Predictive phage therapy for Escherichia coli urinary tract infections: Cocktail selection for therapy based on machine learning models.

This study supports the development of predictive bacteriophage (phage) therapy: the concept of phage cocktail selection to treat a bacterial infection based on machine learning (ML) models. For this purpose, ML models were trained on thousands of measured interactions between a panel of phage and sequenced bacterial isolates. The concept was applied to Escherichia coli associated with urinary tract infections. This is an important common infection in humans and companion animals from which multidrug-resistant (MDR) bloodstream infections can originate. The global threat of MDR infection has reinvigorated international efforts into alternatives to antibiotics including phage therapy. E. coli exhibit extensive genome-level variation due to horizontal gene transfer via phage and plasmids. Associated with this, phage selection for E. coli is difficult as individual isolates can exhibit considerable variation in phage susceptibility due to differences in factors important to phage infection including phage receptor profiles and resistance mechanisms. The activity of 31 phage was measured on 314 isolates with growth curves in artificial urine. Random Forest models were built for each phage from bacterial genome features, and the more generalist phage, acting on over 20% of the bacterial population, exhibited F1 scores of >0.6 and could be used to predict phage cocktails effective against previously untested strains. The study demonstrates the potential of predictive ML models which integrate bacterial genomics with phage activity datasets allowing their use on data derived from direct sequencing of clinical samples to inform rapid and effective phage therapy.

Alternative processing of human HTT mRNA with implications for Huntington's disease therapeutics.

Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington's disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualize HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington's disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.

Identification of bacteriophage-encoded anti-sRNAs in pathogenic Escherichia coli.

In bacteria, Hfq is a core RNA chaperone that catalyzes the interaction of mRNAs with regulatory small RNAs (sRNAs). To determine in vivo RNA sequence requirements for Hfq interactions, and to study riboregulation in a bacterial pathogen, Hfq was UV crosslinked to RNAs in enterohemorrhagic Escherichia coli (EHEC). Hfq bound repeated trinucleotide motifs of A-R-N (A-A/G-any nucleotide) often associated with the Shine-Dalgarno translation initiation sequence in mRNAs. These motifs overlapped or were adjacent to the mRNA sequences bound by sRNAs. In consequence, sRNA-mRNA duplex formation will displace Hfq, promoting recycling. Fifty-five sRNAs were identified within bacteriophage-derived regions of the EHEC genome, including some of the most abundant Hfq-interacting sRNAs. One of these (AgvB) antagonized the function of the core genome regulatory sRNA, GcvB, by mimicking its mRNA substrate sequence. This bacteriophage-encoded "anti-sRNA" provided EHEC with a growth advantage specifically in bovine rectal mucus recovered from its primary colonization site in cattle.

The microbial condition of Scottish wild deer carcasses collected for human consumption and the hygiene risk factors associated with Escherichia coli and total coliforms contamination.

Wild deer hunting is necessary in Scotland to control deer population density, with most carcasses being processed for human consumption. As limited information is available on the microbial condition of Scottish venison, we studied the variation of total coliforms and Escherichia coli (E. coli) on 214 wild deer carcasses collected from six approved establishments. Samples were collected from the hide, body cavity and external surface of each carcass and mean values were determined following bacterial plate counts. The mean log10/cm2 coliforms were 5.78 (hide), 6.80 (body cavity) and 6.36 (external surface). The mean log10/cm2E. coli were 1.82 (hide), 2.27 (body cavity) and 2.17 (external carcass). Significantly higher coliforms counts were associated with storage-to-dressing times above 6 days and with longer transport distances. Risk factors that increased E. coli were red deer species, ambient temperature above 7 °C during hunting, dirty hides, faecal contamination and moisture or slimy film on the carcass. Although the bacterial counts obtained in this study indicated some hygienic processing, for around half of the carcasses, the E. coli counts were above 2 log10/cm2. Therefore, the above risk factors suggest a few handling hygiene practices that should be further improved to enhance quality and safety.

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E.

RNA sequencing studies have identified hundreds of non-coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high-throughput analysis of RNA-RNA interactions in bacteria. Here we demonstrate that in vivo sRNA-mRNA duplexes can be recovered using UV-crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base-paired sRNA-mRNA duplexes in association with RNase E, allowing proximity-dependent ligation and sequencing of cognate sRNA-mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA-mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co-regulated target mRNAs. We identified multiple mRNA targets for the pathotype-specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli Numerous sRNA interactions were also identified with non-coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.

Shiga toxin sub-type 2a increases the efficiency of Escherichia coli O157 transmission between animals and restricts epithelial regeneration in bovine enteroids.

