Fimbrial Gene Cluster Research Articles

Salmonella fimbrial protein StcD induces cyclooxygenase-2 expression via Toll-like receptor 4.

The genome of Salmonella enterica serovar Typhimurium contains 13 operons with homology to fimbrial genes. To investigate the involvement of these fimbrial gene clusters in the expression of cyclooxygenase-2 (COX-2), which is an inducible enzyme involved in the synthesis of prostanoids, in J774 macrophages infected with S. enterica serovar Typhimurium, we constructed strains carrying a mutation in genes encoding the putative subunit proteins in 12 fimbrial operons. The level of COX-2 expression was lower in macrophages infected with fimA or stcA mutant Salmonella than in those infected with wild-type Salmonella. Therefore, we focused on putative subunit protein StcA and adhesive like protein StcD encoded in the stc operon. Treatment of macrophages with purified recombinant StcD protein, but not StcA, resulted in the activation of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways, leading to the expression of not only COX-2 but also of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha. The expression of StcD-induced COX-2 was inhibited by treatment with the Toll-like receptor 4 (TLR4) inhibitor TAK-242, but not by treatment with the lipopolysaccharide (LPS) antagonist polymyxin B. Furthermore, StcD treatment stimulated HEK293cells expressing TLR4 in the presence of CD14 and MD-2. StcD is a pathogen-associated molecular pattern of S. enterica serovar Typhimurium that is recognized by TLR4 and plays a significant role in the induction of COX-2 expression in macrophages.

Salmonella enterica serovar Brandenburg abortions in dairy cattle.

Two separate late-term abortion outbreaks in Jersey heifers in July 2020 and December 2020 were investigated by the Iowa State University Veterinary Diagnostic Laboratory. We evaluated 3 whole fetuses and 11 sets of fresh and formalin-fixed fetal tissues during the course of the outbreaks. The late-term abortions were first identified at a heifer development site and subsequently observed at the dairy farm. Aborted fetuses had moderate-to-marked postmortem autolysis with no gross lesions identified. Observed clinical signs in cows at the dairy farm ranged from intermittent loose stools to acute post-abortion pyrexia and reduced feed intake. Routine histopathology and reproductive bacterial culture revealed acute, suppurative placentitis with moderate-to-heavy growth of Salmonella spp. group B from stomach contents, liver, placenta, and heifer fecal contents. Serotyping identified Salmonella enterica subsp. enterica serovar Brandenburg in all 14 fresh tissue cases, as well as individual and pooled heifer feces. Whole-genome sequencing analysis revealed that all isolates belonged to ST type 873 and possessed typhoid toxin genes, several fimbrial gene clusters, type III secretion system genes, and several pathogenicity islands. Abortions caused by Salmonella Brandenburg have not been reported previously in dairy cattle in the United States, to our knowledge.

Functional Insights From KpfR, a New Transcriptional Regulator of Fimbrial Expression That Is Crucial for Klebsiella pneumoniae Pathogenicity

Although originally known as an opportunistic pathogen, Klebsiella pneumoniae has been considered a worldwide health threat nowadays due to the emergence of hypervirulent and antibiotic-resistant strains capable of causing severe infections not only on immunocompromised patients but also on healthy individuals. Fimbriae is an essential virulence factor for K. pneumoniae, especially in urinary tract infections (UTIs), because it allows the pathogen to adhere and invade urothelial cells and to form biofilms on biotic and abiotic surfaces. The importance of fimbriae for K. pneumoniae pathogenicity is highlighted by the large number of fimbrial gene clusters on the bacterium genome, which requires a coordinated and finely adjusted system to control the synthesis of these structures. In this work, we describe KpfR as a new transcriptional repressor of fimbrial expression in K. pneumoniae and discuss its role in the bacterium pathogenicity. K. pneumoniae with disrupted kpfR gene exhibited a hyperfimbriated phenotype with enhanced biofilm formation and greater adhesion to and replication within epithelial host cells. Nonetheless, the mutant strain was attenuated for colonization of the bladder in a murine model of urinary tract infection. These results indicate that KpfR is an important transcriptional repressor that, by negatively controlling the expression of fimbriae, prevents K. pneumoniae from having a hyperfimbriated phenotype and from being recognized and eliminated by the host immune system.

A high-throughput genomic screen identifies a role for the plasmid-borne type II secretion system of Escherichia coli O157:H7 (Sakai) in plant-microbe interactions

Shiga-toxigenic Escherichia coli (STEC) is often transmitted into food via fresh produce plants, where it can cause disease. To identify early interaction factors for STEC on spinach, a high-throughput positive-selection system was used. A bacterial artificial chromosome (BAC) clone library for isolate Sakai was screened in four successive rounds of short-term (2 h) interaction with spinach roots, and enriched loci identified by microarray. A Bayesian hierarchical model produced 115 CDS credible candidates, comprising seven contiguous genomic regions. Of the two candidate regions selected for functional assessment, the pO157 plasmid-encoded type two secretion system (T2SS) promoted interactions, while a chaperone-usher fimbrial gene cluster (loc6) did not. The T2SS promoted bacterial binding to spinach and appeared to involve the EtpD secretin protein. Furthermore, the T2SS genes, etpD and etpC, were expressed at a plant-relevant temperature of 18 °C, and etpD was expressed in planta by E. coli Sakai on spinach plants.

Expression and Functional Characterization of Various Chaperon-Usher Fimbriae, Curli Fimbriae, and Type 4 Pili of Enterohemorrhagic Escherichia coli O157:H7 Sakai.

Enterohemorrhagic Escherichia coli (EHEC) is a highly pathogenic strain leading to hemorrhagic colitis and to the hemolytic-uremic syndrome (HUS) in humans. The mechanisms by which pathogenic E. coli infect and colonize humans leading to the typical disease pattern are in focus of many investigations. The adhesion of EHEC to epithelial cells by the coordinated translocation of receptor Tir and surface expression of corresponding adhesin intimin is a key event in host–pathogen-interaction. However, less is known about other adhesins encoded by EHEC, especially about the complex set of fimbrial adhesins varying among various serotypes. Here, we investigate EHEC serotype O157:H7 strain Sakai possessing at least 16 putative fimbrial gene clusters. Using a synthetic heterologous expression system in a non-pathogenic E. coli strain, a subset of 6 gene clusters for fimbrial adhesins was analyzed. We were able to visualize surface expression of two γ1 class fimbriae (Fim and Ycb), two γ4 class fimbriae (Yad and Yeh), and two fimbrial adhesins which are assembled by the nucleation/precipitation pathway (Curli fimbriae), and by a type 2 secretion system (type 4 pili). Further, we elucidated the impact of these fimbrial adhesins in adhesion to various epithelial cells lines (HeLa, MDCK, and CaCo2), and the contribution on biofilm formation. We demonstrate the ultrastructure of Fim fimbriae and Yad fimbriae of EHEC Sakai, and Yeh fimbriae of E. coli in general. The involvement of Fim fimbriae of EHEC Sakai to adhesion to various epithelial cell lines, and contribution to biofilm formation is reported here. Our approach provides first ultrastructural and functional data for novel EHEC adhesins, and enables further understanding of the involvement of fimbrial adhesins in pathogenesis of EHEC Sakai.

Physical Extraction and Fast Protein Liquid Chromatography for Purifying Flagella Filament From Uropathogenic Escherichia coli for Immune Assay.

