Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions

Abstract

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.

Proteomic Analysis of the Mycobacterium tuberculosis Outer Membrane for Potential Implications in Uptake of Small Molecules

“…Previous studies have shown that proteins present on the outer surface of cells may get stripped off due to the presence of detergents. 36 By growing cells in the absence of tyloxapol, we aimed to retain capsular components and the natural abundance of the surface-exposed proteins. 37 While cell surface accessibility of mycobacterial OMPs has been previously shown by flow cytometry, direct microscopic evidence of their surface localization is lacking.…”

“…Cell surface accessibility of outer membrane proteins is what distinguishes them from inner membrane or periplasmic proteins and was assessed by probing the accessibility of the C-terminal FLAG-tag with Alexa Fluor 488-tagged antibody without permeabilization. Previous studies have shown that proteins present on the outer surface of cells may get stripped off due to the presence of detergents . By growing cells in the absence of tyloxapol, we aimed to retain capsular components and the natural abundance of the surface-exposed proteins .…”

Outer membrane proteins inMycobacterium tuberculosisand its potential role in small molecule permeation

“…Cell surface accessibility of outer membrane proteins is what distinguishes them from inner membrane or periplasmic proteins and was assessed by probing the accessibility of the C-terminal FLAG-tag with Alexa Fluor 488-tagged antibody. Previous studies have shown that proteins present on the outer surface of cells may get stripped off due to presence of detergents (62). By growing cells in the absence of tyloxapol, we aimed to retain capsular components and the natural abundance of the surface-exposed proteins (63).…”

Minimum Information for Conducting and Reporting In Vitro Intracellular Infection Assays

“…Although several methods are used to minimize this problem (e.g., use of detergents in the culture media and physical methods for disaggregation), these can influence macrophage responses and intracellular bacterial survival and should therefore be reported. 46 When reporting, researchers should describe the species of bacterial strain used, including strain number or reference with details on strain type used, and the bacterial identity confirmation by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy or genotyping under the Methods section. If using a clinical isolate, all available information should be included, including method of isolation and media used for maintenance.…”

“…An important consideration when working with M. tuberculosis is the lipid-rich nature of the mycobacterial cell wall; this makes cultures very prone to aggregation, which can impact the accuracy and reproducibility of bacterial numbers delivered during intracellular infections. Although several methods are used to minimize this problem (e.g., use of detergents in the culture media and physical methods for disaggregation), these can influence macrophage responses and intracellular bacterial survival and should therefore be reported …”