Disrupting Biofilm Formation to Improve TB Drug Treatment

描述（由申请人提供）：结核分枝杆菌（Mtb）感染导致肺部和其他组织的慢性炎症，目前的结核病（TB）药物难以治疗。有效治疗活动性结核病患者的挑战是由于在肉芽肿中持续存在的耐药杆菌存在干酪样坏死或空化。现有的治疗方案往往因毒副作用而复杂化，包括数月或数年的联合抗微生物药物，这被认为是有效根除耐药杆菌所必需的。我们提出了一种新的结核病治疗策略，利用一类小分子量抗生物膜化合物与传统结核病药物联合治疗耐药结核。这一策略可以通过更快地根除持久性耐药杆菌，显著提高目前结核病药物治疗的疗效。我们的体外和体内数据表明，结核分枝杆菌形成细胞外生物膜样微生物群落，作为结核药物治疗存活的一种策略。通过对豚鼠结核模型的研究，我们确定这些耐药杆菌在与裂解或坏死的白细胞相关时采用生物膜样模式存在。为了模拟这种独特的体内微环境，我们开发了一种新的体外实验，将细胞外的生物膜样结核分枝杆菌群落培养在从裂解的人白细胞中提取的附着基质上。与在体内观察到的情况类似，我们的数据显示结核分枝杆菌在体外表达极端的药物耐受性，这种耐受性被抗生物膜药物逆转，使杆菌再次对结核药物敏感。我们的数据表明，这些新型化合物分散或抑制了包括耻垢分枝杆菌在内的多种细菌形成的生物膜的形成，并增加了Mtb对异烟肼的敏感性。该策略的有效性将通过我们的体外结核分枝杆菌耐药试验进一步测试，以显示1)抗生物膜

DESCRIPTION (provided by applicant): Infection with Mycobacterium tuberculosis (Mtb) results in chronic inflammation of the lung and other tissues, which is difficult to treat with current tuberculosis (TB) drugs. The challenge to effectively curing patients with active TB is due to the persistence of drug-tolerant bacilli sequestered in granulomas with caseous necrosis or cavitation. Existing treatment regimes that are often complicated by toxic side effects consist of months or years of combination antimicrobial drugs, which are deemed necessary to effectively eradicate drug-tolerant bacilli. We propose a novel TB treatment strategy, targeting drug-tolerant Mtb using a class of small molecular weight, anti-biofilm compounds to be combined with conventional TB drugs. This strategy could significantly improve the efficacy of current TB drug therapy by more rapidly eradicating, persistent, drug-tolerant bacilli. Our in vitr and in vivo data show that Mtb forms extracellular, biofilm-like microbial communities as a strategy to survive TB drug therapy. From studying the Guinea pig TB model we determined that these drug-tolerant bacilli adopt the biofilm-like mode of existence when associated with lysed or necrotic leukocytes. To mimic this unique in vivo microenvironment we developed a novel in vitro assay in which extracellular, biofilm-like communities of Mtb are cultured on an attachment matrix derived from lysed human leukocytes. Similar to what is seen in vivo, our data show that Mtb expresses extreme in vitro drug tolerance, which is reversed by anti-biofilm drugs, rendering bacilli again susceptible to TB drugs. Our data show that these novel compounds disperse or inhibit the formation of biofilms formed by a variety of bacteria including M. smegmatis and increases the susceptibility of Mtb to isoniazid. The effectiveness of this strategy will be further tested using our in vitro Mtb drug tolerance assay to show 1) anti-biofilm compounds disperse and inhibit the formation of Mtb biofilm-like communities in vitro and 2) that combining anti-biofilm compounds with TB drugs effectively eradicates drug-tolerant Mtb associated with host-derived macromolecules. We will then determine whether combining anti-biofilm and TB drugs in the Guinea pig Mtb infection model is more effective at eliminating drug- tolerant Mtb than TB or biofilm drugs alone. In a series of mechanistic studies, we will use biotinylated, anti- biofilm compounds as molecular probes to identify Mtb specific virulence factors unique to the drug-tolerant phenotype. By determining the identity of Mtb adhesins or protein intermediates of metabolic pathways unique to drug-tolerant Mtb, additional compounds will be designed, synthesized and tested for biological activity. Successful completion of these highly innovative, translational studies will establish the feasibility of using anti- biofilm compounds as adjunct therapy to treat chronic Mtb in conjunction with existing TB drugs. We will also determine if anti-biofilm compounds combined with TB drugs are more effective at targeting drug-tolerant bacilli compared to TB drugs alone. Finally, we will reveal the mechanisms of action of our current anti-biofilm compounds and the identity of other druggable targets unique to drug-tolerant Mtb.