Flagella are expressed on the surface of a wide range of bacteria, conferring motility and contributing to virulence and innate immune stimulation. Host-pathogen interaction studies of the roles of flagella in infection, including due to uropathogenic Escherichia coli (UPEC), have used various methods to purify and examine the biology of the major flagella subunit protein, FliC. These studies have offered insight into the ways in which flagella proteins interact with host cells. However, previous methods used to extract and purify FliC, such as mechanical shearing, ultracentrifugation, heterologous expression in laboratory E. coli strains, and precipitation-inducing chemical treatments have various limitations; as a result, there are few observations based on highly purified, non-denatured FliC in the literature. This is especially relevant to host-pathogen interaction studies such as immune assays that are designed to parallel, as closely as possible, naturally-occurring interactions between host cells and flagella. In this study, we sought to establish a new, carefully optimized method to extract and purify non-denatured, native FliC from the reference UPEC strain CFT073 to be suitable for immune assays. To achieve purification of FliC to homogeneity, we used a mutant CFT073 strain containing deletions in four major chaperone-usher fimbriae operons (type 1, F1C and two P fimbrial gene clusters; CFT073Δ4). A sequential flagella extraction method based on mechanical shearing, ultracentrifugation, size exclusion chromatography, protein concentration and endotoxin removal was applied to CFT073Δ4. Protein purity and integrity was assessed using SDS-PAGE, Western blots with anti-flagellin antisera, and native-PAGE. We also generated a fliC-deficient strain, CFT073Δ4ΔfliC, to enable the concurrent preparation of a suitable carrier control to be applied in downstream assays. Innate immune stimulation was examined by exposing J774A.1 macrophages to 0.05-1 μg of purified FliC for 5 h; the supernatants were analyzed for cytokines known to be induced by flagella, including TNF-α, IL-6, and IL-12; the results were assessed in the context of prior literature. Macrophage responses to purified FliC encompassed significant levels of several cytokines consistent with prior literature reports. The purification method described here establishes a new approach to examine highly purified FliC in the context of host-pathogen interaction model systems.

Organization and architecture of AggR‐dependent promoters from enteroaggregative Escherichia coli

SummaryEnteroaggregative Escherichia coli (EAEC), is a diarrhoeagenic human pathogen commonly isolated from patients in both developing and industrialized countries. Pathogenic EAEC strains possess many virulence determinants, which are thought to be involved in causing disease, though, the exact mechanism by which EAEC causes diarrhoea is unclear. Typical EAEC strains possess the transcriptional regulator, AggR, which controls the expression of many virulence determinants, including the attachment adherence fimbriae (AAF) that are necessary for adherence to human gut epithelial cells. Here, using RNA‐sequencing, we have investigated the AggR regulon from EAEC strain 042 and show that AggR regulates the transcription of genes on both the bacterial chromosome and the large virulence plasmid, pAA2. Due to the importance of fimbriae, we focused on the two AAF/II fimbrial gene clusters in EAEC 042 (afaB‐aafCB and aafDA) and identified the promoter elements and AggR‐binding sites required for fimbrial expression. In addition, we examined the organization of the fimbrial operon promoters from other important EAEC strains to understand the rules of AggR‐dependent activation. Finally, we generated a series of semi‐synthetic promoters to define the minimal sequence required for AggR‐mediated activation and show that the correct positioning of a single AggR‐binding site is sufficient to confer AggR‐dependence.

Secretome profiling of Propionibacterium freudenreichii reveals highly variable responses even among the closely related strains.

SummaryThis study compared the secretomes (proteins exported out of the cell) of Propionibacterium freudenreichii of different origin to identify plausible adaptation factors. Phylosecretomics indicated strain‐specific variation in secretion of adhesins/invasins (SlpA, InlA), cell‐wall hydrolysing (NlpC60 peptidase, transglycosylase), protective (RpfB) and moonlighting (DnaK, GroEL, GaPDH, IDH, ENO, ClpB) enzymes and/or proteins. Detailed secretome comparison suggested that one of the cereal strains (JS14) released a tip fimbrillin (FimB) in to the extracellular milieu, which was in line with the electron microscopy and genomic analyses, indicating the lack of surface‐associated fimbrial‐like structures, predicting a mutated type‐2 fimbrial gene cluster (fimB‐fimA‐srtC2) and production of anchorless FimB. Instead, the cereal strain produced high amounts of SlpB that tentatively mediated adherent growth on hydrophilic surface and adherence to hydrophobic material. One of the dairy strains (JS22), producing non‐covalently bound surface‐proteins (LspA, ClpB, AraI) and releasing SlpA and InlA into the culture medium, was found to form clumps under physiological conditions. The JS22 strain lacked SlpB and displayed a non‐clumping and biofilm‐forming phenotype only under conditions of increased ionic strength (300 mM NaCl). However, this strain cultured under the same conditions was not adherent to hydrophobic support, which supports the contributory role of SlpB in mediating hydrophobic interactions. Thus, this study reports significant secretome variation in P. freudenreichii and suggests that strain‐specific differences in protein export, modification and protein–protein interactions have been the driving forces behind the adaptation of this bacterial species.

