Aug 2026· Pathogens· Vol 15, pp. 857· 0 citations· 35 references
Medicine
TL;DR
The findings demonstrate the feasibility of functional pro-apoptotic protein delivery using a mycobacterial vector, providing a robust foundation for the future development of recombinant BCG-based vaccine candidates.
Abstract
Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major global health threat. A key virulence mechanism involves the pathogen’s ability to inhibit apoptosis in infected macrophages, thereby facilitating immune evasion and persistent infection. The pro-apoptotic protein Bcl-2-interacting killer (BIK) represents a critical mediator of macrophage apoptosis. In this study, we engineered a recombinant Mycobacterium smegmatis strain (rMs-BIK) designed to secrete BIK via fusion with the Ag85B signal peptide (ASP). Secretion and expression of the BIK protein were confirmed by Western blotting. Quantitative flow cytometric analysis revealed that rMs-BIK induced significant apoptosis in RAW264.7 macrophages, with rates of 14.49%, 14.22%, and 22.24% at 12, 24, and 48 h post-infection, respectively. These values were markedly higher than those observed in cells infected with the wild-type strain (7.68%, 9.24%, and 10.90%) or the empty vector control (8.80%, 11.19%, and 14.43%). Our findings demonstrate the feasibility of functional pro-apoptotic protein delivery using a mycobacterial vector, providing a robust foundation for the future development of recombinant BCG-based vaccine candidates.
ABSTRACT The serine/threonine protein kinase F (PknF) of Mycobacterium tuberculosis (Mtb) has poorly defined targets and functions but is involved in limiting NLRP3 inflammasome activation in murine macrophages and dendritic cells in vitro. The importance of PknF for the virulence of Mtb in vivo is not known. Here, we demonstrate that the Mtb CDC1551 deletion mutant of pknF (∆pknF) expresses significantly increased levels of the lipid pthiocerol dimycoserosate (PDIM), a polyketide lipid with pro-virulence properties. The ∆pknF mutant strain compared with the Mtb and complemented strains showed a 100-fold increase in growth at day 28 and about a 10-fold increase in growth at days 90–98 in the lungs of mice. The increase in pulmonary bacterial loads after infection with the ∆pknF strain was conserved even in Nlrp3-deficient mice, arguing that PknF modulates Mtb virulence independently of NLRP3-inflammasome activation in mice. Staining of lung sections revealed increased inflammation in the lungs of ∆pknF strain-infected mice when compared with Mtb and the complemented mutant strain. Highly susceptible B6.Sst1S mice displayed decreased host resistance, with significantly decreased survival when infected with the ∆pknF strain compared to the complemented strain or Mtb. In conclusion, our data suggest that expression of PknF, as a modulator of multiple downstream effector proteins, restricts the virulence of Mtb in the lungs of mice through an NLRP3 inflammasome-independent mechanism, but potentially via suppressing production of the virulence lipid, PDIM.
Flor Torres-Juárez, Shivangi Rastogi, David C. Young et al.· Infection and Immunity· 0 citations
Alveolar macrophages (AMs) are the first innate immune cells to encounter Mycobacterium tuberculosis (M.tb) following natural infection. AMs are potent phagocytes, and they are permissive to M.tb survival, replication, and dissemination. Endoplasmic reticulum stress (ERS) is triggered by the accumulation of misfolded/unfolded proteins during cellular stress, and ERS-mediated apoptosis plays a critical role in eliminating intracellular pathogens. While ERS-induced apoptosis in M1 macrophages can effectively clear M.tb, in M2 macrophages it can favor bacterial persistence. AMs exhibit unique molecular and metabolic features, expressing a hybrid M1/M2 phenotype. However, the role of ERS in M.tb infected AMs and its impact on bacterial survival remain poorly understood.
In this study, alveolar macrophage-like (AML) cells derived from peripheral blood mononuclear cells were infected with M.tb to characterize the ERS response over time. We analyzed the expression of key ERS-related genes and proteins following infection with either live M.tb or its virulence factors, HBHA and ESAT-6. Additionally, ERS was induced in AML cells to assess its influence on bacterial survival.
