Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile.

Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA-seq and differential 5'-end RNA-seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

Identification of commensal Escherichia coli genes involved in biofilm resistance to pathogen colonization.

Protection provided by host bacterial microbiota against microbial pathogens is a well known but ill-understood property referred to as the barrier effect, or colonization resistance. Despite recent genome-wide analyses of host microbiota and increasing therapeutic interest, molecular analysis of colonization resistance is hampered by the complexity of direct in vivo experiments. Here we developed an in vitro-to-in vivo approach to identification of genes involved in resistance of commensal bacteria to exogenous pathogens. We analyzed genetic responses induced in commensal Escherichia coli upon entry of a diarrheagenic enteroaggregative E. coli or an unrelated Klebsiella pneumoniae pathogen into a biofilm community. We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm. We tested the in vivo relevance of our findings by comparing the extent of intestinal colonization by enteroaggregative E. coli and K. pneumoniae pathogens in mice pre-colonized with E. coli wild type commensal strain, or mutants corresponding to identified colonization resistance genes. We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut. This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.

The AfaR small RNA controls expression of the AfaD-VIII invasin in pathogenic Escherichia coli strains.

Pathogenic Escherichia coli strains carrying the afa-8 gene cluster are frequently associated with extra-intestinal infections in humans and animals. The afa-8 A to E genes determine the formation of an afimbrial adhesive sheath consisting of the AfaD-VIII invasin and the AfaE-VIII adhesin at the bacterial cell surface. This structure is thought to be required for host colonization. We characterized a new gene encoding the small RNA AfaR, which is transcribed in cis from the complementary strand of the 3' untranslated region of the afaD messenger RNA, within the afaD-afaE intercistronic region. AfaR is a trans-acting Hfq-dependent antisense small RNA that binds the 5' untranslated region of the afaD messenger RNA, initiating several ribonuclease E-dependent cleavages, thereby downregulating production of the AfaD-VIII invasin. AfaR transcription is dependent on σ(E), a member of the stress response family of extracytoplasmic alternative sigma factors. We found that the AfaR-dependent regulatory pathway was controlled by temperature, allowing the production of the AfaD-VIII invasin at temperatures above 37 °C. Our findings suggest that the entry of afa-8-positive pathogenic E. coli strains into epithelial cells is tightly regulated by the AfaR small RNA.

Virulent bacteriophages can target O104:H4 enteroaggregative Escherichia coli in the mouse intestine.

In vivo bacteriophage targeting of enteroaggregative Escherichia coli (EAEC) was assessed using a mouse intestinal model of colonization with the O104:H4 55989Str strain and a cocktail of three virulent bacteriophages. The colonization model was shown to mimic asymptomatic intestinal carriage found in humans. The addition of the cocktail to drinking water for 24 h strongly decreased ileal and weakly decreased fecal 55989Str concentrations in a dose-dependent manner. These decreases in ileal and fecal bacterial concentrations were only transient, since 55989Str concentrations returned to their original levels 3 days later. These transient decreases were independent of the mouse microbiota, as similar results were obtained with axenic mice. We studied the infectivity of each bacteriophage in the ileal and fecal environments and found that 55989Str bacteria in the mouse ileum were permissive to all three bacteriophages, whereas those in the feces were permissive to only one bacteriophage. Our results provide the first demonstration that bacterial permissivity to infection with virulent bacteriophages is not uniform throughout the gut; this highlights the need for a detailed characterization of the interactions between bacteria and bacteriophages in vivo for the further development of phage therapy targeting intestinal pathogens found in the gut of asymptomatic human carriers.

Role of the vpe carbohydrate permease in Escherichia coli urovirulence and fitness in vivo.

Uropathogenic Escherichia coli (UPEC) strains are a leading cause of infections in humans, but the mechanisms governing host colonization by this bacterium remain poorly understood. Previous studies have identified numerous gene clusters encoding proteins involved in sugar transport, in pathogen-specific islands. We investigated the role in fitness and virulence of the vpe operon encoding an EII complex of the phosphotransferase (PTS) system, which is found more frequently in human strains from infected urine and blood (45%) than in E. coli isolated from healthy humans (15%). We studied the role of this locus in vivo, using the UPEC E. coli strain AL511, mutants, and complemented derivatives in two experimental mouse models of infection. Mutant strains displayed attenuated virulence in a mouse model of sepsis. A role in kidney colonization was also demonstrated by coinfection experiments in a mouse model of pyelonephritis. Electron microscopy examinations showed that the vpeBC mutant produced much smaller amounts of a capsule-like surface material than the wild type, particularly when growing in human urine. Complementation of the vpeBC mutation led to an increase in the amount of exopolysaccharide, resistance to serum killing, and virulence. It was therefore clear that the loss of vpe genes was responsible for all the observed phenotypes. We also demonstrated the involvement of the vpe locus in gut colonization in the streptomycin-treated mouse model of intestinal colonization. These findings confirm that carbohydrate transport and metabolism underlie the ability of UPEC strains to colonize the host intestine and to infect various host sites.

Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice.

Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem.

An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains.

Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli.

Maternal/fetal mortality and fetal growth restriction: role of nitric oxide and virulence factors in intrauterine infection in rats.

OBJECTIVE: The mechanism of infection-related deaths of pregnant rats and intrauterine growth restriction are not understood. We assessed whether nitric oxide (NO) has differential effects on infection with Escherichia coli Dr/Afa mutants that lack either AfaE or AfaD invasins. STUDY DESIGN: Sprague-Dawley rats were infected intrauterinally with the clinical strain of E coli AfaE(+)D(+) or 1 of its isogenic mutants in the presence or absence of the NO synthesis inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Maternal/fetal mortality rates, fetoplacental weight, and infection rates were evaluated. RESULTS: Maternal and/or fetal death was associated with the presence of at least 1 virulence factor (AfaE(+)D(+)>AfaE(+)D(-)>AfaE(-)D(+)) and was increased by L-NAME treatment. The fetal and placental weights were lower than controls and were further reduced by L-NAME treatment. CONCLUSION: These results demonstrate that NO enhanced AfaE- and AfaD-mediated virulence and plays an important role in Dr/Afa(+)E coli gestational tropism.

Sugar metabolism, an additional virulence factor in enterobacteria.

Enterobacteria display a high level of flexibility in their fermentative metabolism. Biotyping assays have thus been developed to discriminate between clinical isolates. Each biotype uses one or more sugars more efficiently than the others. Recent studies show links between sugar metabolism and virulence in enterobacteria. In particular, mechanisms of carbohydrate utilization differ substantially between pathogenic and commensal E. coli strains. We are now starting to gain insight into the importance of this variability in metabolic function. Studies using various animal models of intestinal colonization showed that the presence of the fos and deoK loci involved in the metabolism of short-chain fructoligosaccharides and deoxyribose, respectively, help avian and human pathogenic E. coli to outcompete with the normal flora and colonize the intestine. Both PTS and non-PTS sugar transporters have been found to modulate virulence of extraintestinal pathogenic E. coli strains. The vpe, GimA, and aec35-37 loci contribute to bacterial virulence in vivo during experimental septicemia and urinary tract infection, meningitis, and colibacillosis, respectively. However, in most cases, the sugars metabolized, and the precise role of their utilization in the expression of bacterial virulence is still unknown. The massive development of powerful analytical methods over recent years will allow establishing the knowledge of the metabolic basis of bacterial pathogenesis that appears to be the next challenge in the field of infectious diseases.

Repression of invasion genes and decreased invasion in a high-level fluoroquinolone-resistant Salmonella typhimurium mutant.

BACKGROUND: Nalidixic acid resistance among Salmonella Typhimurium clinical isolates has steadily increased, whereas the level of ciprofloxacin resistance remains low. The main objective of this study was to characterize the fluoroquinolone resistance mechanisms acquired in a S. Typhimurium mutant selected with ciprofloxacin from a susceptible isolate and to investigate its invasion ability. METHODOLOGY/PRINCIPAL FINDINGS: Three different amino acid substitutions were detected in the quinolone target proteins of the resistant mutant (MIC of ciprofloxacin, 64 microg/ml): D87G and G81C in GyrA, and a novel mutation, E470K, in ParE. A protein analysis revealed an increased expression of AcrAB/TolC and decreased expression of OmpC. Sequencing of the marRAB, soxRS, ramR and acrR operons did not show any mutation and neither did their expression levels in a microarray analysis. A decreased percentage of invasion ability was detected when compared with the susceptible clinical isolate in a gentamicin protection assay. The microarray results revealed a decreased expression of genes which play a role during the invasion process, such as hilA, invF and the flhDC operon. Of note was the impaired growth detected in the resistant strain. A strain with a reverted phenotype (mainly concerning the resistance phenotype) was obtained from the resistant mutant. CONCLUSIONS/SIGNIFICANCE: In conclusion, a possible link between fluoroquinolone resistance and decreased cell invasion ability may exist explaining the low prevalence of fluoroquinolone-resistant S. Typhimurium clinical isolates. The impaired growth may appear as a consequence of fluoroquinolone resistance acquisition and down-regulate the expression of the invasion genes.

The flagella of an atypical enteropathogenic Escherichia coli strain are required for efficient interaction with and stimulation of interleukin-8 production by enterocytes in vitro.

The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.

Uropathogenic Escherichia coli AL511 requires flagellum to enter renal collecting duct cells.

Escherichia coli is the leading cause of urinary tract infections, but the mechanisms governing renal colonization by this bacterium remain poorly understood. We investigated the ability of 13 E. coli strains isolated from the urine of patients with pyelonephritis and cystitis and normal stools to invade collecting duct cells, which constitute the first epithelium encountered by bacteria ascending from the bladder. The AL511 clinical isolate adhered to mouse collecting duct mpkCCD(cl4) cells, used as a model of renal cell invasion, and was able to enter and persist within these cells. Previous studies have shown that bacterial flagella play an important role in host urinary tract colonization, but the role of flagella in the interaction of E. coli with renal epithelial cells remains unclear. An analysis of the ability of E. coli AL511 mutants to invade renal cells showed that flagellin played a key role in bacterial entry. Both flagellum filament assembly and the motor proteins MotA and MotB appeared to be required for E. coli AL511 uptake into collecting duct cells. These findings indicate that pyelonephritis-associated E. coli strains may invade renal collecting duct cells and that flagellin may act as an invasin in this process.

Role of deoxyribose catabolism in colonization of the murine intestine by pathogenic Escherichia coli strains.

We previously suggested that the ability to metabolize deoxyribose, a phenotype encoded by the deoK operon, is associated with the pathogenic potential of Escherichia coli strains. Carbohydrate metabolism is thought to provide the nutritional support required for E. coli to colonize the intestine. We therefore investigated the role of deoxyribose catabolism in the colonization of the gut, which acts as a reservoir, by pathogenic E. coli strains. Molecular and biochemical characterization of 1,221 E. coli clones from various collections showed this biochemical trait to be common in the E. coli species (33.6%). However, multivariate analysis evidenced a higher prevalence of sugar-metabolizing E. coli clones in the stools of patients from countries in which intestinal diseases are endemic. Diarrhea processes frequently involve the destruction of intestinal epithelia, so it is plausible that such clones may be positively selected for in intestines containing abundant DNA, and consequently deoxyribose. Statistical analysis also indicated that symptomatic clinical disorders and the presence of virulence factors specific to extraintestinal pathogenic E. coli were significantly associated with an increased risk of biological samples and clones testing positive for deoxyribose. Using the streptomycin-treated-mouse model of intestinal colonization, we demonstrated the involvement of the deoK operon in gut colonization by two pathogenic isolates (one enteroaggregative and one uropathogenic strain). These results, indicating that deoxyribose availability promotes pathogenic E. coli growth during host colonization, suggest that the acquisition of this trait may be an evolutionary step enabling these pathogens to colonize and persist in the mammalian intestine.

Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths.

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the approximately 18,000 families of orthologous genes, we found approximately 2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome.

Renal collecting duct epithelial cells react to pyelonephritis-associated Escherichia coli by activating distinct TLR4-dependent and -independent inflammatory pathways.

TLR4 plays a central role in resistance to pyelonephritis caused by uropathogenic Escherichia coli (UPEC). It has been suggested that renal tubule epithelial cells expressing TLRs may play a key role in inflammatory disorders and in initiating host defenses. In this study we used an experimental mouse model of ascending urinary tract infection to show that UPEC isolates preferentially adhered to the apical surface of medullary collecting duct (MCD) intercalated cells. UPEC-infected C3H/HeJ (Lps(d)) mice carrying an inactivating mutation of tlr4 failed to clear renal bacteria and exhibited a dramatic slump in proinflammatory mediators as compared with infected wild-type C3H/HeOuJ (Lps(n)) mice. However, the level of expression of the leukocyte chemoattractants MIP-2 and TNF-alpha still remained greater in UPEC-infected than in naive C3H/HeJ (Lps(d)) mice. Using primary cultures of microdissected Lps(n) MCDs that expressed TLR4 and its accessory molecules MD2, MyD88, and CD14, we also show that UPECs stimulated both a TLR4-mediated, MyD88-dependent, TIR domain-containing adaptor-inducing IFN-beta-independent pathway and a TLR4-independent pathway, leading to bipolarized secretion of MIP-2. Stimulation by UPECs of the TLR4-mediated pathway in Lps(n) MCDs leads to the activation of NF-kappaB, and MAPK p38, ERK1/2, and JNK. In addition, UPECs stimulated TLR4-independent signaling by activating a TNF receptor-associated factor 2-apoptosis signal-regulatory kinase 1-JNK pathway. These findings demonstrate that epithelial collecting duct cells are actively involved in the initiation of an immune response via several distinct signaling pathways and suggest that intercalated cells play an active role in the recognition of UPECs colonizing the kidneys.

The solution structure of the invasive tip complex from Afa/Dr fibrils.

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.

Diffusely adherent Escherichia coli strains expressing Afa/Dr adhesins (Afa/Dr DAEC): hitherto unrecognized pathogens.

Diffusely adherent Escherichia coli (DAEC) strains are currently considered to constitute a putative sixth group of diarrheagenic E. coli. However, on the basis of their diffuse adherence to HEp-2 and HeLa cells, the detection of afa/dra/daa-related operons encoding this adherence phenotype, and the mobilization of decay-accelerating factor, both commensal and pathogenic strains can be classified as Afa/Dr DAEC isolates. Furthermore, strains associated with diarrheal diseases and strains causing extra-intestinal infections can also be identified as Afa/Dr DAEC strains. Although several cell signaling events that occur after epithelial cells have been infected by Afa/Dr DAEC have been reported, the pathophysiological processes that allow intestinal and extra-intestinal infections to develop are not fully understood. This review focuses on the genetic organization of the afa/dra/daa-related operons and on the virulence factors that trigger cellular responses, some of which are deleterious for the host cells. Finally, this review suggests future lines of research that could help to elucidate these questions.

Adhesins and invasins of pathogenic Escherichia coli.

Pathogenic E. coli cause both intestinal and extra-intestinal infections in humans and animals. Bacteria must be able to adhere to host cells if they are to colonize and to invade their hosts. Numerous E. coli adhesins with different morphological features and receptor specificities have been identified. Many bacteria produce several adhesins with different receptor specificities. Although not all adhesin receptors have been identified yet, it appears that adhesins generally behave as lectins, recognizing oligosaccharide residues of glycoproteins or glycolipids. This review summarizes recent advances concerning host tissue colonization properties, providing new insights into adhesive organelle biogenesis in pathogenic E. coli and into the development of reservoirs of pathogenic bacteria in the host. To limit the length of this review, I will use examples of structural characteristics and invasive properties of a few bacterial adherence factors: type 1 pili, Afa adhesive sheath and some outer membrane adhesins.