RT Journal Article SR Electronic T1 Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.06.16.496372 DO 10.1101/2022.06.16.496372 A1 Ekansh Mittal A1 Andrew T. Roth A1 Anushree Seth A1 Srikanth Singamaneni A1 Wandy Beatty A1 Jennifer A. Philips YR 2022 UL http://biorxiv.org/content/early/2022/06/16/2022.06.16.496372.abstract 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 envelop integrity, macrophage inflammatory responses, intracellular Mtb survival, and virulence in mice. We found that both gentle sonication and filtering damaged the mycobacterial cell envelope and markedly impacted the outcome of macrophage infections. Unexpectedly, sonicated bacilli were hyperinflammatory, eliciting dramatically higher expression of TLR2-dependent genes 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 highly 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.Competing Interest StatementSS is an inventor on a provisional patent related to plasmonic-fluor technology, and the technology has been licensed by the Office of Technology Management at Washington University in St. Louis to Auragent Bioscience LLC. SS is a co-founder/shareholder of Auragent Bioscience LLC. SS along with Washington University may have financial gain through Auragent Bioscience LLC through this licensing agreement. AS is currently employed with Auragent Bioscience LLC. These potential conflicts of interest have been disclosed and are being managed by Washington University in St. Louis.