Functional Analysis of the Chaperone-Usher Fimbrial Gene Clusters of Salmonella enterica serovar Typhi.

The human-specific pathogen Salmonella enterica serovar Typhi causes typhoid, a major public health issue in developing countries. Several aspects of its pathogenesis are still poorly understood. S. Typhi possesses 14 fimbrial gene clusters including 12 chaperone-usher fimbriae (stg, sth, bcf, fim, saf, sef, sta, stb, stc, std, ste, and tcf). These fimbriae are weakly expressed in laboratory conditions and only a few are actually characterized. In this study, expression of all S. Typhi chaperone-usher fimbriae and their potential roles in pathogenesis such as interaction with host cells, motility, or biofilm formation were assessed. All S. Typhi fimbriae were better expressed in minimal broth. Each system was overexpressed and only the fimbrial gene clusters without pseudogenes demonstrated a putative major subunits of about 17 kDa on SDS-PAGE. Six of these (Fim, Saf, Sta, Stb, Std, and Tcf) also show extracellular structure by electron microscopy. The impact of fimbrial deletion in a wild-type strain or addition of each individual fimbrial system to an S. Typhi afimbrial strain were tested for interactions with host cells, biofilm formation and motility. Several fimbriae modified bacterial interactions with human cells (THP-1 and INT-407) and biofilm formation. However, only Fim fimbriae had a deleterious effect on motility when overexpressed. Overall, chaperone-usher fimbriae seem to be an important part of the balance between the different steps (motility, adhesion, host invasion and persistence) of S. Typhi pathogenesis.

Complete Genome Sequence of Salmonella enterica subsp. enterica serovar Minnesota Strain

Mango has been implicated as food vehicle in several Salmonella-causing foodborne outbreaks. Here, Salmonella enterica subsp. enterica serovar Minnesota was isolated from fresh mango fruit imported from Mexico in 2014. The complete genome sequence of S. Minnesota CFSAN017963 was sequenced using single-molecule real-time DNA sequencing. Distinct prophage regions, Salmonella pathogenicity islands, and fimbrial gene clusters were observed in comparative genomic analysis on S. Minnesota CFSAN017963 with other phylogenetically closely related Salmonella serovars. Core genome multilocus sequencing typing analysis of all the S. Minnesota isolates in the Genbank and Enterobase also revealed a high genomic diversity among the genomes analyzed.

Genome sequencing and comparative genomics of enterohemorrhagic Escherichia coli O145:H25 and O145:H28 reveal distinct evolutionary paths and marked variations in traits associated with virulence & colonization

BackgroundEnterohemorrhagic Escherichia coli (EHEC) O145 are among the top non-O157 serogroups associated with severe human disease worldwide. Two serotypes, O145:H25 and O145:H28 have been isolated from human patients but little information is available regarding the virulence repertoire, origin and evolutionary relatedness of O145:H25. Hence, we sequenced the complete genome of two O145:H25 strains associated with hemolytic uremic syndrome (HUS) and compared the genomes with those of previously sequenced O145:H28 and other EHEC strains.ResultsThe genomes of the two O145:H25 strains were 5.3 Mbp in size; slightly smaller than those of O145:H28 and other EHEC strains. Both strains contained three nearly identical plasmids and several prophages and integrative elements, many of which differed significantly in size, gene content and organization as compared to those present in O145:H28 and other EHECs. Furthermore, notable variations were observed in several fimbrial gene cluster and intimin types possessed by O145:H25 and O145:H28 indicating potential adaptation to distinct areas of host colonization. Comparative genomics further revealed that O145:H25 are genetically more similar to other non-O157 EHEC strains than to O145:H28.ConclusionPhylogenetic analysis accompanied by comparative genomics revealed that O145:H25 and O145:H28 evolved from two separate clonal lineages and that horizontal gene transfer and gene loss played a major role in the divergence of these EHEC serotypes. The data provide further evidence that ruminants might be a possible reservoir for O145:H25 but that they might be impaired in their ability to establish a persistent colonization as compared to other EHEC strains.

The KP1_4563 gene is regulated by the cAMP receptor protein and controls type 3 fimbrial function in Klebsiella pneumoniae NTUH-K2044.

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen that can adhere to host cells or extracellular matrix via type 1 and type 3 fimbriae. KP1_4563 is a gene encoding a hypothetical protein in K. pneumoniae NTUH-K2044. KP1_4563 is located between the type 1 and type 3 fimbrial gene clusters and is likely associated with fimbrial function given its putative conserved domains of unknown function (DUF1471). Cyclic AMP receptor protein (CRP) regulates virulence-related gene expression and is a crucial transcriptional regulator in many bacteria. The predicted DNA recognition motif of CRP is present in the KP1_4563 promoter region. This study aimed to investigate the function of KP1_4563 in fimbriae and its transcriptional regulation mechanism by CRP. We generated Kp-Δ4563 mutant and complementation strains. We utilized phenotype and adhesion assays to evaluate the role of KP1_4563 in fimbriae. We conducted quantitative RT-PCR (qRT-PCR), LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays to study the transcriptional regulation of KP1_4563 gene by CRP. We found that KP1_4563 negatively regulates the function of type 3 fimbriae. Compared with NTUH-K2044, the absence of KP1_4563 enhanced the ability of Kp-Δ4563 to adhere to A549 cells. CRP negatively regulates KP1_4563 by directly binding to its promoter region. KP1_4563 plays an important role in type 3 fimbrial function. This novel insight will assist in the development of strategies for preventing K. pneumoniae infection.

PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella pneumoniae CG43

In the Klebsiella pneumoniae CG43 genome, the divergently transcribed genes coding for PecS, the MarR-type transcription factor, and PecM, the drug metabolite transporter, are located between the type 1 and type 3 fimbrial gene clusters. The intergenic sequence pecO between pecS and pecM contains three putative PecS binding sites and a CpxR box. Electrophoretic mobility shift assay revealed that the recombinant PecS and CpxR could specifically bind to the pecO sequence, and the specific interaction of PecS and pecO could be attenuated by urate. The expression of pecS and pecM was negatively regulated by CpxAR and PecS, and was inducible by exogenous urate in the absence of cpxAR. Compared with CG43S3ΔcpxAR, the derived mutants CG43S3ΔcpxARΔpecS and CG43S3ΔcpxARΔpecSΔpecM exerted similar levels of sensitivity to H2O2 or paraquat, but higher levels of mannose-sensitive yeast agglutination activity and FimA production. The promoter activity and transcript levels of fimA in CG43S3ΔcpxAR were also increased by deleting pecS. However, no binding activity between PecS and the fimA promoter could be observed. Nevertheless, PecS deletion could reduce the expression of the global regulator HNS and release the negative effect of HNS on FimA expression. In CG43S3ΔcpxAR, the expression of FimA as well as PecS was inducible by urate, whilst urate-induced FimA expression was inhibited by the deletion of pecS. Taken together, we propose that K. pneumoniae PecS indirectly and negatively regulates the expression of type 1 fimbriae, and the regulation is urate-inducible in the absence of CpxAR.

Analysis of yeh Fimbrial Gene Cluster in Escherichia coli O157:H7 in Order to Find a Genetic Marker for this Serotype.

yeh fimbrial gene cluster encodes a type of putative fimbrial complex belonging to chaperone-usher assembly pathway. Recent studies have shown that yeh fimbrial gene cluster is present in 94% of Escherichia coli isolates and responsible for adhesion to some abiotic surfaces. Our preliminary comparative genomic analysis of 96 complete genomes of different E. coli strains revealed that the major region of this gene cluster is unique to E. coli O157:H7 strains. To investigate the detail of the analysis, we BLAST the sequence of this gene cluster against the existing complete and draft genome sequences of different E. coli strains and other genera belonging to Enterobacteriaceae family in NCBI database. The results showed that this gene cluster is properly unique to E. coli O157:H7 strains and could be used as a stable and specific genetic signature for the identification of this serotype. In this respect, we also experimentally validated the specificity of this gene cluster for the identification of E. coli O157:H7 strains by loop-mediated isothermal amplification method.

Pilus hijacking by a bacterial coaggregation factor critical for oral biofilm development

The formation of dental plaque, a highly complex biofilm that causes gingivitis and periodontitis, requires specific adherence among many oral microbes, including the coaggregation of Actinomyces oris with Streptococcus oralis that helps to seed biofilm development. Here, we report the discovery of a key coaggregation factor for this process. This protein, which we named coaggregation factor A (CafA), is one of 14 cell surface proteins with the LPXTG motif predicted in A. oris MG1, whose function was hitherto unknown. By systematic mutagenesis of each of these genes and phenotypic characterization, we found that the Actinomyces/Streptococcus coaggregation is only abolished by deletion of cafA. Subsequent biochemical and cytological experiments revealed that CafA constitutes the tip of a unique form of the type 2 fimbria long known for its role in coaggregation. The direct and predominant role of CafA in adherence is evident from the fact that CafA or an antibody against CafA inhibits coaggregation, whereas the shaft protein FimA or a polyclonal antibody against FimA has no effect. Remarkably, FimA polymerization was blocked by deletion of genes for both CafA and FimB, the previously described tip protein of the type 2 fimbria. Together, these results indicate that some surface proteins not linked to a pilus gene cluster in Gram-positive bacteria may hijack the pilus. These unique tip proteins displayed on a common pilus shaft may serve distinct physiological functions. Furthermore, the pilus shaft assembly in Gram-positive bacteria may require a tip, as is true for certain Gram-negative bacterial pili.

The Fimbrial Protein FlfA from Gallibacterium anatis Is a Virulence Factor and Vaccine Candidate

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg production worldwide. Widespread multidrug resistance largely prevents treatment of this organism using traditional antimicrobial agents, while antigenic diversity hampers disease prevention by classical vaccines. Thus, insight into its pathogenesis and knowledge about important virulence factors is urgently required. A key event during the colonization and invasion of mucosal surfaces is adherence, and recently, at least three F17-like fimbrial gene clusters were identified in the genomes of several G. anatis strains. The objective of this study was to characterize the putative F17-like fimbrial subunit protein FlfA from G. anatis 12656-12 and determine its importance for virulence. In vitro expression and surface exposure of FlfA was demonstrated by flow cytometry and immunofluorescence microscopy. The predicted function of FlfA as a fimbrial subunit protein was confirmed by immunogold electron microscopy. An flfA deletion mutant (ΔflfA) was generated in G. anatis 12656-12, and importantly, this mutant was significantly attenuated in the natural chicken host. Furthermore, protection against G. anatis 12656-12 could be induced by immunizing chickens with recombinant FlfA. Finally, in vitro expression of FlfA homologs was observed in a genetically diverse set of G. anatis strains, suggesting the potential of FlfA as a serotype-independent vaccine candidate This is the first study describing a fimbrial subunit protein of G. anatis with a clear potential as a vaccine antigen.

