Complete Genome Sequence of a CTX-M-15-Producing Escherichia coli Strain from the H30Rx Subclone of Sequence Type 131 from a Patient with Recurrent Urinary Tract Infections, Closely Related to a Lethal Urosepsis Isolate from the Patient\u27s Sister.

We report here the complete genome sequence, including five plasmid sequences, of Escherichia coli sequence type 131 (ST131) strain JJ1887. The strain was isolated in 2007 in the United States from a patient with recurrent cystitis, whose caregiver sister died from urosepsis caused by a nearly identical strain

Evolution of Salmonella enterica Virulence via Point Mutations in the Fimbrial Adhesin

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella

Identification of Mannheimia haemolytica Adhesins Involved in Binding to Bovine Bronchial Epithelial Cells▿

Mannheimia haemolytica, a commensal organism of the upper respiratory tract in cattle, is the principal bacterial pathogen associated with the bovine respiratory disease complex. Adherence to the respiratory mucosa is a crucial event in its pathogenesis. However, the bacterial components that contribute to this process are not fully characterized. In this study, we demonstrated that M. haemolytica adhered to bovine bronchial epithelial cells (BBEC) in vitro and that adherence was inhibited by anti-M. haemolytica antibody. Western blot analysis of M. haemolytica proteins that bind to BBEC showed a dominant protein band with an apparent molecular mass of ∼30 kDa. Peptide sequences for the 30-kDa BBEC-binding proteins, as determined by liquid chromatography-tandem mass spectrometry, matched two M. haemolytica surface proteins: heat-modifiable outer membrane protein A (OmpA) and lipoprotein 1 (Lpp1). Western blotting showed that the 30-kDa protein band is recognized by both anti-M. haemolytica OmpA and anti-Lpp1 antibodies. Furthermore, incubation with anti-OmpA and anti-Lpp1 antibodies significantly inhibited M. haemolytica binding to BBEC monolayers. In summary, these results suggest that OmpA and Lpp1 contribute to adherence of M. haemolytica to bovine respiratory epithelial cells

Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function

Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic <i>E</i>. <i>coli</i>

<div><p>Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic <i>Escherichia coli</i>, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.</p></div

DNA-based protein phylogram of <i>S. enterica</i> FimH, derived from ZP analysis.

<p>The tree was built based on the 50 <i>fimH</i> sequences of <i>S. enterica</i> subsp. I. Each circle represents a unique structural variant, and the size of the circle is proportional to the number of representative sequences. The dashed line separates the long-term (green) from the recently emerged variants (black). Branches marked in blue indicate branches containing synonymous mutations. The length of each branch is proportional to the number of non-synonymous mutations that were acquired. The strain tags of systemically invasive serovars are in red and the non-invasive serovars in black.</p

Schematic representation of evolutionary changes in the FimH binding phenotype of selected <i>S. enterica</i> serovars.

<p>The evolutionary changes in the FimH of <i>S.</i> Enteritidis, <i>S.</i> Pullorum, <i>S.</i> Gallinarum and <i>S.</i> Dublin (A), <i>S.</i> Indiana, <i>S.</i> Paratyphi C and <i>S.</i> Choleraesuis (B), <i>S.</i> Paratyphi B (C) and <i>S.</i> Paratyphi A, <i>S.</i> Sendai and <i>S.</i> Typhi (D). Low (green)-FimH with low-binding phenotype; High (blue)-FimH with high-binding phenotype; None (orange)-inactive variant of FimH. The strain tags of systemically non-invasive serovars are in black and the systemically invasive serovars in red. Structural mutations are given along each arrow. Structural hot-spot mutations are underlined. The activating mutations are in blue and the inactivating mutations are in orange. The FimH variants from strains with phylogenetic relatedness supported by MLST are shown in tan boxes.</p

Inhibitory potency of mAb926 and mAb475.

<p>Binding of FimH^wt-expressing <i>E</i>. <i>coli</i> to surface-immobilized yeast mannan in the presence of different concentrations of anti-FimH antibodies. Data are mean ± SEM (n = 5 independent experiments). The IC₅₀ values were calculated from the fitted curves shown using Prism GraphPad 6 software. **, <i>P</i> ≤ 0.005 (t-test).</p