Identification of sigB inhibitors in L. monocytogenes

The extensively studied intracellular pathogen, L. monocytogenes, is an ideal model for identifying small-molecule agents for treating bacterial infections. By selecting specific biological targets in L. monocytogenes, which are common to Gram-positive pathogens, we could extrapolate drug discovery information derived from this well-studied bacterium. Attenuating the pathogen’s virulence and stress response attributes without killing it, eliminates selective pressure caused by disruption of essential gene functions (as done by current antibiotics) and reduces the likelihood of developing microbes that are impervious to the effects of antibiotics. To this end, we have assessed multiple libraries of small organic compounds to identify inhibitors of L. monocytogenes σB, the alternative sigma factor common to several clinically relevant Gram-positive pathogens, such as Staphylococcus aureus, Bacillus cereus, and Bacillus anthracis. The role of σB as a transcriptional regulator of stress response and virulence makes it an ideal, well conserved target for chemotherapeutic development. Independent RNA isolations were performed for each growth experiment (log phase cells exposed to BHI+0.3M NaCl and BHI+0.3M NaCl + CMPD). Four biological replicates were used in competitive whole-genome microarray experiments. For the hybridizations, RNA from the wildtype parent strain L. monocytogenes 10403S were hybridized to RNA from the wildtype parent strain 10403S treated with CMPD.

被广泛研究的胞内致病菌单核细胞增生李斯特菌（Listeria monocytogenes）是筛选治疗细菌感染小分子制剂的理想模型。通过选取该菌中存在且在革兰氏阳性致病菌间保守的特定生物学靶点，我们可从这一被充分研究的菌株中推导药物开发相关信息。在不杀灭致病菌的前提下削弱其毒力与应激响应能力，可消除现有抗生素通过破坏必需基因功能所带来的选择压力，并降低微生物产生抗生素耐药性的概率。为此，我们对多个小分子有机化合物库进行了筛选，以鉴定单核细胞增生李斯特菌σB（sigma B）的抑制剂；σB是多种临床相关革兰氏阳性致病菌（如金黄色葡萄球菌、蜡样芽孢杆菌、炭疽芽孢杆菌）共有的替代sigma因子。σB作为调控应激响应与毒力的转录因子，其功能高度保守，是化疗药物开发的理想靶点。针对每一组生长实验——即分别暴露于脑心浸液培养基（BHI）+0.3M氯化钠，以及BHI+0.3M氯化钠+CMPD的对数生长期菌体——我们均独立完成了RNA提取。在竞争性全基因组微阵列实验中，我们设置了四次生物学重复。杂交环节中，我们将野生型亲本菌株单核细胞增生李斯特菌10403S的RNA，与经CMPD处理的同一野生型亲本菌株10403S的RNA进行杂交。