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Abstract Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella Typhimurium increases OMV production inside the acidic vacuoles of host cells by changing expression of its outer membrane proteins and modifying the composition of lipid A. However, the molecular mechanisms that translate pH changes into OMV production are not completely understood. Here, we show that the outer membrane protein PagC promotes OMV production through pH-dependent interactions between its extracellular loops and surrounding lipopolysaccharide (LPS). Structural comparisons and mutational studies indicate that a pH-responsive amino acid motif in PagC extracellular loops, containing PagC-specific histidine residues, is crucial for OMV formation. Molecular dynamics simulations suggest that protonation of histidine residues leads to changes in the structure and flexibility of PagC extracellular loops and their interactions with the surrounding LPS, altering membrane curvature. Consistent with that hypothesis, mimicking acidic pH by mutating those histidine residues to lysine increases OMV production. Thus, our findings reveal a mechanism for sensing and responding to environmental pH and for control of membrane dynamics by outer membrane proteins.

Abstract Background The role of Salmonella virulence factor (VF) allelic variation in modulating pathogenesis or host specificity has only been demonstrated in a few cases, mostly through serendipitous findings. Virulence factor (VF) alleles from Salmonella enterica subsp. enterica genomes were compared to identify potential associations with the host-adapted invasive serovars Typhi, Dublin, Choleraesuis, and Gallinarum, and with the broad host-range intestinal serovars Typhimurium, Enteritidis, and Newport. Results Through a bioinformatics analysis of 500 Salmonella genomes, we have identified allelic variants of 70 VFs, many of which are associated with either one of the four host-adapted invasive Salmonella serovars or one of the three broad host-range intestinal serovars. In addition, associations between specific VF alleles and intra-serovar clusters, sequence types (STs) and/or host-adapted FimH adhesins were identified. Moreover, new allelic VF associations with non-typhoidal S. Enteritidis and S. Typhimurium (NTS) or invasive NTS (iNTS) were detected. Conclusions By analogy to the previously shown association of specific FimH adhesin alleles with optimal binding by host adapted Salmonella serovars, lineages or strains, we predict that some of the identified association of other VF alleles with host-adapted serovars, lineages or strains will reflect specific contributions to host adaptation and/or pathogenesis. The identification of these allelic associations will support investigations of the biological impact of VF alleles and better characterize the role of allelic variation in Salmonella pathogenesis. Most relevant functional experiments will test the potential causal contribution of the detected FimH-associated VF variants in host adapted virulence.

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.

While advances in genomic sequencing have highlighted significant strain variability between and within Salmonella serovars, only a few protein variants have been directly related to evolutionary adaptation for survival, such as host specificity or differential virulence. The current study investigated whether allelic variation of the Salmonella adhesin/invasin PagN influences bacterial interaction with their receptors. The Salmonella enterica, subspecies enterica serovar Typhi (S. Typhi) allelic variant of PagN was found to bind significantly better to different enterocytes as well as to the extracellular matrix protein laminin than did the major Salmonella enterica, subspecies enterica serovar Typhimurium (S. Typhimurium) allele. The two alleles differed at amino acid residues 49 and 109 in two of the four predicted PagN surface loops, and residue substitution analysis revealed that a glutamic acid at residue 49 increased the adhesive and invasive properties of S. Typhi PagN. PagN sequence comparisons from 542 Salmonella strains for six representative S. enterica serovars and S. diarizonae further supported the role of glutamic acid at residues 49 and 109 in optimizing adhesion to cells and laminin, as well as for cell invasion. In summary, this study characterized unique residues in allelic variants of a virulence factor that participates in the colonization and invasive properties of different Salmonella stains, subspecies and serovars.

