Bacillus anthracis Targets Involved in Chemokine-Mediated Antimicrobial Activity

描述（由申请人提供）：炭疽芽孢杆菌参与趋化因子介导的抗菌活性的靶标趋化因子是趋化性细胞因子，通过协调白细胞迁移至感染部位而在宿主防御中发挥作用。然而，还发现许多趋化因子通过尚未确定的机制直接杀死一系列病原微生物。我们先前报道了干扰素诱导的CXC趋化因子CXCL 9、CXCL 10和CXCL 11阻断炭疽芽孢杆菌孢子萌发，降低孢子活力，并杀死营养细胞，其中CXCL 10最有效。我们还报道了对肺B.炭疽菌Sterne菌株感染的小鼠在孢子攻击后在其肺中产生显著水平的CXCL 9、CXCL 10和CXCL 11;而高度易感的A/J小鼠在肺部感染期间不产生显著水平的这些趋化因子。CXCL 9、CXCL 9/CXCL 10或CXCL 9/CXCL 10/CXCL 11的体内中和使C57 BL/6小鼠对肺炭疽易感，而它们的共享受体CXCR 3（其是在被CXCL 9、CXCL 10、CXCL 11募集到感染部位的白细胞上表达的受体）的中和对存活没有影响。这些发现支持CXCL 9、CXCL 10和CXCL 11对B具有直接的抗微生物作用。炭疽病的研究。为了鉴定CXCL 10的营养细胞靶标，我们筛选了B。anthracis转座子突变体文库中，发现ftsX，其编码广泛保守的原核ABC转运蛋白的跨膜蛋白的破坏，导致CXCL 10抗性表型。B中ftsX基因缺失。炭疽菌（即，DftsX）导致营养细胞对CXCL 10的抗性，而ftsX的互补恢复了CXCL 10的敏感性。相反，DftsX孢子对CXCL 10仍然敏感，表明孢子具有不同的CXCL 10靶标。进一步研究FtsX以及其他B的作用。为了研究CXCL 10的炭疽靶标，我们提出了三个具体目的：1）确定FtsX在营养细胞对CXCL 10的易感性中的作用; 2）鉴定CXCL 10的孢子靶标;和3）确定CXCL 10的孢子和营养细菌靶标在体内感染期间的作用。这些研究将为开发创新的治疗策略提供关键基础，以治疗不仅由B引起的感染。炭疽病，而且还有一系列致病的、潜在的多重耐药微生物。

DESCRIPTION (provided by applicant): Bacillus anthracis Targets Involved in Chemokine-Mediated Antimicrobial Activity Chemokines are chemotactic cytokines that function in host defense by orchestrating leukocyte migration to sites of infection. However, a number of chemokines have also been found to directly kill a range of pathogenic microorganisms through an as yet undefined mechanism. We previously reported that the interferon-inducible CXC chemokines, CXCL9, CXCL10, and CXCL11, block Bacillus anthracis spore germination, reduce spore viability, and kill vegetative cells, with CXCL10 being the most effective. We also reported that C57BL/6 mice, which are resistant to pulmonary B. anthracis Sterne strain infection, produced significant levels of CXCL9, CXCL10, and CXCL11 in their lungs following spore challenge; whereas, highly susceptible A/J mice did not generate significant levels of these chemokines during pulmonary infection. In vivo neutralization of CXCL9, CXCL9/CXCL10, or CXCL9/CXCL10/CXCL11 rendered C57BL/6 mice susceptible to pulmonary anthrax whereas neutralization of their shared receptor CXCR3, which is the receptor expressed on leukocytes recruited to the site of infection by CXCL9, CXCL10, CXCL11, had no impact on survival. These findings support that CXCL9, CXCL10, and CXCL11 have direct antimicrobial effects against B. anthracis both in vitro and in vivo. To identify the vegetative cell target(s) of CXCL10, we screened a B. anthracis transposon mutant library and found that disruption of ftsX, which encodes the transmembrane protein of a widely conserved prokaryotic ABC transporter, resulted in a CXCL10-resistant phenotype. Deletion of the ftsX gene in B. anthracis (i.e., DftsX) resulted in resistance of vegetative cells to CXCL10, and complementation of ftsX restored CXCL10 susceptibility. In contrast, DftsX spores remained susceptible to CXCL10, suggesting that spores have a different CXCL10 target. To further investigate the role of FtsX, as well as other B. anthracis targets of CXCL10, we propose three Specific Aims: 1) Determine the role of FtsX in susceptibility of vegetative cells to CXCL10; 2) Identify spore target(s) of CXCL10; and 3) Determine the role of spore and vegetative bacterial targets of CXCL10 during in vivo infection. These studies will provide a key foundation for the development of innovative therapeutic strategies for treating infections caused by not only B. anthracis but also a range of pathogenic, potentially multi-drug resistant microorganisms.