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium.

Site Specific Relationships between COVID-19 Cases and SARS-CoV-2 Viral Load in Wastewater Treatment Plant Influent.

Wastewater based epidemiology (WBE) has become an important tool during the COVID-19 pandemic, however the relationship between SARS-CoV-2 RNA in wastewater treatment plant influent (WWTP) and cases in the community is not well-defined. We report here the development of a national WBE program across 28 WWTPs serving 50% of the population of Scotland, including large conurbations, as well as low-density rural and remote island communities. For each WWTP catchment area, we quantified spatial and temporal relationships between SARS-CoV-2 RNA in wastewater and COVID-19 cases. Daily WWTP SARS-CoV-2 influent viral RNA load, calculated using daily influent flow rates, had the strongest correlation (ρ > 0.9) with COVID-19 cases within a catchment. As the incidence of COVID-19 cases within a community increased, a linear relationship emerged between cases and influent viral RNA load. There were significant differences between WWTPs in their capacity to predict case numbers based on influent viral RNA load, with the limit of detection ranging from 25 cases for larger plants to a single case in smaller plants. SARS-CoV-2 viral RNA load can be used to predict the number of cases detected in the WWTP catchment area, with a clear statistically significant relationship observed above site-specific case thresholds.

A high-throughput genomic screen identifies a role for the plasmid-borne type II secretion system of Escherichia coli O157:H7 (Sakai) in plant-microbe interactions.

Shiga-toxigenic Escherichia coli (STEC) is often transmitted into food via fresh produce plants, where it can cause disease. To identify early interaction factors for STEC on spinach, a high-throughput positive-selection system was used. A bacterial artificial chromosome (BAC) clone library for isolate Sakai was screened in four successive rounds of short-term (2 h) interaction with spinach roots, and enriched loci identified by microarray. A Bayesian hierarchical model produced 115 CDS credible candidates, comprising seven contiguous genomic regions. Of the two candidate regions selected for functional assessment, the pO157 plasmid-encoded type two secretion system (T2SS) promoted interactions, while a chaperone-usher fimbrial gene cluster (loc6) did not. The T2SS promoted bacterial binding to spinach and appeared to involve the EtpD secretin protein. Furthermore, the T2SS genes, etpD and etpC, were expressed at a plant-relevant temperature of 18 °C, and etpD was expressed in planta by E. coli Sakai on spinach plants.

The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components.

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively.

Mechanisms involved in the adaptation of Escherichia coli O157:H7 to the host intestinal microenvironment.

Host adaptation of pathogens may increase intra- and interspecies transmission. We showed previously that the passage of a clinically isolated enterohemorrhagic Escherichia coli (EHEC) O157 strain (125/99) through the gastrointestinal tract of mice increases its pathogenicity in the same host. In this work, we aimed to elucidate the underlying mechanism(s) involved in the patho-adaptation of the stool-recovered (125RR) strain. We assessed the global transcription profile by microarray and found almost 100 differentially expressed genes in 125RR strain compared with 125/99 strain. We detected an overexpression of Type Three Secretion System (TTSS) proteins at the mRNA and protein levels and demonstrated increased adhesion to epithelial cell lines for the 125RR strain. Additional key attributes of the 125RR strain were: increased motility on semisolid agar, which correlated with an increased fliC mRNA level; reduced Stx2 production at the mRNA and protein levels; increased survival at pH 2.5, as determined by acid resistance assays. We tested whether the overexpression of the LEE-encoded regulator (ler) in trans in the 125/99 strain could recreate the increased pathogenicity observed in the 125RR strain. As anticipated ler overexpression led to increased expression of TTSS proteins and bacterial adhesion to epithelial cells in vitro but also increased mortality and intestinal colonization in vivo. We conclude that this host-adaptation process required changes in several mechanisms that improved EHEC O157 fitness in the new host. The research highlights some of the bacterial mechanisms required for horizontal transmission of these zoonotic pathogens between their animal and human populations.

An RNA-dependent mechanism for transient expression of bacterial translocation filaments.

