Core and Accessory FGCs of the Salmonella Fimbriome

Whereas only 15 Salmonella usher proteins were described in 2007 [34], the current study detected 35 unique types of usher in April 2011 by taking advantage of 90 available Salmonella genomes (27 distinct serovars). All the ORFs encoding fimbrial usher proteins had neighboring ORFs for chaperone proteins and two or more fimbrial subunits, as recognized by protein sequence similarities to known fimbrial subunits. Interestingly, there were no orphan ushers for a total of 950 ushers (with ≥85% amino acid sequence identities), and 35 different types of fimbrial gene clusters as shown in Figure 1. The 35 FGCs were clearly different from each other (<35% amino acid identity) and only 22 were previously named. On average, Salmonella were found to have 11.8 FGCs per strain with over 85% of the strains or serovars having between 11 and 13 FGCs (Figure S1). Notably, S. enterica subspecies arizonae had the least number of FGCs, (only 5), and S. bongori and S. enterica subspecies diarizonae both had 8 FGCs. Subspecies enterica serovar Pullorum and Weltevreden had 10 FGCs, whereas serovars Virchow and Kentucky carried the most FGCs with 14. Since the collection of 90 Salmonella strains studied (i) doesn’t include all the known (and unknown) serovars, and (ii) is skewed by having over- and underrepresented serovars, subspecies and species (most being S. enterica subsp. I), models evaluating the number of different FGCs in the Salmonella pangenome remain too speculative. However, a model based on the 26 FGCs from strains of serovars that had at least two different complements of FGCs predicted that the pangenome of S. enterica subsp. I has 42 to 43 different FGCs (Figure S2).

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Figure 1. Chaperone-usher fimbrial gene clusters (FGCs) of Salmonella.

A phylogenetic tree was built for 35 types of FGCs by using the amino acid sequences of the combined 950 usher proteins from 90 genomes (MEGA 5.0, as described in method). The FGCs were divided into five clades. The scale indicates the number of substitutions per amino acid. The bottom box lists different protein domain families. The asterisk indicates that some subunits were not picked by CDD or InterPro Scan, but (i) showed sequence similarity with other subunit(s) in the same gene clusters and (ii) were typically β-sheet-rich, as are all fimbrial subunits. Framed arrows are either known or predicted adhesins (as described in the text). C, V, VV, VVV were used to define the level of amino sequence variability for each subunit. C indicates subunits for which there was only one sequence available, or subunits lacking variants; V, VV or VVV indicated respectively ≤1, 1–10 or >10 detected variations per 100 amino acids.

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To address evolutionary questions about all the 35 individual FGCs, it was necessary to determine which FGCs were present in each Salmonella strain. For this, a phylogenomic tree based on 45 highly conserved housekeeping genes of the 90 sequenced Salmonella strains was compared to the panel of FGCs present in each strain (Figure S3). This tree classifies the serovars in five phylogenic clades numbered 1 to 5. Clade 5 represents Salmonella serovars that characteristically associate with cold-blooded animals. The analysis established that the bcf, fim, stb, sth, std, saf and sti FGCs were present in most Salmonella strains and serovars (>80% for both), albeit as pseudogenes in some serovars (core FGCs, Figure S4). The bcf FGC was the only one that was conserved in all Salmonella strains. The predominance of these FGCs suggests that they exert some important or even essential function for Salmonella survival, such as colonization, virulence and/or transmission. The absence of some of these core FGCs seemed to be partially serovar-specific, such as the association of std with serovar Pullorum and Gallinarum. However, such observations need to be confirmed with higher numbers of strains per serovar. The saf FGC was also absent in the avian-linked serovars Javiana and Heidelberg. There were some serovar biases with pseudogene distribution for these core fimbriae, and pseudogenes were more likely to be present in the FGCs of host-restricted serovars. In contrast to the core FGCs, the stf, lpf, ste, stc, stj FGCs were only partially conserved (i.e. present in 40%–80% of the serovars) and revealed different serovar distributions (Figure S4). The stf, lpf, stj FGCs were absent in serovar Typhi and clade 4 Salmonella, and stj was also absent in some clade 1 members (Figure S3). Both ste and stc were absent in clade 4, and ste was also absent in clade 1a Salmonella, which is mainly represented by serovar Typhimurium. Finally, more than half of the FGCs (peg, tcf, sef, stk, fae, sdg, sta, pef, sdf, peh, stg, sdh, mrk, sde, sdi, sdj, sdk, sdl, sdd, sba, sbb, sbc, sdc) were only found in a few Salmonella (Figure S4). Notably, some newly identified FGCs were present in only one serovar. The sba, sbb, sbc FGCs were only found in S. bongori, sdc and sdd only in S. enterica subspecies arizonae, sdi, sdj, sdk, sdl only in subspecies diarizonae, mrk only in serovar Montevideo and sde only in serovar Tennessee. These results suggest that these FGCs were acquired more recently than the FGCs present in most Salmonella, as discussed later.

Using the currently available data, Salmonella have an average of 12 FGCs per strain. The collection of Salmonella fimbriae is represented by three groups of FGCs. A group of core FGCs that are shared by over 80% of the strains or serovars can be distinguished from a group of partially conserved FGCs that are shared by 40%∼80% of the serovars. A third group of FGCs consisted of sporadic FGCs that were shared by less than 40% of the serovars and included more than half of all the Salmonella FGCs.

Classification of the FGCs in the Salmonella Fimbriome

As usher proteins are the most conserved FGC proteins, a phylogenetic tree was built by comparing the usher proteins from all the Salmonella FGCs. This tree, which was based on 950 proteins, was consistent with a tree built previously for 189 usher proteins that originated primarily from Proteobacteria [34]. This study confirmed that the Salmonella ushers were distributed in all the described clades of fimbriae (γ, κ, π, β, α, σ), missing only participants in the γ2 sub-clade (Figure 1). All of the prevalent FGCs, namely bcf, fim, lpf, sth and sti belonged to the γ1 sub-clade. The sde FGC is a new addition to the γ3 sub-clade previously described to include only sef and saf in Salmonella. Many new members of the γ4 clade were identified (peh, sdj, peg, sdg, sdh, sba, stk, sdd, sdf, sdl, mrk) that with the known stc, sta, stb FGCs make this clade the most diverse in Salmonella. Notably, the mrk FGC was only detectable in serovar Montevideo. The mrk designation was used because its gene cluster organization mimics the one of the Klebsiella pneumonia mrk FGC, which suggests that this FGC has moved by HGT. The fimbriae encoded by K. pneumonia mrk were characterized by their capacity to mediate mannose-resistant bacterial agglutination of tannic acid-treated erythrocytes [60].

σ clade fimbria includes the sdc FGC, which was found only in S. arizonae. While its usher protein showed low sequence similarity with others in Salmonella, the Blastp search the NCBI non-redundant database indicated that its closest relative was present in the genomes of a few other Enterobacteriaceae, such as Citrobacter rodentium, Escherichia fergusonii and Enterobacter hormaechei, suggesting that this FGC was acquired by HGT.

The κ clade consisted of three FGCs, one located on a plasmid, pef, and two new FGCs, fae and sbc, that share similarities with the plasmid-encoded K88 and K99 FGCs from E. coli [61]. The pef FGC was only found in serovar Typhimurium and a few other serovars such as Choleraesuis, Paratyphi C and I,4, [5],12:i:-. The Salmonella fae usher protein shares 85% identity with the E. coli orthologous usher and is found only on S. bongori. The sbc FGC shares both a similar FGC organization and an usher protein that is 43% identical to the E. coli K99 usher. The average pairwise differences between the κ-fimbrial ushers of S. enterica isolates were similar to those observed for the E. coli usher proteins. Thus the E. coli and Salmonella κ-fimbriae most likely share the same ancestor. The data also suggested that over time, E. coli and Salmonella have exchanged FGCs belonging to the κ-fimbrial clade by interspecies conjugative transfer of plasmids that afforded some selective advantage.

Six different types of Salmonella FGCs belonged to the π-fimbriae, named for the protoype E. coli P fimbriae [34], stf, ste and std being new ones. Gene cluster organizations were shared between std, sdk, sbb and sdl, and between stf and ste, albeit the latter had additional distal subunit genes. The std FGC, which was the predominant one in Salmonella, was reported to be involved in bacterial binding to murine cecal mucosa and intestinal persistence [51], [62]. The ste FGC, which was only prevalent in clades 1, 2 and 3 of S. enterica subsp. I (Figure S3), has been described to participate in Salmonella colonization of chicken intestines [52]. The role of the other π FGCs and their fimbriae remains unknown. The newly identified sdk, sbb, and sdl FGCs were only present in serovars typically isolated from cold-blooded animals (Figure S3, clade 5), suggesting that their function might be host specific.

The stj FGC was the only representative for the β-fimbriae, which was previously defined as fimbrial usher protein cluster 7 [63]. No structural or functional information is available for this small but distinct clade of FGCs, which lacks a typical adhesin gene, a characteristic assigned to fimbriae that assemble as thin fibrillae or nonfimbrial surface structures [34]. The α- fimbrial clade includes the alternative chaperone/usher FGC that was known as class 5 fimbriae [34]. The tcf is the only known α-fimbrial clade found in Salmonella and is present in several serovars.

Compared to the usher phylogenetic tree, the chaperone tree showed mostly similar lineages, particularly for the γ4, π and κclades (Figure S5). Some FGCs (e.g. bcf and stb) had two independent chaperone genes. One of the two bcf chaperone genes was located in another lineage, suggesting that the latter gene was once acquired by some recombination event. Curiously, the two stb chaperone genes remained together in another lineage, resulting most likely from both duplication and recombination events. Interestingly the pehB chaperone gene has two separate lineages for different serovars, suggesting divergent evolution in different serovar-specific environments or replacement by horizontal gene transfer.

In summary, the Salmonella pangenome carries a large diversity of fimbrial types, considering that it has fimbrial representatives of all six known fimbrial clades. Most core fimbriae belonged to the γclade, particularly the γ1 subclade, highlighting the adaptability and potential usefulness of this clade of FGCs for Salmonella survival and infection.

Extensive Acquisition and Loss of FGCs during Salmonella Evolution

A variety of studies have proposed phylogenetic trees to visualize the evolutionary history of Salmonella species and subspecies [24], [64], [65]. By integrating such data from previous microarray studies [66], [67] with our analysis of FGCs, we propose that an incremental set of steps can illustrate the acquisitions and losses of FGCs in Salmonella (Figure 2). When compared to E. coli, Salmonella bongori maintained five FGCs in the same genetic locus with few modifications by mutation and positive selection. The genes of these FGCs (fim, stg, sba, peg, lpf) were orthologs of the ones of the E. coli FGCs (sfm, lpf2, yad, yeh, lpf1), with bcf (ycb/elf in E. coli) having acquired an additional chaperone [34] (Table S1). A major event that resulted in the divergence of Salmonella from E. coli was the acquisition of SPI-1 [68]. This event enabled Salmonella to efficiently invade mammalian intestinal epithelial cells. The presence of new FGCs in Salmonella as compared to E. coli, suggests that the sth, sbb, sbc (Figure 2) clusters were also selected at this evolutionary step. Moreover, the similarity of the sbc FGC with the fan FGC, which is located on plasmids and encodes the K99 fimbriae of bovine or porcine enterotoxigenic E. coli, is suggestive of either an ancestral HGT gain accompanied of a loss during S. enterica evolution, or FGC acquisition after Salmonella speciation. Only S. enterica carries the fim FGC, as suggested by the currently available genomes and DNA hybrization data for S. enterica subsp. VII and IV [66]. Recent sequencing data (GenBank: FN298495.1) located the FGCs sdi and sbb in the integrative and conjugative element ICESe3 region of S. enterica subsp. VII, suggesting the two gene clusters were acquired by HGT (Table S2) [65]. Compared to S. bongori, S. enterica subsp. IIIa harbors two new FGCs, sdc and sdd, and lacks the sth, lpf, sdb, sda, stg FGCs indicating that these gains and losses occurred either at this step of evolution, or previously with subsp. VII or IV. S. enterica subsp. IIIb gained two new FGCs, std and stb. Whereas std was adjacent to a tRNA gene, stb had a sequence composition bias specific for pathogenicity islands, as determined by SIGI-HMM [69] (Table S2). As observed with subsp. IIIa, subsp. IIIb lost several FGCs. The sth, sbc and stg FGCs were lost by both subspecies, suggesting that they were lost before the speciation of IIIa and IIIb. The major evolutionary step that separates these two subspecies is the acquisition of a second flagellin locus by subsp. IIIb, allowing it to express either one of two flagellins thanks to a coordinated mechanism of flagellar phase variation between the two antigens (antigenic variation) [64], [66]. This more sophisticated system represents a selective advantage that likely occurred after the loss of these three FGCs.

