The Pic Protease of Enteroaggregative \u3cem\u3eEscherichia coli\u3c/em\u3e Promotes Intestinal Colonization and Growth in the Presence of Mucin

Enteroaggregative Escherichia coli (EAEC) is increasingly being recognized as a cause of diarrheal disease in diverse populations. No small animal model is currently available to study this pathogen. We report here that conventional mice orally inoculated with prototype EAEC strain 042 generally became colonized, though the abundance of organisms cultured from their stool varied substantially among individual animals. In contrast, mice whose water contained 5 g/liter streptomycin consistently became colonized at high levels (ca. 108 CFU/g of stool). Neither conventional nor streptomycin-treated mice developed clinical signs or histopathologic abnormalities. Using specific mutants in competition with the wild-type strain, we evaluated the contribution of several putative EAEC virulence factors to colonization of streptomycin-treated mice. Our data suggest that the dispersin surface protein and Pic, a serine protease autotransporter secreted by EAEC and Shigella flexneri, promote colonization of the mouse. In contrast, we found no role for the aggregative adherence fimbriae, the transcriptional activator AggR, or the surface factor termed Air (enteroaggregative immunoglobulin repeat protein). To study Pic further, we constructed a single nucleotide mutation in strain 042 which altered only the Pic catalytic serine (strain 042PicS258A). Fractionation of the tissue at 24 h and 3 days demonstrated an approximate 3-log10 difference between 042 and 042PicS258A in the lumen and mucus layer and adherent to tissue. Strains 042 and 042PicS258A adhered similarly to mouse tissue ex vivo. While no growth differences were observed in a continuous-flow anaerobic intestinal simulator system, the wild-type strain exhibited a growth advantage over 042PicS258A in a culture of cecal mucus and in cecal contents in vitro; this difference was manifest only after 6 h of growth. Moreover, enhanced growth of the wild type was observed in comparison with that of the mutant in minimal medium containing mucin but not in the absence of mucin. The data suggest a novel metabolic role for the Pic mucinase in EAEC colonization

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Background \ud Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. \ud \ud Methods \ud In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. \ud \ud Conclusion \ud This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies

Microbiota that affect risk for shigellosis in children in low-income countries

Pathogens in the gastrointestinal tract exist within a vast population of microbes. We examined associations between pathogens and composition of gut microbiota as they relate to Shigella spp./enteroinvasive Escherichia coli infection. We analyzed 3,035 stool specimens (1,735 nondiarrheal and 1,300 moderate-to-severe diarrheal) from the Global Enteric Multicenter Study for 9 enteropathogens. Diarrheal specimens had a higher number of enteropathogens (diarrheal mean 1.4, nondiarrheal mean 0.95; p<0.0001). Rotavirus showed a negative association with Shigella spp. in cases of diarrhea (odds ratio 0.31, 95% CI 0.17–0.55) and had a large combined effect on moderate-to-severe diarrhea (odds ratio 29, 95% CI 3.8–220). In 4 Lactobacillus taxa identified by 16S rRNA gene sequencing, the association between pathogen and disease was decreased, which is consistent with the possibility that Lactobacillus spp. are protective against Shigella spp.–induced diarrhea. Bacterial diversity of gut microbiota was associated with diarrhea status, not high levels of the Shigella spp. ipaH gene.publishedVersio

The Pic Protease of Enteroaggregative Escherichia coli Promotes Intestinal Colonization and Growth in the Presence of Mucin▿