The prokaryotic RNA chaperone Hfq mediates sRNA-mRNA interactions and plays a significant role in post-transcriptional regulation of the type III secretion (T3S) system produced by a range of Escherichia coli pathotypes. UV-crosslinking was used to map Hfq-binding under conditions that promote T3S and multiple interactions were identified within polycistronic transcripts produced from the locus of enterocyte effacement (LEE) that encodes the T3S system. The majority of Hfq binding was within the LEE5 and LEE4 operons, the latter encoding the translocon apparatus (SepL-EspADB) that is positively regulated by the RNA binding protein, CsrA. Using the identified Hfq-binding sites and a series of sRNA deletions, the sRNA Spot42 was shown to directly repress translation of LEE4 at the sepL 5' UTR. In silico and in vivo analyses of the sepL mRNA secondary structure combined with expression studies of truncates indicated that the unbound sepL mRNA is translationally inactive. Based on expression studies with site-directed mutants, an OFF-ON-OFF toggle model is proposed that results in transient translation of SepL and EspA filament assembly. Under this model, the nascent mRNA is translationally off, before being activated by CsrA, and then repressed by Hfq and Spot42.

Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen that colonizes humans and its reservoir host, cattle. Colonization requires the expression of a type 3 secretion (T3S) system that injects a mixture of effector proteins into host cells to promote bacterial attachment and disease progression. The T3S system is tightly regulated by a complex network of transcriptional and post-transcriptional regulators. Using transposon mutagenesis, here we identified the ybeZYX-Int operon as being required for normal T3S levels. Deletion analyses localized the regulation to the endoribonuclease YbeY, previously linked to 16S rRNA maturation and small RNA (sRNA) function. Loss of ybeY in EHEC had pleiotropic effects on EHEC cells, including reduced motility and growth and cold sensitivity. Using UV cross-linking and RNA-Seq (CRAC) analysis, we identified YbeY-binding sites throughout the transcriptome and discovered specific binding of YbeY to the "neck" and "beak" regions of 16S rRNA but identified no significant association of YbeY with sRNA, suggesting that YbeY modulates T3S by depleting mature ribosomes. In E. coli, translation is strongly linked to mRNA stabilization, and subinhibitory concentrations of the translation-initiation inhibitor kasugamycin provoked rapid degradation of a polycistronic mRNA encoding needle filament and needle tip proteins of the T3S system. We conclude that T3S is particularly sensitive to depletion of initiating ribosomes, explaining the inhibition of T3S in the ΔybeY strain. Accessory virulence transcripts may be preferentially degraded in cells with reduced translational capacity, potentially reflecting prioritization in protein production.

Influence of Plant Species, Tissue Type, and Temperature on the Capacity of Shiga-Toxigenic Escherichia coli To Colonize, Grow, and Be Internalized by Plants.

Contamination of fresh produce with pathogenic Escherichia coli, including Shiga-toxigenic E. coli (STEC), represents a serious risk to human health. Colonization is governed by multiple bacterial and plant factors that can impact the probability and suitability of bacterial growth. Thus, we aimed to determine whether the growth potential of STEC for plants associated with foodborne outbreaks (two leafy vegetables and two sprouted seed species) is predictive of the colonization of living plants, as assessed from growth kinetics and biofilm formation in plant extracts. The fitness of STEC isolates was compared to that of environmental E. coli isolates at temperatures relevant to plant growth. Growth kinetics in plant extracts varied in a plant-dependent and isolate-dependent manner for all isolates, with spinach leaf lysates supporting the highest rates of growth. Spinach extracts also supported the highest levels of biofilm formation. Saccharides were identified to be the major driver of bacterial growth, although no single metabolite could be correlated with growth kinetics. The highest level of in planta colonization occurred on alfalfa sprouts, though internalization was 10 times more prevalent in the leafy vegetables than in sprouted seeds. Marked differences in in planta growth meant that the growth potential of STEC could be inferred only for sprouted seeds. In contrast, biofilm formation in extracts related to spinach colonization. Overall, the capacity of E. coli to colonize, grow, and be internalized within plants or plant-derived matrices was influenced by the isolate type, plant species, plant tissue type, and temperature, complicating any straightforward relationship between in vitro and in planta behaviors.IMPORTANCE Fresh produce is an important vehicle for STEC transmission, and experimental evidence shows that STEC can colonize plants as secondary hosts, but differences in the capacity to colonize occur between different plant species and tissues. Therefore, an understanding of the impact that these plant factors have on the ability of STEC to grow and establish is required for food safety considerations and risk assessment. Here, we determined whether growth and the ability of STEC to form biofilms in plant extracts could be related to specific plant metabolites or could predict the ability of the bacteria to colonize living plants. Growth rates for sprouted seeds (alfalfa and fenugreek) but not those for leafy vegetables (lettuce and spinach) exhibited a positive relationship between plant extracts and living plants. Therefore, the detailed variations at the level of the bacterial isolate, plant species, and tissue type all need to be considered in risk assessment.

Correction: Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation.