AML cells displayed distinct ERS signatures upon infection, with significant differences in gene and protein expression compared to ERS-induced and virulence factor only controls. Notably, cells without ERS harbored higher viable bacterial counts.
The permissive nature of AMs to M.tb could be in part due to the down-regulation of ERS and related apoptosis. The results of our study highlight a potential protective role of ERS in restricting M.tb survival.
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Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Mohanapriya Kumaran, F. Quinn, Kate Rzepka· Journal of Immunology· 0 citations
Brucella is a Gram-negative facultative intracellular pathogen that causes widespread zoonotic infections. Macrophages are crucial immune and antigen-presenting cells that differentiate into proinflammatory M1-type or anti-inflammatory M2-type cells in different microenvironments. The BtpB protein of Brucella is a type IV secretion system effector protein that modulates host inflammatory responses by inhibiting Toll-like receptor signaling and controlling dendritic cell activation. A high expression of intracellular proinflammatory factors is induced in B. suis deficient in BtpB (B. suis mutant strain ΔbtpB). However, the role of BtpB in macrophage polarization triggered by Brucella infection is uncertain. In this study, RAW264.7 macrophages were utilized as a model to investigate the impact of BtpB on macrophage polarization. The cells were infected with wild-type B. suis strain S2, B. suis mutant strain ΔbtpB, or the B. suis complemented strain C-ΔbtpB followed by flow cytometry, reverse transcription quantitative PCR (RT-qPCR), western blotting, immunohistochemistry, and metabolic detection analyses. The expression of BtpB suppressed M1 polarization and promoted M2 macrophage polarization. Moreover, BtpB upregulated the expression of signal transducer and activator of transcription 3 (STAT3) in host cells. Inhibition of STAT3 promoted the expression of nitric oxide synthase (NOS2) in cells infected with either B. suis or C-ΔbtpB, with no observable differences compared to cells infected with ΔbtpB. Conversely, STAT3 overexpression resulted in a downregulation of NOS2 levels in both wild-type and mutant cells. These findings were corroborated by messenger RNA (mRNA) assay and enzyme-linked immunosorbent assay (ELISA) that confirmed the regulatory role of STAT3 in modulating NOS2 expression. Additionally, intracellular proliferation assays under STAT3-modulated conditions indicated that STAT3 suppresses replication of Brucella. In conclusion, the results demonstrate that BtpB inhibits M1 polarization in macrophages by regulating STAT3 expression which provides a strong foundation for improved understanding of Brucella infection mechanisms.
Ye Yuan, Minghui Wang, Mingyue Hao et al.· Veterinary Research· 0 citations
ABSTRACT Bovine tuberculosis (bTB), caused by Mycobacterium bovis, causes major economic losses in the global livestock industry and threatens public health. Macrophages are essential for pathogen clearance, yet serve as the primary intracellular niche for mycobacterial survival. These characteristics emphasize the importance of identifying host factors that regulate macrophage functions during infection. To systematically uncover such factors, we constructed a genome-wide CRISPR-KO library in Bomac cells (the bovine macrophage cell line) and performed iterative infection screening under high selective pressure. Here, the host protein SH3PXD2B was identified as a key host factor to promote mycobacterial invasion: SH3PXD2B was significantly enriched in the screening. M. bovis infection significantly increases the expression of SH3PXD2B. The phagocytic activity of macrophages is enhanced in an SH3PXD2B-dependent manner, and the high expression of SH3PXD2B inhibits the migration of macrophages, causing macrophages to remain at the site of infection and promoting the entry of more mycobacteria into macrophages to establish infection. Further results indicate that macrophage migration inhibitory factor is positively regulated by SH3PXD2B and plays an important role in SH3PXD2B-mediated macrophage migration and phagocytic activity. These results indicate that SH3PXD2B is a critical host factor manipulated by M. bovis to promote infection. It provides a new potential target for genetic breeding against bTB. IMPORTANCE There is a complex interaction between Mycobacterium bovis and the host, which has an important impact on the occurrence and development of bTB. However, the host gene regulatory network for M. bovis infection is still incomplete, and the specific genes that play a key role in the infection process are still unclear. Bovine tuberculosis remains a major economic and public health burden worldwide. This study identifies SH3PXD2B as a novel host factor that is exploited by M. bovis to promote macrophage phagocytosis and inhibit migration, thereby facilitating bacterial invasion. These findings reveal a new pathogenic strategy and suggest that SH3PXD2B may represent a potential target for host-directed therapy and genetic breeding against bTB. There is a complex interaction between Mycobacterium bovis and the host, which has an important impact on the occurrence and development of bTB. However, the host gene regulatory network for M. bovis infection is still incomplete, and the specific genes that play a key role in the infection process are still unclear. Bovine tuberculosis remains a major economic and public health burden worldwide. This study identifies SH3PXD2B as a novel host factor that is exploited by M. bovis to promote macrophage phagocytosis and inhibit migration, thereby facilitating bacterial invasion. These findings reveal a new pathogenic strategy and suggest that SH3PXD2B may represent a potential target for host-directed therapy and genetic breeding against bTB.
Donghui Liu, Ting Zhang, Yu-jie Sheng et al.· Applied and Environmental Mi...· 0 citations
BACKGROUND
Tuberculosis is the leading cause of death among chronic infectious diseases caused by a single pathogen. Macrophages serve as the first line of immune defense, and pulmonary epithelial cells are essential for lung structural integrity and function. However, these cells are highly susceptible to death following Mycobacterium tuberculosis (Mtb) infection. Preserving cell viability has been shown to significantly enhance host anti-infective capacity.
PURPOSE
This study aimed to screen an in-house natural product library derived from Lingnan-region herbal medicines in China for cell-protective activity in H37Rv-infected cells and investigate the underlying mechanisms.
METHODS
Cell viability in macrophages and epithelial cells was assessed using CCK-8 and LDH assays. Pathways involved in cell death inhibition were explored via RNA-seq. Natural product target proteins were identified using DARTS and LC-MS. Gene and protein expression were validated by qRT-PCR and western blotting. Intracellular H37Rv viability was determined by CFU counting.
RESULTS
A novel natural compound, cleistoperlone J (CJ), was identified as a cell viability-protective agent. CJ inhibited ferroptosis by modulating cellular antioxidant balance. Glucose-6-phosphate dehydrogenase (G6PD), a key enzyme in glutathione metabolism, acted as a critical mediator of CJ. By binding to G6PD, CJ enhanced its level and activity via inhibition of ubiquitination-mediated degradation, thereby suppressing ferroptosis in Mtb-infected cells. Notably, both CJ and the G6PD activator AG1 potentiated the antibacterial activity of isoniazid in Mtb-infected macrophages.
CONCLUSION
These findings suggest that CJ holds promise as an adjunctive therapeutic agent for tuberculosis chemotherapy by inhibiting ferroptosis in Mtb-infected cells.
Ke Wang, Xue Li, Wen-Xin Huang et al.· Phytomedicine· 0 citations
Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis secretes proteins during the host-pathogen interaction that facilitate its survival and/or virulence within host cells. Key examples include EsxA and EsxB, CpnT/TNT, phosphatases (SapM, PtpA, PtpB), protein kinase G (PknG), the antigen 85 (ag85) complex, acyl Carrier Protein (AcpM), lipoproteins, and heat shock proteins (Hsps). Together, these secreted factors disrupt host immune responses such as phagosome maturation, antigen presentation, cytokine production, and autophagy thereby aiding in survival of these obligate intracellular bacteria. Several secreted proteins are emerging as therapeutic targets to help meet the need for shorter, more effective, and better tolerated treatment regimens for tuberculosis. Here, we review the current understanding of the major secreted proteins of M. tuberculosis. We also discuss progress in the discovery and development of drugs targeting these proteins. Overall, targeting M.tuberculosis secreted proteins remains largely in the preclinical stages of drug development. Current limitations include low potency, off-target activity, and redundancy among secreted proteins.
Anthony Musaazi Kaweesa, Divya Tiwari· Frontiers in tuberculosis· 0 citations
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