GrlR, a negative regulator in enteropathogenic E. coli, also represses the expression of LEE virulence genes independently of its interaction with its cognate partner GrlA.

Introduction: Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) belong to a group of pathogens that share the ability to form "attaching and effacing" (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates ler expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood. Methods: To further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and cat transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis. Results and discussion: We showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR's alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.

Conduction System Pacing vs Biventricular Pacing in Heart Failure and Wide QRS Patients: LEVEL-AT Trial.

BACKGROUND: Conduction system pacing (CSP) has emerged as an alternative to biventricular pacing (BiVP). Randomized studies comparing both therapies are scarce and do not include left bundle branch pacing. OBJECTIVES: This study aims to compare ventricular resynchronization achieved by CSP vs BiVP in patients with cardiac resynchronization therapy indication. METHODS: LEVEL-AT (Left Ventricular Activation Time Shortening with Conduction System Pacing vs Biventricular Resynchronization Therapy) was a randomized, parallel, controlled, noninferiority trial. Seventy patients with cardiac resynchronization therapy indication were randomized 1:1 to BiVP or CSP, and followed up for 6 months. Crossover was allowed when primary allocation procedure failed. Primary endpoint was the change in left ventricular activation time, measured using electrocardiographic imaging. Secondary endpoints were left ventricular reverse remodeling and the combined endpoint of heart failure hospitalization or death at 6-month follow-up. RESULTS: Thirty-five patients were allocated to each group. Eight (23%) patients crossed over from CSP to BiVP; 2 patients (6%) crossed over from BiVP to CSP. Electrocardiographic imaging could not be performed in 2 patients in each group. A similar decrease in left ventricular activation time was achieved by CSP and BiVP (-28 ± 26 ms vs -21 ± 20 ms, respectively; mean difference -6.8 ms; 95% CI: -18.3 ms to 4.6 ms; P < 0.001 for noninferiority). Both groups showed a similar change in left ventricular end-systolic volume (-37 ± 59 mL CSP vs -30 ± 41 mL BiVP; mean difference: -8 mL; 95% CI: -33 mL to 17 mL; P = 0.04 for noninferiority) and similar rates of mortality or heart failure hospitalizations (2.9% vs 11.4%, respectively) (P = 0.002 for noninferiority). CONCLUSIONS: Similar degrees of cardiac resynchronization, ventricular reverse remodeling, and clinical outcomes were attained by CSP as compared to BiVP. CSP could be a feasible alternative to BiVP. (LEVEL-AT [Left Ventricular Activation Time Shortening With Conduction System Pacing vs Biventricular Resynchronization Therapy]; NCT04054895).

Klf10 favors Mycobacterium tuberculosis survival by impairing IFN-γ production and preventing macrophages reprograming to macropinocytosis.

Mycobacterium tuberculosis has developed diverse mechanisms to survive inside phagocytic cells, such as macrophages. Phagocytosis is a key process in eliminating invading pathogens; thus, M. tuberculosis efficiently disrupts phagosome maturation to ensure infection. However, inflammatory cytokines produced by macrophages in response to early M. tuberculosis infection are key to promoting bacterial clarification. IFN-γ enhances M. tuberculosis engulfment and destruction by reprogramming macrophages from phagocytosis to macropinocytosis. Here, we show that the transcription factor Krüppel-like factor 10 (Klf10) plays a positive role in M. tuberculosis survival and infection by negatively modulating IFN-γ levels. Naïve Klf10-deficient macrophages produce more IFN-γ upon stimulation than wild-type macrophages, thus enhancing bacterial uptake and bactericidal activity achieved by macropinocytosis. Moreover, Klf10⁻/ ⁻ macrophages showed cytoplasmic distribution of coronin 1 correlated with increased pseudopod count and length. In agreement with these observations, Klf10⁻/ ⁻ mice showed improved bacterial clearance from the lungs and increased viability. Altogether, our data indicate that Klf10 plays a critical role in M. tuberculosis survival by preventing macrophage reprogramming from phagocytosis to macropinocytosis by negatively regulating IFN-γ production upon macrophage infection.

