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

Examining transmission of gut bacteria to preserved carcass via anal secretions in Nicrophorus defodiens.

Direct transmission of bacteria to subsequent generations highlights the beneficial nature of host-bacteria relationships. In insects, this process is often mediated by the production of microbe-containing secretions. The objective of this study was to determine if the burying beetle, Nicrophorus defodiens, utilizes anal secretions to transmit adult digestive tract bacteria onto a small vertebrate carcass; thus creating the potential to aid in carcass preservation or pass digestive tract bacteria to their larval offspring. Using high-throughput Illumina sequencing of the 16S rRNA gene, we characterized bacterial communities of adult beetle digestive tracts, their anal secretions, and prepared mouse carcasses. We also examined unprepared carcass bacterial communities as a means to interpret community shifts that take place during carcass preservation. We found a vast reduction in diversity on prepared carcasses after anal secretion application. Overall, there was little similarity in bacterial communities among adult digestive tracts, anal secretions, and prepared carcasses, suggesting bacterial communities found in adult digestive tracts do not successfully colonize and achieve dominance on prepared carcasses by way of beetle anal secretions. We concluded that N. defodiens does not transmit their digestive tract bacterial communities to prepared carcasses in a wholesale manner, but may transmit key microbes, including core microbiome members, to preserved carcasses that may ultimately act to sustain larvae and serve as inocula for larval digestive tracts

Production of Outer Membrane Vesicles by the Plague Pathogen <i>Yersinia pestis</i>

<div><p>Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that <i>Yersinia pestis</i>, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by <i>Y. pestis</i> contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by <i>Y. pestis</i> is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that <i>Y. pestis</i> OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that <i>Y. pestis</i> may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand.</p></div

<i>Y. pestis</i> produces membrane blebs consistent with OMVs.

<p><i>Y. pestis</i> bacteria cultured for 6 h at 37°C were fixed and imaged via SEM. (A and B) Images reveal round membrane protrusions on the bacterial surface (arrows) that are consistent with OMVs. (C) TEM of OMVs purified from <i>Y. pestis</i> supernatants. Bar represents size in nanometers. (D) Size distribution of OMVs purified from <i>Y. pestis</i>. One hundred OMVs were measured and diameters are shown as a percent of the total. The average OMV diameter is 93.07+/−11.75 nm. Bars represent size in nanometers as indicated.</p

<i>Y. pestis</i> culture supernatants contain active Pla.

<p>(A) Plg-activating ability of whole bacteria, 0.2 µm-filtered culture supernatants, or the filtrate of 100 kDa-passed culture supernatants, from wild-type or Δ<i>pla Y. pestis</i>, respectively. Materials were incubated with human glu-plg and a fluorescent substrate of plasmin for 3 h at 37°C. One experiment representative of 3 independent biological replicates is shown.</p

OMV-bound Pla is catalytically active and interacts with components of the ECM.

<p>(A) Plg-activating ability of wild-type or Δ<i>pla Y. pestis</i> bacteria or OMVs. Whole bacteria or purified OMVs were incubated with human glu-plg and a fluorescent substrate of plasmin for 3 hours at 37°C. (B) Degradation of α2-antiplasmin by wild-type or Δ<i>pla Y. pestis</i> bacteria or OMVs. Whole bacteria or purified OMVs were incubated with purified human α2-antiplasmin at 37°C and at the times indicated, the presence of uncleaved α2-antiplasmin was determined by immunoblot analysis.</p

Effects of temperature and stress response factors on OMV production.

<p>(A) OMVs were isolated from <i>Y. pestis</i> cultured at either 26°C or 37°C, and total protein abundance associated with the OMVs was measured. Protein concentrations were normalized to the OD₆₂₀ of the bacterial cultures. (B) Wild-type <i>Y. pestis</i> or strains lacking the genes for RseA, Hfq, or Lpp were cultured at 37°C as above and OMVs were isolated and total associated protein was measured and normalized to the OD₆₂₀. For cold shock experiments, bacteria were placed in an ice water bath for one h before proceeding. One experiment representative of two biological replicates is shown. The mean and SE are shown. *p<0.05, **p<0.005 (student's t–test).</p