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Figure 2. Salmonella and FGCs co-evolution model.

Proposed tree that includes E. coli and the two Salmonella species, S. bongori and S. enterica, the latter being divided into seven subspecies (monophasic IIIa, IV, VII and diphasic I, VI, II, and IIIb; subsp. V is now S. bongori). FGCs shown in red are suggested to have been acquired by HGT. FGCs shown in blue have diverged from orthologous E. coli or other Salmonella FGCs. In purple are FGCs that were lost. In green are FGCs that were duplicated. A dotted line separates the subspecies based on the presence of one or two flagellin genes. A dotted frame includes all the S. enterica subsp. I. The 5 clades correspond to the ones shown in Figure S3. The asterisks indicate that sdi and sbb were found in the integrative and conjugative element ICESe3 region of Salmonella enterica subsp. VII strain SARC16, suggesting independent acquisitions of these FGCs.

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Finally, the adaptive evolution of Salmonella from cold-blooded to warm-blooded animal hosts is characterized by the acquisition of SPI-2, which is only present in S. enterica subsp. I, as previously reported [15], [50]. Based on their acquisition and deletion of FGCs, we suggest an evolutionary pathway that has led to four clades of S. enterica subsp. I. First, seven FGCs were acquired (mrk, tcf, sdi, saf, stj, fae, stk), forming clade 4, which also lost three FGCs (lpf, stg and sta) and had three replaced, as determined by their analogous genomic loci (sdd, sba and peg for sdf, sdg and peh, respectively). As shown in Figure 2, clades 3, 2 and 1, were characterized by the gain of four or one, and the loss of five, four or two FGCs. Clades 3 and 4 include relatively rare serovars that were involved in recent food-borne disease outbreaks in the USA, such as Serovar Tennessee and Schwarzengrund in 2007, Agona in 2008 and Montevideo in 2009 to 2010 [70], [71](Figure S3). Clade 2 includes mainly the human-restricted Serovar Typhi and Parayphi A, and the serovar Newport strain SL254 which carries a multiple drug resistance plasmid [72]. This clade acquired only sef, while the sti, lpf, stj and stf FGCs were deleted. Clade 1 includes a greater diversity of serovars and FGCs, although serovar lineages tended to carry the same set of FGCs (Figure S3). This clade includes serovars Typhimurium and Enteriditis, the most common serovars responsible for human foodborne disease. A feature of the clade 1 serovars was the lack of stg and sta and for some them, the gain of pef.

The variable collections of FGCs in the different Salmonella species, subspecies and serovars resulted from complex changes that mainly involved FGC deletion and acquisition or replacement mediated by HGT, and to a lesser extent, duplication, and divergence. Additional genomic data, particularly for the Salmonella groups currently represented by only one genome, will help to improve evolutionary models in the future.

Salmonella FGCs’ Evolution by Gene Duplication, Rearrangement and Divergence

A minimum of three genes is essential for the expression of a fimbria. These genes have to encode a periplasmic chaperone and an outer membrane usher protein required for fimbrial biogenesis, as well as a fimbrial subunit capable of assembling as a homopolymeric organelle on the bacterial surface [39]. Based on the phylogenetic tree of usher proteins, it is suggested that the different clades of Salmonella FGCs have evolved from such an ancestral FGC (Figure S6). Unlike other Enterobacteriaceae, none of the sequenced Salmonella harbors a minimal FGC with only three genes. The smallest Salmonella FGCs consist of four genes, the additional gene encoding a second fimbrial subunit with the characteristics of an adhesin. As shown with the tcf FGC of the α- clade (Figure S6), a subunit gene that has twice the size of other subunits typically encodes a minor fimbrial tip adhesin. Subunit duplication and recombination events might explain the relocation of the fimbrial adhesin gene at the 3′ end of the operon. Such a genetic organization is consistent with an evolutionary process for low-level expression of minor fimbrial subunits, including fimbrial tip adhesins that are predictably expressed in equimolar concentration with the usher. The Salmonella α-clade has only one representative resulting conceivably from gene rearrangement. In general, trans-complementation between genes of different FGCs in the same genome does not occur, due to the constraints of specific protein interactions during fimbrial biogenesis. Thus, the accumulation of new genes in FGCs is most likely the result of internal gene duplication and not of the acquisition of genes from other FGCs. The tcf FGC has an unusual gene organization with its chaperone at its 5′-end, suggesting a unique type of discriminatory gene regulation to ensure sufficient expression of the fimbrial subunit. Similarly, FGCs of the γ1, γ3 and β clades have evolved using gene duplication and sequence divergence. In addition the inversion of the subunit, chaperone and usher genes in the sde FGC found only in serovar Tennessee highlights an organizational trend that was maintained in the π and κ clades. The switched location of the fimbrial subunit gene at the end of the FGC suggests the presence of an additional promoter to ensure efficient subunit expression, as described for the Salmonella sda-like Escherichia coli K99 (fan) FGC [73]. Evolution of a basic fimbrial operon design into a more complex FGC with additional promoters might have benefited regulatory fine-tuning of fimbrial biogenesis. Such an evolutionary step is more likely the by-product of duplication, recombination and divergence than horizontal gene transfer, which would require structural adaptation of a foreign subunit to an evolutionary separate FGC. Whereas some π clade members maintained an ancestral type of FGC with only four genes, the ste and stf showed dramatic subunit duplication of distal subunits. The κ clade together with one branch of the γ4 highlight an evolutionary process of multiple subunit duplication and divergence steps at the 3′ end of FGCs. Interestingly, the other γ4 branch maintained a basic 4 gene operon-like organization, whereas one FGC, stb, gained an additional chaperone and subunit at the distal end of the FGC. The presence of such additions suggests further evolutionary specialization towards subunit-specific chaperones, as first described for the E. coli 987P fimbriae [74].

