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1.  Control of Bacterial Virulence by the RalR Regulator of the Rabbit-Specific Enteropathogenic Escherichia coli Strain E22 
Infection and Immunity  2013;81(11):4232-4243.
Atypical enteropathogenic Escherichia coli (aEPEC) causes endemic diarrhea, diarrheal outbreaks, and persistent diarrhea in humans, but the mechanism by which aEPEC causes disease is incompletely understood. Virulence regulators and their associated regulons, which often include adhesins, play key roles in the expression of virulence factors in enteric pathogenic bacteria. In this study we identified a transcriptional regulator, RalR, in the rabbit-specific aEPEC strain, E22 (O103:H2) and examined its involvement in the regulation of virulence. Microarray analysis and quantitative real-time reverse transcription-PCR demonstrated that RalR enhances the expression of a number of genes encoding virulence-associated factors, including the Ral fimbria, the Aap dispersin, and its associated transport system, and downregulates several housekeeping genes, including fliC. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins and by adherence and motility assays. To investigate the mechanism of RalR-mediated activation, we focused on its most highly upregulated target operons, ralCDEFGHI and aap. By using primer extension, electrophoretic mobility shift assay, and mutational analysis, we identified the promoter and operator sequences for these two operons. By employing promoter-lacZ reporter systems, we demonstrated that RalR activates the expression of its target genes by binding to one or more 8-bp palindromic sequences (with the consensus of TGTGCACA) located immediately upstream of the promoter core regions. Importantly, we also demonstrated that RalR is essential for virulence since infection of rabbits with E22 carrying a knockout mutation in the ralR gene completely abolished its ability to cause disease.
PMCID: PMC3811808  PMID: 24002063
3.  Contribution of Secretory Antibodies to Intestinal Mucosal Immunity against Helicobacter pylori 
Infection and Immunity  2013;81(10):3880-3893.
The natural immune response to Helicobacter pylori neither clears infection nor prevents reinfection. However, the ability of secretory antibodies to influence the course of H. pylori infection has not been determined. We compared the natural progression of H. pylori infection in wild-type C57BL/6 mice with that in mice lacking the polymeric immunoglobulin receptor (pIgR) that is essential for the secretion of polymeric antibody across mucosal surfaces. H. pylori SS1-infected wild-type and pIgR knockout (KO) mice were sampled longitudinally for gastrointestinal bacterial load, antibody response, and histological changes. The gastric bacterial loads of wild-type and pIgR KO mice remained constant and comparable at up to 3 months postinfection (mpi) despite SS1-reactive secretory IgA in the intestinal contents of wild-type mice at that time. Conversely, abundant duodenal colonization of pIgR KO animals contrasted with the near-total eradication of H. pylori from the intestine of wild-type animals by 3 mpi. H. pylori was cultured only from the duodenum of those animals in which colonization in the distal gastric antrum was of sufficient density for immunohistological detection. By 6 mpi, the gastric load of H. pylori in wild-type mice was significantly lower than in pIgR KO animals. While there was no corresponding difference between the two mouse strains in gastric pathology results at 6 mpi, reductions in gastric bacterial load correlated with increased gastric inflammation together with an intestinal secretory antibody response in wild-type mice. Together, these results suggest that naturally produced secretory antibodies can modulate the progress of H. pylori infection, particularly in the duodenum.
PMCID: PMC3811774  PMID: 23918779
4.  Laboratory Diagnostic Challenges in Case/Control Studies of Diarrhea in Developing Countries 
Case/control studies of acute infectious diarrhea require accurate and dependable laboratory tests to detect pathogens in samples from both symptomatic patients and healthy control subjects. The methods used to detect these pathogens have usually been evaluated on patient samples only, and their performance on samples from control subjects is mostly unknown. Because many pathogens occur at a high overall frequency in developing countries and thus may be present in a notable proportion of control subjects as well as patients, the relative ability of a diagnostic test to detect these pathogens in diarrheic and normal stools can have a profound effect on the interpretation of case/control data.
