Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). RESULTS: By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power. CONCLUSION: Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service

The Mycobacterial Adhesins Heparin-Binding Hemagglutinin (HBHA) and Laminin-Binding Protein (LBP) are involved in Mycobacterium avium subsp. paratuberculosis attachment to epithelial cells

International audienceBackground: The current model of biology of paratuberculose proposes that after ingestion into the host, Mycobacterium avium subsp. paratuberculosis (MAP) crosses the intestinal barrier via internalization by the M cells. However, MAP may also transcytose the intestinal wall via the enterocytes, but the mechanisms and the bacterial factors involved in this process remain poorly understood. Adhesins such as Heparin-Binding HemAgglutinin (HBHA) and Laminin-Binding Protein (LBP), have been characterized in MAP. Objective: The aim of this study is to determine how these adhesins may promote the bacterial attachment to host cells. To achieve this, we examined the in vitro interaction between MAP and epithelial cells as well as the interaction between the adhesins and extracellular matrix molecules. Methods: MAP cytoadherence assays were performed on epithelial cells A549 in presence or absence of inhibitors. The binding activity of recombinants HBHA and LBP was investigated both by heparin-Sepharose chromatography and by in vitro adherence assays. Results: Cytoadherence assays revealed that pre-incubation of the bacteria in medium containing heparin inhibits the adhesion of MAP to A549 cells. In contrast, addition of laminin to the cell culture supernatant of the infected cells increased the percentage of bacterial adhesion. The in vitro assays confirmed the capacity of HBHA and LBP to promote the attachment of MAP to the extracellular matrix molecules of epithelial cells. Interestingly the adhesins HBHA and LBP expressed by the S and C types of MAP strains are different. This difference may be related to an adaptation to the host preference. Conclusion: We demonstrated that HBHA and LBP express by MAP are involved in its adherence to epithelial cells by two different mechanisms. However, role of these adhesins on MAP entry and survival into the cells remain to be investigated

Profil d’expression d’adhésines chez <em>Mycobacterium intracellulare</em>

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Post-translational modifications of the HBHA of <em></em>Mycobacterium avium<em></em> subsp. <em></em>paratuberculosis<em></em> revealed by mass spectrometry

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Molecular characterization of <it>Coxiella burnetii </it>isolates by infrequent restriction site-PCR and MLVA typing

Abstract Background Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). Results By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power. Conclusion Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service.</p

Specific IgG Response against <em>Mycobacterium avium paratuberculosis</em> in children and adults with Crohn's disease

International audienceBackground and Aims: Presence of serum antibodies against Mycobacterium avium paratuberculosis (MAP) in Crohn's Disease (CD) as a disease characteristic remains controversial. In the present work, we assessed antibody reactivity of serum and intestinal fluid against four distinct MAP-antigens, including the recently identified MAP-specific lipopentapeptide (L5P). Methods: Immunoglobulin concentrations and specificity against 3 non MAP-specific antigens: glycosyl-transferase-d (GSD), purified protein derivative from MAP (Johnin-PPD), heparin binding haemagglutinin (MAP-HBHA) and one MAP-specific antigen: synthetic L5P were determined by ELISA in gut lavage fluids from adult controls or patients with CD, and in sera of children or adult controls or patients with CD, ulcerative colitis or celiac disease. Results: Total IgA and IgG concentrations were increased in sera of children with CD but were decreased in sera of adults with CD, thereof specificity against MAP antigens was assessed by normalizing immunoglobulin concentrations between samples. In CD patients, IgG reactivity was increased against the four MAP antigens, including L5P in gut lavage fluids but it was only increased against L5P in sera. By contrast, anti-L5P IgG were not increased in patients with ulcerative colitis or celiac disease. Conclusions: A significant increase in anti-L5P IgG is observed in sera of children and adults with CD but not in patients with other intestinal inflammatory diseases. Anti-L5P antibodies may serve as serological marker for CD

Heterogeneity of subspecies <em>Mycobacterium avium paratuberculosis</em> from genotype to phenotype

International audienceBackground - In the subspecies M. avium ssp. paratuberculosis (Map) two groups, known as Cattle (C) and Sheep (S), have been defined by genotyping. Recent studies show that Map C and S have different phenotypes with respect to infection of macrophages and iron metabolism. Map is adapted to the gastrointestinal tract of ruminant, but the mechanism of entry is currently unknown. In this study, we investigated the phenotype of the Map-host interaction, involving the virulence factor heparin-binding hemagglutinin (HBHA), for both groups of Map. HBHA is described in M. tuberculosis as a major adhesin required for extra pulmonary dissemination of the tubercle bacillus. Method - A large collection of Map isolates (types C & S) were genotyped by MIRU-VNTR and RFLP-IS900. The polymorphism of the hbha gene was investigated by fragment analysis using GcneN:apper technology. Structure-functions properties of recombinant HBHA (types C & S) were analyzed by Heparin Sepharose chromatography and SPR analysis based on Biacore technology. Results - In silico analyses of both types of Map have revealed two forms of hbha. This observation, showing that hbha is distinct according to the group, was confirmed using GeneMapper on 83 Map strains (65 Map C & 18 Map S) with various genotypes. We found that Map type C produces HBHA with a short C-terminal domain, while that of typeS presents a long (-terminal domain, similar to that of HBHA produced by M. tuberculosis. The purification of HBHA from Map type C and S by Heparin-sepharose chromatography highlighted a correlation between their affinities to heparin and the length of their C-terminal domain confirmed by Biacore analysis. Conclusion - We show for the first time that the types C and S of Map may be distinguished by the type of HBHA they produce, which differs in size and adherence properties. Thus, HBHA participates in the genotypic and phenotypic differences observed between the C and S types of Map

Adherence phenotype of HBHA produced by <em>Mycobacterium avium</em> subsp <em>paratuberculosis</em> type C and S

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IgG and IgA specificity against MAP-antigens in GLF from adults with or without CD.

<p><b>(A)</b> IgG specificity assessed by ELISA after normalizing IgG concentration for GSD (7 controls, 22 CD), MAP-HBHA (6 controls, 20 CD), Johnin-PPD (7 controls, 22 CD), L5P (9 controls, 22 CD). <b>(B)</b> IgA specificity assessed by ELISA after normalizing IgA concentration for GSD (16 controls, 20 CD), MAP-HBHA (20 controls, 23 CD), Johnin-PPD (19 controls, 18 CD), L5P (19 controls, 23 CD). Horizontal dashed lines indicate the threshold for specificity corresponding to 3 blanks.</p