Novel feature of Mycobacterium avium subsp. paratuberculosis, highlighted by characterization of the heparin-binding hemagglutinin adhesin.

Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be distinguished by the types of HBHA they produce, which differ in size and adherence properties, thereby providing new evidence that strengthens the genotypic differences between the C and S types of M. avium subsp. paratuberculosis.

Specific IgG response against Mycobacterium avium paratuberculosis in children and adults with Crohn's disease.

BACKGROUND 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.

Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map. FINDINGS: For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris-HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein. CONCLUSIONS: An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays.

Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease.

Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease.

Differential expression of homing receptors and vascular addressins in tonsils and draining lymph nodes: Effect of Brucella infection in sheep.

The differential expression of homing receptors (HR) and complementary vascular addressins was studied in T and B lymphocytes from ovine tonsils and draining lymph nodes (LN) in uninfected and Brucella melitensis-infected sheep. In uninfected sheep, CD4+CD25+ T cells expressed proportionally more L-selectin and beta1 integrin than beta7 integrin in pharyngeal and palatine tonsils and in parotid LN (PLN), retropharyngeal LN (RLN) and the peripheral prescapular LN (PSLN). In contrast, memory CD4+CD45RA- T cells expressed an equivalent proportion of the three HR in PLN and PSLN, whereas beta1 and beta7 integrins were proportionally more expressed than L-selectin in pharyngeal tonsil. beta7 integrin was proportionally more expressed than beta1 integrin or L-selectin in palatine tonsils, RLN and the mucosal mesenteric LN (MLN). beta1 integrin was proportionally more expressed in IgG+ and IgA+ cells than beta7 integrin and L-selectin in tonsils, PLN and RLN. The main endothelial addressin expressed on venules in both pharyngeal and palatine tonsils, the PLN and RLN, as well as in the PSLN, was the peripheral PNAd, while in the MLN it was MAdCAM-1. Conjunctival infection by Brucella resulted in an increase of CD4+CD25+ and CD4+CD45RA- T cell subsets, which was associated to modifications of HR expression. CD4+CD45RA- T cells expressed proportionally more beta1 and beta7 integrins than L-selectin in regional PLN and RLN, but also in PSLN. The infection induced an increase of IgG+ and IgA+ cell percentages expressing beta1 integrin in all LN, and also beta7 integrin in the RLN. PNAd continued to be expressed on venules of tonsils and draining LN after Brucella infection, and MAdCAM-1 was also weakly expressed on RLN venules. These results suggest that lymphocyte trafficking through tonsils and draining LN could involve L-selectin/PNAd interactions, as well as beta1 or beta7 integrin, possibly in interaction with VCAM-1 or MAdCAM-1. The homing of antigen-specific lymphocytes in these tissues could be modulated after conjunctival infection with Brucella, which induces the recruitment of lymphocytes that express both beta1 and/or beta7 integrin in regional and more distant LN.

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.