Cell death mechanisms play a fundamental role in mycobacterial pathogenesis. We critically reviewed 94 research manuscripts, 44 review articles, and 4 book chapters to analyze important discoveries, background literature, and potential shortcomings in the field. The focus of this review is the pathogen Mycobacterium tuberculosis (Mtb) and other Mtb and Mycobacterium avium complex microorganisms. Virulent strains hijack cell death processes by inhibiting autophagy, apoptosis, and pyroptosis while eliciting necrosis and ferroptosis to multiply intracellularly and spread within and between hosts. In addition, virulent strains may induce apoptosis in epithelial cells or secondary infected macrophages to spread. Autophagy does not control Mtb intracellular replication in vivo but suppresses macrophage and T cell responses in Mtb infections, with a predominant role in preventing neutrophil infiltration. In contrast, attenuated vaccine strains promote apoptosis in macrophages, leading to the activation of innate immunity and, eventually, the acquired immune response. Although Mtb infection activates necroptosis, studies with mutant cell lines have indicated that this process is not essential for cell lysis and that Mtb promotes unprogrammed necrosis. Ferroptosis is discussed in the context of necrotic processes involving lipid peroxidation. Recent research indicated that pyroptosis is more akin to apoptosis as Mtb proteins induce cell membrane repair to prevent inflammasome activation. In the supplementary tables, homologs of mycobacterial cell death pathways and virulence factors were identified using a basic local alignment search tool protein followed by a conserved domain database search to determine the presence of functional domains. Finally, prospects for therapeutic interventions are discussed.

Freund’s adjuvants have been used in vaccine and autoimmune settings, and their effects can be overlapping or unique to each. While both incomplete Freund’s adjuvants (IFA) and complete Freund’s adjuvants (CFA) influence antibody and T cell responses, the robust T helper 1 cytokines induced by the mycobacterial components make CFA the powerful immunostimulating adjuvant. In these studies, the adjuvant effects are investigated in a select population of cells, and the changes, if any, with the metabolic alterations in the systemic compartment are unclear. We investigated whether the effects of IFA and CFA can be influenced by the metabolic shifts in mice immunized with saline, IFA, or CFA using Mycobacterium tuberculosis var. bovis Bacillus Calmette–Guérin (BCG) as a positive control. After seven days of immunization, we analyzed the serum metabolite profiles using liquid chromatography coupled with high-resolution mass spectrometry and multivariate statistical analysis to identify metabolic features between the groups. The data revealed that, in the scores space, the CFA and BCG groups were more closely aligned compared to the saline group, while the IFA group displayed an intermediate profile. Furthermore, comparisons between the CFA and BCG groups showed more significant perturbations in lipid and amino acid metabolism, particularly involving glycerophospholipids, cysteine, and aromatic amino acids. In contrast, comparisons between the BCG and IFA groups indicated a more pronounced disruption in central energy metabolism pathways, such as the citric acid cycle and pyruvate metabolism. Together, the data suggest that the serum metabolite profiles in response to IFA and CFA might play a role in modulating the immune responses.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis. Two wild-type strains, a transposon mutant, and two deletion mutant MAP strains (MOI of 10 with 1.2 × 10⁶ CFU) were tested in murine RAW 264.7 macrophages to determine if they induce apoptosis and/or necrosis. Both deletion mutants were previously shown to be attenuated and immunogenic in primary bovine macrophages. All strains had similar growth rates, but cell morphology indicated that both deletion mutants were elongated with cell wall bulging. Cell death kinetics were followed by a real-time cellular assay to measure luminescence (apoptosis) and fluorescence (necrosis). A 6 h infection period was the appropriate time to assess apoptosis that was followed by secondary necrosis. Apoptosis was also quantified via DAPI-stained nuclear morphology and validated via flow cytometry. The combined analysis confirmed the hypothesis that candidate vaccine deletion mutants are pro-apoptotic in RAW 264.7 cells. In conclusion, the increased apoptosis seen in the deletion mutants correlates with the attenuated phenotype and immunogenicity observed in bovine macrophages, a property associated with good vaccine candidates.

Mycobacterium avium subspecies paratuberculosis (Map) is the etiologic agent of Johne’s disease in ruminants and has been associated with Crohn’s disease in humans. An effective control of Map by either vaccines or chemoprophylaxis is a paramount need for veterinary and possibly human medicine. Given the importance of fatty acids in the biosynthesis of mycolic acids and the mycobacterial cell wall, we tested novel amphiphilic C₁₀ and C₁₈ cyclobutene and cyclobutane fatty acid derivatives for Map inhibition. Microdilution minimal inhibitory concentrations (MIC) with 5 or 7 week endpoints were measured in Middlebrook 7H9 base broth media. We compared the Map MIC results with those obtained previously with Mycobacterium tuberculosis and Mycobacterium smegmatis. Several of the C₁₈ compounds showed moderate efficacy (MICs 392 to 824 µM) against Map, while a higher level of inhibition (MICs 6 to 82 µM) was observed for M. tuberculosis for select analogs from both the C₁₀ and C₁₈ groups. For most of these analogs tested in M. smegmatis, their efficacy decreased in the presence of bovine or human serum albumin. Compound 5 (OA-CB, 1-(octanoic acid-8-yl)-2-octylcyclobutene) was identified as the best chemical lead against Map, which suggests derivatives with better pharmacodynamics may be of interest for evaluation in animal models.

Background/Objectives: Freund’s adjuvants induce different immunomodulatory effects, but their underlying molecular mechanisms are unclear. In this study, we investigated whether the immune-stimulating effects of the complete Freund’s adjuvant (CFA) involve the mechanisms of trained immunity (TI). Methods: We examined bone marrow cells (BMCs) isolated from CFA-immunized A/J mice to address this question. Incomplete Freund’s adjuvant (IFA) and Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin (BCG) served as negative and positive controls, respectively. We evaluated cytokine profiles, metabolic, and epigenetic changes. Results: First, BMCs from all groups except saline showed varied levels of IL-1β, IL-6, and TNF-α. But expression of CCL5 and CXCL10 was significantly elevated only in the CFA and BCG groups. Transcriptionally, significant elevations were noted for TNF-α and IL-1β in the CFA and BCG groups, whereas CXCL10, IL-6, and IL-10 were upregulated in the CFA and BCG groups, respectively. Second, while BMCs from the BCG group expressed the markers of both the M1 and M2 macrophages, no clear trends were noted in the CFA and IFA groups. Third, cell lysates from the CFA group revealed metabolic reprogramming in the BMCs. Specifically, we observed an increased level of lactate, indicative of aerobic glycolysis, which is implicated in TI, and this was also detected in the IFA group. Fourth, epigenetic analysis revealed histone enrichment in the promoter region of TNF-α, in the CFA group, but to a lesser degree than the BCG group. However, no epigenetic changes were observed in the IFA group. Conclusions: Our data provide new insights into the mechanisms of Freund’s adjuvants and the immunomodulatory effects of CFA could involve the features of TI.