Although FGC-specific regulatory genes frequently flank a FGC, they can be found elsewhere on the genome. FGC-specific regulatory proteins are very diverse both in structure and mechanism of function, even for orthologous FGCs, indicating less stringent evolutionary linkage with the FGCs they regulate [75]. FGCs are frequently transcribed as one operational unit, an operon, with the first gene encoding the fimbrial subunit followed in sequence by the genes for the chaperone and usher proteins. One could speculate that FGCs have evolved out of an adhesive autotransporter protein [76], whereby gene fragmentation would have separated its three domains into an exported adhesive amino-terminal end (passenger domain) a central region (autochaperone domain) and a carboxy-terminal outer membrane channel (translocator domain). The genetic organization of FGCs permits basic regulatory mechanisms, such as rho-independent stem-loops located at the 3′-end of the subunit gene, to ensure that the structural subunit is expressed in larger amounts than the biogenesis proteins [75].

In summary, FGC comparisons present evidence of subunit gene duplication and gene order reorganization within FGCs as important mechanisms of FGC evolution. Subunit gene duplications can be associated with environmental adaptation and increased function fitness [77], [78]. The varied organization of FGCs is consistent with the selfish operon concept, whereby HGT of complete FGCs together with reorganization of operons increased the efficiency of gene co-regulation to benefit FGC survival [79].

Functional Diversity Mediated by the Strain-specific Collection of FGCs

Over 99% of the human cases of salmonellosis are due to serovars that belong to the four clades of subsp. I. Clade 4 (Figure 3) includes serovar Montevideo, Schwarzengrund and Javiana that are commonly isolated in association with edible plants, such as red and black pepper, dehydrated chili, and tomato [70], [80]. This suggests that these serovars might have efficient mechanisms such as specific adhesive properties for long-term survival in the environment. Several clade 3 serovars are frequently isolated from edible products. For example, serovar Weltevreden strain 2007-60-3289-1 was isolated from a vegetable [81], [82], and serovar Tennessee was linked to a peanut butter outbreak in 2006–7 [83]. Curiously, serovar Tennessee has often been linked to urinary tract infections [84]. Several FGCs were specific for certain clade 4 serovars. For example, even though the sdg and sdf FGCs were typically present in all clade 4 serovars, the mrk, peh, fae were more specifically found in serovar Montevideo. The K. pneumoniae Mrk fimbriae has been described to bind to plant roots [85] as well as to extracellular matrix proteins and epithelial cells from the respiratory and urinary tracts. Serovar Montevideo and Kentucky carry the fae FGC, so designated for its similarity with the K88 FGC of enterotoxigenic E. coli [61] known to bind to several calf intestinal receptors [86]. With the exception of mrk, whether any of the other clade 3 and 4 specific FGCs encode plant-adhesive fimbriae that are expressed in an agricultural ecosystem is not known. Clade 2 of subsp. I. includes the human-specific serovar Typhi and Paratyphi A. Among serovar Typhi- and Paratyphi A-specific FGCs, including tcf, sta, sef and stg, only the tcf FGC was not degraded in some or all the strains. Genome degradation is a general outcome in both serovars and has been related to human host-restriction. The clade 1 includes both host-restricted and non-restricted serovars involved in human and animal gastroenteritis and septicemia. Most of these serovars share core FGCs (saf, bcf, fim, stb, sth and std), together with sti, stf and lpf, which are prominently absent in the clade 2 serovar Typhi.

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Figure 3. Correlation of Salmonella phylogenomic groups with specific collections of FGCs.

On the left, phylogenomic tree of 90 Salmonella and two E. coli control strains, based on 45 highly conserved house-keeping genes totaling ∼43 Kb. Clade 1 to 4 correspond to the clades shown in Figure 2, and clade 5 includes the few sequenced genomes from strains that were not S. enterica, subsp. I. The scale indicates the number of substitutions per nucleotide. On the top and heat map, hierarchical clustering support tree for the FGCs (MeV, complete linkage method with an euclidean distance threshold of 9.525, http://www.tm4.org). FGCs with or without pseudogenes were shown as green or red rectangles, respectively. On the right, Salmonella serovars (somatic O and flagellar H antigens).

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Based on FGC sets, clade 1 (Figure 3) was subdivided in three subclades. Most of the broad host range serovar Typhimurium strains and strains of the two close relatives I,4, [5],12:i:- str. CVM23701 and serovar Saintpaul str. SARA23 shared essentially the same FGCs, with only pef lacking in a few strains, to constitute subclade 1a. This collection of FGCs might participate in the broad-host range of subclade 1a serovars. The stc and lpf FGCs were found to participate in long-term persistence in infected mice [87], which should promote Salmonella transmission. A separate branch of the clade 1 cluster that consisted of serovars Virchow, Hadar, and Heidelberg had the highest numbers of FGCs among all the serovars. These serovars carried the additional ste, stk, fae and tcf FGCs. Accumulation of a large number of different FGCs may benefit survival and transmission and broaden the host and environment range that can be colonized. It may also improve the efficiency of specific host colonization and result from active HGT and recombination events in such hosts.

Serovar Dublin, Enteritidis, Pullorum and Gallinarum, share similar O-antigens and FGCs, forming subclade 1b. As reported for many genes of host-restricted serovars, such as serovar Typhi [88], extensive gene degradation was also detected in the FGCs of the avian-restricted serovar Gallinarum, and to a lesser extent Pullorum (Figure 3). Although serovar Dublin is known to cause diarrhea, septicemia and abortion in cattle and serovar Enteritidis is a major avian colonizer, both serovars infect other animals including humans. These two serovars showed less gene degradation in their FGCs than the avian-restricted serovars and included the additional FGC std and peg. Only serovar Dublin and Enteritidis had a non-degraded sef FGC. The fimbriae of this FGC bind to murine intestinal epithelial cells and are involved in systemic murine and avian infections. Whether Sef also acts as a virulence factor in cattle remains to be determined. Since serovar Dublin and Enteritidis share the same FGCs, other factors might contribute to their respective preferential hosts. Comparisons of gene degradation in the FGCs of subclade 1c isolates indicated more similarities between the human isolates of serovar Choleraesuis and Paratyphi C [89], [90] than with the swine isolate of serovar Choleraesuis [91]. Even though all strains of Choleraesuis are thought to be capable of causing generalized disease in both human and swine, it would interesting to determine whether a subpopulation of serovar Choleraesuis, together with a specific panel of FGCs, are host specialists for swine. Thus, the same host specificity by different serovars might be the result of convergent evolution [90].

