Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance.

Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Δeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.

Repeated Oral Vaccination of Cattle with Shiga Toxin-Negative Escherichia coli O157:H7 Reduces Carriage of Wild-Type E. coli O157:H7 after Challenge.

Subcutaneous vaccination of cattle for enterohemorrhagic Escherichia coli O157:H7 reduces the magnitude and duration of fecal shedding, but the often-required, repeated cattle restraint can increase costs, deterring adoption by producers. In contrast, live oral vaccines may be repeatedly administered in feed, without animal restraint. We investigated whether oral immunization with live stx-negative LEE+E. coli O157:H7 reduced rectoanal junction (RAJ) colonization by wild-type (WT) E. coli O157:H7 strains after challenge. Two groups of cattle were orally dosed twice weekly for 6 weeks with 3 × 109 CFU of a pool of three stx-negative LEE+E. coli O157:H7 strains (vaccine group) or three stx-negative LEE- non-O157:H7 E. coli strains (control group). Three weeks following the final oral dose, animals in both groups were orally challenged with a cocktail of four stx+ LEE+E. coli O157:H7 WT strains. Subsequently, WT strains at the RAJ were enumerated weekly for 4 weeks. Serum antibodies against type III secretion protein (TTSP), the translocated intimin receptor (Tir), and EspA were determined by enzyme-linked immunosorbent assay (ELISA) at day 0 (preimmunization), day 61 (postimmunization, prechallenge), and day 89 (postchallenge). Vaccine group cattle had lower numbers of WT strains at the RAJ than control group cattle on postchallenge days 3 and 7 (P ≤ 0.05). Also, vaccine group cattle shed WT strains for a shorter duration than control group cattle. All cattle seroconverted to TTSP, Tir, and EspA, either following immunization (vaccine group) or following challenge (control group). Increased antibody titers against Tir and TTSP postimmunization were associated with decreased numbers of WT E. coli O157:H7 organisms at the RAJ.IMPORTANCE The bacterium E. coli O157:H7 causes foodborne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A detoxified E. coli O157:H7 strain, missing genes that cause disease, was fed to cattle as an oral vaccine to reduce carriage of pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first 7 days postchallenge and shed the bacteria for a shorter duration than the nonvaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.

Escherichia coli O157:H7 Curli Fimbriae Promotes Biofilm Formation, Epithelial Cell Invasion, and Persistence in Cattle.

Escherichia coli O157:H7 (O157) is noninvasive and a weak biofilm producer; however, a subset of O157 are exceptions. O157 ATCC 43895 forms biofilms and invades epithelial cells. Tn5 mutagenesis identified a mutation responsible for both phenotypes. The insertion mapped within the curli csgB fimbriae locus. Screening of O157 strains for biofilm formation and cell invasion identified a bovine and a clinical isolate with those characteristics. A single base pair A to T transversion, intergenic to the curli divergent operons csgDEFG and csgBAC, was present only in biofilm-producing and invasive strains. Using site-directed mutagenesis, this single base change was introduced into two curli-negative/noninvasive O157 strains and modified strains to form biofilms, produce curli, and gain invasive capability. Transmission electron microscopy (EM) and immuno-EM confirmed curli fibers. EM of bovine epithelial cells (MAC-T) co-cultured with curli-expressing O157 showed intracellular bacteria. The role of curli in O157 persistence in cattle was examined by challenging cattle with curli-positive and -negative O157 and comparing carriage. The duration of bovine colonization with the O157 curli-negative mutant was shorter than its curli-positive isogenic parent. These findings definitively demonstrate that a single base pair stably confers biofilm formation, epithelial cell invasion, and persistence in cattle.

Complete Genome Sequence of Broad-Host-Range Shiga Toxin-Converting Bacteriophage SH2026Stx1, Isolated from Escherichia coli O157:H7.

The Shiga toxin-encoding phage SH2026Stx1 was isolated from Escherichia coli O157:H7 strain 2026. SH2026Stx1 and its detoxified derivative can infect a broad range of E. coli strains, including commensal, enteropathogenic, and enteroaggregative strains. We report here the complete genome sequence of phage SH2026Stx1 and its important features.

High-Quality Complete Genome Sequences of Three Bovine Shiga Toxin-Producing Escherichia coli O177:H- (fliCH25) Isolates Harboring Virulent stx2 and Multiple Plasmids.

Shiga toxin-producing Escherichia coli (STEC) bacteria are zoonotic pathogens. We report here the high-quality complete genome sequences of three STEC O177:H- (fliCH25) strains, SMN152SH1, SMN013SH2, and SMN197SH3. The assembled genomes consisted of one optical map-verified circular chromosome for each strain, plus two plasmids for SMN013SH2 and three plasmids for SMN152SH1 and SMN197SH3, respectively.

Escherichia coli O157:H7 suppresses host autophagy and promotes epithelial adhesion via Tir-mediated and cAMP-independent activation of protein kinase A.

Autophagy is a pivotal innate immune response that not only degrades cytosolic components, but also serves as one of the critical antimicrobial mechanisms eliminating intracellular pathogens. However, its role in host defense against extracellular pathogens is largely unknown. Here we showed that E. coli O157:H7 altered autophagy to evade host defense and facilitate adhesion. Enhancing host cell autophagy with tumor necrosis factor (TNF), host starvation or rapamycin reduced the adherence of E. coli O157:H7 to HT-29 cells. As a key regulator of autophagy, protein kinase A (PKA) was activated by E. coli O157:H7 infection. PKA inhibition by H89 abrogated E. coli O157:H7 inhibition of autophagy and prevented bacterial epithelial adhesion. Thus, PKA had a mediatory role in blocking autophagy and E. coli O157:H7 epithelial adhesion. Furthermore, deletion of translocated intimin receptor (tir) prevented PKA activation, whereas ectopic tir expression in a Δtir mutant strain restored its ability to activate PKA and inhibited autophagy in host cells. This indicated that Tir and PKA played pivotal roles in manipulating host autophagy during infection. Consistent with autophagy inhibition, E. coli O157:H7 infection inhibited endoplasmic reticulum (ER) stress in HT-29 cells, which was reversed by TNF, starvation, or H89 treatment. Additionally, E. coli O157:H7-induced PKA activation suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) activation and enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling, thereby repressing autophagic signaling. Conversely, PKA inhibition prevented downregulation of ERK1/2 signaling due to E. coli O157:H7 infection. In summary, E. coli O157:H7 inhibited host autophagy via Tir-mediated PKA activation that favored bacterial persistence on intestinal epithelial cell surfaces.

Standardized Escherichia coli O157:H7 Exposure Studies in Cattle Provide Evidence that Bovine Factors Do Not Drive Increased Summertime Colonization.

The increased summertime prevalence of cattle carriage of enterohemorrhagic Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) is associated with the increased summertime incidence of human infection. The mechanism driving the seasonality of STEC O157 carriage among cattle is unknown. We conducted experimental challenge trials to distinguish whether factors extrinsic or intrinsic to cattle underlie the seasonality of STEC O157 colonization. Holstein steers (n = 20) exposed to ambient environmental conditions were challenged with a standardized pool of STEC O157 strains four times at 6-month intervals. The densities and durations of rectoanal junction mucosa (RAJ) colonization with STEC O157 were compared by season (winter versus summer), dose (10(9) CFU versus 10(7) CFU), and route of challenge (oral versus rectal). Following summer challenges, the RAJ STEC O157 colonization density was significantly lower (P = 0.016) and the duration was shorter (P = 0.052) than for winter challenges, a seasonal pattern opposite to that observed naturally. Colonization was unaffected by the challenge route, indicating that passage through the gastrointestinal microbiome did not significantly affect the infectious dose to the RAJ. A 2-log reduction of the challenge doses in the second-year trials was accompanied by similarly reduced RAJ colonization in both seasons (P < 0.001). These results refute the hypothesis that cattle are predisposed to STEC O157 colonization during the summer months, either due to intrinsic factors or indirectly due to gastrointestinal tract microbiome effects. Instead, the data support the hypothesis that the increased summertime STEC O157 colonization results from increased seasonal oral exposure to this pathogen.

The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a ß-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006). The E. coli O157 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract.

The acyl-homoserine lactone synthase YenI from Yersinia enterocolitica modulates virulence gene expression in enterohemorrhagic Escherichia coli O157:H7.

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes the rectoanal junction (RAJ) in cattle, its natural reservoir. Colonization at the RAJ poses a serious risk for fecal shedding and contamination of the environment. We previously demonstrated that EHEC senses acyl-homoserine lactones (AHLs) produced by the microbiota in the rumen to activate the gad acid resistance genes necessary for survival through the acidic stomachs in cattle and to repress the locus of enterocyte effacement (LEE) genes important for colonization of the RAJ, but unnecessary in the rumen. Devoid of AHLs, the RAJ is the prominent site of colonization of EHEC in cattle. To determine if the presence of AHLs in the RAJ could repress colonization at this site, we engineered EHEC to express the Yersinia enterocolitica AHL synthase gene yenI, which constitutively produces AHLs, to mimic a constant exposure of AHLs in the environment. The yenI(+) EHEC produces oxo-C6-homoserine lactone (oxo-C6-HSL) and had a significant reduction in LEE expression, effector protein secretion, and attaching and effacing (A/E) lesion formation in vitro compared to the wild type (WT). The yenI(+) EHEC also activated expression of the gad genes. To assess whether AHL production, which decreases LEE expression, would decrease RAJ colonization by EHEC, cattle were challenged at the RAJ with WT or yenI(+) EHEC. Although the yenI(+) EHEC colonized the RAJ with efficiency equal to that of the WT, there was a trend for the cattle to shed the WT strain longer than the yenI(+) EHEC.

SdiA aids enterohemorrhagic Escherichia coli carriage by cattle fed a forage or grain diet.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and life-threatening complications. The main reservoirs for EHEC are healthy ruminants. We reported that SdiA senses acyl homoserine lactones (AHLs) in the bovine rumen to activate expression of the glutamate acid resistance (gad) genes priming EHEC's acid resistance before they pass into the acidic abomasum. Conversely, SdiA represses expression of the locus of enterocyte effacement (LEE) genes, whose expression is not required for bacterial survival in the rumen but is necessary for efficient colonization at the rectoanal junction (RAJ) mucosa. Our previous studies show that SdiA-dependent regulation was necessary for efficient EHEC colonization of cattle fed a grain diet. Here, we compared the SdiA role in EHEC colonization of cattle fed a forage hay diet. We detected AHLs in the rumen of cattle fed a hay diet, and these AHLs activated gad gene expression in an SdiA-dependent manner. The rumen fluid and fecal samples from hay-fed cattle were near neutrality, while the same digesta samples from grain-fed animals were acidic. Cattle fed either grain or hay and challenged with EHEC orally carried the bacteria similarly. EHEC was cleared from the rumen within days and from the RAJ mucosa after approximately one month. In competition trials, where animals were challenged with both wild-type and SdiA deletion mutant bacteria, diet did not affect the outcome that the wild-type strain was better able to persist and colonize. However, the wild-type strain had a greater advantage over the SdiA deletion mutant at the RAJ mucosa among cattle fed the grain diet.

Internalization of Escherichia coli o157:h7 by bovine rectal epithelial cells.

Escherichia coli O157:H7 (O157) causes human diarrheal disease and healthy cattle are its primary reservoir. O157 colonize the bovine epithelial mucosa at the recto-anal junction (RAJ). Previous studies show that O157 at this site are not eliminated by aggressive interventions including applications of O157-specific lytic bacteriophages and other bactericidal agents. We hypothesize that some O157 at the RAJ mucosa are protected from these killing agents by host cell internalization. To test this hypothesis, rectal biopsies from O157 culture positive and negative cattle were analyzed by fluorescent microscopy and subjected to gentamicin protection assays. GFP-labeled bacteria were found located deep within the tissue crypts and a small number of O157 were recovered from rectal biopsies after gentamicin treatment. Primary bovine rectal epithelial (PBRE) cell cultures were incubated with O157 and subjected to gentamicin protection assays. Strains ATCC 43895, 43894, Sakai, and WSU180 entered the PBRE cells with different levels of efficiency ranging from 0.18 to 19.38% of the inocula. Intracellular bacteria were confirmed to be within membrane-bounded vacuoles by electron microscopy. Cytochalasin D curtailed internalization of O157 indicating internalization was dependent on eukaryotic microfilament assembly. Strain ATCC 43895 exhibited the highest efficiency of internalization and survived for at least 24 h within PBRE cells. Deletion mutation of intimin or its receptor in ATCC 43895 did not reduce bacterial internalization. This strain produced more biofilm than the others tested. Retrospective analysis of cattle challenged with two O157 strains, showed ATCC 43895, the most efficient at host cell internalization, was most persistent.

Phenotypic diversity of Escherichia coli O157:H7 strains associated with the plasmid O157.

Escherichia coli O157:H7, a food-borne pathogen, causes hemorrhagic colitis and the hemolytic-uremic syndrome. A putative virulence factor of E. coli O157:H7 is a 60-MDa plasmid (pO157) found in 99% of all clinical isolates and many bovine-derived strains. The well characterized E. coli O157:H7 Sakai strain (Sakai) and its pO157-cured derivative (Sakai-Cu) were compared for phenotypic differences. Sakai-Cu had enhanced survival in synthetic gastric fluid, did not colonize cattle as well as wild-type Sakai, and had unchanged growth rates and tolerance to salt and heat. These results are consistent with our previous findings with another E. coli O157:H7 disease outbreak isolate ATCC 43894 and its pO157-cured (43894-Cu). However, despite the essentially sequence identical pO157 in these strains, Sakai-Cu had changes in antibiotic susceptibility and motility that did not occur in the 43894-Cu strain. This unexpected result was systematically analyzed using phenotypic microarrays testing 1,920 conditions with Sakai, 43894, and the plasmid-cured mutants. The influence of the pO157 differed between strains on a wide number of growth/survival conditions. Relative expression of genes related to acid resistance (gadA, gadX, and rpoS) and flagella production (fliC and flhD) were tested using quantitative real-time PCR and gadA and rpoS expression differed between Sakai-Cu and 43894-Cu. The strain-specific differences in phenotype that resulted from the loss of essentially DNA-sequence identical pO157 were likely due to the chromosomal genetic diversity between strains. The O157:H7 serotype diversity was further highlighted by phenotypic microarray comparisons of the two outbreak strains with a genotype 6 bovine E. coli O157:H7 isolate, rarely associated with human disease.