[This corrects the article DOI: 10.1371/journal.ppat.1004627.].

Elucidation of the RamA regulon in Klebsiella pneumoniae reveals a role in LPS regulation.

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides.

A subset of Gram-negative bacterial pathogens uses a type III secretion system (T3SS) to open up a conduit into eukaryotic cells in order to inject effector proteins. These modulate pathways to enhance bacterial colonization. In this study, we screened established bioactive compounds for any that could repress T3SS expression in enterohemorrhagic Escherichia coli (EHEC) O157. The ketolides telithromycin and, subsequently, solithromycin both demonstrated repressive effects on expression of the bacterial T3SS at sub-MICs, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Preincubation of epithelial cells with solithromycin resulted in significantly less attachment of E. coli O157. Moreover, bacteria expressing the T3SS were more susceptible to solithromycin, and there was significant preferential killing of E. coli O157 bacteria when they were added to epithelial cells that had been preexposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide that has accumulated in epithelial cells may traffic back into the bacteria via the T3SS. Considering that neither ketolide induces the SOS response, nontoxic members of this class of antibiotics, such as solithromycin, should be considered for future testing and trials evaluating their use for treatment of EHEC infections. These antibiotics may also have broader significance for treating infections caused by other pathogenic bacteria, including intracellular bacteria, that express a T3SS.

Predicting the public health benefit of vaccinating cattle against Escherichia coli O157.

Identifying the major sources of risk in disease transmission is key to designing effective controls. However, understanding of transmission dynamics across species boundaries is typically poor, making the design and evaluation of controls particularly challenging for zoonotic pathogens. One such global pathogen is Escherichia coli O157, which causes a serious and sometimes fatal gastrointestinal illness. Cattle are the main reservoir for E. coli O157, and vaccines for cattle now exist. However, adoption of vaccines is being delayed by conflicting responsibilities of veterinary and public health agencies, economic drivers, and because clinical trials cannot easily test interventions across species boundaries, lack of information on the public health benefits. Here, we examine transmission risk across the cattle-human species boundary and show three key results. First, supershedding of the pathogen by cattle is associated with the genetic marker stx2. Second, by quantifying the link between shedding density in cattle and human risk, we show that only the relatively rare supershedding events contribute significantly to human risk. Third, we show that this finding has profound consequences for the public health benefits of the cattle vaccine. A naïve evaluation based on efficacy in cattle would suggest a 50% reduction in risk; however, because the vaccine targets the major source of human risk, we predict a reduction in human cases of nearly 85%. By accounting for nonlinearities in transmission across the human-animal interface, we show that adoption of these vaccines by the livestock industry could prevent substantial numbers of human E. coli O157 cases.

Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella.

Flagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response.

Strain-dependent cellular immune responses in cattle following Escherichia coli O157:H7 colonization.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic diarrhea and potentially fatal renal failure in humans. Ruminants are considered to be the primary reservoir for human infection. Vaccines that reduce shedding in cattle are only partially protective, and their underlying protective mechanisms are unknown. Studies investigating the response of cattle to colonization generally focus on humoral immunity, leaving the role of cellular immunity unclear. To inform future vaccine development, we studied the cellular immune responses of cattle during EHEC O157:H7 colonization. Calves were challenged either with a phage type 21/28 (PT21/28) strain possessing the Shiga toxin 2a (Stx2a) and Stx2c genes or with a PT32 strain possessing the Stx2c gene only. T-helper cell-associated transcripts at the terminal rectum were analyzed by reverse transcription-quantitative PCR (RT-qPCR). Induction of gamma interferon (IFN-γ) and T-bet was observed with peak expression of both genes at 7 days in PT32-challenged calves, while upregulation was delayed, peaking at 21 days, in PT21/28-challenged calves. Cells isolated from gastrointestinal lymph nodes demonstrated antigen-specific proliferation and IFN-γ release in response to type III secreted proteins (T3SPs); however, responsiveness was suppressed in cells isolated from PT32-challenged calves. Lymph node cells showed increased expression of the proliferation marker Ki67 in CD4(+) T cells from PT21/28-challenged calves, NK cells from PT32-challenged calves, and CD8(+) and γδ T cells from both PT21/28- and PT32-challenged calves following ex vivo restimulation with T3SPs. This study demonstrates that cattle mount cellular immune responses during colonization with EHEC O157:H7, the temporality of which is strain dependent, with further evidence of strain-specific immunomodulation.

Lysogeny with Shiga toxin 2-encoding bacteriophages represses type III secretion in enterohemorrhagic Escherichia coli.

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.