TY - JOUR TI - Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions AU - Mittal, Ekansh AU - Roth, Andrew T AU - Seth, Anushree AU - Singamaneni, Srikanth AU - Beatty, Wandy AU - Philips, Jennifer A A2 - Kana, Bavesh D VL - 12 PY - 2023 DA - 2023/02/28 SP - e85416 C1 - eLife 2023;12:e85416 DO - 10.7554/eLife.85416 UR - https://doi.org/10.7554/eLife.85416 AB - For decades, investigators have studied the interaction of Mycobacterium tuberculosis (Mtb) with macrophages, which serve as a major cellular niche for the bacilli. Because Mtb are prone to aggregation, investigators rely on varied methods to disaggregate the bacteria for these studies. Here, we examined the impact of routinely used preparation methods on bacterial cell envelope integrity, macrophage inflammatory responses, and intracellular Mtb survival. We found that both gentle sonication and filtering damaged the mycobacterial cell envelope and markedly impacted the outcome of infections in mouse bone marrow-derived macrophages. Unexpectedly, sonicated bacilli were hyperinflammatory, eliciting dramatically higher TLR2-dependent gene expression and elevated secretion of IL-1β and TNF-α. Despite evoking enhanced inflammatory responses, sonicated bacilli replicated normally in macrophages. In contrast, Mtb that had been passed through a filter induced little inflammatory response, and they were attenuated in macrophages. Previous work suggests that the mycobacterial cell envelope lipid, phthiocerol dimycocerosate (PDIM), dampens macrophage inflammatory responses to Mtb. However, we found that the impact of PDIM depended on the method used to prepare Mtb. In conclusion, widely used methodologies to disaggregate Mtb may introduce experimental artifacts in Mtb-host interaction studies, including alteration of host inflammatory signaling, intracellular bacterial survival, and interpretation of bacterial mutants. KW - mycobacterium tuberculosis KW - macrophage KW - host-pathogen interactions JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -