Novel Feature of Mycobacterium avium subsp. paratuberculosis, Highlighted by Characterization of the Heparin-Binding Hemagglutinin Adhesin

Abstract

j 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.

Coordinate regulation of virulence and metabolic genes by the transcription factor HbhR in Mycobacterium marinum

“…In addition to the M. tuberculosis complex, HBHA‐like proteins have been detected in M. avium (Lefrancois et al , ), M. leprae (de Lima et al , ), Mycobacterium smegmatis (Biet et al , ) and hbhA homologs are found in all sequenced mycobacterial genomes, suggesting that this protein plays an important role in the physiology or virulence of most mycobacteria. Despite this, the biogenesis of HBHA remains poorly understood.…”

Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review

“…The heparin binding haemagglutinin (HBHG) is one such adhesion located on the surface of the mycobacterium, which has been shown to mediate the binding of the bacterium to epithelial cells and fibroblasts [ 82 ]. The C-terminal regions of the hbhA gene differ between Type S and Type C Map strains mainly in deletions or differences in the lysine-rich repeats, which are important for the binding of HBHA to HS-GAG [ 83 ]. No differences could be found between the Type S subtypes I and III.…”

“…No differences could be found between the Type S subtypes I and III. The HBHA proteins from Map Type S and Type C strains were found to exhibit different binding activity with sulphated glycoconjugates, with Type S strains having the highest binding affinity correlating with a greater number of lysine-rich repeats [ 83 ]. Since HS-GAG structures differ according to both hosts and organs, it is possible that mycobacterial pathogens use heparin-binding domain variability to define their host preference or tropism for the intestine.…”

Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide

“…On the transcriptional level, C‐ and S‐type strains exposed to low iron or heat stress conditions had different mRNA expression patterns (Gumber and Whittington, ). Furthermore, iron storage in low iron conditions was only observed in the C‐type but not S‐type strains (Janagama et al ., ) and virulence adhesin differences were characterized (Lefrancois et al ., ). In this study, differences in a lipopeptide that is a component of the mycobacterial cell envelope were identified between C‐ and S‐type strains.…”

“…The S‐type isolates are readily distinguishable from C‐type isolates based on genome sequencing studies (Li et al ., ; Bannantine et al ., ). But these two lineages can also be readily discriminated by genotyping methods due to single nucleotide polymorphisms (Marsh et al ., ) as well as deletions/insertions of large DNA segments (termed large sequence polymorphisms or LSP) using phylogenetic techniques such as variable number tandem repeats (Lefrancois et al ., ), single sequence repeats (Amonsin et al ., ; Thibault et al ., ), representational difference analysis (Dohmann et al ., ) and hsp65 sequencing (Turenne et al ., ). Furthermore, genomic hybridization of S‐type strains on a C‐type microarray revealed a large 23‐gene deletion in S‐type strains (Marsh et al ., ).…”