Enteroaggregative Escherichia coli (EAEC) is increasingly being recognized as a cause of diarrheal disease in diverse populations. No small animal model is currently available to study this pathogen. We report here that conventional mice orally inoculated with prototype EAEC strain 042 generally became colonized, though the abundance of organisms cultured from their stool varied substantially among individual animals. In contrast, mice whose water contained 5 g/liter streptomycin consistently became colonized at high levels (ca. 108 CFU/g of stool). Neither conventional nor streptomycin-treated mice developed clinical signs or histopathologic abnormalities. Using specific mutants in competition with the wild-type strain, we evaluated the contribution of several putative EAEC virulence factors to colonization of streptomycin-treated mice. Our data suggest that the dispersin surface protein and Pic, a serine protease autotransporter secreted by EAEC and Shigella flexneri, promote colonization of the mouse. In contrast, we found no role for the aggregative adherence fimbriae, the transcriptional activator AggR, or the surface factor termed Air (enteroaggregative immunoglobulin repeat protein). To study Pic further, we constructed a single nucleotide mutation in strain 042 which altered only the Pic catalytic serine (strain 042PicS258A). Fractionation of the tissue at 24 h and 3 days demonstrated an approximate 3-log10 difference between 042 and 042PicS258A in the lumen and mucus layer and adherent to tissue. Strains 042 and 042PicS258A adhered similarly to mouse tissue ex vivo. While no growth differences were observed in a continuous-flow anaerobic intestinal simulator system, the wild-type strain exhibited a growth advantage over 042PicS258A in a culture of cecal mucus and in cecal contents in vitro; this difference was manifest only after 6 h of growth. Moreover, enhanced growth of the wild type was observed in comparison with that of the mutant in minimal medium containing mucin but not in the absence of mucin. The data suggest a novel metabolic role for the Pic mucinase in EAEC colonization

Nitazoxanide Inhibits Biofilm Production and Hemagglutination by Enteroaggregative Escherichia coli Strains by Blocking Assembly of AafA Fimbriae▿

Enteroaggregative Escherichia coli (EAEC) strains have emerged as common causes of persistent diarrhea and malnutrition among children and HIV-infected persons. During infection, EAEC typically adheres to the intestinal mucosa via fimbrial adhesins, which results in a characteristic aggregative pattern. In the study described here we investigated whether the broad-spectrum antiparasitic and antidiarrheal drug nitazoxanide (NTZ) might be active against EAEC in vitro. While E. coli strains were resistant to NTZ in rich Luria-Bertani medium (MIC > 64 μg/ml), the drug was slightly inhibitory in a minimal medium supplemented with glucose (MinA-G medium; MIC, ∼32 μg/ml). NTZ also inhibited biofilm production by strain EAEC 042 in both Dulbecco's modified Eagle's medium and MinA-G medium with a 50% inhibitory concentration of ∼12 μg/ml. Immunofluorescence and immunoblot analyses with antibody against the major fimbrial subunit AafA of aggregative adherence fimbriae vaariant II (AAF/II) established that the numbers of AAF/II filaments on bacteria grown in the presence of NTZ were dramatically reduced. Comparative quantitative reverse transcription-PCR and reporter gene fusions (aafA::phoA) indicated that aafA expression was unaffected by NTZ, while aggR transcript levels and aggR::lacZ expression were increased ∼10- and 2.5-fold, respectively, compared with that for the untreated controls. More generally, NTZ inhibited hemagglutination (HA) of red blood cells by the non-biofilm-producing strain JM221 expressing either AAF/I or type I fimbriae. Our findings suggest that the inhibitory action of NTZ on biofilm formation and HA is likely due to inhibition of fimbrial assembly. Antimicrobial agents that inhibit the assembly or function of fimbrial filaments should be good candidates for the prevention of infection

Experimental infection of healthy volunteers with enterotoxigenic Escherichia coli wild-type strain TW10598 in a hospital ward

Background Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea in resource-limited regions. It is also an important cause of diarrhea in travellers to these areas. To evaluate the protective efficacy of new ETEC vaccines that are under development, there is a need to increase the capacity to undertake Phase IIB (human challenge) clinical trials and to develop suitable challenge models. Methods An in-hospital study was performed where fasting adult volunteers were experimentally infected with 1 × 106 to 1 × 109 colony forming units (CFUs) of the wild-type ETEC strain TW10598, which had been isolated from a child with diarrhea in West Africa in 1997. We recorded symptoms and physical signs and measured serum immune response to the TW10598 bacterium. Results We included 30 volunteers with mean age 22.8 (range 19.8, 27.4) years. The most common symptoms were diarrhea (77%), abdominal pain (67%), nausea (63%), and abdominal cramping (53%). Seven subjects (23%) experienced fever, none were hypotensive. Most of the volunteers responded with a substantial rise in the level of serum IgA antibodies against the challenge strain. Conclusions We established the capacity and methods for safely undertaking challenge studies to measure the efficacy of ETEC vaccine candidates in a hospital ward. Strain TW10598 elicited both clinical symptoms and an immune response across the doses given

The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization.

Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells