C. Dozois - Academia.edu (original) (raw)

Papers by C. Dozois

Research paper thumbnail of Lack of a Role of Cytotoxic Necrotizing Factor 1 Toxin from Escherichia coli in Bacterial Pathogenicity and Host Cytokine Response in Infected Germfree Piglets

Infection and Immunity, 2000

Some Escherichia coli strains isolated from intestinal or extraintestinal infections in pigs prod... more Some Escherichia coli strains isolated from intestinal or extraintestinal infections in pigs produce cytotoxic necrotizing factor 1 (CNF1). In order to analyze the role of CNF1 in the pathogenesis of porcine colibacillosis, newborn colostrum-deprived germfree piglets were orally inoculated with a wild-type CNF1-producing strain (M623) or with an isogenic cnf1 mutant (M623⌬CNF1). The two isogenic strains induced a high mortality with similar lung and serosal inflammatory lesions, indicating that both strains were pathogenic in these piglets.

Research paper thumbnail of Dynamics of Escherichia coli Infection in Experimentally Inoculated Chickens

Avian Diseases, 1997

SUMMARY. In order to study the dynamics of avian colibacillosis, commercial broiler chickens were... more SUMMARY. In order to study the dynamics of avian colibacillosis, commercial broiler chickens were inoculated with a pathogenic Escherichia coli strain (01:K1:H7) into the left caudal thoracic air sac. Chickens were euthanatized at different times from 3 to 48 hr postinoculation and ...

Research paper thumbnail of Metals: Ironing out copper toxicity

Research paper thumbnail of Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences

Proceedings of the National Academy of Sciences, 2006

The cDNA obtained by selective capture of transcribed sequences is a complex mixture that can be ... more The cDNA obtained by selective capture of transcribed sequences is a complex mixture that can be used in conjunction with microarrays to determine global gene expression by a pathogen during infection. We used this method to study genes expressed by Salmonella enterica serovar Typhi, the etiological agent of typhoid fever, within human macrophages. Global expression profiles of Typhi grown in vitro and within macrophages at different time points were obtained and compared. Known virulence factors, such as the SPI-1-and SPI-2-encoded type III secretion systems, were found to be expressed as predicted during infection by Salmonella, which validated our data. Typhi inside macrophages showed increased expression of genes encoding resistance to antimicrobial peptides, used the glyoxylate bypass for fatty acid utilization, and did not induce the SOS response or the oxidative stress response. Genes coding for the flagellar apparatus, chemotaxis, and iron transport systems were down-regulated in vivo. Many cDNAs corresponding to genes with unknown functions were up-regulated inside human macrophages and will be important to consider for future studies to elucidate the intracellular lifestyle of this human-specific pathogen. Real-time quantitative PCR was consistent with the microarray results. The combined use of selective capture of transcribed sequences and microarrays is an effective way to determine the bacterial transcriptome in vivo and could be used to investigate transcriptional profiles of other bacterial pathogens without the need to recover many nanograms of bacterial mRNA from host and without increasing the multiplicity of infection beyond what is seen in nature.

Research paper thumbnail of Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells

Microbiology, 2013

Iron is an essential element but can be toxic at high concentrations. Therefore, its acquisition ... more Iron is an essential element but can be toxic at high concentrations. Therefore, its acquisition and storage require tight control. Salmonella encodes the global regulator Fur (ferric uptake regulator) and the small regulatory non-coding RNAs (sRNAs) RfrA and RfrB, homologues of RyhB. The role of these iron homeostasis regulators was investigated in Salmonella enterica serovar Typhi (S. Typhi). Strains containing either single or combined deletions of these regulators were obtained. The mutants were tested for growth in low and high iron conditions, resistance to oxidative stress, expression and production of siderophores, and during interaction with host cells. The fur mutant showed a growth defect and was sensitive to hydrogen peroxide. The expression of the sRNAs was responsible for these defects. Siderophore expression by S. Typhi and both sRNAs were regulated by iron and by Fur. Fur contributed to invasion of epithelial cells, and was shown for the first time to play a role in phagocytosis and intracellular survival of S. Typhi in human macrophages. The sRNAs RfrA and RfrB were not required for interaction with epithelial cells, but both sRNAs were important for optimal intracellular replication in macrophages. In S. Typhi, Fur is a repressor of both sRNAs, and loss of either RfrA or RfrB resulted in distinct phenotypes, suggesting a non-redundant role for these regulatory RNAs.

Research paper thumbnail of Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter jejuni

Journal of Biological Chemistry, 2006

Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins... more Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins carry diacetamidobacillosamine (DAB), a sugar that is not found in humans. Hence, the enzymes responsible for DAB synthesis represent potential therapeutic targets. We describe the biochemical characterization of Cj1121c, a putative aminotransferase encoded by the general protein glycosylation locus, to assess its role in DAB biosynthesis. By using overexpressed and affinity-purified enzyme, we demonstrate that Cj1121c has pyridoxal phosphate- and glutamate-dependent UDP-4-keto-6-deoxy-GlcNAc C-4 transaminase activity and produces UDP-4-amino-4,6-dideoxy-GlcNAc. This is consistent with a role in DAB biosynthesis and distinguishes Cj1121c from Cj1294, a homologous UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-4-hexulose C-4 aminotransferase that we characterized previously. We show that Cj1121c can also use this 4-keto-arabino sugar indirectly as a substrate, that Cj1121c and Cj1294 are active simultaneously in C. jejuni, and that the activity of Cj1121c is preponderant under standard growth conditions. Kinetic data indicate that Cj1121c has a slightly higher catalytic efficiency than Cj1294 with regard to the 4-keto-arabino substrate. By site-directed mutagenesis, we show that residues Glu-158 and Leu-131 are not essential for catalysis or for substrate specificity contrary to expectations. We further demonstrate that a cj1121c knock-out mutant is impaired for flagella-mediated motility, for invasion of intestinal epithelial cells, and for persistence in the chicken intestine, clearly demonstrating that Cj1121c is essential for host colonization and virulence. Finally, we show that cj1121c is necessary for protein glycosylation by lectin Western blotting. Collectively, these results validate Cj1121c as a promising drug target and provide the means to assay for inhibitors.

Research paper thumbnail of Autotransporter-Encoding Sequences Are Phylogenetically Distributed among Escherichia coli Clinical Isolates and Reference Strains

Applied and Environmental Microbiology, 2007

Autotransporters are secreted bacterial proteins exhibiting diverse virulence functions. Various ... more Autotransporters are secreted bacterial proteins exhibiting diverse virulence functions. Various autotransporters have been identified among Escherichia coli associated with intestinal or extraintestinal infections; however, the specific distribution of autotransporter sequences among a diversity of E. coli strains has not been investigated. We have validated the use of a multiplex PCR assay to screen for the presence of autotransporter sequences. Herein, we determined the presence of 13 autotransporter sequences and five allelic variants of antigen 43 (Ag43) among 491 E. coli isolates from human urinary tract infections, diarrheagenic E. coli, and avian pathogenic E. coli (APEC) and E. coli reference strains belonging to the ECOR collection. Clinical isolates were also classified into established phylogenetic groups. The results indicated that Ag43 alleles were significantly associated with clinical isolates (93%) compared to commensal isolates (56%) and that agn43K12 was the most common and widely distributed allele. agn43 allelic variants were also phylogenetically distributed. Sequences encoding espC, espP, and sepA and agn43 alleles EDL933 and RS218 were significantly associated with diarrheagenic E. coli strains compared to other groups. tsh was highly associated with APEC strains, whereas sat was absent from APEC. vat, sat, and pic were associated with urinary tract isolates and were identified predominantly in isolates belonging to either group B2 or D of the phylogenetic groups based on the ECOR strain collection. Overall, the results indicate that specific autotransporter sequences are associated with the source and/or phylogenetic background of strains and suggest that, in some cases, autotransporter gene profiles may be useful for comparative analysis of E. coli strains from clinical, food, and environmental sources.

Research paper thumbnail of pap-and pil-related DNA sequences and other virulence determinants associated with Escherichia coli isolated from septicemic chickens and turkeys

Infection and immunity, 1992

Escherichia coli isolates from septicemic or healthy chickens and turkeys from Quebec were seroty... more Escherichia coli isolates from septicemic or healthy chickens and turkeys from Quebec were serotyped, examined genotypically by using DNA probes specific for the pil and pap fimbrial systems and the aerobactin siderophore system, and examined phenotypically for lethality in day-old chicks, hemagglutination, serum resistance, and aerobactin production. Serogroups O78 and O1 were most common in septicemic chickens and turkeys. pap+ isolates from chickens were associated with septicemia, and pap+ isolates from turkeys were associated with lethality in day-old chicks. Four of nine pap+ isolates from septicemic turkeys expressed P adhesin, whereas all pap+ isolates from septicemic chickens were negative for P adhesin. The pil+ genotype was associated with septicemia in chickens and with serum resistance in isolates from turkeys. Mannose-sensitive hemagglutination of guinea pig erythrocytes was associated with septicemia in chickens and turkeys, although this phenotype was not associated ...

Research paper thumbnail of A reverse transcription-polymerase chain reaction method to analyze porcine cytokine gene expression

Veterinary immunology and immunopathology, Jan 19, 1997

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provi... more A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provide a highly sensitive, rapid, and simple means of simultaneously measuring the expression of porcine cytokines in immune cell populations. Oligonucleotide primers were designed to amplify porcine cytokine cDNA from genes encoding IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, TNF-beta and the housekeeping genes beta-actin and cyclophilin by PCR. Primers were chosen from different exons to detect for possible genomic DNA contamination of samples. To validate RT-PCR, unstimulated and concanavalin A (ConA) stimulated porcine peripheral blood mononuclear cells (PBMCs) were cultured from 2 h to 72 h, RNA was extracted and reverse transcribed, and cDNA was amplified using the different primer sets. Band intensities of PCR products were quantified by densitometric scanning and values were normalized against cyclophilin. For each of the cytokines, the kinetics...

Research paper thumbnail of Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages

Infection and immunity, 1997

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a pow... more Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. TDM primes murine macrophages (Mphi) to produce nitric oxide (NO) and to develop antitumoral activity upon activation with low doses of lipopolysaccharide (LPS). In this study, we investigated the ability of TDM to induce interleukin 12 (IL-12) and the role of this cytokine in TDM-induced activation of murine Mphi. RNA isolated from peritoneal exudate cells (PEC) collected at different times after TDM injection was used to determine IL-12 (p35 and p40 subunits) and gamma interferon (IFN-gamma) mRNA levels by semiquantitative reverse transcriptase-PCR. Constitutive expression of IL-12p35 was observed in PEC from untreated as well as from TDM-injected mice. In contrast, expression of the IL-12p40 subunit was almost undetectable in control PEC but was dramatically upregulated in PEC from TDM-injected mice. IL-12p40 expression peaked at 8 h and subsided to baseline lev...

Research paper thumbnail of Dynamics of Escherichia coil infection in experimentally inoculated chickens

Avian diseases

In order to study the dynamics of avian colibacillosis, commercial broiler chickens were inoculat... more In order to study the dynamics of avian colibacillosis, commercial broiler chickens were inoculated with a pathogenic Escherichia coli strain (01:K1:H7) into the left caudal thoracic air sac. Chickens were euthanatized at different times from 3 to 48 hr postinoculation and examined for bacterial counts and macroscopic and microscopic lesions. The E. coli strain colonized the air sacs, lungs, and trachea and was recovered from blood and all tested extrarespiratory organs of inoculated birds. A gradual increase in bacterial counts in the trachea, lungs, air sacs, and liver was observed from 3 to 12 hr. Clinical signs and macroscopic lesions of colibacillosis were observed in all inoculated birds. Moderate to severe lesions of airsacculitis, pericarditis, perihepatitis, and splenic hypertrophy were observed. Microscopically, inflammatory cell infiltration, serious to fibrinous exudate, and cellular debris on serosal surfaces were present in the liver, spleen, and air sacs. In air sacs,...

Research paper thumbnail of Expression of P and type 1 (F1) fimbriae in pathogenic Escherichia coli from poultry

Veterinary microbiology, 1995

To investigate the expression of P and type 1 (F1) fimbriae in pathogenic avian Escherichia coli,... more To investigate the expression of P and type 1 (F1) fimbriae in pathogenic avian Escherichia coli, fourteen pap+/fim+ E. coli isolates pathogenic for poultry were grown on four complex or minimal media, and examined for the presence of mannose resistant (MR) and mannose sensitive (MS) hemagglutination (HA), and for P or for type 1 (F1) fimbriae using immunofluorescence, immunodot, and immunoblot. In addition, isolates grown under different culture conditions were examined for adherence to frozen sections of chicken trachea. Twelve of the 14 isolates were divided into three groups based on adhesin expression in the different media. Isolates of all three groups exhibited strong MSHA reactions when cultures were grown serially in static broth, and expressed a subunit protein with an apparent molecular mass of 17 to 18.5 kDa, serologically related to the FIA major fimbrial subunit. There was a good correlation between MSHA and adherence to chicken tracheal sections. Isolates of group I o...

Research paper thumbnail of A small RNA promotes siderophore production through transcriptional and metabolic remodeling

Proceedings of the National Academy of Sciences, 2010

Siderophores are essential factors for iron (Fe) acquisition in bacteria during colonization and ... more Siderophores are essential factors for iron (Fe) acquisition in bacteria during colonization and infection of eukaryotic hosts, which restrain iron access through iron-binding protein, such as lactoferrin and transferrin. The synthesis of siderophores by Escherichia coli is considered to be fully regulated at the transcriptional level by the Fe-responsive transcriptional repressor Fur. Here we characterized two different pathways that promote the production of the siderophore enterobactin via the action of the small RNA RyhB. First, RyhB is required for normal expression of an important enterobactin biosynthesis polycistron, entCEBAH. Second, RyhB directly represses the translation of cysE, which encodes a serine acetyltransferase that uses serine as a substrate for cysteine biosynthesis. Reduction of CysE activity by RyhB allows serine to be used as building blocks for enterobactin synthesis through the nonribosomal peptide synthesis pathway. Thus, RyhB plays an essential role in siderophore production and may modulate bacterial virulence through optimization of siderophore production. enterobactin | iron | RyhB | sRNA

Research paper thumbnail of Cytokine mRNA expression in pigs infected with Schistosoma japonicum

Parasitology, 2001

The pig is a natural host of Schistosoma japonicum and a useful animal model of human disease. In... more The pig is a natural host of Schistosoma japonicum and a useful animal model of human disease. In the present study mRNA levels of Th1 (IFN-gamma) and Th2 (IL-10 and IL-4) cytokines were assessed by RT-PCR within tissues from infected pigs. Twelve Danish crossbred pigs were infected by intramuscular injection or orally with 1000 cercariae. Six other pigs served as non-infected controls. Liver and intestinal tissues were collected 10 weeks post-infection, and analysed for their relative levels of cytokine mRNA. Infected pigs developed a Th2 response as characterized by the increased level of mRNA encoding for IL-4 and IL-10 in their large intestine (caecum and colon). In contrast, levels of IFN-gamma did not differ between control and infected animals although variation between animals was observed. When comparing the immune response of orally and intramuscularly infected animals, we found that orally infected pigs produced higher IL-4 and IL-10 levels in their caecum and colon respectively. This stronger Th2 response correlated with a previously reported delay in maturation of infection following oral infection. The cytokine expression levels in tissue samples taken from lesion sites and in nearby areas, without obvious lesions, were then compared. Subsequent to an oral infection, the Th2 type cytokine production was higher in the lesion sites of the liver. In conclusion, this study is the first demonstration of IL-4 and IL-10 cytokine response in pig tissues during S. japonicum infection.

Research paper thumbnail of MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium

Molecular Microbiology, 2001

Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimuriu... more Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium ) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coli x7122 conferred mannoseresistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR-like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain x7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA-lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild-type S. typhimurium SL1344 or SR-11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a lowcopy-number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperatureand RpoS-independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by E. coli and S. typhimurium.

Research paper thumbnail of P-fimbriae-producing septicaemic Escherichia coli from poultry possess fel-related gene clusters whereas pap-hybridizing P-fimbriae-negative strains have partial or divergent P fimbrial gene clusters

Microbiology, 1996

CP 5000, Sa i nt-H yaci nt he, Quebec, Canada J2S 7C6

Research paper thumbnail of Characterization of Stg Fimbriae from an Avian Pathogenic Escherichia coli O78:K80 Strain and Assessment of Their Contribution to Colonization of the Chicken Respiratory Tract

Journal of Bacteriology, 2006

In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 stra... more In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 strain 7122, was found to be expressed in vivo in infected chicken tissues. The region encompassing ecs-3 carries a fimbrial gene cluster that is a putative ortholog of the stg fimbrial gene cluster of Salmonella enterica serovar Typhi. This APEC fimbrial gene cluster, which we have termed stg, is a member of a distinct group of related fimbriae that are located in the glmS-pstS intergenic region of certain E. coli and S. enterica strains. Under the control of the pBAD promoter, the production of Stg fimbriae was demonstrated by Western blotting and immunogold electron microscopy with E. coli K-12. Transcriptional fusions suggest that stg expression is influenced by the carbohydrate source and decreased by the addition of iron and that Fur plays a role in the regulation of stg expression. stg sequences were associated with APEC O78 isolates, and stg was phylogenetically distributed among E. coli reference strains and clinical isolates from human urinary tract infections. Stg fimbriae contributed to the adherence of a nonfimbriated E. coli K-12 strain to avian lung sections and human epithelial cells in vitro. Coinfection experiments with APEC strain 7122 and an isogenic ⌬stg mutant demonstrated that compared to the wild-type parent, the ⌬stg mutant was less able to colonize air sacs, equally able to colonize lungs, and able to more effectively colonize tracheas of infected chickens. Stg fimbriae, together with other adhesins, may therefore contribute to the colonization of avian respiratory tissues by certain APEC strains.

Research paper thumbnail of Klebsiella pneumoniae Yersiniabactin Promotes Respiratory Tract Infection through Evasion of Lipocalin 2

Infection and Immunity, 2011

Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especi... more Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especially due to K. pneumoniae carbapenemases (KPCs). K. pneumoniae must acquire iron to replicate, and it utilizes iron-scavenging siderophores, such as enterobactin (Ent). The innate immune protein lipocalin 2 (Lcn2) is able to specifically bind Ent and disrupt iron acquisition. To determine whether K. pneumoniae must produce Lcn2-resistant siderophores to cause disease, we examined siderophore production by clinical isolates (n = 129) from respiratory, urine, blood, and stool samples and by defined siderophore mutants through genotyping and liquid chromatography-mass spectrometry. Three categories of K. pneumoniae isolates were identified: enterobactin positive (Ent(+)) (81%), enterobactin and yersiniabactin positive (Ent(+) Ybt(+)) (17%), and enterobactin and salmochelin (glycosylated Ent) positive (Ent(+) gly-Ent(+)) with or without Ybt (2%). Ent(+) Ybt(+) strains were significantly overrepresented among respiratory tract isolates (P = 0.0068) and β-lactam-resistant isolates (P = 0.0019), including the epidemic KPC-producing clone multilocus sequence type 258 (ST258). In ex vivo growth assays, gly-Ent but not Ybt allowed evasion of Lcn2 in human serum, whereas siderophores were dispensable for growth in human urine. In a murine pneumonia model, an Ent(+) strain was an opportunistic pathogen that was completely inhibited by Lcn2 but caused severe, disseminated disease in Lcn2(-/-) mice. In contrast, an Ent(+) Ybt(+) strain was a frank respiratory pathogen, causing pneumonia despite Lcn2. However, Lcn2 retained partial protection against disseminated disease. In summary, Ybt is a virulence factor that is prevalent among KPC-producing K. pneumoniae isolates and promotes respiratory tract infections through evasion of Lcn2.

Research paper thumbnail of Increased Pho Regulon Activation Correlates with Decreased Virulence of an Avian Pathogenic Escherichia coli O78 Strain

Infection and Immunity, 2010

Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, sept... more Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, septicemia, cellulitis, peritonitis, and other conditions, since colibacillosis manifests in many ways. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the specific roles of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain 7122, different phoBR and pst mutant strains were tested in vivo in chickens and in vitro for virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were outcompeted by the wild-type parent, including strains containing a functional Pst system. Cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. In addition, loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of the APEC O78 strain 7122.

Research paper thumbnail of Relationship between the Tsh Autotransporter and Pathogenicity of Avian Escherichia coli and Localization and Analysis of the tsh Genetic Region

Infection and Immunity, 2000

The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins ... more The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain 7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the tsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with 7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain 7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain 7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of the Enterobacteriaceae. These results demonstrate that the tsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.

Research paper thumbnail of Lack of a Role of Cytotoxic Necrotizing Factor 1 Toxin from Escherichia coli in Bacterial Pathogenicity and Host Cytokine Response in Infected Germfree Piglets

Infection and Immunity, 2000

Some Escherichia coli strains isolated from intestinal or extraintestinal infections in pigs prod... more Some Escherichia coli strains isolated from intestinal or extraintestinal infections in pigs produce cytotoxic necrotizing factor 1 (CNF1). In order to analyze the role of CNF1 in the pathogenesis of porcine colibacillosis, newborn colostrum-deprived germfree piglets were orally inoculated with a wild-type CNF1-producing strain (M623) or with an isogenic cnf1 mutant (M623⌬CNF1). The two isogenic strains induced a high mortality with similar lung and serosal inflammatory lesions, indicating that both strains were pathogenic in these piglets.

Research paper thumbnail of Dynamics of Escherichia coli Infection in Experimentally Inoculated Chickens

Avian Diseases, 1997

SUMMARY. In order to study the dynamics of avian colibacillosis, commercial broiler chickens were... more SUMMARY. In order to study the dynamics of avian colibacillosis, commercial broiler chickens were inoculated with a pathogenic Escherichia coli strain (01:K1:H7) into the left caudal thoracic air sac. Chickens were euthanatized at different times from 3 to 48 hr postinoculation and ...

Research paper thumbnail of Metals: Ironing out copper toxicity

Research paper thumbnail of Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences

Proceedings of the National Academy of Sciences, 2006

The cDNA obtained by selective capture of transcribed sequences is a complex mixture that can be ... more The cDNA obtained by selective capture of transcribed sequences is a complex mixture that can be used in conjunction with microarrays to determine global gene expression by a pathogen during infection. We used this method to study genes expressed by Salmonella enterica serovar Typhi, the etiological agent of typhoid fever, within human macrophages. Global expression profiles of Typhi grown in vitro and within macrophages at different time points were obtained and compared. Known virulence factors, such as the SPI-1-and SPI-2-encoded type III secretion systems, were found to be expressed as predicted during infection by Salmonella, which validated our data. Typhi inside macrophages showed increased expression of genes encoding resistance to antimicrobial peptides, used the glyoxylate bypass for fatty acid utilization, and did not induce the SOS response or the oxidative stress response. Genes coding for the flagellar apparatus, chemotaxis, and iron transport systems were down-regulated in vivo. Many cDNAs corresponding to genes with unknown functions were up-regulated inside human macrophages and will be important to consider for future studies to elucidate the intracellular lifestyle of this human-specific pathogen. Real-time quantitative PCR was consistent with the microarray results. The combined use of selective capture of transcribed sequences and microarrays is an effective way to determine the bacterial transcriptome in vivo and could be used to investigate transcriptional profiles of other bacterial pathogens without the need to recover many nanograms of bacterial mRNA from host and without increasing the multiplicity of infection beyond what is seen in nature.

Research paper thumbnail of Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells

Microbiology, 2013

Iron is an essential element but can be toxic at high concentrations. Therefore, its acquisition ... more Iron is an essential element but can be toxic at high concentrations. Therefore, its acquisition and storage require tight control. Salmonella encodes the global regulator Fur (ferric uptake regulator) and the small regulatory non-coding RNAs (sRNAs) RfrA and RfrB, homologues of RyhB. The role of these iron homeostasis regulators was investigated in Salmonella enterica serovar Typhi (S. Typhi). Strains containing either single or combined deletions of these regulators were obtained. The mutants were tested for growth in low and high iron conditions, resistance to oxidative stress, expression and production of siderophores, and during interaction with host cells. The fur mutant showed a growth defect and was sensitive to hydrogen peroxide. The expression of the sRNAs was responsible for these defects. Siderophore expression by S. Typhi and both sRNAs were regulated by iron and by Fur. Fur contributed to invasion of epithelial cells, and was shown for the first time to play a role in phagocytosis and intracellular survival of S. Typhi in human macrophages. The sRNAs RfrA and RfrB were not required for interaction with epithelial cells, but both sRNAs were important for optimal intracellular replication in macrophages. In S. Typhi, Fur is a repressor of both sRNAs, and loss of either RfrA or RfrB resulted in distinct phenotypes, suggesting a non-redundant role for these regulatory RNAs.

Research paper thumbnail of Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter jejuni