The Fis Nucleoid Protein Negatively Regulates the Phase Variation fimS Switch of the Type 1 Pilus Operon in Enteropathogenic Escherichia coli.

In Escherichia coli the expression of type 1 pili (T1P) is determined by the site-specific inversion of the fimS ON-OFF switch located immediately upstream of major fimbrial subunit gene fimA. Here we investigated the role of virulence (Ler, GrlR, and GrlA) and global regulators (H-NS, IHF, and Fis) in the regulation of the fimS switch in the human enteropathogenic E. coli (EPEC) O127:H6 strain E2348/69. This strain does not produce detectable T1P and PCR analysis of the fimS switch confirmed that it is locked in the OFF orientation. Among the regulator mutants analyzed, only the ∆fis mutant produced significantly high levels of T1P on its surface and yielded high titers of agglutination of guinea pig erythrocytes. Expression analysis of the fimA, fimB, and fimE promoters using lacZ transcriptional fusions indicated that only PfimA activity is enhanced in the absence of Fis. Collectively, these data demonstrate that Fis is a negative regulator of T1P expression in EPEC and suggest that it is required for the FimE-dependent inversion of the fimS switch from the ON-to-OFF direction. It is possible that a similar mechanism of T1P regulation exists in other intestinal and extra-intestinal pathogenic classes of E. coli.

Conduction system pacing vs. biventricular pacing in patients with ventricular dysfunction and AV block.

BACKGROUND: It is unknown whether His-Purkinje conduction system pacing (HPCSP), as either His bundle or left bundle branch pacing, could be an alternative to cardiac resynchronization therapy (BiVCRT) for patients with left ventricular dysfunction needing ventricular pacing due to atrioventricular block. The aim of the study is to compare the echocardiographic response and clinical improvement between HPCSP and BiVCRT. METHODS: Consecutive patients who successfully received HPCSP were compared with a historical cohort of BiVCRT patients. Patients were 1:1 matched by age, LVEF, atrial fibrillation, renal function and cardiomyopathy type. Responders were defined as patients who survived, did not require heart transplantation and increased LVEF ≥5 points at 6-month follow-up. RESULTS: HPCSP was successfully achieved in 92.5% (25/27) of patients. During follow-up, 8% (2/25) of HPCSP patients died and 4% (1/25) received a heart transplant, whereas 4% (1/25) of those in the BiVCRT cohort died. LVEF improvement was 10% ± 8% HPCSP versus 7% ± 5% BiVCRT (p = .24), and the percentage of responders was 76% (19/25) HPCSP versus 64% (16/25) BiVCRT (p = .33). Among survivors, the percentage of patients who improved from baseline II-IV mitral regurgitation (MR) to 0-I MR was 9/11 (82%) versus 2/8 (25%) (p = .02). Compared to those with BiVCRT, patients with HPCSP achieved better NYHA improvement: 1 point versus 0.5 (OR 0.34; p = .02). CONCLUSION: HPCSP in patients with LVEF ≤45% and atrioventricular block improved the LVEF and induced a response similar to that of BiVCRT. HPCSP significantly improved MR and NYHA functional class. HPCSP may be an alternative to BiVCRT in these patients. (Figure 1. Central Illustration). [Figure: see text].

Population analysis of D6-like plasmid prophage variants associated with specific IncC plasmid types in the emerging Salmonella Typhimurium ST213 genotype.

The Salmonella enterica serovar Typhimurium sequence type 213 (ST213) emerged as a predominant genotype in Mexico. It is characterized by harboring multidrug resistance (MDR) IncC plasmids (previously IncA/C) and the lack of the Salmonella virulence plasmid (pSTV). Here we show that the D6-like plasmid prophage is present in most of the ST213 strains. We used the reported nucleotide sequence of YU39 plasmid (pYU39_89) to design a PCR typing scheme for the D6-like plasmid prophages, and determined the complete nucleotide sequences for the D6-like prophages of three additional ST213 strains (YU07-18, SL26 and SO21). Two prophage variants were described: i) a complete prophage, containing homologous sequences for most of the genetic modules described in P1 and D6 phages, which most likely allow for the lytic and lysogenic lifestyles; and ii) an incomplete prophage, lacking a 15 kb region containing morphogenesis genes, suggesting that it is defective. The tail fiber gene inversion region was the most divergent one between D6 and pYU39_89 genomes, suggesting the production of a distinct set of tail fibers, which could be involved in host range preferences. A glutaminyl-tRNA synthetase gene (glnS), which could be involved in providing host cell increased fitness or plasmid maintenance functions, was found in all D6-like genomes. Population level analysis revealed a biogeographic pattern of distribution of these plasmid-phages and specific associations with variants of MDR IncC plasmids. Statistically significant associations were found between the two prophage variants (p75 or p89), the type of IncC plasmids (I or II) and geographic isolation regions (Sonora, San Luis Potosí, Michoacán and Yucatán). This work integrates results from molecular typing, genomics and epidemiology to provide a broad overview for the evolution of an emergent Salmonella genotype.

Characterization of Salmonella enterica isolates causing bacteremia in Lima, Peru, using multiple typing methods.

In this study, different molecular typing tools were applied to characterize 95 Salmonella enterica blood isolates collected between 2008 and 2013 from patients at nine public hospitals in Lima, Peru. Combined results of multiplex PCR serotyping, two- and seven-loci multilocus sequence typing (MLST) schemes, serotyping, IS200 amplification and RAPD fingerprints, showed that these infections were caused by eight different serovars: Enteritidis, Typhimurium, Typhi, Choleraesuis, Dublin, Paratyphi A, Paratyphi B and Infantis. Among these, Enteritidis, Typhimurium and Typhi were the most prevalent, representing 45, 36 and 11% of the isolates, respectively. Most isolates (74%) were not resistant to ten primarily used antimicrobial drugs; however, 37% of the strains showed intermediate susceptibility to ciprofloxacin (ISC). Antimicrobial resistance integrons were carried by one Dublin (dfra1 and aadA1) and two Infantis (aadA1) isolates. The two Infantis isolates were multidrug resistant and harbored a large megaplasmid. Amplification of spvC and spvRA regions showed that all Enteritidis (n = 42), Typhimurium (n = 34), Choleraesuis (n = 3) and Dublin (n = 1) isolates carried the Salmonella virulence plasmid (pSV). We conclude that the classic serotyping method can be substituted by the multiplex PCR and, when necessary, sequencing of only one or two loci of the MLST scheme is a valuable tool to confirm the results. The effectiveness and feasibility of different typing tools is discussed.

Self-Conjugation of the Enteropathogenic Escherichia coli Adherence Factor Plasmid of Four Typical EPEC Isolates.

The enteropathogenic Escherichia coli (EPEC) adherence factor plasmid (pEAF) encodes the proteins involved in the biogenesis of the bundle-forming pilus (BFP), a key virulence factor that mediates microcolony formation and the localized adherence phenotype on the surface of the host enterocytes. The presence or absence of this plasmid defines typical EPEC (tEPEC) and atypical EPEC (aEPEC), respectively. Although lateral transfer of pEAF has been evidenced by phylogenetic studies, conjugal transfer ability has been experimentally established only for two pEAF plasmids from strains isolated in the late 60s. In the present work, we tested the self-conjugation ability of four pEAF plasmids from tEPEC strains isolated between 2007 and 2008 from children in Peru and the potential of aEPEC to receive them. A kanamycin resistance cassette was inserted into donor pEAF plasmids in order to provide a selectable marker in the conjugation experiments. Two aEPEC isolated from the same geographic region were used as recipient strains along with the laboratory E. coli DH5α strain. Here we show that the four pEAF plasmids tested are self-conjugative, with transfer frequencies in the range of 10-6 to 10-9. Moreover, the generation of aEPEC strains harboring pEAF plasmids provides valuable specimens to further perform functional studies.

Locus of Adhesion and Autoaggregation (LAA), a pathogenicity island present in emerging Shiga Toxin-producing Escherichia coli strains.

Shiga Toxin-producing Escherichia coli (STEC) are a group of foodborne pathogens associated with diarrhea, dysentery, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxins are the major virulence factor of these pathogens, however adhesion and colonization to the human intestine is required for STEC pathogenesis. A subset of STEC strains carry the Locus of Enterocyte Effacement (LEE) pathogenicity island (PAI), which encodes genes that mediate the colonization of the human intestine. While LEE-positive STEC strains have traditionally been associated with human disease, the burden of disease caused by STEC strains that lacks LEE (LEE-negative) has increased recently in several countries; however, in the absence of LEE, the molecular pathogenic mechanisms by STEC strains are unknown. Here we report a 86-kb mosaic PAI composed of four modules that encode 80 genes, including novel and known virulence factors associated with adherence and autoaggregation. Therefore, we named this PAI as Locus of Adhesion and Autoaggregation (LAA). Phylogenomic analysis using whole-genome sequences of STEC strains available in the NCBI database indicates that LAA PAI is exclusively present in a subset of emerging LEE-negative STEC strains, including strains isolated from HC and HUS cases. We suggest that the acquisition of this PAI is a recent evolutionary event, which may contribute to the emergence of these STEC.

Effects of static magnetic fields on the enteropathogenic Escherichia coli.

This study reports the effects of exposing cells of the prototypical enteropathogenic Escherichia coli (EPEC) strain E2348/69 to static magnetic fields (SMF) of varying intensities to observe their capacity to autoaggregate and the effect on cell adherence. The results showed that bacteria exposure over the course of 5 min to an intensity of 53 mT reduced autoaggregation by 28%. However, with intensities of up to 100 mT with the same exposure time, bacteria autoaggregation was reduced by approximately 50%; and after 30 min at the same intensity, it was indistinguishable from that observed in a non-autoaggregative strain. Furthermore, it was observed that SMF treatment also modified the typical localized adherence pattern of EPEC E2348/69. The observed effects are not related to bacteria damage. The above was confirmed because, after a 107 mT SMF treatment over the course of 30 min, cell viability and membrane permeability were the same to that observed in untreated controls. The obtained results suggest that the SMF effect on the E2348/69 EPEC strain alters the expression of the bundle-forming pilus (BFP), due to the fact that the same strain without the EPEC adherence factor plasmid that encodes the BFP operon was unable to autoaggregate. Electron microscopic analyses revealed structural differences between cells exposed to SMF with respect to untreated controls. In conclusion, the SMF treatment of 107 mT for 30 min reduced EPEC E2348/69 autoaggregation and modified its adherence pattern, with both events likely being associated with changes in BFP expression. Bioelectromagnetics. 38:570-578, 2017. © 2017 Wiley Periodicals, Inc.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Infantis Strain SPE101, Isolated from a Chronic Human Infection.

We report a 4.99-Mb draft genome sequence of Salmonella enterica subsp. enterica serovar Infantis strain SPE101, isolated from feces of a 5-month-old breast-fed female showing diarrhea associated with severe dehydration and malnutrition. The infection prolonged for 6 months despite antibiotic treatment.

IL-22 Controls Iron-Dependent Nutritional Immunity Against Systemic Bacterial Infections.