ABSTRACT Allelic combinations and host specificities for three fimbrial adhesins, FimH, BcfD, and StfH, were compared for 262 strains of Salmonella enterica serovar Newport, a frequent human and livestock pathogen. Like FimH, BcfD had two major alleles (designated A and B), whereas StfH had two allelic groups, each with two alleles (subgroup A1 and A2 and subgroup B1 and B2). The most prevalent combinations of FimH/BcfD/StfH alleles in S. Newport were A/A/A1 and B/B/B1. The former set was most frequently found in bovine and porcine strains, whereas the latter combination was most frequently found in environmental and human isolates. Bacteria genetically engineered to express Fim, Bcf, or Stf fimbriae on their surface were tested with the different alleles for binding to human, porcine, and bovine intestinal epithelial cells. The major allelic combinations with bovine and porcine strains (A/A/A1) or with human isolates (B/B/B1) provided at least two alleles capable of binding significantly better than the other alleles to an intestinal epithelial cell line from the respective host(s). However, each combination of alleles kept at least one allele mediating binding to an intestinal epithelial cell from another host. These findings indicated that allelic variation in multiple adhesins of S. Newport contributes to bacterial adaptation to certain preferential hosts without losing the capacity to maintain a broad host range. IMPORTANCE Salmonella enterica remains a leading foodborne bacterial pathogen in the United States; infected livestock serve often as the source of contaminated food products. A study estimated that over a billion Salmonella gastroenteritis cases and up to 33 million typhoid cases occur annually worldwide, with 3.5 million deaths. Although many Salmonella strains with a broad host range present preferential associations with certain host species, it is not clear what determines the various levels of host adaptation. Here, causal properties of host associations were determined with allelic variants of three colonization factors of S. enterica serovar Newport, a most frequent zoonotic serovar. This is the first study that related not only individual but also a small group of host-associated gene variants with functional properties that cooperate to determine the level of host-adapted virulence. The detected associations should help to identify sources of Salmonella infections in both humans and animals.

ABSTRACT Salmonella enterica serovar Typhimurium is one of the most common S. enterica serovars associated with U.S. foodborne outbreaks. S. Typhimurium bacteria isolated from humans exhibit wide-ranging virulence phenotypes in inbred mice, leading to speculation that some strains are more virulent in nature. However, it is unclear whether increased virulence in humans is related to organism characteristics or initial treatment failure due to antibiotic resistance. Strain diversity and genetic factors contributing to differential human pathogenicity remain poorly understood. We reconstructed phylogeny, resolved genetic population structure, determined gene content and nucleotide variants, and conducted targeted phenotyping assays for S. Typhimurium strains collected between 1946 and 2012 from humans and animals in the United States and abroad. Strains from recent U.S. salmonellosis cases were associated with five S. Typhimurium lineages distributed within three phylogenetic clades, which are not restricted by geography, year of acquisition, or host. Notably, two U.S. strains and four Mexican strains are more closely related to strains associated with human immunodeficiency virus (HIV)-infected individuals in sub-Saharan Africa than to other North American strains. Phenotyping studies linked variants specific to these strains in hmpA and katE to loss of fitness under nitrosative and oxidative stress, respectively. These results suggest that U.S. salmonellosis is caused by diverse S. Typhimurium strains circulating worldwide. One lineage has mutations in genes affecting fitness related to innate immune system strategies for fighting pathogens and may be adapting to immunocompromised humans by a reduction in virulence capability, possibly due to a lack of selection for its maintenance as a result of the worldwide HIV epidemic. IMPORTANCE Nontyphoidal Salmonella bacteria cause an estimated 1.2 million illnesses annually in the United States, 80 million globally, due to ingestion of contaminated food or water. Salmonella Typhimurium is one of the most common serovars associated with foodborne illness, causing self-limiting gastroenteritis and, in approximately 5% of infected patients, systemic infection. Although some S. Typhimurium strains are speculated to be more virulent than others, it is unknown how strain diversity and genetic factors contribute to differential human pathogenicity. Ours is the first study to examine the diversity of S. Typhimurium associated with recent cases of U.S. salmonellosis and to provide some initial correlation between observed genotypes and phenotypes. Definition of specific S. Typhimurium lineages based on such phenotype/genotype correlations may identify strains with greater capability of associating with specific food sources, allowing outbreaks to be more quickly identified. Additionally, defining simple correlates of pathogenesis may have predictive value for patient outcome.

Nature Communications 6 Article: 8754 (2015); Published: 30 Octomber 2015; Updated: 8 August 2017 In Fig. 3 of this Article, the numbers of isolates studied for the fimH41_Newp, fimH44_Newp and fimH45_Newp alleles were inadvertently swapped. The correct version of Fig. 3 appears below as Fig. 1.

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.

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.