Bacteria of the genus Salmonella comprise a large and evolutionary related population of zoonotic pathogens that can infect mammals, including humans and domestic animals, birds, reptiles and amphibians. Salmonella carries a plethora of virulence genes, including fimbrial adhesins, some of them known to participate in mammalian or avian host colonization. Each type of fimbria has its structural subunit and biogenesis genes encoded by one fimbrial gene cluster (FGC). The accumulation of new genomic information offered a timely opportunity to better evaluate the number and types of FGCs in the Salmonella pangenome, to test the use of current classifications based on phylogeny, and to infer potential correlations between FGC evolution in various Salmonella serovars and host niches. This study focused on the FGCs of the currently deciphered 90 genomes and 60 plasmids of Salmonella. The analysis highlighted a fimbriome consisting of 35 different FGCs, of which 16 were new, each strain carrying between 5 and 14 FGCs. The Salmonella fimbriome was extremely diverse with FGC representatives in 8 out of 9 previously categorized fimbrial clades and subclades. Phylogenetic analysis of Salmonella suggested macroevolutionary shifts detectable by extensive FGC deletion and acquisition. In addition, microevolutionary drifts were best depicted by the high level of allelic variation in predicted or known adhesins, such as the type 1 fimbrial adhesin FimH for which 67 different natural alleles were identified in S. enterica subsp. I. Together with strain-specific collections of FGCs, allelic variation among adhesins attested to the pathoadaptive evolution of Salmonella towards specific hosts and tissues, potentially modulating host range, strain virulence, disease progression, and transmission efficiency. Further understanding of how each Salmonella strain utilizes its panel of FGCs and specific adhesin alleles for survival and infection will support the development of new approaches for the control of Salmonellosis.

Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts.

Yersinia pestis, the causative agent of plague, expresses the Psa fimbriae (pH 6 antigen) in vitro and in vivo. To evaluate the potential virulence properties of Psa for pneumonic plague, anEscherichia colistrain expressing Psa was engineered and shown to adhere to three types of human respiratory tract epithelial cells. Psa binding specificity was confirmed with Psa-coated polystyrene beads and by inhibition assays. IndividualY. pestiscells were found to be able to express the capsular antigen fraction 1 (F1) concomitantly with Psa on their surface when analyzed by flow cytometry. To better evaluate the separate effects of F1 and Psa on the adhesive and invasive properties ofY. pestis, isogenic Δcaf(F1 genes), Δpsa, and ΔcafΔpsamutants were constructed and studied with the three respiratory tract epithelial cells. The Δpsamutant bound significantly less to all three epithelial cells compared to the parental wild-type strain and the Δcafand Δcaf Δpsamutants, indicating that Psa acts as an adhesin for respiratory tract epithelial cells. An antiadhesive effect of F1 was clearly detectable only in the absence of Psa, underlining the dominance of the Psa⁺phenotype. Both F1 and Psa inhibited the intracellular uptake ofY. pestis. Thus, F1 inhibits bacterial uptake by inhibiting bacterial adhesion to epithelial cells, whereas Psa seems to block bacterial uptake by interacting with a host receptor that doesn't direct internalization. The Δcaf Δpsadouble mutant bound and invaded all three epithelial cell types well, revealing the presence of an undefined adhesin(s) and invasin(s).

Inhaled Yersinia pestis produces a severe primary pneumonia known as pneumonic plague, which is contagious and highly lethal to humans and animals. In this study, we first determined the susceptibility of Y. pestis KIM6 to antimicrobial molecules of the airways. We found that (i) rat bronchoalveolar lavage fluid (rBALF) effectively killed KIM6 cells growing at 37°C; (ii) the antibacterial components of rBALF were small peptides (<10 kDa) that included two cationic antimicrobial peptides (CAMPs), the rat cathelicidin rCRAMP, and β-defensin RBD-1; (iii) the human cathelicidin LL-37 killed KIM6 cells as well as rBALF did; and (iv) the bactericidal property of LL-37 was synergistically amplified by human β-defensin 1, another constitutively expressed pulmonary CAMP. Second, the effects of three major surface proteins of Y. pestis , namely, the capsular antigen fraction 1 (F1), the pH 6 antigen (Psa fimbriae), and the outer membrane protease Pla, on the bactericidal effect of the antimicrobial rBALF peptides was determined with corresponding deletion mutants. We showed that (i) a Y. pestis psa mutant was only slightly more susceptible to rBALF than the parental KIM6 strain, (ii) a caf (F1 gene) mutant and a caf psa mutant were resistant to rBALF or LL-37, (iii) a caf pla mutant was as susceptible to the effect of rBALF or LL-37 as KIM6 was ( caf ⁺ pla ⁺ ), and (iv) only the single caf mutant ( pla ⁺ ), but not KIM6 or the caf pla double mutant, degraded LL-37. The activity of Pla toward LL-37 was confirmed with pla mutants carrying a single-residue substitution affecting plasminogen cleavage. Taken together, our data indicated that Pla might act as a virulence factor not only by processing plasminogen but also by inactivating CAMPs, particularly when F1 is not expressed.

The pH 6 antigen (Psa) of Yersinia pestis consists of fimbriae with adhesive properties of potential importance for the pathogenesis of plague, including pneumonic plague. The Psa fimbriae mediate bacterial binding to human alveolar epithelial cells. The Psa fimbriae bound mostly to one component present in the total lipid extract from type II alveolar epithelial cells of the cell line A549 separated by thin-layer chromatography (TLC). The Psa receptor was identified as phosphatidylcholine (PC) by TLC using alkali treatment, molybdenum blue staining, and Psa overlays. The Psa fimbriae bound to PC in a dose-dependent manner, and binding was inhibited by phosphorylcholine (ChoP) and choline. Binding inhibition was dose dependent, although only high concentrations of ChoP completely blocked Psa binding to PC. In contrast, less than 1 μM of a ChoP-polylysine polymer inhibited specifically the adhesion of Psa-fimbriated Escherichia coli to PC, and type I (WI-26 VA4) and type II alveolar epithelial cells. These results indicated that the homopolymeric Psa fimbriae are multimeric adhesins. Psa also bound to pulmonary surfactant, which covers the alveolar surface as a product of type II alveolar epithelial cells and includes PC as the major component. The observed dose-dependent interaction of Psa with pulmonary surfactant was blocked by ChoP. Interestingly, surfactant did not inhibit Psa-mediated bacterial binding to alveolar cells, suggesting that both surfactant and cell membrane PC retain Psa-fimbriated bacteria on the alveolar surface. Altogether, the results indicate that Psa uses the ChoP moiety of PC as a receptor to mediate bacterial binding to pulmonary surfactant and alveolar epithelial cells.

Bacterial outer membrane vesicles (OMVs) mediate critical bacterium-bacterium and host-microbial interactions that influence pathogenesis through multiple mechanisms, including the elicitation of inflammatory responses, delivery of virulence factors, and enhancement of biofilm formation. As such, there is a growing interest in understanding the underlying mechanisms of OMV production.