The role of galacturonic acid in outer membrane stability in Klebsiella pneumoniae.

In most members of the Enterobacteriaceae, including Escherichia coli and Salmonella, the lipopolysaccharide core oligosaccharide backbone is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. Mutants lacking the core heptose region and the phosphate residues display pleiotrophic defects collectively known as the deep-rough phenotype, characterized by changes in outer membrane structure and function. Klebsiella pneumoniae lacks phosphoryl residues in its core, but instead contains galacturonic acid. The goal of this study was to determine the contribution of galacturonic acid as a critical source of negative charge. A mutant was created lacking all galacturonic acid by targeting UDP-galacturonic acid precursor synthesis through a mutation in gla(KP). Gla(KP) is a K. pneumoniae UDP-galacturonic acid C4 epimerase providing UDP-galacturonic acid for core synthesis. The gla(KP) gene was inactivated and the structure of the mutant lipopolysaccharide was determined by mass spectrometry. The mutant displayed characteristics of a deep-rough phenotype, exhibiting a hypersensitivity to hydrophobic compounds and polymyxin B, an altered outer membrane profile, and the release of the periplasmic enzyme beta-lactamase. These results indicate that the negative charge provided by the carboxyl groups of galacturonic acid do play an equivalent role to the core oligosaccharide phosphate residues in establishing outer membrane integrity in E. coli and Salmonella

Peptidoglycan-Modifying Enzyme Pgp1 Is Required for Helical Cell Shape and Pathogenicity Traits in Campylobacter jejuni

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

Characterization of Gla(KP), a UDP-Galacturonic Acid C4-Epimerase from Klebsiella pneumoniae with Extended Substrate Specificity

In Escherichia coli and Salmonella enterica, the core oligosaccharide backbone of the lipopolysaccharide is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. In contrast, Klebsiella pneumoniae lacks phosphoryl groups in its core oligosaccharide but instead contains galacturonic acid residues that are proposed to serve a similar function in outer membrane stability. Gla(KP) is a UDP-galacturonic acid C4-epimerase that provides UDP-galacturonic acid for core synthesis, and the enzyme was biochemically characterized because of its potentially important role in outer membrane stability. High-performance anion-exchange chromatography was used to demonstrate the UDP-galacturonic acid C4-epimerase activity of Gla(KP), and capillary electrophoresis was used for activity assays. The reaction equilibrium favors UDP-galacturonic acid over UDP-glucuronic acid in a ratio of 1.4:1, with the K(m) for UDP-glucuronic acid of 13.0 μM. Gla(KP) exists as a dimer in its native form. NAD(+)/NADH is tightly bound by the enzyme and addition of supplementary NAD(+) is not required for activity of the purified enzyme. Divalent cations have an unexpected inhibitory effect on enzyme activity. Gla(KP) was found to have a broad substrate specificity in vitro; it is capable of interconverting UDP-glucose/UDP-galactose and UDP-N-acetylglucosamine/UDP-N-acetylgalactosamine, albeit at much lower activity. The epimerase GalE interconverts UDP-glucose/UDP-galactose. Multicopy plasmid-encoded gla(KP) partially complemented a galE mutation in S. enterica and in K. pneumoniae; however, chromosomal gla(KP) could not substitute for galE in a K. pneumoniae galE mutant in vivo

Biosynthesis of a novel 3-deoxy-D-mannooct-2-ulosonic acid-containing outer core oligosaccharide in the lipopolysaccharide of Klebsiella pneumoniae.

NRC publication: Ye

Data_Sheet_1_Multiple Campylobacter jejuni proteins affecting the peptidoglycan structure and the degree of helical cell curvature.PDF

Campylobacter jejuni is a Gram-negative helical bacterium. Its helical morphology, maintained by the peptidoglycan (PG) layer, plays a key role in its transmission in the environment, colonization, and pathogenic properties. The previously characterized PG hydrolases Pgp1 and Pgp2 are important for generating C. jejuni helical morphology, with deletion mutants being rod-shaped and showing alterations in their PG muropeptide profiles in comparison to the wild type. Homology searches and bioinformatics were used to identify additional gene products involved in C. jejuni morphogenesis: the putative bactofilin 1104 and the M23 peptidase domain-containing proteins 0166, 1105, and 1228. Deletions in the corresponding genes resulted in varying curved rod morphologies with changes in their PG muropeptide profiles. All changes in the mutants complemented except 1104. Overexpression of 1104 and 1105 also resulted in changes in the morphology and in the muropeptide profiles, suggesting that the dose of these two gene products influences these characteristics. The related helical ε-Proteobacterium Helicobacter pylori has characterized homologs of C. jejuni 1104, 1105, and 1228 proteins, yet deletion of the homologous genes in H. pylori had differing effects on H. pylori PG muropeptide profiles and/or morphology compared to the C. jejuni deletion mutants. It is therefore apparent that even related organisms with similar morphologies and homologous proteins can have diverse PG biosynthetic pathways, highlighting the importance of studying PG biosynthesis in related organisms.</p

Structures of lipopolysaccharides from Klebsiella pneumoniae : Elucidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains

In press: YesNRC publication: Ye

Effects of sequential Campylobacter jejuni 81-176 lipooligosaccharide core truncations on biofilm formation, stress survival, and pathogenesis

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 \u394galT and \u394cstII mutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (\u394waaF and \u394lgtF but not \u394galT or \u394cstII 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 (\u394cstII), 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 \u394waaF 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.Peer reviewed: YesNRC publication: Ye

HPLC elution profile of <i>C. jejuni</i> muropeptides and proposed muropeptide structures.

<p>Purified PG was digested with cellosyl and the resulting muropeptides were reduced with sodium borohydride and separated on a Prontosil 120-3-C18 AQ reverse-phase column. HPLC profiles are shown for <b>A, </b><i>C. jejuni</i> wild-type 81-176; <b>B,</b> Δ<i>pgp1</i>; <b>C,</b> the complement Δ<i>pgp1</i>c; <b>D,</b> the <i>pgp1</i> overexpressing strain, 81-176+<i>pgp1</i>. Peak numbers correspond to the main muropeptide peak fractions of <i>C. jejuni</i> 81-176 analyzed by LTQ-FT-MS (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002602#ppat.1002602.s005" target="_blank">Table S3</a>) to determine the structures shown in <b>E</b>. G, N-acetylglucosamine; M, reduced N-acetylmuramic acid; L-Ala, L-alanine; D-iGlu, D-isoglutamic acid; D-Glu, D-glutamic acid; <i>meso-</i>DAP, <i>meso-</i>diaminopimelic acid; Gly. Glycine; Ac, O-acetyl groups at the C-6 hydroxyl group of MurNAc; Anh, 1,6-anhydro group at MurNAc. The asterisk (*) indicates that it is not known on which MurNAc residue the modification occurs.</p