PMCID: PMC3502310  PMID: 23169943
5.  Transcriptional Activation of the mrkA Promoter of the Klebsiella pneumoniae Type 3 Fimbrial Operon by the c-di-GMP-Dependent MrkH Protein 
PLoS ONE  2013;8(11):e79038.
The Gram-negative bacterial pathogen Klebsiella pneumoniae forms biofilms to facilitate colonization of biotic and abiotic surfaces. The formation of biofilms by K. pneumoniae requires the expression of type 3 fimbriae: elongate proteinaceous filaments extruded by a chaperone-usher system in the bacterial outer membrane. The expression of the mrkABCDF cluster that encodes this fimbrial system is strongly positively regulated by MrkH, a transcriptional activator that responds to the second messenger, c-di-GMP. In this study, we analyzed the mechanism by which the MrkH protein activates transcriptional initiation from the mrkA promoter. A mutational analysis supported by electrophoretic mobility shift assays demonstrated that a 12-bp palindromic sequence (the MrkH box) centered at −78.5 is the binding site of MrkH. Deletion of half a turn, but not a full turn, of DNA located between the MrkH box and the mrkA promoter destroyed the ability of MrkH to activate mrkA transcription. In addition, a 10-bp AT-rich sequence (the UP element) centered at −63.5 contributed significantly to MrkH-dependent mrkA transcription. In vivo analysis of rpoA mutants showed that the R265 and E273 determinants in the C-terminal domain of RNA polymerase α subunit are needed for MrkH-mediated activation of mrkA transcription. Furthermore, results from mutagenesis of the mrkH gene suggest that the N-terminal region of the protein is involved in transcriptional activation. Taken together, our results suggest that MrkH activates mrkA expression by interacting directly with RNA polymerase, to overcome the inefficient transcriptional initiation caused by the presence of defective core promoter elements.
PMCID: PMC3828302  PMID: 24244411
6.  RegR Virulence Regulon of Rabbit-Specific Enteropathogenic Escherichia coli Strain E22 
Infection and Immunity  2013;81(4):1078-1089.
AraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli, enteroaggregative E. coli, and Citrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, of C. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target, sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression of sefA by binding to a region upstream of the sefA promoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22.
PMCID: PMC3639590  PMID: 23340312
7.  Involvement of PatE, a Prophage-Encoded AraC-Like Regulator, in the Transcriptional Activation of Acid Resistance Pathways of Enterohemorrhagic Escherichia coli Strain EDL933 
Applied and Environmental Microbiology  2012;78(15):5083-5092.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a lethal human intestinal pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. EHEC is transmitted by the fecal-oral route and has a lower infectious dose than most other enteric bacterial pathogens in that fewer than 100 CFU are able to cause disease. This low infectious dose has been attributed to the ability of EHEC to survive in the acidic environment of the human stomach. In silico analysis of the genome of EHEC O157:H7 strain EDL933 revealed a gene, patE, for a putative AraC-like regulatory protein within the prophage island, CP-933H. Transcriptional analysis in E. coli showed that the expression of patE is induced during stationary phase. Data from microarray assays demonstrated that PatE activates the transcription of genes encoding proteins of acid resistance pathways. In addition, PatE downregulated the expression of a number of genes encoding heat shock proteins and the type III secretion pathway of EDL933. Transcriptional analysis and electrophoretic mobility shift assays suggested that PatE also activates the transcription of the gene for the acid stress chaperone hdeA by binding to its promoter region. Finally, assays of acid tolerance showed that increasing the expression of PatE in EHEC greatly enhanced the ability of the bacteria to survive in different acidic environments. Together, these findings indicate that EHEC strain EDL933 carries a prophage-encoded regulatory system that contributes to acid resistance.
PMCID: PMC3416394  PMID: 22582067
8.  Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS 
PLoS Pathogens  2013;9(1):e1003117.
The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS.
Author Summary
The type 2 secretion system (T2SS) is a sophisticated, multi-component molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. In Vibrio cholerae, for example, the T2SS mediates the secretion of cholera toxin. We find that there are three distinct forms of T2SS, based on the sequence characteristics of the secretin. A targeting paradigm, developed for the Klebsiella-type secretin PulD, could not previously be applied to the T2SS in Vibrio cholerae and many other bacterial species whose genomes encode no homolog of the crucial targeting factor PulS (also called OutS, EtpO or GspS). Using bioinformatics we find, remarkably, that these bacteria have instead evolved a structurally distinct protein to serve in place of PulS. We crystallized and solved the structure of this distinct factor, AspS, measured its activity in novel assays for T2SS assembly, and show that the protein is essential for the function of the Vibrio-type T2SS. A structural homolog of AspS found here in Pseudomonas suggests widespread use of the pilotin-secretin targeting paradigm for T2SS assembly.
PMCID: PMC3542185  PMID: 23326233
9.  Factors That Explain Excretion of Enteric Pathogens by Persons Without Diarrhea 
Excretion of enteropathogens by subjects without diarrhea influences our appreciation of the role of these pathogens as etiologic agents. Characteristics of the pathogens and host and environmental factors help explain asymptomatic excretion of diarrheal pathogens by persons without diarrhea. After causing acute diarrhea followed by clinical recovery, some enteropathogens are excreted asymptomatically for many weeks. Thus, in a prevalence survey of persons without diarrhea, some may be excreting pathogens from diarrheal episodes experienced many weeks earlier. Volunteer challenges with Vibrio cholerae O1, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, Campylobacter jejuni, and Giardia lamblia document heterogeneity among enteropathogen strains, with some inexplicably not eliciting diarrhea. The immune host may not manifest diarrhea following ingestion of a pathogen but may nevertheless asymptomatically excrete. Some human genotypes render them less susceptible to symptomatic or severe diarrheal infection with certain pathogens such as Vibrio cholerae O1 and norovirus. Pathogens in stools of individuals without diarrhea may reflect recent ingestion of inocula too small to cause disease in otherwise susceptible hosts or of animal pathogens (eg, bovine or porcine ETEC) that do not cause human illness.
PMCID: PMC3502317  PMID: 23169942
10.  The Type II Secretion System and Its Ubiquitous Lipoprotein Substrate, SslE, Are Required for Biofilm Formation and Virulence of Enteropathogenic Escherichia coli 
Infection and Immunity  2012;80(6):2042-2052.
Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenic E. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease.
PMCID: PMC3370571  PMID: 22451516
11.  Genome-Wide Analysis of the Pho Regulon in a pstCA Mutant of Citrobacter rodentium 
PLoS ONE  2012;7(11):e50682.
The phosphate-specific transport operon, pstSCAB-phoU, of Gram-negative bacteria is an essential part of the Pho regulon. Its key roles are to encode a high-affinity inorganic phosphate transport system and to prevent activation of PhoB in phosphate-rich environments. In general, mutations in pstSCAB-phoU lead to the constitutive expression of the Pho regulon. Previously, we constructed a pstCA deletion mutant of Citrobacter rodentium and found it to be attenuated for virulence in mice, its natural host. This attenuation was dependent on PhoB or PhoB-regulated gene(s) because a phoB mutation restored virulence for mice to the pstCA mutant. To investigate how downstream genes may contribute to the virulence of C. rodentium, we used microarray analysis to investigate global gene expression of C. rodentium strain ICC169 and its isogenic pstCA mutant when grown in phosphate-rich medium. Overall 323 genes of the pstCA mutant were differentially expressed by at least 1.5-fold compared to the wild-type C. rodentium. Of these 145 were up-regulated and 178 were down-regulated. Differentially expressed genes included some involved in phosphate homoeostasis, cellular metabolism and protein metabolism. A large number of genes involved in stress responses and of unknown function were also differentially expressed, as were some virulence-associated genes. Up-regulated virulence-associated genes in the pstCA mutant included that for DegP, a serine protease, which appeared to be directly regulated by PhoB. Down-regulated genes included those for the production of the urease, flagella, NleG8 (a type III-secreted protein) and the tad focus (which encodes type IVb pili in Yersinia enterocolitica). Infection studies using C57/BL6 mice showed that DegP and NleG8 play a role in bacterial virulence. Overall, our study provides evidence that Pho is a global regulator of gene expression in C. rodentium and indicates the presence of at least two previously unrecognized virulence determinants of C. rodentium, namely, DegP and NleG8.
PMCID: PMC3511308  PMID: 23226353
12.  Effect of Pneumococcal Vaccination on Nasopharyngeal Carriage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian Children 
Journal of Clinical Microbiology  2012;50(3):1034-1038.
The 7-valent pneumococcal conjugate vaccine (PCV7) reduces carriage of vaccine type Streptococcus pneumoniae but leads to replacement by nonvaccine serotypes and may affect carriage of other respiratory pathogens. We investigated nasopharyngeal carriage of S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian infants participating in a pneumococcal vaccine trial using quantitative PCR. Vaccination did not affect pathogen carriage rates or densities, whereas significant differences between the two major ethnic groups were observed.
PMCID: PMC3295152  PMID: 22170924
13.  Randomized control trials using a tablet formulation of hyperimmune bovine colostrum to prevent diarrhea caused by enterotoxigenic Escherichia coli in volunteers 
Objective. Enterotoxigenic Escherichia coli (ETEC) is the leading cause of travelers' diarrhea. The aim of this study was to investigate the ability of a powdered extract of hyperimmune bovine colostrum to protect against diarrhea in volunteers challenged with ETEC. Materials and methods. Tablets were manufactured from a colostrum extract from cattle immunized with 14 ETEC strains, including serogroup O78. Two separate randomized, double-blind, placebo-controlled trials involving 90 healthy adult volunteers were performed to investigate the ability of different tablet formulations to protect against diarrhea following an oral challenge with an O78 ETEC strain. Results. The first study with 30 participants evaluated the efficacy of tablets, containing 400 mg of colostrum protein, taken thrice daily with bicarbonate buffer. This regimen conferred 90.9% protection against diarrhea in the group receiving the active preparation compared with the placebo group (p = 0.0005). The second study examined the efficacy of tablets containing 400 mg colostrum protein given with buffer (83.3% protection;p = 0.0004) or without buffer (76.7% protection;p =0.007), and tablets containing 200 mg colostrum protein given without buffer (58.3% protection; p = 0.02), compared with placebo. The difference between buffered and unbuffered treatments was not significant (p > 0.1). Conclusions. Active tablet formulations were significantly more effective than placebo in protecting volunteers against the development of diarrhea caused by ETEC. These results suggest that administration of a tablet formulation of hyperimmune bovine colostrum containing antibodies against ETEC strains may reduce the risk of travelers' diarrhea.
PMCID: PMC3154584  PMID: 21526980
Enterotoxigenic Escherichia coli; hyperimmune bovine colostrum; travelers' diarrhea; volunteers
15.  Fimbrial Adhesins Produced by Atypical Enteropathogenic Escherichia coli Strains ▿ 
Applied and Environmental Microbiology  2011;77(23):8391-8399.
Atypical enteropathogenic Escherichia coli (aEPEC) has emerged as a significant cause of pediatric diarrhea worldwide; however, information regarding its adherence mechanisms to the human gut mucosa is lacking. In this study, we investigated the prevalence of several (fimA, ecpA, csgA, elfA, and hcpA) fimbrial genes in 71 aEPEC strains isolated from children with diarrhea (54 strains) and healthy individuals (17 strains) in Brazil and Australia by PCR. These genes are associated with adhesion and/or biofilm formation of pathogenic and commensal E. coli. Here, the most prevalent fimbrial genes found, in descending order, were hcpA (98.6%), ecpA (86%), fimA (76%), elfA (72%), and csgA (19.7%). Phenotypic expression of pili in aEPEC strains was assessed by several approaches. We were not able to detect the hemorrhagic coli pilus (HCP) or the E. coli laminin-binding fimbriae (ELF) in these strains by using immunofluorescence. Type 1 pili and curli were detected in 59% (by yeast agglutination) and 2.8% (by Congo red binding and immunofluorescence) of the strains, respectively. The E. coli common pilus (ECP) was evidenced in 36.6% of the strains on bacteria adhering to HeLa cells by immunofluorescence, suggesting that ECP could play an important role in cell adherence for some aEPEC strains. This study highlights the complex nature of the adherence mechanisms of aEPEC strains involving the coordinated function of fimbrial (e.g., ECP) and nonfimbrial (e.g., intimin) adhesins and indicates that these strains bear several pilus operons that could potentially be expressed in different niches favoring colonization and survival in and outside the host.
PMCID: PMC3233042  PMID: 21926222
16.  Multilocus Sequence Typing of Streptococcus pneumoniae by Use of Mass Spectrometry ▿  
Journal of Clinical Microbiology  2011;49(11):3756-3760.
Multilocus sequence typing (MLST) is an important tool for the global surveillance of bacterial pathogens that is performed by comparing the sequences of designated housekeeping genes. We developed and tested a novel mass spectrometry-based method for MLST of Streptococcus pneumoniae. PCR amplicons were subjected to in vitro transcription and base-specific cleavage, followed by analysis of the resultant fragments by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Comparison of the cleavage fragment peak patterns to a reference sequence set permitted automated identification of alleles. Validation experiments using 29 isolates of S. pneumoniae revealed that the results of MALDI-TOF MS MLST matched those obtained by traditional sequence-based MLST for 99% of alleles and that the MALDI-TOF MS method accurately identified two single-nucleotide variations. The MADLI-TOF MS method was then used for MLST analysis of 43 S. pneumoniae isolates from Papua New Guinean children. The majority of the isolates present in this population were not clonal and contained seven new alleles and 30 previously unreported sequence types.
PMCID: PMC3209096  PMID: 21880964
17.  Phasevarion Mediated Epigenetic Gene Regulation in Helicobacter pylori 
PLoS ONE  2011;6(12):e27569.
Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a “phasevarion”), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis.
PMCID: PMC3230613  PMID: 22162751
18.  Autogenous Transcriptional Regulation of the regA Gene, Encoding an AraC-Like, Essential Virulence Regulator in Citrobacter rodentium▿  
Journal of Bacteriology  2011;193(7):1777-1782.
We identified several promoters responsible for the expression of regA, which encodes a global virulence regulator in Citrobacter rodentium. Expression of some of the promoters was strongly autoactivated by RegA in conjunction with bicarbonate. Biochemical and mutational analyses were used to determine the consensus sequence of the RegA-binding sites.
PMCID: PMC3067653  PMID: 21278290
19.  Molecular Epidemiology of Streptococcus pneumoniae Serogroup 6 Isolates from Fijian Children, Including Newly Identified Serotypes 6C and 6D▿  
Journal of Clinical Microbiology  2010;48(11):4298-4300.
Multilocus sequence typing (MLST) was applied to all unique serotype 6C and 6D isolates and a random selection of serotype 6B and 6A isolates from nasopharyngeal swabs from Fijian children enrolled in a recent vaccine trial. The results suggest that Fijian serotype 6D has arisen independently from both serotypes 6A/C and 6B.
PMCID: PMC3020807  PMID: 20810769
20.  Transcriptional Analysis of the grlRA Virulence Operon from Citrobacter rodentium▿  
Journal of Bacteriology  2010;192(14):3722-3734.
The locus for enterocyte effacement (LEE) is the virulence hallmark of the attaching-and-effacing (A/E) intestinal pathogens, namely, enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium. The LEE carries more than 40 genes that are arranged in several operons, e.g., LEE1 to LEE5. Expression of the various transcriptional units is subject to xenogeneic silencing by the histone-like protein H-NS. The LEE1-encoded regulator, Ler, plays a key role in relieving this repression at several major LEE promoters, including LEE2 to LEE5. To achieve appropriate intracellular concentrations of Ler in different environments, A/E pathogens have evolved a sophisticated regulatory network to control ler expression. For example, the LEE-encoded GrlA and GrlR proteins work as activator and antiactivator, respectively, of ler transcription. Thus, control of the transcriptional activities of the LEE1 (ler) promoter and the grlRA operon determines the rate of transcription of all of the LEE-encoded virulence factors. To date, only a single promoter has been identified for the grlRA operon. In this study, we showed that the non-LEE-encoded AraC-like regulatory protein RegA of C. rodentium directly stimulates transcription of the grlRA promoter by binding to an upstream region in the presence of bicarbonate ions. In addition, in vivo and in vitro transcription assays revealed a σ70 promoter that is specifically responsible for transcription of grlA. Expression from this promoter was strongly repressed by H-NS and its paralog StpA but was activated by Ler. DNase I footprinting demonstrated that Ler binds to a region upstream of the grlA promoter, whereas H-NS interacts specifically with a region extending from the grlA core promoter into its coding sequence. Together, these findings provide new insights into the environmental regulation and differential expressions of the grlR and grlA genes of C. rodentium.
PMCID: PMC2897351  PMID: 20472788
21.  Meningitis after cochlear implantation 
BMJ : British Medical Journal  2007;335(7629):1058.
The risk is low, and preventive measures can reduce this further
PMCID: PMC2094185  PMID: 18033888
22.  Antibody-Mediated Protection against Infection with Helicobacter pylori in a Suckling Mouse Model of Passive Immunity▿  
Infection and Immunity  2009;77(11):5116-5129.
Studies of active immunization against Helicobacter pylori indicate that antibodies play a minor role in immunity. There is also evidence, however, that the translocation of antibodies in the stomach may be insufficient to achieve functional antibody levels in the gastric lumen. We have used a suckling mouse model of passive immunity to determine if perorally delivered antibodies can protect against infection with H. pylori. Female C57BL/6 mice were immunized parenterally with formalin-fixed cells of three clinical isolates of H. pylori (3HP) or the mouse-adapted H. pylori strain SS1 before mating. Their pups were challenged with the SS1 strain at 4 days of age and left to suckle before determination of bacterial loads 14 days later. Compared to age-matched controls, pups suckled by 3HP-vaccinated dams were significantly protected against infection (>95% reduction in median bacterial load; P < 0.0001). Pups suckled by SS1-vaccinated dams were also significantly protected in terms of both median bacterial load (>99.5% reduction; P < 0.0001) and the number of culture-negative pups (28% versus 2% for immune and nonimmune cohorts, respectively; P < 0.0001). Similar results were obtained with pups suckled by dams immunized with a urease-deficient mutant of SS1. Fostering experiments demonstrated that protection was entirely attributable to suckling from an immunized dam, and antibody isotype analysis suggested that protection was mediated by the immunoglobulin G fraction of immune milk. Analysis of the bacterial loads in pups sampled before and after weaning confirmed that infection had been prevented in culture-negative animals. These data indicate that antibodies can prevent colonization by H. pylori and suppress the bacterial loads in animals that are colonized.
PMCID: PMC2772510  PMID: 19737910
23.  Virulence regulation in Citrobacter rodentium: the art of timing 
Microbial biotechnology  2010;3(3):259-268.