Although there are over 2600 Salmonella serovars based on the O- and H- antigens, few are known to be host species specialists (i.e. specific for individual animal host species). This property has historically been emphasized to support the concept of host-serovar specificity, despite a lack of experimental data to explain at the molecular level how individual O- or H-antigens would determine host species specificity. The fact that these antigens are always expressed in vitro presented the opportunity to use this trait for diagnostic classification and epidemiology surveys. However, the exclusive focus on these antigens to identify a link between host specificity and population diversity in Salmonella can be misleading. Many serovars are not host specialists, indicating that affinity for specific host species may involve alternative bacterial pathways and surface molecules other than O- and H-antigens. These other surface molecules might serve as better genotypic predictors of host specificity. Bacterial surface ligands that interact only with specific host receptors have been particularly well documented in E. coli, such as the K88 and 987P fimbrial adhesins that mediate bacterial adhesion and colonization of pig intestines. We propose that the affinity of Salmonella strains for certain host species and tissues might be best determined by a collection of surface proteins with host-, tissue- and cell specific binding properties, such as the fimbriae. The variability in the number, identity and organization of FGCs in each Salmonella strain correlated with evolutionary processes that result from differential adaptation to a large variety of niches for survival. The high numbers of pseudogenes in otherwise undeleted FGCs suggests either recent inactivation of unnecessary or deleterious functions (particularly in host-restricted serovars), or the accumulation of FGCs for potential future use upon reactivation in a new environment. Although Salmonella have been reported to adhere to or invade plants by using non-specific binding factors such as cellulose and curli [92]–[94], Salmonella is better adapted to the intestinal environment of animals, particularly warm-blooded animals, where Salmonella multiplication is poorly hindered. Although contaminated edible plants might benefit the transmission of Salmonella, mammalian and avian intestines are likely a better location for the development of efficient adaptive evolutionary activities. The relatively low number of FGCs found in Salmonella bongori and in serovars that are mainly associated with cold-blooded animals, as shown for clade 5 in Figure 3, is in agreement with this. Clade 5 Salmonella have nine out of 17 FGCs that are unique by being not found in other clades. These FGCs, sba, sbb and sbc for Salmonella bongori, sdc and sdd for S. enterica subspecies IIIa, and sdl, sdk, sdj and sdi for S. enterica subspecies IIIb might be expressed at lower temperatures and be specific for intestinal receptors of reptiles or other cold- blooded animals. As more clade 5 Salmonella are sequenced, it is likely that other unique FGCs will be identified. In contrast, the bcf FGC was conserved in all Salmonella clades, albeit in a degraded form for the human serovars Typhi and Paratyphi A. Even though bcf expression might be deleterious to the maintenance of the serovars Typhi and Paratyphi A in humans, experimental evidence has indicated that it contributes to the colonization of bovine Peyer’s patches (PPs) and participates in gastrointestinal and long-term systemic infection in mice without murine PPs colonization [51], [95]. Presumably, either Bcf or Bcf receptor expression varies, depending on the different mammalian hosts and tissues encountered by Salmonella. Most of the other fimbriae that are partially shared between S. bongori and S. enterica (such as fim, stb, sth, std, lpf, peg, and stg) were shown to bind to intestinal epithelial cells or to participate in the colonization of mammalian or avian intestines [51], [52], [96]. The sta FGC was unable to direct bacterial adhesion or invasion of the human intestinal cell line INT-407. However, it remains possible that Sta fimbriae recognize mammalian or avian intestinal cell receptors that are absent on INT407. Even though cold-blooded animals are a reservoir for S. bongori, and S. enterica subsp. arizonae and diarizonae, these organisms may still have ligands that can colonize mammals. Whether any of these fimbriae play a role in human colonization or infection remains unclear.

Taken together, the phylogenomic analysis of all the sequenced Salmonella strains was mostly consistent with serovar and FGC profiles. However, the observed discrepancies that highlighted macro-evolutionary processes such as HGT-mediated acquisition of FGCs and functional (and likely structural) loss of FGCs that lead to host species specificity was more interesting. The data presented support the hypothesis that specific fimbriae are involved in determining preferential niches or hosts for Salmonella survival or transmission.

Functional Diversity Mediated by the Strain-specific Collection of Adhesin Alleles

In addition to the macro-evolutionary mechanisms of FGC gain and loss in Salmonella, the detection of allelic variants among the known or predicted functional molecules of fimbriae, namely their adhesins, attests to the presence of additional adaptive micro-evolutionary pathways. Accordingly, we suggest that in addition to the phenotype mediated by serovar- or strain-specific sets of FGCs, the allelic variants of fimbrial adhesins influence the preferential or specific colonization of certain host species and possibly the form and extent of the disease (carrier state, gastrointestinal or systemic symptoms). This assertion is supported by several studies on FimH, the type 1 fimbrial adhesin of Salmonella. The original definition for the type 1 fimbriae was based on the lectin-like affinity for mannose residues. Since mannose is a carbohydrate that frequently participates in the decoration of animal glycoproteins, including membrane glycoproteins, these fimbriae have been observed to bind to many cell types. A recently described FimH receptor is glycoprotein 2, which is expressed on the apical plasma membrane of M cells, where it serves as a bacterial transcytotic receptor [97]. As previously determined with small sets of Salmonella strains and serovars, the sequence of the type 1 fimbrial adhesin FimH demonstrates allelic variation. These FimH variants modulate the binding properties of the fimbriae, not only by changing the affinity for mannose, but also by substituting mannose for other receptors [59]. For example, the serine of serovar Enteritidis in place of phenylalanine of serovar Typhimurium at residue 96 of the mature protein altered the mannose-binding properties of FimH from a low to a high adhesive form [56], whereas two different strains of serovar Typhimurium with asparagine or tyrosine at position 136 presented different mammalian cell binding properties [59], that corresponded to mannosylated substrate binding [98]. Furthermore, a threonine to isoleucine substitution of residue 56 in the FimH of serovar Gallinarum and Pullorum could explain why this protein didn’t bind mannose [58]. This and other substitutions in the FimH residue(s) of serovar Gallinarum and Pullorum correlated with an improved FimH-mediated bacterial binding of these serovars to avian leucocytes [59]. Allelic variation of FimH has also recently been shown to influence the catch-bond adhesive properties of the Salmonella type 1 fimbriae [98].

This study compared the FimH sequences from the 90 available full genomes, 17 individual sequences in GenBank and the recently sequenced FimH from clinical isolates [99], [100] and from the Duguid et al. collection [101]. A total of 67 different FimH alleles carrying amino acid substitutions were identified in S. enterica subsp. I (Figure S7). Even though many residue substitutions were randomly distributed, others clearly identified hotspots. An average distance tree separated the FimH alleles into six groups (color-coded in Figure S7). Group one consisted exclusively of one allele found in the 10 Typhi strains that could be distinguished from all the other serovars by having unique FimH substitutions at positions 35, 36, 39, 137 and 195. Group two included FimH alleles characterized by substitutions at positions 49, 52, 67 and 295. Group three is less well-defined and included FimH alleles that had frequently substitutions at positions 10, 67, 115, 212 and/or 226. The fifth cysteine at position 104 of the sequenced serovar Abortusovis may be the result of a sequencing error, given that fimbrial subunits typically include even numbers of cysteine residues paired as cystines, consistent with the oxidized environment of a bacterial surface. Group four included FimH alleles characterized by substitutions at positions 104, 109 and/or 115. Unlike serovar Typhi, broad host range serovars such as Typhimurium and Enteritidis were distributed in both group three and four, and showed extensive allelic variability for FimH, highlighting phylogenetic incongruence for broad host range serovars and FimH. Group five consisted exclusively of the four serovar Paratyphi B FimH alleles that could be distinguished from the other serovars by unique substitutions at position 267. Group six consisted of a single strain of serovars Aluchua, which was the only allele with a substitution at position 288.

A three-dimensional model for the mature Salmonella FimH adhesin is proposed, based on the structure of the E. coli FimH protein (Figure 4). Both the amino-terminal residues 1 to 173 predicted to carry the binding pocket and the carboxy-terminal half predicted to function as the fimbrial assembly domain (residue 177 to 313) had similar numbers of substituted positions (29 versus 25, respectively). The linker region had two positions with substitutions. Even though most of these substitutions did not include the residues predicted by Phyre2 to interact with mannose in the binding pocket, residues 52, 56 and 155 were located in the loops that form the pocket (Figure 4). Only a few FimH with substitutions in a total of 56 variable positions from 67 natural alleles (Figure S7) were studied for their effects on adhesion. These FimH alleles had substitutions in 2, 3 or 5 different positions and their adhesiveness was increased, decreased or unaffected respectively.

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Figure 4. Predicted structural model of the Salmonella FimH fimbrial adhesin.

The structure of the Salmonella FimH protein was based on the template structure 1klf (Protein Data Bank) from the E. coli FimH adhesin [119]. On the left, ribbon model of the predicted structure of Salmonella FimH with its lectin and pilin domains, each with one disulfide bond. ˜β -barrel are shown in yellow and α-helices are shown in pink. On the right, the variable amino acid positions are shown in a tube-rendering model of the FimH backbone structure, with a color gradation from blue (most conserved residues) to red (most variable positions). None of the natural variable positions were located in the predicted binding pocket, shown as green circles on both models.

https://doi.org/10.1371/journal.pone.0038596.g004

Findings with natural FimH alleles were in agreement with those of a recent study that engineered a library of random fimH mutants by PCR [98]. In this study 15 out of 38 single mutants bound 2 to 7 times better than the parental strain to mannose-BSA, 10 of these mutants being dispersed along the amino-terminal half of FimH, with only one apparent hotspot at positions 136–138. Only two mutations (N136D and R232W) targeted residues that vary naturally (N136Y in Typhimurium strain AJB3, and R232W in Paratyphi C strain RK54954). Although most of the substitutions were in the lectin domain of FimH, only one was in a loop for the binding pocket (Y15F) and none included residues predicted to participate in the mannose-specific binding pocket itself. Since enhanced binding was mainly observed with substitutions located further away from the mannose-binding pocket, and more proximal to the predicted interface between the lectin and pilin domains of FimH, the authors suggested that allosteric effects were the prevalent modifiers of binding affinities [98]. Consistent with the E. coli FimH catch bond adhesin model [102], the binding affinity of a Salmonella FimH was increased by extending the binding molecule through shear force. Studies on a few other fimbriae supported this model [103], [104], suggesting that the evolution of fimbrial adhesins by allelic variation has benefited bacteria not only by increasing the range of receptor and host specificities, but also by modulating binding strengths to better resist environmental or host clearing mechanisms. Antibodies to the E. coli FimH lectin domain mainly recognize the extended high-affinity conformer of the protein and accordingly enhance binding of the fimbriae [105]. Consistent data were obtained with Salmonella [98], suggesting that new approaches will be needed to induce anti-adhesive antibodies, such as antibodies that bind preferentially to low-affinity conformers of adhesins known to function by a catch-bond mechanism of binding. The linear epitopes of FimH predicted to be most immunogenic include residues 49–56 and 115–155 [106], and thus include the binding pocket residues of FimH. The high-affinity conformers, by being more extended, may present more efficiently linear epitopes than the more compact low-affinity conformer, which may be better recognized by conformation-specific antibodies.

In contrast to a large body of studies on FimH, no other Salmonella fimbrial adhesin subunit has been identified and characterized functionally. Nevertheless, several fimbrial structures of Salmonella have been shown to provide adhesive properties with corresponding FGCs carrying one (or 2 for ste FGCs) predicted adhesin gene (Table S3). In addition to the fim FGC, the other Salmonella core FGCs showed a range of 19 to 25 allelic variants of their predicted adhesins for a total of 70 to 80 sequences (Table S4, in red). For the partially conserved FGCs (stc, ste, stf, lpf, stj) (Table S4, in green), 6 to 22 different alleles were found in the 18–36 available sequences, with SteG and LpfD showing the most variation at the protein sequence level. Most of the remaining FGCs had few detectable adhesin alleles, due to insufficient numbers of available sequences or serovars (Table S4, sporadic adhesins, in blue). Nevertheless, some of these adhesin alleles (SdbD, FaeG, TcfD, StgD) showed tremendous sequence variations between themselves, indicating that orthologous chaperone-usher genes share synteny with genes that encode highly variable subunits predicted to be adhesins. Interestingly, one FGC (ste) frequently carried two predicted adhesin genes in tandem, suggestive of gene duplication. In several strains, ste carried only one predicted adhesin gene with a size and composition that suggested the result of a recombination event between the two genes.

Taken together, studies on allelic variation of the FimH Salmonella adhesin have began to unravel how structure variability intervenes in the function of this ligand. Despite a great number of substituted amino acids in FimH, the restricted number of different alleles is likely representative of adaptive mutation and recombination events guided by beneficial functions of FimH for Salmonella survival in specific or diverse environments. Phylogenetic incongruence between serovars and FimH adhesins is consistent with evolutionary pathways that have been impacted by HGT and recombination. Allelic variation of other fimbrial ligands in Salmonella, as determined here with predicted fimbrial adhesins, should guide new studies aimed at determining the role of ligand diversity for the lifestyle and pathogenesis of Salmonella.

Evolution of a Large Fimbriome and Adhesinome

The comparative analysis of Salmonella fimbrial adhesins undertaken in this study suggests that macroevolutionary pathways led to the gain of new adhesins by FGC duplication or/and HGT. Microevolutionary adaptation processes directed both the diversification of adhesin specificity and affinity by substitution mutagenesis, and the inactivation of genes that were incompatible with the new lifestyle of the strain [107]. Gene disruption was mostly detected in host-restricted serovars by the large number of fimbrial pseudogenes, as a result of frameshifts. Consistent with the interpretation of our data, lineage-specific positive selection of genes, including adaptive gene loss, has been documented to contribute to the evolution of host restricted Salmonella serovars [53], [88], [90], [108]. This study offered a glimpse into the genetic diversity of the Salmonella fimbriome and adhesinome (the collection of adhesins and adhesin alleles in the Salmonella pangenome) and evaluated the phylogeny of a large number of new Salmonella fimbriae (one third) that had not been detected and catalogued previously [34], [39]. More sequence data from Salmonella strains will be needed, particularly from underrepresented serovars, to explore the relationship between Salmonella adhesin type or allele and host or niche specialization. The current analysis should also instigate new in vitro and in vivo studies to improve our understanding of the role of most Salmonella fimbriae on this bacterium’s binding properties, lifestyle and choice of hosts. Intestinal adhesion mechanisms together with other virulence factors profit efficient Salmonella residence, multiplication and transmission to new hosts [32], [52], [62]. The detection of associations of collections of strain-specific fimbriae and adhesin alleles with host species and potential disease progression should support the development of new rational diagnostic and therapeutic approaches.

Data Collection and Annotation for Salmonella DNA

The available Salmonella chromosomal and plasmid genome sequences were obtained from NCBI (http://www.ncbi.nlm.nih.gov/bioproject/12302), the Welcome Trust Sanger Institute (http://www.sanger.ac.uk/resources/downloads/bacteria/salmonella.html), the Genome Institute at Washington University (http://genome.wustl.edu/genomes/P180/), and Salmonella.org (http://www.salmonella.org/genomics/). The data were from 90 chromosomal and 60 plasmid sequences from 115 Salmonella strains (Table S5). All the sequences not found in NCBI (Genbank) were submitted to the RAST annotation server (http://rast.nmpdr.org/) for standard genome annotation and targeted sequence extraction [109]. The latter sequences were from S. bongori 12149 (ATCC 43975) and S. enterica serovars Hadar, Infantis, Typhimurium DT104 and DT2 from the Welcome Trust Sanger Institute, serovar Pullorum from Salmonella.org, and S. enterica subspecies diarizonae CDC 01–0005, from the Genome Institute at Washington University. The list includes 114 S. enterica strains and only one S. bongori strain. 112 of the 114 S. enterica belong to the subspecies enterica (I), one to the subsp. arizonae (IIIa) and one to the subsp. diarizonae (IIIb). The list carries a total of 27 different serovars. From the 115 strains, only 90 have chromosomal sequences, 19 of them with 30 identified plasmids (1–3 plasmids per strain). The remaining 30-plasmid sequences originate from strains with unknown chromosomal sequences. In addition, the genomes of two Escherichia coli strains, E. coli K-12 substrain MG1655 and enterohemorrhagic E. coli O157:H7 EDL933 with accession number NC_000913.2 and NC_002655.2, respectively, were collected from NCBI to be used as control genomes to assemble a Salmonella phylogenomic tree.

Identification of Salmonella Fimbrial Usher and Chaperone Proteins

Fimbrial gene clusters always encode a single usher protein, which is the largest and most conserved protein of a fimbrial apparatus. Thus, sequences of usher proteins were used to identify and collect all predicted genes and encoded proteins of fimbrial gene clusters from the available Salmonella genomes. At least one usher representative of each fimbrial gene cluster from the Virulence Factor Database (VFDB) (http://www.mgc.ac.cn/cgi-bin/VFs/genus.cgi?Genus=Salmonella) was used to search all the Salmonella genomes with BLASTp (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?) [110]. A similar search was repeated with chaperone proteins, which are the next most conserved proteins of fimbrial systems. Ushers and chaperones were recognized by alignment coverage of 70% with an E-value below 0.1. Hits that showed shorter alignments were checked manually to detect potential frameshifts and resulting pseudogenes in the context of fimbrial gene clusters. By using the same approach, we identified also the FGCs in two control E. coli strains to detect orthologs (Table S2).