Expression and purification of SfaXII, a protein involved in regulating adhesion and motility genes in extraintestinal pathogenic Escherichia coli

Pathogenic Escherichia coli strains commonly harbor genes involved in formation of fimbriae, such as the sfaII fimbrial gene cluster found in uropathogenic and newborn meningitis isolates. The sfaXII gene, located at the distal end of the sfaII operon, was recently shown to play a role in controlling virulence-related gene expression in extraintestinal pathogenic E. coli (ExPEC). Until now, detailed characterization of the SfaXII protein has been hampered by difficulties in obtaining large quantities of soluble protein. By a rational modeling approach, we engineered a Cys70Ser mutation, which successfully improved solubility of the protein. Here, we present the expression, purification, and initial characterization of the recombinant SfaXIIC70S mutant. The protein was produced in E. coli BL21 (DE3) cells grown in autoinduction culture media. The plasmid vector harbored DNA encoding the SfaXIIC70S protein N-terminally fused with a six histidine (H6) sequence followed by a ZZ tag (a derivative of the Staphylococcus protein A) (H6-ZZ tag). The H6-ZZ tag was cleaved off with Tobacco Etch Virus (TEV) protease and the 166 amino acid full-length homo-dimeric protein was purified using affinity and size-exclusion chromatography. Electrophoretic mobility gel shift assays and atomic force microscopy demonstrated that the protein possesses DNA-binding properties, suggesting that the transcriptional regulatory activity of SfaXII can be mediated via direct binding to DNA.

Diversification of the Salmonella Fimbriae: A Model of Macro- and Microevolution

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.

Klebsiella pneumoniae type 3 fimbriae agglutinate yeast in a mannose-resistant manner

The ability of bacterial pathogens to express different fimbrial adhesins plays a significant role in virulence. Thus, specific detection of fimbrial expression is an important task in virulence characterization and epidemiological studies. Most clinical Klebsiella pneumoniae isolates express type 1 and type 3 fimbriae, which are characterized by mediation of mannose-sensitive agglutination of yeast cells and agglutination of tannic acid-treated ox red blood cells (RBCs), respectively. It has been observed that K. pneumoniae isolates agglutinate yeast cells and commercially available sheep RBCs in a mannose-resistant manner. Thus, this study was initiated to identify the adhesin involved. Screening of a mutant library surprisingly revealed that the mannose-resistant agglutination of yeast and sheep RBCs was mediated by type 3 fimbriae. Specific detection of type 1 fimbriae expression in K. pneumoniae was feasible only by the use of guinea pig RBCs. This was further verified by the use of isogenic fimbriae mutants and by cloning and expressing K. pneumoniae fimbrial gene clusters in Escherichia coli. Yeast agglutination assays are commonly used to detect type 1 fimbriae expression but should not be used for bacterial species able to express type 3 fimbriae. For these species, the use of guinea pig blood for specific type 1 fimbriae detection is essential. The use of commercially available sheep RBCs or yeast is an easy alternative to traditional methods to detect type 3 fimbriae expression. Easy and specific detection of expression of type 1 and type 3 fimbriae is essential in the continuous characterization of these important adhesive virulence factors present in members of the Enterobacteriaceae.

Reproducible gene targeting in recalcitrant Escherichia coli isolates

BackgroundA number of allele replacement methods can be used to mutate bacterial genes. For instance, the Red recombinase system of phage Lambda has been used very efficiently to inactivate chromosomal genes in E. coli K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical E. coli isolates.FindingsThe procedure was modified by using longer homologous regions (85 bp and 500-600 bp), to inactivate genes in the uropathogenic E. coli strain UTI89. An lrhA regulator mutant, and deletions of the lac operon as well as the complete type 1 fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant E. coli, like the avian pathogenic E. coli strain APEC1. The lrhA regulator and lac operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic E. coli strains (APEC3E, APEC11A and APEC16A) it was very difficult or impossible to construct these mutants, with the original Red recombinase-based method, with a Red recombinase-based method using longer (85 bp) homologous regions or with our modified protocol, using 500 - 600 bp homologous regions.ConclusionsThe method using 500-600 bp homologous regions can be used reliably in some clinical isolates, to delete single genes or entire operons by homologous recombination. However, it does not invariably show a greater efficiency in obtaining mutants, when compared to the original Red-mediated gene targeting method or to the gene targeting method with 85 bp homologous regions. Therefore the length of the homology regions is not the only limiting factor for the construction of mutants in these recalcitrant strains.