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

Differential Stability and Trade-Off Effects of Pathoadaptive Mutations in the Escherichia coli FimH Adhesin▿ †

FimH is the tip adhesin of mannose-specific type 1 fimbriae of Escherichia coli, which are critical to the pathogenesis of urinary tract infections. Point FimH mutations increasing monomannose (1M)-specific uroepithelial adhesion are commonly found in uropathogenic strains of E. coli. Here, we demonstrate the emergence of a mixed population of clonally identical E. coli strains in the urine of a patient with acute cystitis, where half of the isolates carried a glycine-to-arginine substitution at position 66 of the mature FimH. The R66 mutation induced an unusually strong 1M-binding phenotype and a 20-fold advantage in mouse bladder colonization. However, E. coli strains carrying FimH-R66, but not the parental FimH-G66, had disappeared from the patient's rectal and urine samples collected from 29 to 44 days later, demonstrating within-host instability of the R66 mutation. No FimH variants with R66 were identified in a large (>600 strains) sequence database of fimH-positive E. coli strains. However, several strains carrying genes encoding FimH with either S66 or C66 mutations appeared to be relatively stable in the E. coli population. Relative to FimH-R66, the FimH-S66 and FimH-C66 variants mediated only moderate increases in 1M binding but preserved the ability to enhance binding under flow-induced shear conditions. In contrast, FimH-R66 completely lost shear-enhanced binding properties, with bacterial adhesion being inhibited by shear forces and lacking a rolling mode of binding. These functional trade-offs may determine the natural populational instability of this mutation or other pathoadaptive FimH mutations that confer dramatic increases in 1M binding strength

Analysis of the Genome of the Escherichia coli O157:H7 2006 Spinach-Associated Outbreak Isolate Indicates Candidate Genes That May Enhance Virulence ▿ †

In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx1. The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx1, indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx1 and a propensity for HUS

List of bacterial strains used in this study.

1<p>Harborview Medical Center, Seattle, WA, USA.</p>2<p>Institute for Environmental Health, Lake Forest Park, WA, USA.</p>3<p>University of Iowa, Iowa City, IA, USA.</p>4<p>Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland.</p>5<p>Ohio State University, Columbus, OH, USA.</p>6<p>University of Illinois, Urbana, IL, USA.</p

Amino acid variation in <i>S. enterica</i> subspecies I FimH.

<p>Residues identical to the amino acid sequence of <i>S.</i> Typhimurium SL1344 (Thm1) FimH are indicated by dots. Systemically invasive serovars are shown in red and non-invasive serovars are shown in black. Position -10 represents the position in the signal peptide of unprocessed FimH upstream of the cleavage site.</p

FimH-mediated bacterial interaction with epithelial and macrophage cell lines.

<p>Bacterial adhesion to (A and C) and invasion of (B and D) Hep-2 cells (A and B) and RAW264.7 cells (C and D). Different variants of FimH were expressed in <i>S.</i> Typhimurium SL1344H3 and bacterial binding was tested in the absence and presence of 50 mM methyl-D-mannopyranoside (α-mm). Data are the means ± SD of triplicates from one representative experiment of three experiments that were performed.</p

Binding phenotypes of natural <i>S. enterica</i> subspecies I FimH variants.

<p>Static adhesion of <i>S.</i> Typhimurium LBH4 transformed with plasmids encoding different variants of FimH or plasmids carrying <i>fimH</i> deletion (fimHΔ) to Man-BSA (red bars) and anti-FimH^SE antibody (grey bars). The binding of ³H- labeled bacteria was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002733#s4" target="_blank">Material and Methods</a>. Data are the means ± SD of triplicates from one representative experiment of three experiments that were performed. The strain tags of systemically invasive serovars are in red and the non-invasive ones in black. Bacterial binding was >95% inhibitable in the presence of 50 mM methyl-D-mannopyranoside (not shown). * The non-binding FimH variants of <i>S.</i> Gallinarum were not tested in this study.</p

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