HAL (Le Centre pour la Communication Scientifique Directe), Dec 1, 1999
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. We have developed an original model of macrophage activation where TDM is injected in vivo to prime peritoneal macrophages. These primed macrophages do not express inducible NO synthase (NOS II), however, they can be fully activated, i.e. induced to express NOS II and to develop a NOS II-dependent antiproliferative activity, following in vitro exposure to low concentrations of LPS. In a previous paper, we have shown that TDM-priming of mouse peritoneal macrophages is mediated by the sequential production of IL-12 and IFN-gamma. In the present paper, we investigated the role of TNF in the priming of macrophages by TDM. By semi-quantitative RT-PCR, we have shown that TDM injection induced transcription of TNF-alpha in peritoneal cells. TNF-mRNA levels peaked 5 hours after TDM injection and remained elevated for at least 32 hours. TNF expression was absolutely necessary for macrophage priming, as injection of an anti-TNF monoclonal antibody, 4 h before and 20 hours after TDM injection, prevented LPS-dependent activation of macrophages in vitro. This result was confirmed by the inability of TDM to prime macrophages from LT-alpha/TNF-alpha knockout (LT/TNFKO) mice. In addition, analysis of LT/TNFKO mice treated with TDM revealed that induction of the IL-12 transcript in their peritoneal cells and expression of a functional NADPH oxidase in macrophages are TNF-independent events.
Veterinary Immunology and Immunopathology, Sep 1, 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 ...
Applied and Environmental Microbiology, Sep 1, 2012
Genetic complementation in many bacteria is commonly achieved by reintroducing functional copies ... more Genetic complementation in many bacteria is commonly achieved by reintroducing functional copies of the mutated or deleted genes on a recombinant plasmid. Chromosomal integration systems using the Tn7 transposon have the advantage of providing a stable single-copy integration that does not require selective pressure. Previous Tn7 systems have been developed, although none have been shown to work effectively in a variety of enterobacteria. We have developed several mini-Tn7 and transposase vectors to provide a more versatile system. Transposition of Tn7 at the chromosomal attTn7 site was achieved by a classical conjugation approach, wherein the donor strain harbored the mini-Tn7 vector and the recipient strain possessed the transposase vector. This approach was efficient for five different pathogenic enterobacterial species. Thus, this system provides a useful tool for single-copy complementation at an episomal site for research in bacterial genetics and microbial pathogenesis. Furthermore, these vectors could also be used for the introduction of foreign genes for use in biotechnology applications, vaccine development, or gene expression and gene fusion constructs.
The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein ... more The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDAassociated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA-I, which comprises imperfect repeats of a 19-amino-acid consensus sequence and is predicted to form a b-helix. Here, we show that Aah homologues can be found in many Gram-negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA-like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19-amino-acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of b-helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a b-helical polypeptide lacking the 19-amino-acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif.
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 (M) 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 M. 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-␥) 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 levels at 39 h postinjection. TDM was also able to induce IFN-␥ expression; however, kinetics of induction of IFN-␥ was different from that of IL-12p40. Maximal levels of IFN-␥ mRNA were reached by 24 h and did not return to baseline by 4 days. In addition, pretreatment of mice with neutralizing monoclonal antibodies directed against IL-12 (C15.6.7 and C15.1.2) blocked IFN-␥ mRNA induction in PEC from TDM-treated mice. We further determined if the induction of IL-12 and/or IFN-␥ contributes to the in vivo priming effect of TDM on peritoneal M. TDM-injected mice were treated in vivo with anti-IL-12 or anti-IFN-␥ (XMG.1.6) monoclonal antibodies. TDM-primed M were then activated in vitro with LPS and tested for their ability to produce NO and to develop cytostatic activity toward cocultivated L1210 tumor cells. Priming of M by TDM was completely blocked by in vivo neutralization of either IL-12 or IFN-␥ as demonstrated by an absence of tumoricidal activity and NO production by TDM-elicited M in the presence of LPS. Taken together our results show that TDM, a defined molecule from M. tuberculosis, induces in vivo production of IL-12. Moreover, synthesis of IL-12 mediates TDM priming of mouse peritoneal M through IFN-␥ induction.
Schistosomiasis threatens hundreds of millions of people worldwide. The larval stage of Schistoso... more Schistosomiasis threatens hundreds of millions of people worldwide. The larval stage of Schistosoma mansoni migrates through the lung and adult worms reside adjacent to the colonic mucosa. Several candidate vaccines are in preclinical development, but none is designed to elicit both systemic and mucosal responses. We have repurposed an attenuated Salmonella enterica Typhimurium strain (YS1646) to express Cathepsin B (CatB), a digestive enzyme important for the juvenile and adult stages of the S. mansoni life cycle. Previous studies have demonstrated the prophylactic and therapeutic efficacy of our plasmid-based vaccine. Here, we have generated chromosomally integrated (CI) YS1646 strains that express CatB to produce a viable candidate vaccine for eventual human use (stability, no antibiotic resistance). 6-8-week-old C57BL/6 mice were vaccinated in a multimodal oral (PO) and intramuscular (IM) regimen, and then sacrificed 3 weeks later. The PO + IM group had significantly higher anti-CatB IgG titers with greater avidity and mounted significant intestinal anti-CatB IgA responses compared to PBS control mice (all P < 0.0001). Multimodal vaccination generated balanced T H 1/T H 2 humoral and cellular immune responses. Production of IFNγ by both CD4 + and CD8 + T cells was confirmed by flow cytometry (P < 0.0001 & P < 0.01). Multimodal vaccination reduced worm burden by 80.4%, hepatic egg counts by 75.2%, and intestinal egg burden by 78.4% (all P < 0.0001). A stable and safe vaccine that has both prophylactic and therapeutic activity would be ideal for use in conjunction with praziquantel mass treatment campaigns.
Since its first description in 1982, the zoonotic life-threatening Shiga toxin-producing Escheric... more Since its first description in 1982, the zoonotic life-threatening Shiga toxin-producing Escherichia coli O157:H7 has emerged as an important food- and water-borne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. In the last decade, increases in E. coli O157:H7 outbreaks were associated with environmental contamination in water and through fresh produce such as green leaves or vegetables. Both intrinsic (genetic adaptation) and extrinsic factors may contribute and help E. coli O157:H7 to survive in adverse environments. This makes it even more difficult to detect and monitor food and water safety for public health surveillance. E. coli O157:H7 has evolved in behaviors and strategies to persist in the environment.
Extraintestinal pathogenic E. coli (ExPEC) are responsible for many infectious diseases in livest... more Extraintestinal pathogenic E. coli (ExPEC) are responsible for many infectious diseases in livestock, such as airsacculitis in poultry, acute mastitis in dairy animals and neonatal septicaemia and urinary tract infections (UTI) in pigs and cattle. In their animal hosts, ExPEC have to cope with low iron availability. By using different strategies, ExPEC strains are able to retrieve iron sequestered by host proteins. One of these strategies is the use of siderophores, which are small secreted molecules with high affinity for iron. ExPEC are known to synthesize up to four different types of siderophores: enterobactin, salmochelins, yersiniabactin and aerobactin. Steps required for iron acquisition by siderophores include (1) siderophore synthesis in the cytoplasm, (2) siderophore secretion, (3) ferri-siderophore reception, (4) ferri-siderophore internalization and (5) iron release in the cytoplasm. Each siderophore has specific properties and may be differentially regulated to provide different advantages, potentially allowing ExPEC to adapt to different environmental conditions or to overcome host innate immunity. Iron acquisition by siderophores plays a significant role in ExPEC virulence and, as it requires outer membrane receptors, it constitutes an interesting target for the development of vaccines that could be used to limit the number of infectious diseases due to ExPEC in livestock.
Transposon (TnphoA) mutagenesis was used to study the expression of F165(1) fimbriae, related to ... more Transposon (TnphoA) mutagenesis was used to study the expression of F165(1) fimbriae, related to Prs fimbriae, in the pathogenic Escherichia coli strain 5131 (O115:K &amp;amp;amp;amp;amp;amp;quot;V165&amp;amp;amp;amp;amp;amp;quot;:F165). This strain causes septicemia in swine and also expresses F165(2) fimbriae, related to F1C. Adhesin-defective mutants from the wild-type pathogenic strain were produced and TnphoA insertions were localized either in the f165(1)A gene, which encodes the major fimbrial subunit or in the f165(1)E, gene, which encodes a minor fimbrial subunit. TnphoA gene fusions were used to measure expression of F165(1) fimbrial genes. Similar pattern of regulation of expression was observed in both f165(1)A and f165(1)E genes. Optimal expression of F165(1) fimbriae was obtained on solid minimal medium. Production of F165(1) fimbriae was negatively regulated by addition of glucose, leucine or alanine to the media, by growth at 18 degrees C, and by pH above or below 7.0.
HAL (Le Centre pour la Communication Scientifique Directe), Dec 1, 1999
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. We have developed an original model of macrophage activation where TDM is injected in vivo to prime peritoneal macrophages. These primed macrophages do not express inducible NO synthase (NOS II), however, they can be fully activated, i.e. induced to express NOS II and to develop a NOS II-dependent antiproliferative activity, following in vitro exposure to low concentrations of LPS. In a previous paper, we have shown that TDM-priming of mouse peritoneal macrophages is mediated by the sequential production of IL-12 and IFN-gamma. In the present paper, we investigated the role of TNF in the priming of macrophages by TDM. By semi-quantitative RT-PCR, we have shown that TDM injection induced transcription of TNF-alpha in peritoneal cells. TNF-mRNA levels peaked 5 hours after TDM injection and remained elevated for at least 32 hours. TNF expression was absolutely necessary for macrophage priming, as injection of an anti-TNF monoclonal antibody, 4 h before and 20 hours after TDM injection, prevented LPS-dependent activation of macrophages in vitro. This result was confirmed by the inability of TDM to prime macrophages from LT-alpha/TNF-alpha knockout (LT/TNFKO) mice. In addition, analysis of LT/TNFKO mice treated with TDM revealed that induction of the IL-12 transcript in their peritoneal cells and expression of a functional NADPH oxidase in macrophages are TNF-independent events.
Veterinary Immunology and Immunopathology, Sep 1, 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 ...
Applied and Environmental Microbiology, Sep 1, 2012
Genetic complementation in many bacteria is commonly achieved by reintroducing functional copies ... more Genetic complementation in many bacteria is commonly achieved by reintroducing functional copies of the mutated or deleted genes on a recombinant plasmid. Chromosomal integration systems using the Tn7 transposon have the advantage of providing a stable single-copy integration that does not require selective pressure. Previous Tn7 systems have been developed, although none have been shown to work effectively in a variety of enterobacteria. We have developed several mini-Tn7 and transposase vectors to provide a more versatile system. Transposition of Tn7 at the chromosomal attTn7 site was achieved by a classical conjugation approach, wherein the donor strain harbored the mini-Tn7 vector and the recipient strain possessed the transposase vector. This approach was efficient for five different pathogenic enterobacterial species. Thus, this system provides a useful tool for single-copy complementation at an episomal site for research in bacterial genetics and microbial pathogenesis. Furthermore, these vectors could also be used for the introduction of foreign genes for use in biotechnology applications, vaccine development, or gene expression and gene fusion constructs.
The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein ... more The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDAassociated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA-I, which comprises imperfect repeats of a 19-amino-acid consensus sequence and is predicted to form a b-helix. Here, we show that Aah homologues can be found in many Gram-negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA-like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19-amino-acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of b-helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a b-helical polypeptide lacking the 19-amino-acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif.
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 (M) 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 M. 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-␥) 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 levels at 39 h postinjection. TDM was also able to induce IFN-␥ expression; however, kinetics of induction of IFN-␥ was different from that of IL-12p40. Maximal levels of IFN-␥ mRNA were reached by 24 h and did not return to baseline by 4 days. In addition, pretreatment of mice with neutralizing monoclonal antibodies directed against IL-12 (C15.6.7 and C15.1.2) blocked IFN-␥ mRNA induction in PEC from TDM-treated mice. We further determined if the induction of IL-12 and/or IFN-␥ contributes to the in vivo priming effect of TDM on peritoneal M. TDM-injected mice were treated in vivo with anti-IL-12 or anti-IFN-␥ (XMG.1.6) monoclonal antibodies. TDM-primed M were then activated in vitro with LPS and tested for their ability to produce NO and to develop cytostatic activity toward cocultivated L1210 tumor cells. Priming of M by TDM was completely blocked by in vivo neutralization of either IL-12 or IFN-␥ as demonstrated by an absence of tumoricidal activity and NO production by TDM-elicited M in the presence of LPS. Taken together our results show that TDM, a defined molecule from M. tuberculosis, induces in vivo production of IL-12. Moreover, synthesis of IL-12 mediates TDM priming of mouse peritoneal M through IFN-␥ induction.
