Courtney D. Petro

To discern if there was a particular genotype associated with clinical enteroaggregative Escherichia coli (EAEC) strains isolated from deployed military personnel (DMP) with travelers' diarrhea (TD), we characterized a collection of EAEC from DMP deployed to Afghanistan, Djibouti, Kenya, or Honduras. Although we did not identify a specific EAEC genotype associated with TD in DMP, we found that EAEC isolated at the first clinic visit were more likely to encode the dispersin gene aap than EAEC collected at follow-up visits. A majority of the EAEC isolates were typical EAEC that adhered to HEp-2 cells, formed biofilms, and harbored genes for aggregative adherence fimbriae (AAF), AggR, and serine protease autotransporters of Enterobacteriaceae (SPATEs). A separate subset of the EAEC had aggR and genes for SPATEs but encoded a gene highly homologous to that for CS22, a fimbriae more commonly found in enterotoxigenic E. coli. None of these CS22-encoding EAEC formed biofilms in vitro or adhered to HEp-2 cells. Whole genome sequence and single nucleotide polymorphism analyses demonstrated that most of the strains were genetically diverse, but that a few were closely related. Isolation of these related strains occurred within days to more than a year apart, a finding that suggests a persistent source and genomic stability. In an ampicillin-treated mouse model we found that an agg4A+ aar- isolate formed a biofilm in the intestine and caused reduced weight gain in mice, whereas a strain that did not form an in vivo biofilm caused no morbidity. Our diverse strain collection from DMP displays the heterogeneity of EAEC strains isolated from human patients, and our mouse model of infection indicated the genotype agg4A+ aar– and/or capacity to form biofilm in vivo may correlate to disease severity.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes foodborne outbreaks of bloody diarrhea. There are two major types of immunologically distinct Stxs: Stx1a and Stx2a. Stx1a is more cytotoxic to Vero cells than Stx2a, but Stx2a has a lower 50% lethal dose (LD₅₀) in mice. Epidemiological data suggest that infections by STEC strains that produce only Stx2a progress more often to a life-threatening sequela of infection called hemolytic-uremic syndrome (HUS) than isolates that make Stx1a only or produce both Stx1a and Stx2a. In this study, we found that an E. coli O26:H11 strain that produces both Stx1a and Stx2a was virulent in streptomycin- and ciprofloxacin-treated mice and that mice were protected by administration of an anti-Stx2 antibody. However, we discovered that in the absence of ciprofloxacin, neutralization of Stx1a enhanced the virulence of the strain, a result that corroborated our previous finding that Stx1a reduces the toxicity of Stx2a by the oral route. We further found that intraperitoneal administration of the purified Stx1a B subunit delayed the mean time to death of mice intoxicated with Stx2a and reduced the cytotoxic effect of Stx2a on Vero cells. Taken together, our data suggest that Stx1a reduces both the pathogenicity of Stx2 in vivo and cytotoxicity in vitro.

Type IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenicEscherichia coli(EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, fromPseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer.IMPORTANCESecretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins.