The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics.

In the lipopolysaccharides of Escherichia coli there are five distinct core oligosaccharide (core OS) structures, designated K-12 and R1 to R4. The objective of this work was to determine the prevalences of these core OS types within the species. Unique sequences in the waa (core OS biosynthesis) gene operon were used to develop a PCR-based system that facilitated unequivocal determination of the core OS types in isolates of E. coli. This system was applied to the 72 isolates in the E. coli ECOR collection, a compilation of isolates that is considered to be broadly representative of the genetic diversity of the species. Fifty (69.4%) of the ECOR isolates contained the R1 core OS, 8 (11.1%) were representatives of R2, 8 (11.1%) were R3, 2 (2.8%) were R4, and only 4 (5.6%) were K-12. R1 is the only core OS type found in all four major phylogenetic groups (A, B1, B2, and D) in the ECOR collection. Virulent extraintestinal pathogenic E. coli isolates tend to be closely related to group B2 and, to a lesser extent, group D isolates. All of the ECOR representatives from the B2 and D groups had the R1 core OS. In contrast, commensal E. coli isolates are more closely related to group A, which contains isolates representing each of the five core OS structures. R3 was the only core OS type found in 38 verotoxigenic E. coli (VTEC) isolates from humans and cattle belonging to the common enterohemorrhagic E. coli serogroups O157, O111, and O26. Although isolates from other VTEC serogroups showed more core OS diversity, the R3 type (83.1% of all VTEC isolates) was still predominant. When non-VTEC commensal isolates from cattle were analyzed, it was found that most possessed the R1 core OS type.The lipopolysaccharides (LPSs) of Escherichia coli consist of (i) a hydrophobic lipid A component that forms the outer leaflet of the outer membrane, (ii) a phosphorylated, nonrepetitive hetero-oligosaccharide known as the core oligosaccharide (core OS), and (iii) a polysaccharide (O-PS) that extends from the cell surface and that forms the O antigen detected in serotyping (37). The smooth LPS (S-LPS) molecules found in most clinical isolates of E. coli are composed of this three-part structure, whereas rough LPS (R-LPS) lacks the O antigen and can have a truncated core OS. The extent of structural diversity in E. coli LPS molecules ranges from the highly conserved lipid A to the extreme variations reflected in more than 170 known O antigens (19). The core OS is conceptually divided into inner and outer core regions. The inner core is composed primarily of L-glycero-D-manno-heptose (heptose) and 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) residues, and this part of the core OS is phosphorylated in E. coli. The principal features of the inner core OS structure are conserved among members of the Enterobacteriaceae, presumably reflecting its essential role in outer-membrane stability (16). The inner core OS structure carries additional (often nonstoichiometric) glycosyl substituents, but these vary according to core OS type (16,18). The outer ...

Background: C. jejuni helical shape is important to pathogenesis.Results: Deletion of pgp2 results in loss of C. jejuni helical shape and change in peptidoglycan structure and pathogenic properties.Conclusion: Pgp2 is a ld-carboxypeptidase cleaving peptidoglycan tetrapeptides to tripeptides.Significance: Characterization of enzymes involved in C. jejuni peptidoglycan and cell shape maintenance is crucial to the understanding of fundamental properties of this organism.

Deamination of LPSs from Klebsiella pneumoniae released O-chain polysaccharides together with a fragment of the core oligosaccharide. The structures of the products from serotypes O1, O2a, O2a,c, O3, O4, O5, and O12 were determined by NMR spectroscopy and chemical methods, identifying the linkage region between the O antigens and the core as well as novel residues at the non-reducing ends of the polysaccharides. All serotypes had an identical linkage between the O chain and core.Like other members of the family Enterobacteriaceae, the lipopolysaccharides (LPSs) 1 of Klebsiella pneumoniae consist of three structural domains, (i) the hydrophobic lipid A, which is a major component of the outer leaflet of the Gram-negative outer membrane, and (ii) the core oligosaccharide, which is linked to lipid A and provides the attachment site for (iii) the long chain polysaccharide (O antigen; O chain). Typically, structural diversity is greater in the regions of LPS extending from the cell surface (i.e. the O chains). Varying chemical structures in the O chains gives rise to a number of serologically distinct O antigens. In the Klebsiellae there are 11 known O-chain structures, but structural similarities lead to some serological cross-reactivities, so the actual number of unique O serotypes is less (1, 2). Several of the O-antigens are based on a structure designated D-galactan I with a repeat unit compris-2 When present alone, this structure provides the 2a antigen (3), but it can be capped by additional structural domains or modified by side chain acetyl or galactosyl residues to generate additional unique antigens (3-6). For example, in the most clinically prevalent serotype, the O1 antigen, D-galactan I chains are capped by a domain with a different repeat unit structure, [-. Genetic (7) and chemical (8, 9) analyses indicate that D-galactan I chains are linked directly to the lipid A core structure, whereas D-galactan II is confined to the distal end of some of the available D-galactan I chains (10). D-Galactan II provides the epitope(s) that defines the O1 antigen (9), and its presence is required for the resistance of the bacteria to complement-mediated killing in the host; K. pneumoniae mutants that only produce D-galactan I are therefore serum-sensitive (11, 12). However, not all Klebsiella O serotypes are based on D-galactan I. The prevalent O3 and O5 serotypes comprise mannan O chains with structures identical to the Escherichia coli O9 and O8 antigens, respectively (13,14). The remaining Klebsiella O-chain structures are heteropolymers.The biosynthesis of the polysaccharide O chain has been investigated in some serotypes of K. pneumoniae by biochemical and genetic experimental approaches. Much of the data for the O3 and O5 serotypes is derived from the related equivalent systems in E. coli. However, the genetic loci required for synthesis of the corresponding O chain structures in E. coli (O8 ec and O9 ec ) and K. pneumoniae (O5 kp and O3 kp , respectively) are essentially identical and reflect lateral gene trans...

The enteric pathogen Campylobacter jejuni is a highly prevalent yet fastidious bacterium. Biofilms and surface polysaccharides participate in stress survival, transmission, and virulence in C. jejuni; thus, the identification and characterization of novel genes involved in each process have important implications for pathogenesis. We found that C. jejuni reacts with calcofluor white (CFW), indicating the presence of surface polysaccharides harboring ␤1-3 and/or ␤1-4 linkages. CFW reactivity increased with extended growth, under 42°C anaerobic conditions, and in a ⌬spoT mutant defective for the stringent response (SR). Conversely, two newly isolated dim mutants exhibited diminished CFW reactivity as well as growth and serum sensitivity differences from the wild type. Genetic, biochemical, and nuclear magnetic resonance analyses suggested that differences in CFW reactivity between wild-type and ⌬spoT and dim mutant strains were independent of well-characterized lipooligosaccharides, capsular polysaccharides, and N-linked polysaccharides. Targeted deletion of carB downstream of the dim13 mutation also resulted in CFW hyporeactivity, implicating a possible role for carbamoylphosphate synthase in the biosynthesis of this polysaccharide. Correlations between biofilm formation and production of the CFW-reactive polymer were demonstrated by crystal violet staining, scanning electron microscopy, and confocal microscopy, with the C. jejuni ⌬spoT mutant being the first SR mutant in any bacterial species identified as up-regulating biofilms. Together, these results provide new insight into genes and processes important for biofilm formation and polysaccharide production in C. jejuni.

Campylobacter jejuni is a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that a C. jejuni strain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required for C. jejuni pathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaF and lgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed ⌬galT and ⌬cstII mutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (⌬waaF and ⌬lgtF but not ⌬galT or ⌬cstII mutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (⌬cstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active against C. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The ⌬waaF mutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect of C. jejuni pathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of the C. jejuni LOS in host colonization. Collectively, this study has uncovered novel roles for the C. jejuni LOS, highlights the dynamic nature of the C. jejuni cell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.

The diarrheal pathogen Campylobacter jejuni and other gastrointestinal bacteria encounter changes in osmolarity in the environment, through exposure to food processing, and upon entering host organisms, where osmotic adaptation can be associated with virulence. In this study, growth profiles, transcriptomics, and phenotypic, mutant, and single-cell analyses were used to explore the effects of hyperosmotic stress exposure on C. jejuni. Increased growth inhibition correlated with increased osmotic concentration, with both ionic and nonionic stressors inhibiting growth at 0.620 total osmol liter ؊1 . C. jejuni adaptation to a range of osmotic stressors and concentrations was accompanied by severe filamentation in subpopulations, with microscopy indicating septum formation and phenotypic diversity between individual cells in a filament. Population heterogeneity was also exemplified by the bifurcation of colony morphology into small and large variants on salt stress plates. Flow cytometry of C. jejuni harboring green fluorescent protein (GFP) fused to the ATP synthase promoter likewise revealed bimodal subpopulations under hyperosmotic stress. We also identified frequent hyperosmotic stress-sensitive variants within the clonal wild-type population propagated on standard laboratory medium. Microarray analysis following hyperosmotic upshift revealed enhanced expression of heat shock genes and genes encoding enzymes for synthesis of potential osmoprotectants and cross-protective induction of oxidative stress genes. The capsule export gene kpsM was also upregulated, and an acapsular mutant was defective for growth under hyperosmotic stress. For C. jejuni, an organism lacking most conventional osmotic response factors, these data suggest an unusual hyperosmotic stress response, including likely "bet-hedging" survival strategies relying on the presence of stress-fit individuals in a heterogeneous population.

In the Enterobacteriaceae, the outer membrane is primarily comprised of lipopolysaccharides. The lipopolysaccharide molecule is important in mediating interactions between the bacterium and its environment and those regions of the molecule extending further away from the cell surface show a higher amount of structural diversity. The hydrophobic lipid A is highly conserved, due to its important role in the structural integrity of the outer membrane. Attached to the lipid A region is the core oligosaccharide. The inner core oligosaccharide (lipid A proximal) backbone is also well conserved. However, non-stoichiometric substitutions of the basic inner core structure lead to structural variation and microheterogeneity. These include the addition of negatively charged groups (phosphate or galacturonic acid), ethanolamine derivatives, and glycose residues (Kdo, rhamnose, galactose, glucosamine, N-acetylglucosamine, heptose, Ko). The genetics and biosynthesis of these substitutions is beginning to be elucidated. Modification of heptose residues with negatively charged molecules (such as phosphate in Escherichia coli and Salmonella and galacturonic acid in Klebsiella pneumoniae) has been shown to be involved in maintaining membrane stability. However, the biological role(s) of the remaining substitutions is unknown.