Influence of plasmid pO157 on Escherichia coli O157:H7 Sakai biofilm formation.

The role of plasmid pO157 in biofilm formation was investigated using wild-type and pO157-cured Escherichia coli O157:H7 Sakai. Compared to the wild type, the biofilm formed by the pO157-cured mutant produced fewer extracellular carbohydrates, had lower viscosity, and did not give rise to colony morphology variants that hyperadhered to solid surfaces.

Characterization of an Escherichia coli O157:H7 O-antigen deletion mutant and effect of the deletion on bacterial persistence in the mouse intestine and colonization at the bovine terminal rectal mucosa.

Escherichia coli O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome in humans and transiently colonizes healthy cattle at the terminal rectal mucosa. To investigate the role of the O antigen in persistence and colonization in the animal host, we generated an E. coli O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (per) in the rfb cluster. The lack of O antigen was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anti-O157 antibody. The growth rate and cell membrane permeability of the Deltaper mutant were similar to the growth rate and cell membrane permeability of the wild type. Changes in membrane and secreted proteins were observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Similar to other O-antigen deletion mutants, the Deltaper mutant was pleiotropic for autoaggregation and motility (it was FliC negative as determined by immunoblotting and flagellum negative as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the Deltaper mutant shed lower numbers of bacteria (P < 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the Deltaper mutant than of the wild type colonized the rectoanal junction mucosa, and the duration of the colonization was shorter (P < 0.05). Our previous work showed that flagella do not influence E. coli O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization.

Characterization of an Escherichia coli O157:H7 plasmid O157 deletion mutant and its survival and persistence in cattle.

Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic DeltapO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the DeltapO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the DeltapO157 mutant. The DeltapO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the DeltapO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the DeltapO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed approximately 50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.

Escherichia coli O157:H7 colonization at the rectoanal junction of long-duration culture-positive cattle.

Long-duration consistently Escherichia coli O157:H7 culture-positive cattle were euthanized and necropsied. Tissue and digesta from along the gastrointestinal tract (GIT) were cultured for the bacteria and examined histologically for lymphoid character. E. coli O157:H7 was detected only at the rectoanal junction mucosa and not at any other GIT location.

Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants.

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios > or = 10(2) terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10(10) PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be > or = 10(2). In addition, phages were maintained at 10(6) PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.

Role of Escherichia coli O157:H7 virulence factors in colonization at the bovine terminal rectal mucosa.

The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the approximately 92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, delta tir, and delta eae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.

Proteomics-based expression library screening (PELS): a novel method for rapidly defining microbial immunoproteomes.

Current methodologies for global identification of microbial proteins that elicit host humoral immune responses have several limitations and are not ideally suited for use in the postgenomic era. Here we describe a novel application of proteomics, proteomics-based expression library screening, to rapidly define microbial immunoproteomes. Proteomics-based expression library screening is broadly applicable to any cultivable, sequenced pathogen eliciting host antibody responses and hence is ideal for rapidly mining microbial proteomes for targets with diagnostic, prophylactic, and therapeutic potential. In this report, we demonstrate "proof-of-principle" by identifying 207 proteins of the Escherichia coli O157:H7 immunome in bovine reservoirs in only 3 weeks.

Identification of pseudo attP sites for phage phiC31 integrase in bovine genome.

Streptomyces phage phiC31 integrase was found to mediate site-specific integration of foreign genes at pseudo attP sites of genomes in human, mouse, rat, and Drosophila. This paper reports that phiC31 integrase can also mediate homologous recombination between attB and pseudo attP sites in bovine cells and foreign gene integration was increased at least 2-fold in bovine fibroblasts or Madin-Darby bovine kidney (MDBK) cells. Two intrinsic pseudo attP sites named BpsF1 and BpsM1 located in the inter-gene regions on chromosome 28 and 19, respectively, were identified in bovine genome. These pseudo attP sites shared similar characteristics with those from other species as previously described. Our study demonstrated that the phiC31 integrase system provides a new potential for genetic engineering of the bovine genome and might be beneficial for the research on ruminant.