN3NA.

Pseudomonas aeruginosa bacteria are becoming increasingly resistant against multiple antibiotics. Therefore, the development of vaccines to prevent infections with these bacteria is an urgent medical need. While the immunological activity of lipopolysaccharide O-antigens in P. aeruginosa is well-known, the specific protective epitopes remain unidentified. Herein, we present the first chemical synthesis of highly functionalized aminoglycoside trisaccharide 1 and its acetamido derivative 2 found in the P. aeruginosa serotype O5 O-antigen. The synthesis of the trisaccharide targets is based on balancing the reactivity of disaccharide acceptors and monosaccharide donors. Glycosylations were analyzed by quantifying the reactivity of the hydroxyl group of the disaccharide acceptor using the orbital-weighted Fukui function and dual descriptor. The stereoselective formation of 1,2-cis-α-fucosylamine linkages was achieved through a combination of remote acyl participation and reagent modulation. The simultaneous SN2 substitution of azide groups at C2' and C2″ enabled the efficient synthesis of 1,2-cis-ß-linkages for both 2,3-diamino-D-mannuronic acids. Through a strategic orthogonal modification, the five amino groups on target trisaccharide 1 were equipped with a rare acetamidino (Am) and four acetyl (Ac) groups. Glycan microarray analyses of sera from patients infected with P. aeruginosa indicated that trisaccharides 1 and 2 are key antigenic epitopes of the serotype O5 O-antigen. The acetamidino group is not an essential determinant of antibody binding. The ß-D-ManpNAc3NAcA residue is a key motif for the antigenicity of serotype O5 O-antigen. These findings serve as a foundation for the development of glycoconjugate vaccines targeting P. aeruginosa serotype O5.

Functional Identification and Genetic Analysis of O-Antigen Gene Clusters of Food-Borne Pathogen Yersinia enterocolitica O:10 and Other Uncommon Serotypes, Further Revealing Their Virulence Profiles.

Yersinia enterocolitica is a globally distributed food-borne gastrointestinal pathogen. The O-antigen variation-determined serotype is an important characteristic of Y. enterocolitica, allowing intraspecies classification for diagnosis and epidemiology purposes. Among the 11 serotypes associated with human yersiniosis, O:3, O:5,27, O:8, and O:9 are the most prevalent, and their O-antigen gene clusters have been well defined. In addition to the O-antigen, several virulence factors are involved in infection and pathogenesis of Y. enterocolitica strains, and these are closely related to their biotypes, reflecting pathogenic properties. In this study, we identified the O-AGC of a Y. enterocolitica strain WL-21 of serotype O:10, and confirmed its functionality in O-antigen synthesis. Furthermore, we analyzed in silico the putative O-AGCs of uncommon serotypes, and found that the O-AGCs of Y. enterocolitica were divided into two genetic patterns: (1) O-AGC within the hemH-gsk locus, possibly synthesizing the O-antigen via the Wzx/Wzy dependent pathway, and (2) O-AGC within the dcuC-galU-galF locus, very likely assembling the O-antigen via the ABC transporter dependent pathway. By screening the virulence genes against genomes from GenBank, we discovered that strains representing different serotypes were grouped according to different virulence gene profiles, indicating strong links between serotypes and virulence markers and implying an interaction between them and the synergistic effect in pathogenicity. Our study provides a framework for further research on the origin and evolution of O-AGCs from Y. enterocolitica, as well as on differences in virulent mechanisms among distinct serotypes.

Comprehensive genomics reveals novel sequence types of multidrug resistant Klebsiella oxytoca with uncharacterized capsular polysaccharide K- and lipopolysaccharide O-antigen loci from the National Hospital of Uganda.

The Klebsiella oxytoca complex comprises diverse opportunistic bacterial pathogens associated with hospital and community-acquired infections with growing alarming antimicrobial resistance. We aimed to uncover the genomic features underlying the virulence and antimicrobial resistance of isolates from Mulago National Hospital in Uganda. We coupled whole genome sequencing with Pathogenwatch multilocus sequence typing (MLST) and downstream bioinformatic analysis to delineate sequence types (STs) capsular polysaccharide K- and O-antigen loci, along with antimicrobial resistance (AMR) profiles of eight clinical isolates from the National Referral Hospital of Uganda. Our findings revealed that only two isolates (RSM6774 and RSM7756) possess a known capsular polysaccharide K-locus (KL74). The rest carry various unknown K-loci (KL115, KL128, KLI52, KL161 and KLI63). We also found that two isolates possess unknown loci for the lipopolysaccharide O-antigen (O1/O2v1 type OL104 and unknown O1). The rest possess known O1 and O3 serotypes. From MLST, we found four novel sequence types (STs), carrying novel alleles for the housekeeping genes glyceraldehyde-6-phosphate dehydrogenase A (gapA), glucose-6-phosphate isomerase (pgi), and RNA polymerase subunit beta (rpoB). Our AMR analysis revealed that all the isolates are resistant to ampicillin and ceftriaxone, with varied resistance to other antibiotics, but all carry genes for extended-spectrum beta-lactamases (ESBLs). Notably, one strain (RSM7756) possesses outstanding chromosomal and plasmid-encoded AMR to beta-lactams, cephalosporins, fluoroquinolones and methoprims. Conclusively, clinical samples from Mulago National Referral Hospital harbor novel STs and multidrug resistant K. oxytoca strains, with significant public health importance, which could have been underrated.

Geographical distribution, disease association and diversity of Klebsiella pneumoniae K/L and O antigens in India: roadmap for vaccine development.

Klebsiella pneumoniae poses a significant healthcare challenge due to its multidrug resistance and diverse serotype landscape. This study aimed to explore the serotype diversity of 1072 K. pneumoniae and its association with geographical distribution, disease severity and antimicrobial/virulence patterns in India. Whole-genome sequencing was performed on the Illumina platform, and genomic analysis was carried out using the Kleborate tool. The analysis revealed a total of 78 different KL types, among which KL64 (n=274/1072, 26 %), KL51 (n=249/1072, 24 %), and KL2 (n=88/1072, 8 %) were the most prevalent. In contrast, only 13 distinct O types were identified, with O1/O2v1 (n=471/1072, 44 %), O1/O2v2 (n=353/1072, 33 %), and OL101 (n=66/1072, 6 %) being the predominant serotypes. The study identified 114 different sequence types (STs) with varying serotypes, with ST231 being the most predominant. O serotypes were strongly linked with STs, with O1/O2v1 predominantly associated with ST231. Simpson's diversity index and Fisher's exact test revealed higher serotype diversity in the north and east regions, along with intriguing associations between specific serotypes and resistance profiles. No significant association between KL or O types and disease severity was observed. Furthermore, we found the specific association of virulence factors yersiniabactin and aerobactin (P<0.05) with KL types but no association with O antigen types (P>0.05). Conventionally described hypervirulent clones (i.e. KL1 and KL2) in India lacked typical virulent markers (i.e. aerobactin), contrasting with other regional serotypes (KL51). The cumulative distribution of KL and O serotypes suggests that future vaccines may have to include either ~20 KL or four O types to cover >85 % of the carbapenemase-producing Indian K. pneumoniae population. The results highlight the necessity for comprehensive strategies to manage the diverse landscape of K. pneumoniae strains across different regions in India. Understanding regional serotype dynamics is pivotal for targeted surveillance, interventions, and tailored vaccine strategies to tackle the diverse landscape of K. pneumoniae infections across India. This article contains data hosted by Microreact.

Convergent synthesis of the pentasaccharide repeating unit corresponding to the cell wall O-polysaccharide of Salmonella milwaukee (group U) O:43 strain.

Synthesis of the pentasaccharide repeating unit of the cell O-polysaccharide produced by Salmonella milwaukee O:43 strain (group U) has been achieved in very good yield adopting a convergent stereoselective [3 + 2] block glycosylation strategy. Thioglycosides and glycosyl trichloroacetimidate derivative were used as glycosyl donors in the presence of a combination of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) as thiophilic activator and TMSOTf as trichloroacetimidate activator respectively. The stereochemical outcome of all glycosylation reactions was excellent.

MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria.

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans.

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

Klebsiella pneumoniae O-polysaccharide biosynthesis highlights the diverse organization of catalytic modules in ABC transporter-dependent glycan assembly.

Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multimodule proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain length. In serotype O4, we discovered a CMP-α-3-deoxy-ᴅ-manno-octulosonic acid-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137) and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.

Preclinical validation of an Escherichia coli O-antigen glycoconjugate for the prevention of serotype O1 invasive disease.

A US collection of invasive Escherichia coli serotype O1 bloodstream infection (BSI) isolates were assessed for genotypic and phenotypic diversity as the basis for designing a broadly protective O-antigen vaccine. Eighty percent of the BSI isolate serotype O1 strains were genotypically ST95 O1:K1:H7. The carbohydrate repeat unit structure of the O1a subtype was conserved in the three strains tested representing core genome multi-locus sequence types (MLST) sequence types ST95, ST38, and ST59. A long-chain O1a CRM197 lattice glycoconjugate antigen was generated using oxidized polysaccharide and reductive amination chemistry. Two ST95 strains were investigated for use in opsonophagocytic assays (OPA) with immune sera from vaccinated animals and in murine lethal challenge models. Both strains were susceptible to OPA killing with O1a glycoconjugate post-immune sera. One of these, a neonatal sepsis strain, was found to be highly lethal in the murine challenge model for which virulence was shown to be dependent on the presence of the K1 capsule. Mice immunized with the O1a glycoconjugate were protected from challenges with this strain or a second, genotypically related, and similarly virulent neonatal isolate. This long-chain O1a CRM197 lattice glycoconjugate shows promise as a component of a multi-valent vaccine to prevent invasive E. coli infections. IMPORTANCE: The Escherichia coli serotype O1 O-antigen serogroup is a common cause of invasive bloodstream infections (BSI) in populations at risk such as newborns and the elderly. Sequencing of US BSI isolates and structural analysis of O polysaccharide antigens purified from strains that are representative of genotypic sub-groups confirmed the relevance of the O1a subtype as a vaccine antigen. O polysaccharide was purified from a strain engineered to produce long-chain O1a O-antigen and was chemically conjugated to CRM197 carrier protein. The resulting glycoconjugate elicited functional antibodies and was protective in mice against lethal challenges with virulent K1-encapsulated O1a isolates.

Chemical Synthesis of a Densely Functionalized Aminodisaccharide from Fusobacterium nucleatum ATCC 23726 O-Antigen.

Fusobacterium nucleatum, a colorectal-cancer-associated oncomicrobe, can trigger or accelerate numerous pathologies. We report the first synthesis of a conjugation-ready disaccharide containing six amino groups from F. nucleatum ATCC 23726 O-antigen. Rare 2,3-diamido-d-glucuronic acid amide and 2-acetamido-4-amino-d-fucose were synthesized from d-glucosamine through configuration inversion, nucleophilic substitution, C6 oxidation, and C6 deoxygenation. A judicious choice of protecting groups and reaction conditions enabled the selective installation of N-acetyl, N-propanoyl, N-formyl, and carboxamido groups.

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis.

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria.IMPORTANCEThe genetic basis of enzymatic assembly of structurally diverse O antigen (OAg) repeating units (RUs) in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to confidently assign glycosyltransferase (GT) functions and the order in which they act during assembly of the OAg RU. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.