Host immunity limits iron availability to pathogenic bacteria, but whether immunity limits pathogenic bacteria from accessing host heme, the major source of iron in the body, remains unclear. Using Citrobacter rodentium, a mouse enteric pathogen and Escherichia coli, a major cause of sepsis in humans as models, we find that interleukin-22, a cytokine best known for its ability to promote epithelial barrier function, also suppresses the systemic growth of bacteria by limiting iron availability to the pathogen. Using an unbiased proteomic approach to understand the mechanistic basis of IL-22 dependent iron retention in the host, we have identified that IL-22 induces the production of the plasma hemoglobin scavenger haptoglobin and heme scavenger hemopexin. Moreover, the anti-microbial effect of IL-22 depends on the induction of hemopexin expression, while haptogloblin is dispensable. Impaired pathogen clearance in infected Il22-/- mice was restored by hemopexin administration and hemopexin-deficient mice had increased pathogen loads after infection. These studies reveal a previously unrecognized host defense mechanism regulated by IL-22 that relies on the induction of hemopexin to limit heme availability to bacteria leading to suppression of bacterial growth during systemic infections.

Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome.

The type III secretion system (T3SS) is a supramolecular machine used by many bacterial pathogens to translocate effector proteins directly into the eukaryotic host cell cytoplasm. Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrheal disease in underdeveloped countries. EPEC virulence relies on a T3SS encoded within a chromosomal pathogenicity island known as the locus of enterocyte effacement (LEE). In this work, we pursued the functional characterization of the LEE-encoded protein EscK (previously known as Orf4). We provide evidence indicating that EscK is crucial for efficient T3S and belongs to the SctK (OrgA/YscK/MxiK) protein family, whose members have been implicated in the formation of a sorting platform for secretion of T3S substrates. Bacterial fractionation studies showed that EscK localizes to the inner membrane independently of the presence of any other T3SS component. Combining yeast two-hybrid screening and pulldown assays, we identified an interaction between EscK and the C-ring/sorting platform component EscQ. Site-directed mutagenesis of conserved residues revealed amino acids that are critical for EscK function and for its interaction with EscQ. In addition, we found that T3S substrate overproduction is capable of compensating for the absence of EscK. Overall, our data suggest that EscK is a structural component of the EPEC T3SS sorting platform, playing a central role in the recruitment of T3S substrates for boosting the efficiency of the protein translocation process. IMPORTANCE: The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

The Impact of 18 Ancestral and Horizontally-Acquired Regulatory Proteins upon the Transcriptome and sRNA Landscape of Salmonella enterica serovar Typhimurium.

We know a great deal about the genes used by the model pathogen Salmonella enterica serovar Typhimurium to cause disease, but less about global gene regulation. New tools for studying transcripts at the single nucleotide level now offer an unparalleled opportunity to understand the bacterial transcriptome, and expression of the small RNAs (sRNA) and coding genes responsible for the establishment of infection. Here, we define the transcriptomes of 18 mutants lacking virulence-related global regulatory systems that modulate the expression of the SPI1 and SPI2 Type 3 secretion systems of S. Typhimurium strain 4/74. Using infection-relevant growth conditions, we identified a total of 1257 coding genes that are controlled by one or more regulatory system, including a sub-class of genes that reflect a new level of cross-talk between SPI1 and SPI2. We directly compared the roles played by the major transcriptional regulators in the expression of sRNAs, and discovered that the RpoS (σ38) sigma factor modulates the expression of 23% of sRNAs, many more than other regulatory systems. The impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts is described, and we found 13 sRNAs that are co-regulated with SPI1 and SPI2 virulence genes. We report the first example of an sRNA, STnc1480, that is subject to silencing by H-NS and subsequent counter-silencing by PhoP and SlyA. The data for these 18 regulatory systems is now available to the bacterial research community in a user-friendly online resource, SalComRegulon.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Strain YU15 (Sequence Type 19) Harboring the Salmonella Genomic Island 1 and Virulence Plasmid pSTV.

The complete genome ofSalmonella entericasubsp.entericaserovar Typhimurium sequence type 19 (ST19) strain YU15, isolated in Yucatán, Mexico, from a human baby stool culture, was determined using PacBio technology. The chromosome contains five intact prophages and theSalmonellagenomic island 1 (SGI1). This strain carries theSalmonellavirulence plasmid pSTV.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Strain SO2 (Sequence Type 302) Isolated from an Asymptomatic Child in Mexico.