Characterization of Fimbrial Gene Clusters and Predicted Proteins

Usher and chaperone proteins, as well as proteins encoded by genes neighboring usher and chaperone genes (whether or not annotated as being part of a fimbrial system) were queried with the Conserved Domain Database v2.29 (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and the InterPro release v18.0 database (http://www.ebi.ac.uk/Tools/pfa/iprscan/). In addition, fimbrial proteins from newly identified fimbrial gene clusters were characterized by undertaking BLASTp searches with the non-redundant protein database of Genbank. Genes from clusters that shared synteny with fimbrial clustered genes in other Enterobacteriaceae were given the same designation, whereas genes from previously unnamed fimbrial gene clusters were given new designations (sba, sbb, sbc, sdc, sdd, sde, sdf, sdg, sdh, sdi, sdj, sdk, sdl, peh). To identify putative adhesins within Salmonella FGCs, we utilized unique characteristics of known adhesin genes in Escherichia coli. First, fimbrial subunit genes were recognized as genes flanking the usher or chaperone(s) genes and harboring specific sequence signature recognized by the sequence search tools used above. Second, the adhesin gene is typically larger than other subunit genes to accommodate a binding domain. Genes that encode fimbrial adhesins are frequently twice the size of structural (or pilin) subunit genes. This has been best exemplified with the resolved structure of tip adhesins, as well as with an internal adhesin [36], [111]–[113]. Third, adhesin genes typically follow the usher and chaperone genes in the directional order of transcription of an FGC and are frequently the most distal fimbrial subunit gene. Each identified FGC of Salmonella had at least two fimbrial subunit genes and most FGCs carried only one clearly predictable adhesin, with the exception of 1 FGC (ste), which had 2 ORFS that could encode a potential adhesin. Table S6 lists the genes and pseudogenes (labeled with asterisks) of all the FGCs, as well as their locus numbers.

Evaluation of the Number of different FGCs in Salmonella

A mathematical model was used to evaluate the number of different FGCs in the pangenome of S. enterica subsp I. For this, only FGCs found in serovars that had strains with different sets of FGCs were used, namely, the 26 FGCs of serovars Dublin, Gallinarum, Newport, Saintpaul and Typhimurium. A constructed collector’s curve was found to near an asymptote. The curve was fitted to a logarithmic curve with the equation y = 4.2043 ln(x) +11.655 (r² = 0.99957). The current number of identified S. enterica subsp I serovars being 1531 (x), a total number of different FGCs for this subspecies was predicted to be 42.5 (y).

Phylogenetic Analysis of Fimbrial Usher and Chaperone Proteins

Protein sequences were chosen for phylogenic analysis because they are significantly more conserved than DNA sequences. Trees with DNA sequences include too much noise due to synonymous mutations. From a functional point of view, evolution of the protein sequences is more relevant, considering that fimbrial biogenesis involves many fimbriae-specific protein-protein interactions that restrict the selection of evolutionary mutations. Earlier phylogenic studies of bacterial fimbriae used the same approach [39], [40]. MEGA 5.05 was used to prepare multiple sequence alignments of 950 usher and 1094 chaperone proteins with ClustalW (default parameters) and to construct phylogenetic trees (Neighbor-Joining method with bootstrap replication 1,000, Jones-Taylor-Thornton model).

Phylogenomic Analysis of Salmonella

A phylogenomic tree was prepared from the available 90 Salmonella genomes, along with the two E. coli reference genomes described above. To identify the orthologous genes shared by these bacteria, their predicted encoded protein sequences were retrieved from the GenBank and RAST databases. Pairs of proteins with more than 45% identity, 70% alignment coverage, and an E-value below 1×10⁻²⁰ were considered orthologous. To construct the phylogenomic tree, 45 highly conserved orthologous genes involved in gene regulation and transcription were chosen (cheB, cheR, clpS, cobB, comEA, fis, flgM, flhD, ftsB, ftsY, groS, grxB, helD, holC, hscB, hslR, infB, luxS, metJ, mreC, ndaG, nikR, nrdR, nusA, prmA, rho, rib, rimM, rnhA, rof, rplQ, rplY, rpoN, rpsP, rsd, rsmE, sulA, tsf, ybeA, yciH, yfjA, yhbY, yhhF, yhjY, and yicC). For this, the 45 genes were concatenated into a nucleotide sequence of approximately 43 kb. The 92 concatenated sequences were aligned using ClustalW with default parameters to produce an alignment in MEGA format [114]. Phylogenomic Tree construction was done using the Maximum-likelihood method with a bootstrap value of 1,000 [27]. To evaluate the distribution of fimbrial gene clusters among the Salmonella spp., the MultiExperiment Viewer was used to make a hierarchical clustering support tree (MeV4.7, http://www.tm4.org).

FimH Allele Analysis and Structural Modeling

In addition to the FimH sequences from the 90 available Salmonella genomes, Salmonella FimH sequences were collected from GenBank and recent publications [99] [100], [101]. FimH allele groups were characterized by the average distance tree that was produced with BLOSUM62 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Alignments of the alleles were obtained with ClustalW and further edited manually. Structure modeling of the FimH sequences was done with Phyre-2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) [115], nFOLD3 (http://www.reading.ac.uk/bioinf/nFOLD/) [116], MUSTER (http://zhanglab.ccmb.med.umich.edu/MUSTER/) [117] and I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) [118], respectively. The best structural template (1klf, Protein Data Bank, E. coli FimH) [119] was used to construct the tertiary structure of FimH using Phyre-2. The predicted model was viewed and analyzed using the molecular visualization program Jmol (http://jmol.sourceforge.net/). The protein variability server was used to analyze FimH allele variability within a multiple of sequence alignment and mapping on the structure [120]. Residues involved in the putative binding site of the FimH lectin domain were discerned by using the eF Site database [121] and searching for large cleft with the Pocket program of Phyre-2. A FimH sequence was sent to the IEDB analysis resource for identification of epitope sequences and 3D structural homology mapping [106]. Other adhesion and putative adhesin sequences (Table S7) were sent to the Datamonkey server, using the TOGGLE model to determine the number of different alleles with substitution sites and types [122].