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Dr Jennifer Ritchie


Senior Lecturer in Microbial Pathogenesis
BSc, PhD
+44 (0)1483 686484

Biography

Biography

I have a BSc in Agricultural Science and a PhD in Plant and Soil Science from the Universities of Edinburgh and Aberdeen, respectively. Since then I have competed many years as a Post-doctoral scientist both here in the UK and in the USA studying how food- and water-borne bacterial pathogens survive in the environment and cause disease. I use a combination of molecular and genetic techniques to investigate the biology, pathogenesis and transmission of these pathogens in various in vitro and in vivo models. As such, I have a broad range of skills within the disciplines of agriculture, environmental science, infectious disease, microbiology, molecular biology, pathology and immunology.

After working for 12 years in the USA, I accepted a position as Lecturer in Microbial Pathogenesis at the University of Surrey in the UK.

Research interests

Overview

My research focuses on the biology, pathogenesis and transmission of enteric pathogens and their impact on food safety. This includes studies aimed at developing a better understanding of whether carriage of antimicrobial drug resistance affects how these bacteria survive in the environment and cause disease. The organisms I study include various Vibrio species and E. coli pathotypes including enterohaemorrhagic E. coli, and all are capable of inhabiting two diverse ecological niches, that of the mammalian intestine as well as that of the natural environment.

Impact and transmission of antimicrobial resistance (AMR)

The emergence of antimicrobial resistance (AMR) in pathogens of clinical and veterinary importance is a growing public health concern. How these plasmids spread within the intestinal environment is poorly understood and difficult to study. In collaboration with Dr Jose Jimenez, we are currently developing a new approach to study antimicrobial resistance plasmid transfer within complex microbial communities. Once we establish this methodology, we will use it to study how resistance plasmids are maintained within the intestine and begin to ask questions relating to how antibiotic use affects their distribution.

Host-pathogen interactions

The ability of organisms to colonise, persist and survive in the intestine is a pre-requisite for any enteric pathogen. My group studies the factors and molecular processes that enable these pathogens to persist in the mammalian intestine. To date, my studies have described for the first time how pandemic Vibrio parahaemolyticus interacts with the epithelial surface causing profound ultrastructural changes in the host cell that leads to villi disruption (Ritchie et al., highlighted publication). We are currently examining whether these pathological changes are induced by all virulent strains of this species, with the longer-term aim of identifying key factors that may be used to delineate pathogenic and non-pathogenic isolates.

My studies on enterohaemorrhagic E. coli (EHEC) focus on both E. coli O104:H4, the strain responsible for the particularly deadly food-borne outbreak that occurred in German in 2011, and the more prototypical EHEC serotype O157:H7. Most EHEC outbreaks in the UK are caused by strains belonging to serotype O157:H7 and as a consequence, most research has focused on this pathogen. For instance, our recent findings suggest that E. coli O157:H7 uses fucose, a host-derived signal made available by the microbiota, to modulate virulence gene expression and effectively colonise the intestine (Pacheco et al., highlighted publication). In contrast to E. coli O157:H7, E. coli O104:H4 lacks traditional EHEC colonisation factors (e.g. the LEE pathogenicity island) and instead is genetically similar to enteroaggregative E. coli. My current studies focus on examining the pathogenic mechanisms of E. coli O104:H4 in order to better understand why this organism appears to cause more severe disease than previously reported for E. coli O157:H7 isolates. One possibility is that E. coli O104:H4 is better able to colonise and disrupt the intestinal epithelial barrier, thereby allowing more Shiga toxin to enter the systemic circulation and reach the target cells in the kidney.

Pathogen transmission

We are currently in the midst of the 7th cholera pandemic. Despite many years of research, this ancient disease remains a significant public health threat in countries with poor sanitation systems or in those whose infrastructure has been disrupted by natural disasters or war. Cholera is caused by Vibrio cholerae, a non-invasive enteric pathogen that can also survive in the environment. Individuals infected with V. cholerae develop profuse, watery diarrhoea that while life-threatening, can be treated with adequate and timely hydration therapy. However, the rapid spread of infection within communities can challenge infection control programmes and has puzzled researchers. Recent research has suggested that passage of V. cholerae through the intestine promotes the ability of this organism to cause disease, and thus reduces the number of organisms required for infection. The molecular bases of this phenomenon are largely unknown and current approaches to study this aspect of the organism's lifecycle are limited. Using a novel infection model that I developed (Ritchie et al., highlighted publication), we are beginning to identify the factors that contribute to the transmission of V. cholerae. The longer-term outcomes of this work could lead to the emergence of novel strategies that may slow the spread of this deadly infection during outbreaks.

Impact and transmission of antimicrobial resistance

The emergence of antimicrobial resistance in pathogens of clinical and veterinary importance is a growing public health concern. How these plasmids spread within the intestinal environment is poorly understood and difficult to study. In collaboration with Professor La Ragione, we are beginning to examine the metabolic burden associated with plasmid carriage in different host genetic backgrounds, and gain insights into the factors that influence the frequency of transfer using in vitro intestinal models.

Research collaborations

Dr Jose Jimenez (University of Surrey, England)

Dr Gang Fang (Mount Sinai Hospital, New York, USA)

Dr John Kenny (NERC hub, University Liverpool, England)

Dr Craig Baker-Austin (Cefas, Weymouth, England)

Dr Andrew Roe (University of Glasgow, Scotland)

Dr Robert Atterbury & Professor Paul Barrow (University of Nottingham, England)

Dr Esmeralda Valienta & Professor Brendan Wren (London School of Hygiene and Tropical Medicine)

Professor Nicolas Thomson (LSHTM / Sanger Institute)

Dr William Gaze (University of Exeter)

Teaching

Module organiser for:

  • BMS1026 Microbiology - An Introduction to the Microbial World (FHEQ4)
  • MMVM002 Bacterial Genetics (FHEQ7)

Contributor to numerous other modules.

Publication highlights

  • Valiente E, Davies C, Mills DC, Getino M, Ritchie JM and Wren BW. (2018) Vibrio cholerae accessory colonisation factor AcfC: a chemotactic sulfate-binding protein with a role in hyperinfectivity. Scientific Reports. In press.
  • Giles M, Cawthraw S, AbuOun M, Thomas C, Munera D, Waldor MK, La Ragione RM and Ritchie JM. (2018) Host-specific differences in the contribution of an extended spectrum β-lactamase (ESBL) IncI1 plasmid to intestinal colonisation by Escherichia coli O104:H4. Journal of Antimicrobial Chemotherapy. In press.
  • Le Roux F, Wegner KM, Baker-Austin C et al. (2015) The emergence of Vibrio pathogens in Europe: ecology, evolution and pathogenesis. Frontiers in Microbiology 13:6:830.
  • Ritchie JM (2014) Animal models of Enterohaemorrhagic Escherichia coli infection. Microbiol Spectr 2(4).
  • Munera D, Ritchie JM, Hatzios S, Fang G, Schadt E, Davis B, Bronson R, Waldor MK (2014) Autotransporters but not pAA are critical for rabbit colonization by Shiga toxin-producing E. coli O104:H4. Nature Communications 5:3080.
  • Beckham KS, Connolly JP, Ritchie JM et al. (2014) The metabolic enzyme AdhE controls the virulence of Escherichia coli O157:H7. Molecular Microbiology 93:199-211.
  • Pacheco AR, Curtis MM, Ritchie JM, Munera D, Waldor MK, Moreira CG, Sperandio V (2012) Fucose sensing regulates bacterial intestinal colonization. Nature 492:113-117.
  • Ritchie JM, Rui H, Zhou X, Iida T, Kodoma T, Ito S, Davis BM, Bronson R, Waldor MK. (2012) Inflammation and disintegration of intestinal villi in an experimental model for Vibrio parahaemolyticus-induced diarrhea. Plos Pathogens 8(3):e1002593.
  • Ritchie JM, Rui H, Bronson RT, Waldor MK. (2010) Back to the future: studying cholera pathogenesis using infant rabbits. mBio :1e1:000047.

University roles and responsibilities

  • Chair of the Biomedical Research Facility User group
  • Member of FHMS Ethics committee
  • Member of Animal Welfare and Ethical Review Body
  • Admissions Tutor for MSc Veterinary Microbiology

My publications

Publications

Mandlik A, Livny J, Robins WP, Ritchie JM, Mekalanos JJ, Waldor MK (2011) RNA-seq-based monitoring of infection-linked changes in vibrio cholerae gene expression, Cell Host and Microbe 10 (2) pp. 165-174 Elsevier
Pathogens adapt to the host environment by altering their patterns of geneexpression. Microarray-based and genetic techniques used to characterize bacterial geneexpression during infection are limited in their ability to comprehensively and simultaneously monitor genome-wide transcription. We used massively parallel cDNA sequencing (RNA-seq) techniques to quantitatively catalog the transcriptome of the cholera pathogen, Vibriocholerae, derived from two animal models of infection. Transcripts elevated in infected rabbits and mice relative to laboratory media derive from the major known V. cholerae virulence factors and also from genes and small RNAs not previously linked to virulence. The RNA-seq data was coupled with metabolite analysis of cecal fluid from infected rabbits to yield insights into the host environment encountered by the pathogen and the mechanisms controlling pathogen geneexpression. RNA-seq-based transcriptome analysis of pathogens during infection produces a robust, sensitive, and accessible data set for evaluation of regulatory responses driving pathogenesis.
Zhou X, Ritchie JM, Hiyoshi H, Iida T, Davis BM, Waldor MK, Kodama T (2012) The hydrophilic translocator for Vibrio parahaemolyticus T3SS2 is also translocated., Infect Immun
The pathogenesis of the diarrheal disease caused by Vibrio parahaemolyticus, a leading cause of seafood-associated enteritis worldwide, is dependent upon a type III secretion system, T3SS2. This apparatus enables the pathogen to inject bacterial proteins (effectors) into the cytosol of host cells, and thereby modulate host processes. T3SS effector proteins transit into the host cell via a membrane pore (translocon) typically formed by 3 bacterial proteins. We have identified the third translocon protein for T3SS2: VopW, which was previously classified as an effector protein for a homologous T3SS in V. cholerae. VopW is a hydrophilic translocon protein; like other such proteins, it is not inserted into the host cell membrane, but is required for insertion of the two hydrophobic translocators, VopB2 and VopD2, that constitute the membrane channel. VopW is not required for secretion of T3SS2 effectors into the bacterial culture media; however, it is essential for transfer of these proteins into the host cell cytoplasm. Consequently, deletion of vopW abrogates the virulence of V. parahaemolyticus in several animal models of diarrheal disease. Unlike previously described hydrophilic translocators, VopW is itself translocated into the host cell cytoplasm, raising the possibility that it functions as both a translocator and an effector.
Pacheco AR, Curtis MM, Ritchie JM, Munera D, Waldor MK, Moreira CG, Sperandio V (2012) Fucose sensing regulates bacterial intestinal colonization., Nature 492 (7427) pp. 113-117
The mammalian gastrointestinal tract provides a complex and competitive environment for the microbiota. Successful colonization by pathogens requires scavenging nutrients, sensing chemical signals, competing with the resident bacteria and precisely regulating the expression of virulence genes. The gastrointestinal pathogen enterohaemorrhagic Escherichia coli (EHEC) relies on inter-kingdom chemical sensing systems to regulate virulence gene expression. Here we show that these systems control the expression of a novel two-component signal transduction system, named FusKR, where FusK is the histidine sensor kinase and FusR the response regulator. FusK senses fucose and controls expression of virulence and metabolic genes. This fucose-sensing system is required for robust EHEC colonization of the mammalian intestine. Fucose is highly abundant in the intestine. Bacteroides thetaiotaomicron produces multiple fucosidases that cleave fucose from host glycans, resulting in high fucose availability in the gut lumen. During growth in mucin, B. thetaiotaomicron contributes to EHEC virulence by cleaving fucose from mucin, thereby activating the FusKR signalling cascade, modulating the virulence gene expression of EHEC. Our findings suggest that EHEC uses fucose, a host-derived signal made available by the microbiota, to modulate EHEC pathogenicity and metabolism.
Ritchie JM (2014) Animal Models of Enterohemorrhagic Escherichia coli Infection., Microbiol Spectr 2 (4)
The first major outbreaks caused by enterohemorrhagic Escherichia coli (EHEC) raised public and medical awareness of the risks associated with acquiring this potentially deadly infection. The widespread presence of these organisms in the environment, the severity of the clinical sequelae, and the lack of treatment options and effective preventive measures demand that we obtain a better understanding of how this group of organisms cause disease. Animal models allow study of the processes and factors that contribute to disease and, as such, form a valuable tool in the repertoire of infectious disease researchers. Yet despite more than 30 years of research, it seems that no single model host reproduces the full spectrum of clinical disease induced by EHEC in humans. In the first part of this review, a synopsis of what is known about EHEC infections is garnered from human outbreaks and biopsy specimens. The main features and limitations of EHEC infection models that are based on the three most commonly used species (pigs, rabbits, and mice) are described within a historical context. Recent advances are highlighted, and a brief overview of models based on other species is given. Finally, the impact of the host on moderating EHEC infection is considered in light of growing evidence for the need to consider the biology and virulence strategies of EHEC in the context of its niche within the intestine.
Murphy KC, Ritchie JM, Waldor MK, Løbner-Olesen A, Marinus MG (2008) Dam methyltransferase is required for stable lysogeny of the Shiga toxin (Stx2)-encoding bacteriophage 933W of enterohemorrhagic Escherichia coli O157:H7., J Bacteriol 190 (1) pp. 438-441
Shiga toxin 2 (Stx2), one of the principal virulence factors of enterohemorrhagic Escherichia coli, is encoded by 933W, a lambda-like prophage. 933W prophage induction contributes to Stx2 production, and here, we provide evidence that Dam methyltransferase is essential for maintenance of 933W lysogeny. Our findings are consistent with the idea that the 933W prophage has a relatively low threshold for induction, which may promote Stx2 production during infection.
Ritchie JM, Rui H, Bronson RT, Waldor MK (2010) Back to the future: studying cholera pathogenesis using infant rabbits., MBio 1 (1) American Society for Microbiology
Cholera is a severe diarrheal disease, caused by Vibrio cholerae, for which there has been no reproducible, nonsurgical animal model. Here, we report that orogastric inoculation of V. cholerae into 3-day-old rabbits pretreated with cimetidine led to lethal, watery diarrhea in virtually all rabbits. The appearance and chemical composition of the rabbit diarrheal fluid were comparable to those of the "rice-water stool" produced by cholera patients. As in humans, V. cholerae mutants that do not produce cholera toxin (CT) and toxin-coregulated pilus (TCP) did not induce cholera-like disease in rabbits. CT induced extensive exocytosis of mucin from intestinal goblet cells, and wild-type V. cholerae was predominantly found in close association with mucin. Large aggregates of mucin-embedded V. cholerae were observed, both attached to the epithelium and floating within the diarrheal fluid. These findings suggest that CT-dependent mucin secretion significantly influences V. cholerae's association with the host intestine and its exit from the intestinal tract. Our model should facilitate identification and analyses of factors that may govern V. cholerae infection, survival, and transmission, such as mucin. In addition, our results using nontoxigenic V. cholerae suggest that infant rabbits will be useful for study of the reactogenicity of live attenuated-V. cholerae vaccines.
Ritchie JM, Waldor MK (2009) Vibrio cholerae interactions with the gastrointestinal tract: lessons from animal studies., Curr Top Microbiol Immunol 337 pp. 37-59
Vibrio cholerae is a curved Gram-negative rod that causes the diarrheal disease cholera. One hundred and twenty five years of study of V. cholerae microbiology have made this lethal pathogen arguably the most well-understood non-invasive mucosal pathogen. Over the past 25 years, modern molecular techniques have permitted the identification of many genes and cellular processes that are critical for V. cholerae colonization of the gastrointestinal tract. Review of the literature reveals that there are two classes of genes that influence V. cholerae colonization of the suckling mouse intestine, the most commonly used animal model to study V. cholerae pathogenesis. Inactivation of one class of genes results in profound attenuation of V. cholerae intestinal colonization, whereas inactivation of the other class of genes results in only moderate colonization defects. The latter class of genes suggests that V. cholerae may colonize several intestinal niches that impose distinct requirements and biological challenges, thus raising the possibility that there is physiologic heterogeneity among the infecting population. Efficient V. cholerae intestinal colonization and subsequent dissemination to the environment appears to require temporally ordered expression of sets of genes during the course of infection. Key challenges for future investigations of V. cholerae pathogenicity will be to assess the degree of heterogeneity in the infecting population, whether such heterogeneity has functional significance, and if stochastic processes contribute to generation of heterogeneity in vivo.
Rasko DA, Moreira CG, Li DER, Reading NC, Ritchie JM, Waldor MK, Williams N, Taussig R, Wei S, Roth M, Hughes DT, Huntley JF, Fina MW, Falck JR, Sperandio V (2008) Targeting QseC signaling and virulence for antibiotic development., Science 321 (5892) pp. 1078-1080
Many bacterial pathogens rely on a conserved membrane histidine sensor kinase, QseC, to respond to host adrenergic signaling molecules and bacterial signals in order to promote the expression of virulence factors. Using a high-throughput screen, we identified a small molecule, LED209, that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. LED209 is not toxic and does not inhibit pathogen growth; however, this compound markedly inhibits the virulence of several pathogens in vitro and in vivo in animals. Inhibition of signaling offers a strategy for the development of broad-spectrum antimicrobial drugs.
Ritchie JM, Rui H, Zhou X, Iida T, Kodoma T, Ito S, Davis BM, Bronson RT, Waldor MK (2012) Inflammation and disintegration of intestinal villi in an experimental model for Vibrio parahaemolyticus-induced diarrhea., PLoS Pathog 8 (3) Public Library of Science
Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis in many parts of the world, but there is limited knowledge of the pathogenesis of V. parahaemolyticus-induced diarrhea. The absence of an oral infection-based small animal model to study V. parahaemolyticus intestinal colonization and disease has constrained analyses of the course of infection and the factors that mediate it. Here, we demonstrate that infant rabbits oro-gastrically inoculated with V. parahaemolyticus develop severe diarrhea and enteritis, the main clinical and pathologic manifestations of disease in infected individuals. The pathogen principally colonizes the distal small intestine, and this colonization is dependent upon type III secretion system 2. The distal small intestine is also the major site of V. parahaemolyticus-induced tissue damage, reduced epithelial barrier function, and inflammation, suggesting that disease in this region of the gastrointestinal tract accounts for most of the diarrhea that accompanies V. parahaemolyticus infection. Infection appears to proceed through a characteristic sequence of steps that includes remarkable elongation of microvilli and the formation of V. parahaemolyticus-filled cavities within the epithelial surface, and culminates in villus disruption. Both depletion of epithelial cell cytoplasm and epithelial cell extrusion contribute to formation of the cavities in the epithelial surface. V. parahaemolyticus also induces proliferation of epithelial cells and recruitment of inflammatory cells, both of which occur before wide-spread damage to the epithelium is evident. Collectively, our findings suggest that V. parahaemolyticus damages the host intestine and elicits disease via previously undescribed processes and mechanisms.
Ritchie JM, Brady MJ, Riley KN, Ho TD, Campellone KG, Herman IM, Donohue-Rolfe A, Tzipori S, Waldor MK, Leong JM (2008) EspFU, a type III-translocated effector of actin assembly, fosters epithelial association and late-stage intestinal colonization by E. coli O157:H7., Cell Microbiol 10 (4) pp. 836-847
Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 induces filamentous actin-rich 'pedestals' on intestinal epithelial cells. Pedestal formation in vitro requires translocation of bacterial effectors into the host cell, including Tir, an EHEC receptor, and EspF(U), which increases the efficiency of actin assembly initiated by Tir. While inactivation of espF(U) does not alter colonization in two reservoir hosts, we utilized two disease models to explore the significance of EspF(U)-promoted actin pedestal formation. EHECDeltaespF(U) efficiently colonized the rabbit intestine during co-infection with wild-type EHEC, but co-infection studies on cultured cells suggested that EspF(U) produced by wild-type bacteria might have rescued the mutant. Significantly, EHECDeltaespF(U) by itself was fully capable of establishing colonization at 2 days post inoculation but unlike wild type, failed to expand in numbers in the caecum and colon by 7 days. In the gnotobiotic piglet model, an espF(U) deletion mutant appeared to generate actin pedestals with lower efficiency than wild type. Furthermore, aggregates of the mutant occupied a significantly smaller area of the intestinal epithelial surface than those of the wild type. Together, these findings suggest that, after initial EHEC colonization of the intestinal surface, EspF(U) may stabilize bacterial association with the epithelial cytoskeleton and promote expansion beyond initial sites of infection.
Lu X, Skurnik D, Pozzi C, Roux D, Cywes-Bentley C, Ritchie JM, Munera D, Gening ML, Tsvetkov YE, Nifantiev NE, Waldor MK, Pier GB (2014) A Poly-N-acetylglucosamine-Shiga toxin broad-spectrum conjugate vaccine for Shiga toxin-producing Escherichia coli., MBio 5 (2) pp. e00974-e00914
Many pathogens produce the ²-(1-6)-linked poly-N-acetylglucosamine (PNAG) surface polysaccharide that is being developed as a broadly protective antimicrobial vaccine. However, it is unknown whether systemically injected PNAG vaccines or antibodies would provide protective immunity against pathogens confined to the gastrointestinal tract such as Shiga toxin (Stx)-producing Escherichia coli (STEC), an important group of gastrointestinal (GI) pathogens for which effective immunotherapeutics are lacking. To ascertain whether systemic IgG antibody to PNAG impacts this infectious situation, a vaccine consisting of a synthetic nonamer of nonacetylated PNAG, 9GlcNH2, conjugated to the Shiga toxin 1b subunit (9GlcNH2-Stx1b) was produced. Rabbit antibodies raised to the conjugate vaccine were tested for bacterial killing and toxin neutralization in vitro and protection against infection in infant mice. Cell surface PNAG was detected on all 9 STEC isolates tested, representing 6 STEC serogroups, including E. coli O157:H7. Antibody to the 9GlcNH2-Stx1b conjugate neutralized Stx1 potently and Stx2 modestly. For O157:H7 and O104:H4 STEC strains, antibodies elicited by the 9GlcNH2-Stx1b conjugate possessed opsonic killing and bactericidal activity. Following intraperitoneal injection, antibodies to both PNAG and Stx were needed for infant mouse protection against O157 STEC. These antibodies also mediated protection against the Stx2-producing O104:H4 strain that was the cause of a recent outbreak in Germany, although sufficient doses of antibody to PNAG alone were protective against this strain in infant mice. Our observations suggest that vaccination against both PNAG and Stx, using a construct such as the 9GlcNH2-Stx1b conjugate vaccine, would be protective against a broad range of STEC serogroups. IMPORTANCE The presence of poly-N-acetylglucosamine (PNAG) on many pathogens presents an opportunity to target this one structure with a multispecies vaccine. Whether antibodies to PNAG can protect against pathogens confined to the gastrointestinal tract is not known. As Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are serious causes of infection whose virulence is dependent on elaboration of Stx, we prepared a vaccine containing a synthetic nonamer of PNAG (9GlcNH2) conjugated to Shiga toxin 1b subunit (9GlcNH2-Stx1b) to evaluate bacterial killing, toxin neutralization, and protective efficacy in infant mice. All nine (100%) clinical strains of STEC from dif
Lloyd SJ, Torres AG, Ritchie JM, Torres AG, Lloyd SJ (2012) Fimbriation and curliation in escherichia coli O157:H7 a paradigm of intestinal and environmental colonization, Gut Microbes 3 (3) pp. 1-5
Shiga toxin-producing Escherichia coli (STEC) serotypes, particularly E. coli O157:H7, possess a variety of fimbrial and afimbrial adhesins which have emerged as important contributors to intestinal colonization. E. coli O157:H7 possesses two chromosomal operons encoding long polar fimbriae (Lpf), which have been found to influence adherence in vitro and colonization in vivo. In a recent Infection and Immunity paper, we further explored the role of Lpf in E. coli O157:H7 intestinal colonization by using the infant rabbit model of STEC infection. We found that an E. coli O157:H7 Lpf-deficient mutant was outcompeted in the rabbit intestine by its parental strain, which may suggest that Lpf contributes to colonization. In contrast, the Lpf-deficient mutant showed an increased adherence to cultured intestinal epithelial cells, and we discovered that this strain overexpressed curli fibers. In this addendum article, we provide a continued perspective on the predicted roles of Lpf and curli, both in vivo and in vitro. © 2012 Landes Bioscience.
Lloyd SJ, Ritchie JM, Torres AG Fimbriation and curliation in escherichia coli O157:H7 a paradigm of intestinal and environmental colonization, Gut Microbes 3 (3) pp. 1-5
Shiga toxin-producing Escherichia coli (STEC) serotypes, particularly E. coli O157:H7, possess a variety of fimbrial and afimbrial adhesins which have emerged as important contributors to intestinal colonization. E. coli O157:H7 possesses two chromosomal operons encoding long polar fimbriae (Lpf), which have been found to influence adherence in vitro and colonization in vivo. In a recent Infection and Immunity paper, we further explored the role of Lpf in E. coli O157:H7 intestinal colonization by using the infant rabbit model of STEC infection. We found that an E. coli O157:H7 Lpf-deficient mutant was outcompeted in the rabbit intestine by its parental strain, which may suggest that Lpf contributes to colonization. In contrast, the Lpf-deficient mutant showed an increased adherence to cultured intestinal epithelial cells, and we discovered that this strain overexpressed curli fibers. In this addendum article, we provide a continued perspective on the predicted roles of Lpf and curli, both in vivo and in vitro. © 2012 Landes Bioscience.
Moreira CG, Russell R, Mishra AA, Narayanan S, Ritchie Jennifer M., Waldor MK, Curtis MM, Winter SE, Weinshnker D, Sperandio V (2016) Bacterial adrenergic sensors regulate virulence of enteric pathogens in the gut, mBio 7 (3) e00826-16 pp. 1-14 American Society for Microbiology
Enteric pathogens such as enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium, which is largely used as a surrogate EHEC model for murine infections, are exposed to several host neurotransmitters in the gut. An important chemical exchange within the gut involves the neurotransmitters epinephrine and/or norepinephrine, extensively reported to increase virulence gene expression in EHEC, acting through two bacterial adrenergic sensors: QseC and QseE. However, EHEC is unable to establish itself and cause its hallmark lesions, attaching and effacing (AE) lesions, on murine enterocytes. To address the role of these neurotransmitters during enteric infection, we employed C. rodentium. Both EHEC and C. rodentium harbor the locus of enterocyte effacement (LEE) that is necessary for AE lesion formation. Here we show that expression of the LEE, as well as other virulence genes in C. rodentium is also activated by epinephrine and/or norepinephrine. Both QseC and QseE are required for LEE gene activation in C. rodentium, and the qseC and qseE mutants are attenuated for murine infection. C. rodentium has decreased ability to colonize dopamine ² hydroxylase (Dbh-/-) knockout mice, which do not produce epinephrine and norepinephrine. Both adrenergic sensors are required for C. rodentium to sense these neurotransmitters and activate the LEE genes during infection. These data indicate that epinephrine and norepinephrine are sensed by bacterial adrenergic receptors during enteric infection to promote activation of their virulence repertoire. This is the first report of the role of these neurotransmitters during mammalian gastrointestinal (GI) infection by a non-invasive pathogen.
Shin OS, Tam VC, Suzuki M, Ritchie JM, Bronson RT, Waldor MK, Mekalanos JJ (2011) Type III secretion is essential for the rapidly fatal diarrheal disease caused by non-O1, non-O139 Vibrio cholerae., MBio 2 (3) pp. e00106-e00111
Cholera is a severe diarrheal disease typically caused by O1 serogroup strains of Vibrio cholerae. The pathogenicity of all pandemic V. cholerae O1 strains relies on two critical virulence factors: cholera toxin, a potent enterotoxin, and toxin coregulated pilus (TCP), an intestinal colonization factor. However, certain non-O1, non-O139 V. cholerae strains, such as AM-19226, do not produce cholera toxin or TCP, yet they still cause severe diarrhea. The molecular basis for the pathogenicity of non-O1, non-O139 V. cholerae has not been extensively characterized, but many of these strains encode related type III secretion systems (TTSSs). Here, we used infant rabbits to assess the contribution of the TTSS to non-O1, non-O139 V. cholerae pathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected with V. cholerae O1. Unlike V. cholerae O1 strains, which do not damage the intestinal epithelium in rabbits or humans, AM-19226 caused marked disruptions of the epithelial surface in the rabbit small intestine. TTSS proved to be essential for AM-19226 virulence in infant rabbits; an AM-19226 derivative deficient for TTSS did not elicit diarrhea, colonize the intestine, or induce pathological changes in the intestine. Deletion of either one of the two previously identified or two newly identified AM-19226 TTSS effectors reduced but did not eliminate AM-19226 pathogenicity, suggesting that at least four effectors contribute to this strain's virulence. In aggregate, our results suggest that the TTSS-dependent virulence in non-O1, non-O139 V. cholerae represents a new type of diarrheagenic mechanism.
Pacheco AR, Curtis MM, Ritchie JM, Munera D, Waldor MK, Moreira CG, Sperandio V (2012) Fucose sensing regulates bacterial intestinal colonization, Nature 491 (7427) pp. 113-117
The mammalian gastrointestinal tract provides a complex and competitive environment for the microbiota. Successful colonization by pathogens requires scavenging nutrients, sensing chemical signals, competing with the resident bacteria and precisely regulating the expression of virulence genes. The gastrointestinal pathogen enterohaemorrhagic Escherichia coli (EHEC) relies on inter-kingdom chemical sensing systems to regulate virulence gene expression. Here we show that these systems control the expression of a novel two-component signal transduction system, named FusKR, where FusK is the histidine sensor kinase and FusR the response regulator. FusK senses fucose and controls expression of virulence and metabolic genes. This fucose-sensing system is required for robust EHEC colonization of the mammalian intestine. Fucose is highly abundant in the intestine. Bacteroides thetaiotaomicron produces multiple fucosidases that cleave fucose from host glycans, resulting in high fucose availability in the gut lumen. During growth in mucin, B. thetaiotaomicron contributes to EHEC virulence by cleaving fucose from mucin, thereby activating the FusKR signalling cascade, modulating the virulence gene expression of EHEC. Our findings suggest that EHEC uses fucose, a host-derived signal made available by the microbiota, to modulate EHEC pathogenicity and metabolism. © 2012 Macmillan Publishers Limited. All rights reserved.
Ho TD, Davis BM, Ritchie JM, Waldor MK (2008) Type 2 secretion promotes enterohemorrhagic Escherichia coli adherence and intestinal colonization., Infect Immun 76 (5) pp. 1858-1865
Enterohemorrhagic Escherichia coli (EHEC) is a noninvasive food-borne pathogen that colonizes the distal ileum and colon. Proteins encoded in the EHEC locus of enterocyte effacement (LEE) pathogenicity island are known to contribute to this pathogen's adherence to epithelial cells and intestinal colonization. The role of non-LEE-encoded proteins in these processes is not as clear. We found that the Z2053 gene (designated adfO here), a gene located in a cryptic EHEC prophage, exhibits similarity to adherence and/or colonization factor genes found in several other enteric pathogens. An EHEC adfO mutant exhibited marked reductions in adherence to HeLa cells and in the secretion of several proteins into the supernatant. YodA, one of these secreted proteins, was found to be a substrate of the EHEC pO157-encoded type 2 secretion system (T2SS). Both the T2SS and YodA proved to be essential for EHEC adherence to cultured HeLa cell monolayers. Using an infant rabbit model of infection, we found that the adfO mutation did not affect colonization but that the colonization of an etpC (T2SS) mutant was reduced approximately 5-fold. A strain deficient in YodA had a more severe colonization defect; however, this strain also exhibited a growth defect in vitro. Overall, our findings indicate that the pO157-encoded T2SS contributes to EHEC adherence and intestinal colonization and thus show that EHEC pathogenicity depends on type 2 secretion as well as type 3 secretion.
Rui H, Ritchie JM, Bronson RT, Mekalanos JJ, Zhang Y, Waldor MK (2010) Reactogenicity of live-attenuated Vibrio cholerae vaccines is dependent on flagellins., Proc Natl Acad Sci U S A 107 (9) pp. 4359-4364 NATIONAL ACADEMY OF SCIENCES
Cholera is a severe diarrheal disease caused by the motile Gram-negative rod Vibrio cholerae. Live-attenuated V. cholerae vaccines harboring deletions of the genes encoding cholera toxin have great promise for reducing the global burden of cholera. However, development of live vaccines has been hampered by the tendency of such strains to induce noncholeric reactogenic diarrhea in human subjects. The molecular bases of reactogenicity are unknown, but it has been speculated that reactogenic diarrhea is a response to V. cholerae's flagellum and/or the motility that it enables. Here, we used an infant rabbit model of reactogenicity to determine what V. cholerae factors trigger this response. We found that V. cholerae ctx mutants that produced flagellins induced diarrhea, regardless of whether the proteins were assembled into a flagellum or whether the flagellum was functional. In contrast, approximately 90% of rabbits infected with V. cholerae lacking all five flagellin-encoding genes did not develop diarrhea. Thus, flagellin production, independent of flagellum assembly or motility, is sufficient for reactogenicity. The intestinal colonization and intraintestinal localization of the nonreactogenic flagellin-deficient strain were indistinguishable from those of a flagellated motile strain; however, the flagellin-deficient strain stimulated fewer mRNA transcripts coding for proinflammatory cytokines in the intestine. Thus, reactogenic diarrhea may be a consequence of an innate host inflammatory response to V. cholerae flagellins. Our results suggest a simple genetic blueprint for engineering defined nonreactogenic live-attenuated V. cholerae vaccine strains.
Shifrin Y, Peleg A, Ilan O, Nadler C, Kobi S, Baruch K, Yerushalmi G, Berdichevsky T, Altuvia S, Elgrably-Weiss M, Abe C, Knutton S, Sasakawa C, Ritchie JM, Waldor MK, Rosenshine I (2008) Transient shielding of intimin and the type III secretion system of enterohemorrhagic and enteropathogenic Escherichia coli by a group 4 capsule., J Bacteriol 190 (14) pp. 5063-5074
Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.
Ritchie JM, Greenwich JL, Davis BM, Bronson RT, Gebhart D, Williams SR, Martin D, Scholl D, Waldor MK (2011) An Escherichia coli O157-specific engineered pyocin prevents and ameliorates infection by E. coli O157:H7 in an animal model of diarrheal disease., Antimicrob Agents Chemother 55 (12) pp. 5469-5474
AvR2-V10.3 is an engineered R-type pyocin that specifically kills Escherichia coli O157, an enteric pathogen that is a major cause of food-borne diarrheal disease. New therapeutics to counteract E. coli O157 are needed, as currently available antibiotics can exacerbate the consequences of infection. We show here that orogastric administration of AvR2-V10.3 can prevent or ameliorate E. coli O157:H7-induced diarrhea and intestinal inflammation in an infant rabbit model of infection when the compound is administered either in a postexposure prophylactic regimen or after the onset of symptoms. Notably, administration of AvR2-V10.3 also reduces bacterial carriage and fecal shedding of this pathogen. Our findings support the further development of pathogen-specific R-type pyocins as a way to treat enteric infections.
Zhou X, Gewurz BE, Ritchie JM, Takasaki K, Greenfeld H, Kieff E, Davis BM, Waldor MK (2013) A Vibrio parahaemolyticus T3SS Effector Mediates Pathogenesis by Independently Enabling Intestinal Colonization and Inhibiting TAK1 Activation., Cell Rep 3 (5) pp. 1690-1702
Vibrio parahaemolyticus type III secretion system 2 (T3SS2) is essential for the organism's virulence, but the effectors required for intestinal colonization and induction of diarrhea by this pathogen have not been identified. Here, we identify a type III secretion system (T3SS2)-secreted effector, VopZ, that is essential for V. parahaemolyticus pathogenicity. VopZ plays distinct, genetically separable roles in enabling intestinal colonization and diarrheagenesis. Truncation of VopZ prevents V. parahaemolyticus colonization, whereas deletion of VopZ amino acids 38-62 abrogates V. parahaemolyticus-induced diarrhea and intestinal pathology but does not impair colonization. VopZ inhibits activation of the kinase TAK1 and thereby prevents the activation of MAPK and NF-ºB signaling pathways, which lie downstream. In contrast, the VopZ internal deletion mutant cannot counter the activation of pathways regulated by TAK1. Collectively, our findings suggest that VopZ's inhibition of TAK1 is critical for V. parahaemolyticus to induce diarrhea and intestinal pathology.
Beckham KSH, Connolly JPR, Ritchie JM, Wang D, Gawthorne JA, Tahoun A, Gally DL, Burgess K, Burchmore RJ, Smith BO, Beatson SA, Byron O, Wolfe AJ, Douce GR, Roe AJ (2014) The metabolic enzyme AdhE controls the virulence of Escherichia coliO157: H7, Molecular Microbiology 93 (1) pp. 199-211
Summary: Classical studies have focused on the role that individual regulators play in controlling virulence gene expression. An emerging theme, however, is that bacterial metabolism also plays a key role in this process. Our previous work identified a series of proteins that were implicated in the regulation of virulence. One of these proteins was AdhE, a bi-functional acetaldehyde-CoA dehydrogenase and alcohol dehydrogenase. Deletion of its gene (adhE) resulted in elevated levels of extracellular acetate and a stark pleiotropic phenotype: strong suppression of the Type Three Secretion System (T3SS) and overexpression of non-functional flagella. Correspondingly, the adhE mutant bound poorly to host cells and was unable to swim. Furthermore, the mutant was significantly less virulent than its parent when tested in vivo, which supports the hypothesis that attachment and motility are central to the colonization process. The molecular basis by which AdhE affects virulence gene regulation was found to be multifactorial, involving acetate-stimulated transcription of flagella expression and post-transcriptional regulation of the T3SS through Hfq. Our study reveals fascinating insights into the links between bacterial physiology, the expression of virulence genes, and the underlying molecular mechanism mechanisms by which these processes are regulated. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.
Munera D, Ritchie JM, Hatzios SK, Bronson R, Fang G, Schadt EE, Davis BM, Waldor MK (2014) Autotransporters but not pAA are critical for rabbit colonization by Shiga toxin-producing Escherichia coli O104:H4., Nat Commun 5
The outbreak of diarrhoea and haemolytic uraemic syndrome that occurred in Germany in 2011 was caused by a Shiga toxin-producing enteroaggregative Escherichia coli (EAEC) strain. The strain was classified as EAEC owing to the presence of a plasmid (pAA) that mediates a characteristic pattern of aggregative adherence on cultured cells, the defining feature of EAEC that has classically been associated with virulence. Here we describe an infant rabbit-based model of intestinal colonization and diarrhoea caused by the outbreak strain, which we use to decipher the factors that mediate the pathogen's virulence. Shiga toxin is the key factor required for diarrhoea. Unexpectedly, we observe that pAA is dispensable for intestinal colonization and development of intestinal pathology. Instead, chromosome-encoded autotransporters are critical for robust colonization and diarrhoeal disease in this model. Our findings suggest that conventional wisdom linking aggregative adherence to EAEC intestinal colonization is false for at least a subset of strains.
Kulasekara BR, Jacobs M, Zhou Y, Wu Z, Sims E, Saenphimmachak C, Rohmer L, Ritchie JM, Radey M, McKevitt M, Freeman TL, Hayden H, Haugen E, Gillett W, Fong C, Chang J, Beskhlebnaya V, Waldor MK, Samadpour M, Whittam TS, Kaul R, Brittnacher M, Miller SI (2009) Analysis of the genome of the Escherichia coli O157:H7 2006 spinach-associated outbreak isolate indicates candidate genes that may enhance virulence., Infect Immun 77 (9) pp. 3713-3721
In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx(1). The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx(1), indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx(1) and a propensity for HUS.
Beckham KS, Connolly JP, Ritchie JM, Wang D, Gawthorne JA, Tahoun A, Gally DL, Burgess K, Burchmore RJ, Smith BO, Beatson SA, Byron O, Wolfe AJ, Douce GR, Roe AJ (2014) The metabolic enzyme AdhE controls the virulence of Escherichia coli?O157:H7., Mol Microbiol 93 (1) pp. 199-211
Classical studies have focused on the role that individual regulators play in controlling virulence gene expression. An emerging theme, however, is that bacterial metabolism also plays a key role in this process. Our previous work identified a series of proteins that were implicated in the regulation of virulence. One of these proteins was AdhE, a bi-functional acetaldehyde-CoA dehydrogenase and alcohol dehydrogenase. Deletion of its gene (adhE) resulted in elevated levels of extracellular acetate and a stark pleiotropic phenotype: strong suppression of the Type Three Secretion System (T3SS) and overexpression of non-functional flagella. Correspondingly, the adhE mutant bound poorly to host cells and was unable to swim. Furthermore, the mutant was significantly less virulent than its parent when tested in vivo, which supports the hypothesis that attachment and motility are central to the colonization process. The molecular basis by which AdhE affects virulence gene regulation was found to be multifactorial, involving acetate-stimulated transcription of flagella expression and post-transcriptional regulation of the T3SS through Hfq. Our study reveals fascinating insights into the links between bacterial physiology, the expression of virulence genes, and the underlying molecular mechanism mechanisms by which these processes are regulated.
Le Roux F, Wegner KM, Baker-Austin C, Vezzulli L, Osorio CR, Amaro C, Ritchie JM, Defoirdt T, Destoumieux-Garzón D, Blokesch M, Mazel D, Jacq A, Cava F, Gram L, Wendling CC, Strauch E, Kirschner A, Huehn S (2015) The emergence of Vibrio pathogens in Europe: Ecology, evolution and pathogenesis (Paris, 11-12 March 2015), Frontiers in Microbiology 6 (JUL)
© 2015 Le Roux, Wegner, Austin, Vezzulli, Osorio, Amaro, Ritchie, Defoirdt, Destoumieux-garzon, Blokesch, Mazel, Jacq, Cava, Gram, Wendling, Strauch, Kirschner and Huehn.Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio emergence. A major challenge is to foster cooperation between fundamental and applied research in order to investigate the consequences of pathogen emergence in natural Vibrio populations and answer federative questions that meet societal needs. Here we report the proceedings of the first European workshop dedicated to these specific goals of the Vibrio research community by connecting current knowledge to societal issues related to ocean health and food security.
Lloyd SJ, Ritchie JM, Rojas-Lopez M, Blumentritt CA, Popov VL, Greenwich JL, Waldor MK, Torres AG (2012) A double, long polar fimbria mutant of Escherichia coli O157:H7 expresses Curli and exhibits reduced in vivo colonization., Infect Immun 80 (3) pp. 914-920
Escherichia coli O157:H7 causes food and waterborne enteric infections that can result in hemorrhagic colitis and life-threatening hemolytic uremic syndrome. Intimate adherence of the bacteria to intestinal epithelial cells is mediated by intimin, but E. coli O157:H7 also possess several other putative adhesins, including curli and two operons that encode long polar fimbriae (Lpf). To assess the importance of Lpf for intestinal colonization, we performed competition experiments between E. coli O157:H7 and an isogenic ”lpfA1 ”lpfA2 double mutant in the infant rabbit model. The mutant was outcompeted in the ileum, cecum, and midcolon, suggesting that Lpf contributes to intestinal colonization. In contrast, the ”lpfA1 ”lpfA2 mutant showed increased adherence to colonic epithelial cells in vitro. Transmission electron microscopy revealed curli-like structures on the surface of the ”lpfA1 ”lpfA2 mutant, and the presence of curli was confirmed by Congo red binding, immunogold-labeling electron microscopy, immunoblotting, and quantitative real-time reverse transcription-PCR (qRT-PCR) measuring csgA expression. However, deletion of csgA, which encodes the major curli subunit, does not appear to affect intestinal colonization. In addition to suggesting that Lpf can contribute to EHEC intestinal colonization, our observations indicate that the regulatory pathways governing the expression of Lpf and curli are interdependent.
Vazquez-Juarez RC, Kuriakose JA, Rasko DA, Ritchie JM, Kendall MM, Slater TM, Sinha M, Luxon BA, Popov VL, Waldor MK, Sperandio V, Torres AG (2008) CadA negatively regulates Escherichia coli O157:H7 adherence and intestinal colonization., Infect Immun 76 (11) pp. 5072-5081
Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen's adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun. 71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue-cultured monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. The cadA mutant did not possess lysine decarboxylation activity and was hyperadherent to tissue-cultured cells. Adherence of the cadA mutant was nearly twofold greater than that of the wild type, and the adherence phenotype was independent of pH, lysine, or cadaverine in the media. Additionally, complementation of the cadA defect reduced adherence back to wild-type levels, and it was found that the mutation affected the expression of the intimin protein. Disruption of the eae gene (intimin-encoding gene) in the cadA mutant significantly reduced its adherence to tissue-cultured cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1,332 genes was downregulated and that of 132 genes was upregulated in the mutant compared to the wild-type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins, flagella and F9 fimbria, were upregulated in the cadA mutant, suggestive of their association with adherence in the absence of the Cad regulatory mechanism. In the infant rabbit model, the cadA mutant outcompeted the wild-type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.
Lloyd SJ, Ritchie JM, Torres AG (2012) Fimbriation and curliation in Escherichia coli O157:H7: a paradigm of intestinal and environmental colonization., Gut microbes 3 (3) pp. 272-276
Shiga toxin-producing Escherichia coli (STEC) serotypes, particularly E. coli O157:H7, possess a variety of fimbrial and afimbrial adhesins which have emerged as important contributors to intestinal colonization. E. coli O157:H7 possesses two chromosomal operons encoding long polar fimbriae (Lpf), which have been found to influence adherence in vitro and colonization in vivo. In a recent Infection and Immunity paper, we further explored the role of Lpf in E. coli O157:H7 intestinal colonization by using the infant rabbit model of STEC infection. We found that an E. coli O157:H7 Lpf-deficient mutant was outcompeted in the rabbit intestine by its parental strain, which may suggest that Lpf contributes to colonization. In contrast, the Lpf-deficient mutant showed an increased adherence to cultured intestinal epithelial cells, and we discovered that this strain overexpressed curli fibers. In this addendum article, we provide a continued perspective on the predicted roles of Lpf and curli, both in vivo and in vitro.
Giles M, Cawthraw S, AbuOun M, Thomas C, Munera D, Waldor M, La Ragione Roberto, Ritchie Jennifer (2018) Host-specific differences in the contribution of an extended spectrum ²-lactamase (ESBL) IncI1 plasmid to intestinal colonisation by Escherichia coli O104:H4, Journal of Antimicrobial Chemotherapy 73 (6) pp. 1579-1585 Oxford University Press
Objectives.

To assess stability and contribution of a large extended spectrum ²-lactamase (ESBL)-containing IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts.

Methods.

Specific-pathogen-free 3-day old New Zealand White rabbits and conventionally-reared 6-week-old weaned lambs were orally infected with wild-type E. coli O104:H4 or the ESBL-plasmid cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time.

Results.

Carriage of the ESBL plasmid had differing impacts on E. coli O104:H4 colonisation of the two experimental hosts. The plasmid cured strain was recovered at significantly higher levels than wild type during late-stage colonization of rabbits, but at lower levels than wildtype in sheep. Regardless of the animal host, the ESBL plasmid was stably maintained in virtually all in vivo passaged bacteria that were examined.

Conclusions.

These findings suggest that carriage of ESBL plasmids has distinct effects on the host bacterium depending upon the animal species it encounters and demonstrates that, as for E. coli O157:H7, ruminants could represent a potential transmission reservoir.

Cameron E, Gruber C, Ritchie Jennifer, Waldor M, Sperandio V (2018) The QseG lipoprotein impacts the virulence of enterohemorrhagic E. coli and Citrobacter rodentium and regulates flagellar phase variation in Salmonella enterica serovar Typhimurium, Infection and Immunity 86 (4) e00936-17 American Society for Microbiology
The QseEF histidine kinase/response regulator system modulates expression of enterohemorrhagic E. coli (EHEC) and Salmonella enterica serovar Typhimurium (Stm) virulence genes in response to the host neurotransmitters epinephrine and norepinephrine. QseG, which encodes an outer membrane lipoprotein, is co-transcribed with qseEF in these enteric pathogens, but there is little knowledge of its role in virulence. Here, we found that in EHEC QseG interacts with the type three secretion system (T3SS) gate protein SepL, and modulates the kinetics of attaching and effacing (AE) lesion formation on tissue-cultured cells. Moreover, an EHEC ”qseG mutant had reduced intestinal colonization in the infant rabbit model. Additionally, in Citrobacter rodentium, an AE lesion-forming pathogen like EHEC, QseG is required for full virulence in a mouse model. In Stm, we found that QseG regulates the phase switch between the two flagellin types, FliC and FljB. In an Stm ”qseG mutant, the phase variable promoter for fljB is preferentially switched into the ?on? position, leading to an overproduction of this ?phase two? flagellin. In infection of tissue-cultured cells, the ”qseG Stm mutant provokes increased inflammatory cytokine production vs wild-type; in vivo, in a murine infection model, the ”qseG strain caused a more severe inflammatory response, and was attenuated vs the wild-type strain. Collectively, our findings demonstrate that QseG is important for full virulence in several enteric pathogens and controls flagellar phase variation in Stm, and highlight both the complexity and conservation of the regulatory networks that control the virulence of enteric pathogens.
Rui H, Ritchie Jennifer M, Bronson R, Zhang Y, Mekalanos J, Waldor Matthew K Reactogenicity of live-attenuated Vibrio cholerae vaccines
is dependent on flagellins,
Proceedings of the National Academy of Sciences of the United States of America (PNAS) ISSN 1091-6490 National Academy of Sciences
Neisseria meningitidis causes meningococcal disease, a global life threatening illness with annual incidences of between 1 and 1000 per 100,000 population. Humans are the only known host with approximately 10% of people having asymptomatic nasopharyngeal carriage at any one time. Thus, the ability of meningococci to attach, invade, and grow in the epithelium is crucial for both its commensal and pathogenic properties. In the rare event that meningococci cross the epithelium into the bloodstream, disease may occur. In order to better understand the mechanisms of meningococcal pathogenesis, transposon mutagenesis was used to identify bacterial genes involved in epithelial cell adherence and internalization as well as traversal of the epithelial barrier.
Three epithelial cell lines of respiratory origin, A549 cells, 16HBE14o- cells and Detroit 562 cells were used to examine N. meningitidis L91543 (C:2a:P1.2, ST-11; ET-37) pathogenesis. First, adhesion, invasion and traversal assays were optimized for bacterial uptake to enable the maximum number of mutants to be tested and to avoid stochastic loss from the transposon library. Since the highest level of meningococci adherence and invasion was observed using 16HBE14o- cells, this cell line was chosen for subsequent traversal assays, where an intact epithelial barrier was established on Transwell® membrane inserts. Epithelial barrier integrity was assessed by measuring transepithelial electrical resistance (TEER), permeability of the marker protein, 70 kDa Dextran, and by examining the distribution of the tight junction proteins, occludin and ZO-1, by immunofluorescence.
Next, transposon mutagenesis libraries comprising of approx. 14,500 N. meningitidis L91543 mutants, were used to probe meningococcal interactions with 16HBE14o- epithelial cells. Illumina sequencing of amplified transposon junctions was performed on DNA extracted from both input and output pools obtained from the various assays. Comparative analysis of input/output pools showed reduced fitness, not only of genes associated with type IV pili, but mainly of genes involved in metabolism especially nucleotide and amino acid metabolism. Genes involved in membrane transport, regulatory functions and cellular processes also showed reduced fitness. The function of putative genes of interest was validated by generating insertion knockout mutants and testing them independently for their ability to alter meningococcal-epithelial cell interactions. The knockout mutant assays showed 67-100% agreement to the Tn-Seq analysis prediction. Based on the knock out mutant assays as well the Tn-Seq prediction we can conclude that type IV pili, nucleotide biosynthesis, glucose and amino acid metabolism, as well as resistance to antimicrobial peptide are critical for meningococcal interaction with epithelial cells.
Bhandare Sudhakar, Colom Joan, Baig Abiyad, Ritchie Jennifer M, Bokhari Habib, Shah Muhammad A, Sarkar Banwarilal L, Su Jingliang, Wren Brendan, Barrow Paul A, Atterbury Robert (2018) Reviving phage therapy for the treatment of Cholera, The Journal of Infectious Diseases 219 (5) pp. 786-794 Oxford University Press (OUP)
Cholera remains a major risk in developing countries, particularly after natural or man-made disasters. Vibrio cholerae El Tor is the most important cause of these outbreaks, and is becoming increasingly resistant to antibiotics, so alternative therapies are urgently needed. In this study, a single bacteriophage, Phi_1, was used to control cholera prophylactically and therapeutically in an infant rabbit model. In both cases, phage-treated animals showed no clinical signs of disease, compared with 69% of untreated control animals. Bacterial counts in the intestines of phage-treated animals were reduced by up to 4 log10 colony-forming units/g. There was evidence of phage multiplication only in animals that received a V. cholerae challenge. No phage-resistant bacterial mutants were isolated from the animals, despite extensive searching. This is the first evidence that a single phage could be effective in the treatment of cholera, without detectable levels of resistance. Clinical trials in human patients should be considered.