The mouse enteric pathogen Citrobacter rodentium, like its human counterpart, enteropathogenic Escherichia coli, causes attaching and effacing lesions in the intestinal epithelium of its host. This phenotype requires virulence factors encoded by the locus for enterocyte effacement (LEE) pathogenicity island. For timely expression of these virulence determinants at the site of infection and for efficient delivery of some virulence factors into epithelial cells, C. rodentium utilizes a positive regulatory loop involving the LEE‐encoded regulatory proteins Ler, GrlA and GrlR to control LEE expression. Several transcription factors not encoded by LEE, some of which respond to specific environmental signals, also participate in this regulatory loop. Recently, we identified a non‐LEE encoded, AraC‐like regulatory protein, RegA, which plays a key role in the ability of C. rodentium to colonize the intestine. RegA functions by activating the transcription of a number of horizontally acquired operons encoding virulence‐associated factors, such as autotransporters, fimbriae, a dispersin‐like protein and its transporter. In addition, RegA represses transcription of a number of housekeeping genes. Importantly, RegA requires a gut‐specific environmental signal, bicarbonate, to exert its effects on gene expression. In our proposed model, when C. rodentium senses bicarbonate ions in the gastrointestinal tract, RegA directs the bacterium to reduce the production of proteins involved in normal cellular functions, while enhancing the production of factors required for colonization and virulence.
PMCID: PMC3815369  PMID: 21255326
24.  Contribution of the pst-phoU Operon to Cell Adherence by Atypical Enteropathogenic Escherichia coli and Virulence of Citrobacter rodentium▿  
Infection and Immunity  2009;77(5):1936-1944.
Strains of enteropathogenic Escherichia coli (EPEC) generally employ the adhesins bundle-forming pili (Bfp) and intimin to colonize the intestine. Atypical EPEC strains possess intimin but are negative for Bfp and, yet, are able to cause disease. To identify alternative adhesins to Bfp in atypical EPEC, we constructed a transposon mutant library of atypical EPEC strain E128012 (serotype O114:H2) using TnphoA. Six mutants that had lost the ability to adhere to HEp-2 cells were identified, and in all six mutants TnphoA had inserted into the pstSCAB-phoU (Pst) operon. To determine if the Pst operon is required for adherence, we used site-directed mutagenesis to construct a pstCA mutant of E128012. The resultant mutant showed a reduced ability to adhere to HEp-2 cells and T84 intestinal epithelial cells, which was restored by trans-complementation with intact pstCA. To determine if pst contributes to bacterial colonization in vivo, a pstCA mutation was made in the EPEC-like murine pathogen, Citrobacter rodentium. C57BL/6 mice infected perorally with the pstCA mutant of C. rodentium excreted significantly lower numbers of C. rodentium than those given the wild-type strain. Moreover, colonic hyperplasia and diarrhea, which are features of infections with C. rodentium, were not observed in mice infected with the pstCA mutant but did occur in mice given the trans-complemented mutant. As mutations in pst genes generally lead to constitutive expression of the Pho regulon, our findings suggested that the Pho regulon may contribute to the reduced virulence of the pstCA mutants. To investigate this, we inactivated phoB in the pstCA mutants of EPEC E128012 and C. rodentium and found that the phoB mutation restored the adherent phenotype of both mutant strains. These results demonstrate that Pst contributes to the virulence of atypical EPEC and C. rodentium, probably by causing increased expression of an unidentified, Pho-regulated adhesin.
PMCID: PMC2681749  PMID: 19255191
25.  RegA, an AraC-Like Protein, Is a Global Transcriptional Regulator That Controls Virulence Gene Expression in Citrobacter rodentium▿  
Infection and Immunity  2008;76(11):5247-5256.
Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5α, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.
PMCID: PMC2573378  PMID: 18765720

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