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Differential contribution of the repeats to heparin binding of HBHA, a major adhesin of Mycobacterium tuberculosis.

ABSTRACT: BACKGROUND: Tuberculosis remains one of the most important causes of global mortality and morbidity, and the molecular mechanisms of the pathogenesis are still incompletely understood. Only few virulence factors of the causative agent Mycobacterium tuberculosis are known. One of them is the heparin-binding haemagglutinin (HBHA), an important adhesin for epithelial cells and an extrapulmonary dissemination factor. HBHA mediates mycobacterial adherence to epithelial cells via the interactions of its C-terminal, lysine rich repeat domain with sulfated glycoconjugates on the surface of epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Using defined heparin sulfate (HS) analogs, we determined the minimal heparin fragment length for HBHA binding and structural adaptations of the HBHA heparin-binding domain (HBD) upon binding to heparin. The NMR studies show significant shifts of all residues in the HBD upon interaction with heparin, with stronger shifts in the last repeats compared to the upstream repeats, and indicated that the HS fragments with 14 sugar units cover the entire C-terminal lysine-rich domain of HBHA. The differential implication of the repeats is determined by the relative position of prolines and lysines within each repeat, and may contribute to binding specificity. GAG binding induces a non-homogeneous structural rearrangement in the HBD, with stabilization of a nascent ?-helix only in the last penta-repeats. CONCLUSION/SIGNIFICANCE: Mycobacterial HBHA undergoes structural adaptation upon interaction with GAGs, which is likely involved in binding specificities of the adhesin, and mycobacterial pathogens may use HBD polymorphisms for host or organ specificity. Further studies will aim at decoding the complementarity between HBD repeats and HS sequence.

SUBMITTER: Lebrun P

PROVIDER: S-EPMC3293801 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Differential contribution of the repeats to heparin binding of HBHA, a major adhesin of Mycobacterium tuberculosis.

Lebrun Pierre P Raze Dominique D Fritzinger Bernd B Wieruszeski Jean-Michel JM Biet Franck F Dose Alexander A Carpentier Mathieu M Schwarzer Dirk D Allain Fabrice F Lippens Guy G Locht Camille C

PloS one 20120305 3

<h4>Background</h4>Tuberculosis remains one of the most important causes of global mortality and morbidity, and the molecular mechanisms of the pathogenesis are still incompletely understood. Only few virulence factors of the causative agent Mycobacterium tuberculosis are known. One of them is the heparin-binding haemagglutinin (HBHA), an important adhesin for epithelial cells and an extrapulmonary dissemination factor. HBHA mediates mycobacterial adherence to epithelial cells via the interactio ...[more]

PMID: 22403657

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Project description: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.

Project description:BackgroundThe Mycobacterial heparin-binding hemagglutinin (HBHA) is an important latency-associated antigen that can be used to distinguish between latent tuberculosis infection (LTBI) and active tuberculosis (ATB). Although many studies were explored the efficiency of the HBHA-induced interferon-γ release assay (IGRA) in different populations, the clinical differential value of HBHA-IGRA is still controversial. Therefore, the aim of this study was to determine whether the HBHA-IGRA can be used as an efficient test for the discrimination of LTBI and ATB by a systematic review and meta-analysis.MethodsRelevant articles were retrieved from PubMed, Embase, Web of Science, and the Cochrane Library on Oct 18, 2020, with no start date limitation. The quality of each study was evaluated using Review Manager 5.4. The Stata MP v.14.0 software was used to combine sensitivity, specificity, likelihood ratio (LR), diagnostic odds ratio (DOR), summary receiver operating characteristic (SROC) curve, and area under SROC (AUC) to evaluate the diagnostic value of HBHA-IGRA for discrimination of LTBI and ATB. Meta-regression and subgroup analysis were performed for the sources of heterogeneity based on the selection criteria for active TB, the population, the TB burden, the type of antigen, the type of sample, and the time of antigen stimulation.ResultsA total of 13 studies (14 results) were included in this meta-analysis, including 603 ATB patients and 514 LTBI individuals. The pooled sensitivity and specificity of the HBHA-IGRA for discrimination of the LTBI and ATB were 0.70 (95% CI, 0.57~0.80) and 0.78 (95% CI, 0.71~0.84), respectively. The pooled positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were 3.15 (95%CI, 2.43~4.09), 0.39 (95% CI, 0.27~0.56), and 8.11 (95% CI, 4.81~13.67), respectively. The AUC was 0.81 (95% CI, 0.77~0.84). The subgroup analysis showed that the main source of heterogeneity was due to the HIV-infected population incorporated, and the different selection criteria of active TB subjects would also lead to the variation of the pooled sensitivity and specificity. Different TB burdens, HBHA antigen types, sample types, antigen stimulation time and BCG vaccination did not affect the heterogeneity in this analysis.ConclusionThe HBHA-IGRA is a promising immunodiagnostic test for discrimination of latent and active TB, which can be added in commercial IGRAs to enhance the differential diagnostic performance.

Project description:RD1-based Interferon-? Release Assays (IGRAs) cannot distinguish latent from active tuberculosis (TB) disease. Conversely, a positive response to heparin-binding haemagglutinin (HBHA)-based IGRAs, among TB-infected subjects, correlates with Mycobacterium tuberculosis (Mtb) containment and low risk of TB progression. The aim of this study was to characterize HBHA-immune responses in HIV-infected and uninfected subjects with active TB or latent TB infection (LTBI).49 subjects were prospectively enrolled: 22 HIV-uninfected (13 TB, 9 LTBI) and 27 HIV-infected (12 HIV-TB, 15 HIV-LTBI). Whole blood and peripheral blood mononuclear cells were stimulated with HBHA and RD1 antigens. Interferon (IFN)? release was evaluated by ELISA whereas cytokine profile [IFN?, tumor necrosis (TNF)?, interleukin (IL)2] and phenotype (CD45RA, CCR7) by flow cytometry.Among LTBI individuals, HBHA stimulation induced IFN? release in all the HIV-uninfected, while, only 4/15 HIV-infected responded. Within the active TB, only 5/13 HIV-uninfected and 1/12 HIV-TB patients responded. Interestingly, by cytometry we showed that CD4+ T-cells response to HBHA was significantly impaired in the HIV-infected subjects with TB or LTBI compared to the HIV-uninfected subjects. The phenotype of HBHA-specific CD4 T-cells showed a predominantly central memory (CM) and effector memory (EM) phenotype without differences among the groups. Differently, HBHA-specific CD8+ T-cells, showed mainly a CM and naïve phenotype in LTBI group while TB, HIV-LTBI and HIV-TB groups were characterized by EM or terminally differentiated phenotypes. Interestingly, differently than what observed for RD1, the cytokine profile of HBHA-specific T-cells evaluated by cytometry showed that the CD4+ T-cells were mostly monofunctional. Conversely, CD8-specific T-cells were mostly monofunctional for both HBHA and RD1 stimulations.These results characterize the impact of HIV infection in CD4- and CD8-specific response to HBHA in both LTBI and TB patients. HIV infection impairs the CD4 response to HBHA and likely this may lead to an impairment of TB control.

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Differential contribution of the repeats to heparin binding of HBHA, a major adhesin of Mycobacterium tuberculosis.

Publications

Differential contribution of the repeats to heparin binding of HBHA, a major adhesin of Mycobacterium tuberculosis.

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