INRA-Centre Val de Loire, UMR 1282, Infectiologie, Santé Publique (ISP-311), Nouzilly, France.
J Bacteriol. 2013 Nov;195(21):4844-53. doi: 10.1128/JB.00671-13. Epub 2013 Aug 23.
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.
分支杆菌副结核分枝杆菌包括两个在基因型上定义明确的群体，称为牛（C）和绵羊（S）群体。最近的研究报告称，分支杆菌副结核分枝杆菌 C 组和 S 组之间存在表型差异，包括生长速度、对巨噬细胞的感染力和铁代谢。在这项研究中，我们研究了 C 组和 S 组分支杆菌副结核分枝杆菌的毒力因子肝素结合血凝素粘附素（HBHA）的基因型和生物学特性。在结核分枝杆菌中，HBHA 是一种主要的粘附素，参与分枝杆菌与宿主的相互作用和感染的肺外传播。为了研究分支杆菌副结核分枝杆菌中的 HBHA，我们使用 GeneMapper 技术通过片段分析研究了 hbhA 多态性，该技术跨越了通过分枝杆菌插入重复单元-可变数串联重复（MIRU-VNTR）和 IS900 限制性片段长度多态性（IS900-RFLP）分析进行基因分型的大型分离株。此外，我们通过肝素-琼脂糖色谱和表面等离子体共振（SPR）分析分析了 C 型和 S 型重组 HBHA 蛋白的结构-功能关系。计算机分析显示，有两种形式的 HBHA，分别对应于分支杆菌副结核分枝杆菌 C 型和 S 型的原型基因组。这一观察结果在使用 GeneMapper 对 85 株分支杆菌副结核分枝杆菌菌株进行分析时得到了证实，其中包括 67 株 C 型和 18 株 S 型。我们发现，所有 C 型菌株的 HBHAs 都含有短的 C 末端结构域，而 S 型的 HBHAs 则含有长的 C 末端结构域，类似于分支杆菌副结核分枝杆菌产生的。通过肝素-琼脂糖色谱从两种类型的分支杆菌副结核分枝杆菌中纯化重组 HBHA 突出了它们与肝素亲和力和 C 末端结构域长度之间的相关性，这一相关性通过 SPR 分析得到了证实。因此，分支杆菌副结核分枝杆菌的 C 型和 S 型可能通过它们产生的 HBHA 类型来区分，这些 HBHA 在大小和粘附特性上有所不同，从而提供了新的证据，加强了 C 型和 S 型分支杆菌副结核分枝杆菌之间的基因型差异。
