Bacillus anthracis Targets Involved in Chemokine-Mediated Antimicrobial Activity

描述(申请人提供)：炭疽芽孢杆菌靶标参与趋化因子介导的抗微生物活性趋化因子是趋化细胞因子，通过协调白细胞迁移到感染部位而在宿主防御中发挥作用。然而，一些趋化因子也被发现通过一种尚未确定的机制直接杀死一系列病原微生物。我们以前曾报道过干扰素诱导的CXC趋化因子CXCL9、CXCL10和CXCL11可以阻止炭疽芽孢杆菌的孢子萌发，降低孢子活力，杀死营养细胞，其中CXCL10效果最好。我们还报道了对炭疽杆菌肺炎株感染具有抵抗力的C57BL/6小鼠在孢子攻击后在肺中产生了显著水平的CXCL9、CXCL10和CXCL11；而高度敏感的A/J小鼠在肺部感染过程中不产生显著水平的这些趋化因子。体内中和CXCL9、CXCL9/CXCL10或CXCL9/CXCL10/CXCL11使C57BL/6小鼠对肺炭疽病易感，而中和CXCL9、CXCL10、CXCL11感染部位的白细胞上表达的共同受体CXCR3对小鼠的存活没有影响。这些结果支持CXCL9、CXCL10和CXCL11在体内外对炭疽杆菌具有直接的抗菌作用。为了确定CXCL10的营养细胞靶标(S)，我们筛选了炭疽杆菌转座子突变体文库，发现编码广泛保守的原核生物ABC转运蛋白跨膜蛋白的ftsX的破坏导致了CXCL10抗性的表型。炭疽杆菌中ftsX基因的缺失(即DftsX)导致营养细胞对CXCL10产生抗性，而ftsX的互补恢复了CXCL10的敏感性。相反，DftsX孢子仍然对CXCL10敏感，这表明孢子有不同的CXCL10靶标。为了进一步研究FtsX的作用，以及CXCL10的其他炭疽杆菌靶标，我们提出了三个特定的目标：1)确定FtsX在营养细胞对CXCL10易感性中的作用；2)确定CXCL10的孢子靶标(S)；以及3)确定CXCL10的孢子和营养细菌靶标在体内感染过程中的作用。这些研究将为开发创新的治疗策略提供关键基础，以治疗不仅由炭疽杆菌引起的感染，还包括一系列致病的、可能具有多药耐药性的微生物。

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.