The complete genome sequence ofSalmonella entericaserovar Typhimurium strain SO2, isolated from an asymptomatic child in Mexico, was determined using PacBio single-molecule real-time technology. Strain SO2 has six complete chromosomal prophages, namely, ST104, Gifsy-2, ST64B, Gifsy-1, ELPhiS, and FSL SP-004, and carries aSalmonellavirulence plasmid.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Strain SO3 (Sequence Type 302) Isolated from a Baby with Meningitis in Mexico.

The complete genome of ITALIC! Salmonella entericaserovar Typhimurium strain SO3 (sequence type 302), isolated from a fatal meningitis infection in Mexico, was determined using PacBio technology. The chromosome hosts six complete prophages and is predicted to harbor 51 genomic islands, including 13 pathogenicity islands (SPIs). It carries the ITALIC! Salmonellavirulence plasmid (pSTV).

Complete Genome Sequence of a Human-Invasive Salmonella enterica Serovar Typhimurium Strain of the Emerging Sequence Type 213 Harboring a Multidrug Resistance IncA/C Plasmid and a blaCMY-2-Carrying IncF Plasmid.

Salmonella enterica subsp. enterica serovar Typhimurium strain 33676 was isolated in Mexico City, Mexico, from a patient with a systemic infection, and its complete genome sequence was determined using PacBio single-molecule real-time technology. Strain 33676 harbors an IncF plasmid carrying the extended-spectrum cephalosporin gene blaCMY-2 and a multidrug resistance IncA/C plasmid.

Humoral Immunity in the Gut Selectively Targets Phenotypically Virulent Attaching-and-Effacing Bacteria for Intraluminal Elimination.

Virulence factors expressed by enteric bacteria are pivotal for pathogen colonization and induction of intestinal disease, but the mechanisms by which host immunity regulates pathogen virulence are largely unknown. Here we show that specific antibody responses are required for downregulation of virulence gene expression in Citrobacter rodentium, an enteric pathogen that models human infections with attaching-and-effacing bacteria. In the absence of antibodies against the pathogen, phenotypically virulent C. rodentium, accumulated and infected the epithelium and subsequently invaded the lamina propia, causing host lethality. IgG induced after infection recognized virulence factors and bound virulent bacteria within the intestinal lumen, leading to their engulfment by neutrophils, while phenotypically avirulent pathogens remained in the intestinal lumen and were eventually outcompeted by the microbiota. Thus, the interplay of the innate and adaptive immune system selectively targets virulent C. rodentium in the intestinal lumen to promote pathogen eradication and host survival.

Identification and regulation of a novel Citrobacter rodentium gut colonization fimbria (Gcf).

UNLABELLED: The Gram-negative enteric bacterium Citrobacter rodentium is a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. For C. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novel C. rodentium colonization factor, called gut colonization fimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. A gcfA mutant shows a severe colonization defect within the first 10 days of infection. The gcf promoter is not active in C. rodentium under several in vitro growth conditions; however, it is readily expressed in a C. rodentium Δhns1 mutant lacking the closest ortholog of the Escherichia coli histone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region of gcf, further supporting its direct role as a repressor of the gcf promoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. The gcf operon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures. IMPORTANCE: Fimbriae are surface bacterial structures implicated in a variety of biological processes. Some have been shown to play a critical role during host colonization and thus in disease. Pathogenic bacteria possess the genetic information for an assortment of fimbriae, but their function and regulation and the interplay between them have not been studied in detail. This work provides new insights into the function and regulation of a novel fimbria called Gcf that is important for early establishment of a successful infection by C. rodentium in mice, despite being poorly expressed under in vitro growth conditions. This discovery offers an opportunity to better understand the individual role and the regulatory mechanisms controlling the expression of specific fimbrial operons that are critical during infection.