Schistosomiasis threatens hundreds of millions of people worldwide. The larval stage of Schistoso... more Schistosomiasis threatens hundreds of millions of people worldwide. The larval stage of Schistosoma mansoni migrates through the lung and adult worms reside adjacent to the colonic mucosa. Several candidate vaccines are in preclinical development, but none is designed to elicit both systemic and mucosal responses. We have repurposed an attenuated Salmonella enterica Typhimurium strain (YS1646) to express Cathepsin B (CatB), a digestive enzyme important for the juvenile and adult stages of the S. mansoni life cycle. Previous studies have demonstrated the prophylactic and therapeutic efficacy of our plasmid-based vaccine. Here, we have generated chromosomally integrated (CI) YS1646 strains that express CatB to produce a viable candidate vaccine for eventual human use (stability, no antibiotic resistance). 6-8-week-old C57BL/6 mice were vaccinated in a multimodal oral (PO) and intramuscular (IM) regimen, and then sacrificed 3 weeks later. The PO + IM group had significantly higher anti-CatB IgG titers with greater avidity and mounted significant intestinal anti-CatB IgA responses compared to PBS control mice (all P < 0.0001). Multimodal vaccination generated balanced T H 1/T H 2 humoral and cellular immune responses. Production of IFNγ by both CD4 + and CD8 + T cells was confirmed by flow cytometry (P < 0.0001 & P < 0.01). Multimodal vaccination reduced worm burden by 80.4%, hepatic egg counts by 75.2%, and intestinal egg burden by 78.4% (all P < 0.0001). A stable and safe vaccine that has both prophylactic and therapeutic activity would be ideal for use in conjunction with praziquantel mass treatment campaigns.
Since its first description in 1982, the zoonotic life-threatening Shiga toxin-producing Escheric... more Since its first description in 1982, the zoonotic life-threatening Shiga toxin-producing Escherichia coli O157:H7 has emerged as an important food- and water-borne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. In the last decade, increases in E. coli O157:H7 outbreaks were associated with environmental contamination in water and through fresh produce such as green leaves or vegetables. Both intrinsic (genetic adaptation) and extrinsic factors may contribute and help E. coli O157:H7 to survive in adverse environments. This makes it even more difficult to detect and monitor food and water safety for public health surveillance. E. coli O157:H7 has evolved in behaviors and strategies to persist in the environment.
Extraintestinal pathogenic E. coli (ExPEC) are responsible for many infectious diseases in livest... more Extraintestinal pathogenic E. coli (ExPEC) are responsible for many infectious diseases in livestock, such as airsacculitis in poultry, acute mastitis in dairy animals and neonatal septicaemia and urinary tract infections (UTI) in pigs and cattle. In their animal hosts, ExPEC have to cope with low iron availability. By using different strategies, ExPEC strains are able to retrieve iron sequestered by host proteins. One of these strategies is the use of siderophores, which are small secreted molecules with high affinity for iron. ExPEC are known to synthesize up to four different types of siderophores: enterobactin, salmochelins, yersiniabactin and aerobactin. Steps required for iron acquisition by siderophores include (1) siderophore synthesis in the cytoplasm, (2) siderophore secretion, (3) ferri-siderophore reception, (4) ferri-siderophore internalization and (5) iron release in the cytoplasm. Each siderophore has specific properties and may be differentially regulated to provide different advantages, potentially allowing ExPEC to adapt to different environmental conditions or to overcome host innate immunity. Iron acquisition by siderophores plays a significant role in ExPEC virulence and, as it requires outer membrane receptors, it constitutes an interesting target for the development of vaccines that could be used to limit the number of infectious diseases due to ExPEC in livestock.
Transposon (TnphoA) mutagenesis was used to study the expression of F165(1) fimbriae, related to ... more Transposon (TnphoA) mutagenesis was used to study the expression of F165(1) fimbriae, related to Prs fimbriae, in the pathogenic Escherichia coli strain 5131 (O115:K &amp;amp;amp;amp;amp;amp;quot;V165&amp;amp;amp;amp;amp;amp;quot;:F165). This strain causes septicemia in swine and also expresses F165(2) fimbriae, related to F1C. Adhesin-defective mutants from the wild-type pathogenic strain were produced and TnphoA insertions were localized either in the f165(1)A gene, which encodes the major fimbrial subunit or in the f165(1)E, gene, which encodes a minor fimbrial subunit. TnphoA gene fusions were used to measure expression of F165(1) fimbrial genes. Similar pattern of regulation of expression was observed in both f165(1)A and f165(1)E genes. Optimal expression of F165(1) fimbriae was obtained on solid minimal medium. Production of F165(1) fimbriae was negatively regulated by addition of glucose, leucine or alanine to the media, by growth at 18 degrees C, and by pH above or below 7.0.
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Papers by Charles Dozois