Journal of Biological Chemistry, 2006

Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins... more Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins carry diacetamidobacillosamine (DAB), a sugar that is not found in humans. Hence, the enzymes responsible for DAB synthesis represent potential therapeutic targets. We describe the biochemical characterization of Cj1121c, a putative aminotransferase encoded by the general protein glycosylation locus, to assess its role in DAB biosynthesis. By using overexpressed and affinity-purified enzyme, we demonstrate that Cj1121c has pyridoxal phosphate- and glutamate-dependent UDP-4-keto-6-deoxy-GlcNAc C-4 transaminase activity and produces UDP-4-amino-4,6-dideoxy-GlcNAc. This is consistent with a role in DAB biosynthesis and distinguishes Cj1121c from Cj1294, a homologous UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-4-hexulose C-4 aminotransferase that we characterized previously. We show that Cj1121c can also use this 4-keto-arabino sugar indirectly as a substrate, that Cj1121c and Cj1294 are active simultaneously in C. jejuni, and that the activity of Cj1121c is preponderant under standard growth conditions. Kinetic data indicate that Cj1121c has a slightly higher catalytic efficiency than Cj1294 with regard to the 4-keto-arabino substrate. By site-directed mutagenesis, we show that residues Glu-158 and Leu-131 are not essential for catalysis or for substrate specificity contrary to expectations. We further demonstrate that a cj1121c knock-out mutant is impaired for flagella-mediated motility, for invasion of intestinal epithelial cells, and for persistence in the chicken intestine, clearly demonstrating that Cj1121c is essential for host colonization and virulence. Finally, we show that cj1121c is necessary for protein glycosylation by lectin Western blotting. Collectively, these results validate Cj1121c as a promising drug target and provide the means to assay for inhibitors.

Research paper thumbnail of Autotransporter-Encoding Sequences Are Phylogenetically Distributed among Escherichia coli Clinical Isolates and Reference Strains

Applied and Environmental Microbiology, 2007

Autotransporters are secreted bacterial proteins exhibiting diverse virulence functions. Various ... more Autotransporters are secreted bacterial proteins exhibiting diverse virulence functions. Various autotransporters have been identified among Escherichia coli associated with intestinal or extraintestinal infections; however, the specific distribution of autotransporter sequences among a diversity of E. coli strains has not been investigated. We have validated the use of a multiplex PCR assay to screen for the presence of autotransporter sequences. Herein, we determined the presence of 13 autotransporter sequences and five allelic variants of antigen 43 (Ag43) among 491 E. coli isolates from human urinary tract infections, diarrheagenic E. coli, and avian pathogenic E. coli (APEC) and E. coli reference strains belonging to the ECOR collection. Clinical isolates were also classified into established phylogenetic groups. The results indicated that Ag43 alleles were significantly associated with clinical isolates (93%) compared to commensal isolates (56%) and that agn43K12 was the most common and widely distributed allele. agn43 allelic variants were also phylogenetically distributed. Sequences encoding espC, espP, and sepA and agn43 alleles EDL933 and RS218 were significantly associated with diarrheagenic E. coli strains compared to other groups. tsh was highly associated with APEC strains, whereas sat was absent from APEC. vat, sat, and pic were associated with urinary tract isolates and were identified predominantly in isolates belonging to either group B2 or D of the phylogenetic groups based on the ECOR strain collection. Overall, the results indicate that specific autotransporter sequences are associated with the source and/or phylogenetic background of strains and suggest that, in some cases, autotransporter gene profiles may be useful for comparative analysis of E. coli strains from clinical, food, and environmental sources.

Research paper thumbnail of pap-and pil-related DNA sequences and other virulence determinants associated with Escherichia coli isolated from septicemic chickens and turkeys

Infection and immunity, 1992

Escherichia coli isolates from septicemic or healthy chickens and turkeys from Quebec were seroty... more Escherichia coli isolates from septicemic or healthy chickens and turkeys from Quebec were serotyped, examined genotypically by using DNA probes specific for the pil and pap fimbrial systems and the aerobactin siderophore system, and examined phenotypically for lethality in day-old chicks, hemagglutination, serum resistance, and aerobactin production. Serogroups O78 and O1 were most common in septicemic chickens and turkeys. pap+ isolates from chickens were associated with septicemia, and pap+ isolates from turkeys were associated with lethality in day-old chicks. Four of nine pap+ isolates from septicemic turkeys expressed P adhesin, whereas all pap+ isolates from septicemic chickens were negative for P adhesin. The pil+ genotype was associated with septicemia in chickens and with serum resistance in isolates from turkeys. Mannose-sensitive hemagglutination of guinea pig erythrocytes was associated with septicemia in chickens and turkeys, although this phenotype was not associated ...

Research paper thumbnail of A reverse transcription-polymerase chain reaction method to analyze porcine cytokine gene expression

Veterinary immunology and immunopathology, Jan 19, 1997

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provi... more A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provide a highly sensitive, rapid, and simple means of simultaneously measuring the expression of porcine cytokines in immune cell populations. Oligonucleotide primers were designed to amplify porcine cytokine cDNA from genes encoding IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, TNF-beta and the housekeeping genes beta-actin and cyclophilin by PCR. Primers were chosen from different exons to detect for possible genomic DNA contamination of samples. To validate RT-PCR, unstimulated and concanavalin A (ConA) stimulated porcine peripheral blood mononuclear cells (PBMCs) were cultured from 2 h to 72 h, RNA was extracted and reverse transcribed, and cDNA was amplified using the different primer sets. Band intensities of PCR products were quantified by densitometric scanning and values were normalized against cyclophilin. For each of the cytokines, the kinetics...

Research paper thumbnail of Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages

Infection and immunity, 1997

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a pow... more Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. TDM primes murine macrophages (Mphi) to produce nitric oxide (NO) and to develop antitumoral activity upon activation with low doses of lipopolysaccharide (LPS). In this study, we investigated the ability of TDM to induce interleukin 12 (IL-12) and the role of this cytokine in TDM-induced activation of murine Mphi. RNA isolated from peritoneal exudate cells (PEC) collected at different times after TDM injection was used to determine IL-12 (p35 and p40 subunits) and gamma interferon (IFN-gamma) mRNA levels by semiquantitative reverse transcriptase-PCR. Constitutive expression of IL-12p35 was observed in PEC from untreated as well as from TDM-injected mice. In contrast, expression of the IL-12p40 subunit was almost undetectable in control PEC but was dramatically upregulated in PEC from TDM-injected mice. IL-12p40 expression peaked at 8 h and subsided to baseline lev...

