Laboratory of Bacterial Genomics, Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Infect Immun. 2013 Jun;81(6):2242-57. doi: 10.1128/IAI.01273-12. Epub 2013 Apr 8.
Mycobacterium avium subsp. paratuberculosis causes Johne's disease, an enteric infection in cattle and other ruminants, greatly afflicting the dairy industry worldwide. Once inside the cell, M. avium subsp. paratuberculosis is known to survive harsh microenvironments, especially those inside activated macrophages. To improve our understanding of M. avium subsp. paratuberculosis pathogenesis, we examined phagosome maturation associated with transcriptional responses of M. avium subsp. paratuberculosis during macrophage infection. Monitoring cellular markers, only live M. avium subsp. paratuberculosis bacilli were able to prevent phagosome maturation and reduce its acidification. On the transcriptional level, over 300 M. avium subsp. paratuberculosis genes were significantly and differentially regulated in both naive and IFN-γ-activated macrophages. These genes include the sigma factor H (sigH) that was shown to be important for M. avium subsp. paratuberculosis survival inside gamma interferon (IFN-γ)-activated bovine macrophages. Interestingly, an sigH-knockout mutant showed increased sensitivity to a sustained level of thiol-specific oxidative stress. Large-scale RNA sequence analysis revealed that a large number of genes belong to the sigH regulon, especially following diamide stress. Genes involved in oxidative stress and virulence were among the induced genes in the sigH regulon with a putative consensus sequence for SigH binding that was recognized in a subset of these genes (n = 30), suggesting direct regulation by SigH. Finally, mice infections showed a significant attenuation of the ΔsigH mutant compared to its parental strain, suggesting a role for sigH in M. avium subsp. paratuberculosis virulence. Such analysis could identify potential targets for further testing as vaccine candidates against Johne's disease.
分支杆菌副结核亚种引起的副结核病是一种牛和其他反刍动物的肠感染，对全球的乳制品行业造成了极大的困扰。一旦进入细胞内，分支杆菌副结核亚种已知能够在恶劣的微环境中存活，特别是在活化的巨噬细胞内。为了更好地了解分支杆菌副结核亚种的发病机制，我们研究了吞噬体成熟与分支杆菌副结核亚种在巨噬细胞感染期间的转录反应相关联的情况。通过监测细胞标记物，只有活的分支杆菌副结核亚种才能阻止吞噬体成熟并降低其酸化。在转录水平上，超过 300 个分支杆菌副结核亚种基因在未活化和 IFN-γ 活化的巨噬细胞中均有显著差异的调节。这些基因包括σ因子 H（sigH），它被证明对分支杆菌副结核亚种在 γ干扰素（IFN-γ）活化的牛巨噬细胞中的存活很重要。有趣的是，sigH 敲除突变体对持续的硫醇特异性氧化应激表现出更高的敏感性。大规模 RNA 序列分析表明，大量基因属于 sigH 调控子，尤其是在二酰胺应激后。氧化应激和毒力相关基因是 sigH 调控子中诱导基因的一部分，在这些基因中有一个用于 SigH 结合的假定共有序列（n = 30），这表明 SigH 直接调节这些基因。最后，小鼠感染实验表明，与亲本菌株相比，ΔsigH 突变体的感染明显减弱，这表明 sigH 在分支杆菌副结核亚种的毒力中起作用。这种分析可以确定潜在的目标，进一步测试作为抗副结核病的疫苗候选物。