Research paper thumbnail of Dynamics of Escherichia coil infection in experimentally inoculated chickens

Avian diseases

In order to study the dynamics of avian colibacillosis, commercial broiler chickens were inoculat... more In order to study the dynamics of avian colibacillosis, commercial broiler chickens were inoculated with a pathogenic Escherichia coli strain (01:K1:H7) into the left caudal thoracic air sac. Chickens were euthanatized at different times from 3 to 48 hr postinoculation and examined for bacterial counts and macroscopic and microscopic lesions. The E. coli strain colonized the air sacs, lungs, and trachea and was recovered from blood and all tested extrarespiratory organs of inoculated birds. A gradual increase in bacterial counts in the trachea, lungs, air sacs, and liver was observed from 3 to 12 hr. Clinical signs and macroscopic lesions of colibacillosis were observed in all inoculated birds. Moderate to severe lesions of airsacculitis, pericarditis, perihepatitis, and splenic hypertrophy were observed. Microscopically, inflammatory cell infiltration, serious to fibrinous exudate, and cellular debris on serosal surfaces were present in the liver, spleen, and air sacs. In air sacs,...

Research paper thumbnail of Expression of P and type 1 (F1) fimbriae in pathogenic Escherichia coli from poultry

Veterinary microbiology, 1995

To investigate the expression of P and type 1 (F1) fimbriae in pathogenic avian Escherichia coli,... more To investigate the expression of P and type 1 (F1) fimbriae in pathogenic avian Escherichia coli, fourteen pap+/fim+ E. coli isolates pathogenic for poultry were grown on four complex or minimal media, and examined for the presence of mannose resistant (MR) and mannose sensitive (MS) hemagglutination (HA), and for P or for type 1 (F1) fimbriae using immunofluorescence, immunodot, and immunoblot. In addition, isolates grown under different culture conditions were examined for adherence to frozen sections of chicken trachea. Twelve of the 14 isolates were divided into three groups based on adhesin expression in the different media. Isolates of all three groups exhibited strong MSHA reactions when cultures were grown serially in static broth, and expressed a subunit protein with an apparent molecular mass of 17 to 18.5 kDa, serologically related to the FIA major fimbrial subunit. There was a good correlation between MSHA and adherence to chicken tracheal sections. Isolates of group I o...

Research paper thumbnail of A small RNA promotes siderophore production through transcriptional and metabolic remodeling

Proceedings of the National Academy of Sciences, 2010

Siderophores are essential factors for iron (Fe) acquisition in bacteria during colonization and ... more Siderophores are essential factors for iron (Fe) acquisition in bacteria during colonization and infection of eukaryotic hosts, which restrain iron access through iron-binding protein, such as lactoferrin and transferrin. The synthesis of siderophores by Escherichia coli is considered to be fully regulated at the transcriptional level by the Fe-responsive transcriptional repressor Fur. Here we characterized two different pathways that promote the production of the siderophore enterobactin via the action of the small RNA RyhB. First, RyhB is required for normal expression of an important enterobactin biosynthesis polycistron, entCEBAH. Second, RyhB directly represses the translation of cysE, which encodes a serine acetyltransferase that uses serine as a substrate for cysteine biosynthesis. Reduction of CysE activity by RyhB allows serine to be used as building blocks for enterobactin synthesis through the nonribosomal peptide synthesis pathway. Thus, RyhB plays an essential role in siderophore production and may modulate bacterial virulence through optimization of siderophore production. enterobactin | iron | RyhB | sRNA

Research paper thumbnail of Cytokine mRNA expression in pigs infected with Schistosoma japonicum

Parasitology, 2001

The pig is a natural host of Schistosoma japonicum and a useful animal model of human disease. In... more The pig is a natural host of Schistosoma japonicum and a useful animal model of human disease. In the present study mRNA levels of Th1 (IFN-gamma) and Th2 (IL-10 and IL-4) cytokines were assessed by RT-PCR within tissues from infected pigs. Twelve Danish crossbred pigs were infected by intramuscular injection or orally with 1000 cercariae. Six other pigs served as non-infected controls. Liver and intestinal tissues were collected 10 weeks post-infection, and analysed for their relative levels of cytokine mRNA. Infected pigs developed a Th2 response as characterized by the increased level of mRNA encoding for IL-4 and IL-10 in their large intestine (caecum and colon). In contrast, levels of IFN-gamma did not differ between control and infected animals although variation between animals was observed. When comparing the immune response of orally and intramuscularly infected animals, we found that orally infected pigs produced higher IL-4 and IL-10 levels in their caecum and colon respectively. This stronger Th2 response correlated with a previously reported delay in maturation of infection following oral infection. The cytokine expression levels in tissue samples taken from lesion sites and in nearby areas, without obvious lesions, were then compared. Subsequent to an oral infection, the Th2 type cytokine production was higher in the lesion sites of the liver. In conclusion, this study is the first demonstration of IL-4 and IL-10 cytokine response in pig tissues during S. japonicum infection.

Research paper thumbnail of MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium

Molecular Microbiology, 2001

Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimuriu... more Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium ) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coli x7122 conferred mannoseresistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR-like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain x7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA-lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild-type S. typhimurium SL1344 or SR-11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a lowcopy-number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperatureand RpoS-independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by E. coli and S. typhimurium.

Research paper thumbnail of P-fimbriae-producing septicaemic Escherichia coli from poultry possess fel-related gene clusters whereas pap-hybridizing P-fimbriae-negative strains have partial or divergent P fimbrial gene clusters

Microbiology, 1996

CP 5000, Sa i nt-H yaci nt he, Quebec, Canada J2S 7C6

Research paper thumbnail of Characterization of Stg Fimbriae from an Avian Pathogenic Escherichia coli O78:K80 Strain and Assessment of Their Contribution to Colonization of the Chicken Respiratory Tract

Journal of Bacteriology, 2006

In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 stra... more In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 strain 7122, was found to be expressed in vivo in infected chicken tissues. The region encompassing ecs-3 carries a fimbrial gene cluster that is a putative ortholog of the stg fimbrial gene cluster of Salmonella enterica serovar Typhi. This APEC fimbrial gene cluster, which we have termed stg, is a member of a distinct group of related fimbriae that are located in the glmS-pstS intergenic region of certain E. coli and S. enterica strains. Under the control of the pBAD promoter, the production of Stg fimbriae was demonstrated by Western blotting and immunogold electron microscopy with E. coli K-12. Transcriptional fusions suggest that stg expression is influenced by the carbohydrate source and decreased by the addition of iron and that Fur plays a role in the regulation of stg expression. stg sequences were associated with APEC O78 isolates, and stg was phylogenetically distributed among E. coli reference strains and clinical isolates from human urinary tract infections. Stg fimbriae contributed to the adherence of a nonfimbriated E. coli K-12 strain to avian lung sections and human epithelial cells in vitro. Coinfection experiments with APEC strain 7122 and an isogenic ⌬stg mutant demonstrated that compared to the wild-type parent, the ⌬stg mutant was less able to colonize air sacs, equally able to colonize lungs, and able to more effectively colonize tracheas of infected chickens. Stg fimbriae, together with other adhesins, may therefore contribute to the colonization of avian respiratory tissues by certain APEC strains.

Research paper thumbnail of Klebsiella pneumoniae Yersiniabactin Promotes Respiratory Tract Infection through Evasion of Lipocalin 2

Infection and Immunity, 2011

Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especi... more Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especially due to K. pneumoniae carbapenemases (KPCs). K. pneumoniae must acquire iron to replicate, and it utilizes iron-scavenging siderophores, such as enterobactin (Ent). The innate immune protein lipocalin 2 (Lcn2) is able to specifically bind Ent and disrupt iron acquisition. To determine whether K. pneumoniae must produce Lcn2-resistant siderophores to cause disease, we examined siderophore production by clinical isolates (n = 129) from respiratory, urine, blood, and stool samples and by defined siderophore mutants through genotyping and liquid chromatography-mass spectrometry. Three categories of K. pneumoniae isolates were identified: enterobactin positive (Ent(+)) (81%), enterobactin and yersiniabactin positive (Ent(+) Ybt(+)) (17%), and enterobactin and salmochelin (glycosylated Ent) positive (Ent(+) gly-Ent(+)) with or without Ybt (2%). Ent(+) Ybt(+) strains were significantly overrepresented among respiratory tract isolates (P = 0.0068) and β-lactam-resistant isolates (P = 0.0019), including the epidemic KPC-producing clone multilocus sequence type 258 (ST258). In ex vivo growth assays, gly-Ent but not Ybt allowed evasion of Lcn2 in human serum, whereas siderophores were dispensable for growth in human urine. In a murine pneumonia model, an Ent(+) strain was an opportunistic pathogen that was completely inhibited by Lcn2 but caused severe, disseminated disease in Lcn2(-/-) mice. In contrast, an Ent(+) Ybt(+) strain was a frank respiratory pathogen, causing pneumonia despite Lcn2. However, Lcn2 retained partial protection against disseminated disease. In summary, Ybt is a virulence factor that is prevalent among KPC-producing K. pneumoniae isolates and promotes respiratory tract infections through evasion of Lcn2.

Research paper thumbnail of Increased Pho Regulon Activation Correlates with Decreased Virulence of an Avian Pathogenic Escherichia coli O78 Strain

Infection and Immunity, 2010

Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, sept... more Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, septicemia, cellulitis, peritonitis, and other conditions, since colibacillosis manifests in many ways. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the specific roles of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain 7122, different phoBR and pst mutant strains were tested in vivo in chickens and in vitro for virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were outcompeted by the wild-type parent, including strains containing a functional Pst system. Cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. In addition, loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of the APEC O78 strain 7122.

Research paper thumbnail of Relationship between the Tsh Autotransporter and Pathogenicity of Avian Escherichia coli and Localization and Analysis of the tsh Genetic Region

Infection and Immunity, 2000

The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins ... more The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain 7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the tsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with 7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain 7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain 7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of the Enterobacteriaceae. These results demonstrate that the tsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.