BCG Educates Hematopoietic Stem Cells to Generate Protective Innate Immunity against Tuberculosis.

The dogma that adaptive immunity is the only arm of the immune response with memory capacity has been recently challenged by several studies demonstrating evidence for memory-like innate immune training. However, the underlying mechanisms and location for generating such innate memory responses in vivo remain unknown. Here, we show that access of Bacillus Calmette-Guérin (BCG) to the bone marrow (BM) changes the transcriptional landscape of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), leading to local cell expansion and enhanced myelopoiesis at the expense of lymphopoiesis. Importantly, BCG-educated HSCs generate epigenetically modified macrophages that provide significantly better protection against virulent M. tuberculosis infection than naïve macrophages. By using parabiotic and chimeric mice, as well as adoptive transfer approaches, we demonstrate that training of the monocyte/macrophage lineage via BCG-induced HSC reprogramming is sustainable in vivo. Our results indicate that targeting the HSC compartment provides a novel approach for vaccine development.

Disruption of an antimycobacterial circuit between dendritic and helper T cells in human SPPL2a deficiency.

Human inborn errors of IFN-γ immunity underlie mycobacterial diseases. We describe patients with Mycobacterium bovis (BCG) disease who are homozygous for loss-of-function mutations of SPPL2A. This gene encodes a transmembrane protease that degrades the N-terminal fragment (NTF) of CD74 (HLA invariant chain) in antigen-presenting cells. The CD74 NTF therefore accumulates in the HLA class II+ myeloid and lymphoid cells of SPPL2a-deficient patients. This toxic fragment selectively depletes IL-12- and IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their circulating progenitors. Moreover, SPPL2a-deficient memory TH1* cells selectively fail to produce IFN-γ when stimulated with mycobacterial antigens in vitro. Finally, Sppl2a-/- mice lack cDC2s, have CD4+ T cells that produce small amounts of IFN-γ after BCG infection, and are highly susceptible to infection with BCG or Mycobacterium tuberculosis. These findings suggest that inherited SPPL2a deficiency in humans underlies mycobacterial disease by decreasing the numbers of cDC2s and impairing IFN-γ production by mycobacterium-specific memory TH1* cells.

A noninvasive BCG skin challenge model for assessing tuberculosis vaccine efficacy.

We report here on the characterisation in mice of a noninvasive bacille Calmette-Guérin (BCG) skin challenge model for assessing tuberculosis (TB) vaccine efficacy. Controlled human infection models (CHIMs) are valuable tools for assessing the relevant biological activity of vaccine candidates, with the potential to accelerate TB vaccine development into the clinic. TB infection poses significant constraints on the design of a CHIM using the causative agent Mycobacterium tuberculosis (Mtb). A safer alternative is a challenge model using the attenuated vaccine agent Mycobacterium bovis BCG as a surrogate for Mtb, and intradermal (skin) challenge as an alternative to pulmonary infection. We have developed a unique noninvasive imaging system based on fluorescent reporters (FluorBCG) to quantitatively measure bacterial load over time, thereby determining a relevant biological vaccine effect. We assessed the utility of this model to measure the effectiveness of 2 TB vaccines: the currently licenced BCG and a novel subunit vaccine candidate. To assess the efficacy of the skin challenge model, a nonlinear mixed-effects models was built describing the decline of fluorescence over time. The model-based analysis identified that BCG vaccination reduced the fluorescence readout of both fluorophores compared to unvaccinated mice (p < 0.001). However, vaccination with the novel subunit candidate did not alter the fluorescence decline compared to unvaccinated mice (p > 0.05). BCG-vaccinated mice that showed the reduced fluorescent readout also had a reduced bacterial burden in the lungs when challenged with Mtb. This supports the fluorescence activity in the skin as a reflection of vaccine induced functional pulmonary immune responses. This novel noninvasive approach allows for repeated measurements from the challenge site, providing a dynamic readout of vaccine induced responses over time. This BCG skin challenge model represents an important contribution to the ongoing development of controlled challenge models for TB.

Multiple genetic loci influence vaccine-induced protection against Mycobacterium tuberculosis in genetically diverse mice.

Mycobacterium tuberculosis (M.tb.) infection leads to over 1.5 million deaths annually, despite widespread vaccination with BCG at birth. Causes for the ongoing tuberculosis endemic are complex and include the failure of BCG to protect many against progressive pulmonary disease. Host genetics is one of the known factors implicated in susceptibility to primary tuberculosis, but less is known about the role that host genetics plays in controlling host responses to vaccination against M.tb. Here, we addressed this gap by utilizing Diversity Outbred (DO) mice as a small animal model to query genetic drivers of vaccine-induced protection against M.tb. DO mice are a highly genetically and phenotypically diverse outbred population that is well suited for fine genetic mapping. Similar to outcomes in people, our previous studies demonstrated that DO mice have a wide range of disease outcomes following BCG vaccination and M.tb. challenge. In the current study, we used a large population of BCG-vaccinated/M.tb.-challenged mice to perform quantitative trait loci mapping of complex infection traits; these included lung and spleen M.tb. burdens, as well as lung cytokines measured at necropsy. We found sixteen chromosomal loci associated with complex infection traits and cytokine production. QTL associated with bacterial burdens included a region encoding major histocompatibility antigens that are known to affect susceptibility to tuberculosis, supporting validity of the approach. Most of the other QTL represent novel associations with immune responses to M.tb. and novel pathways of cytokine regulation. Most importantly, we discovered that protection induced by BCG is a multigenic trait, in which genetic loci harboring functionally-distinct candidate genes influence different aspects of immune responses that are crucial collectively for successful protection. These data provide exciting new avenues to explore and exploit in developing new vaccines against M.tb.

Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens.

Neutrophils are critical for antifungal defense, but the mechanisms that clear hyphae and other pathogens that are too large to be phagocytosed remain unknown. We found that neutrophils sensed microbe size and selectively released neutrophil extracellular traps (NETs) in response to large pathogens, such as Candida albicans hyphae and extracellular aggregates of Mycobacterium bovis, but not in response to small yeast or single bacteria. NETs were fundamental in countering large pathogens in vivo. Phagocytosis via dectin-1 acted as a sensor of microbe size and prevented NET release by downregulating the translocation of neutrophil elastase (NE) to the nucleus. Dectin-1 deficiency led to aberrant NET release and NET-mediated tissue damage during infection. Size-tailored neutrophil responses cleared large microbes and minimized pathology when microbes were small enough to be phagocytosed.

Intravenous Bacillus Calmette-Guérin (BCG) Induces a More Potent Airway and Lung Immune Response than Intradermal BCG in Simian Immunodeficiency Virus-infected Macaques.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of death due to an infectious agent. Coinfection with HIV exacerbates M. tuberculosis infection outcomes in people living with HIV. Bacillus Calmette-Guérin (BCG), the only approved TB vaccine, is effective in infants, but its efficacy in adolescents and adults is limited. In this study, we investigated the immune responses elicited by BCG administered via i.v. or intradermal (i.d.) routes in SIV-infected Mauritian cynomolgus macaques (MCM) without the confounding effects of M. tuberculosis challenge. We assessed the impact of vaccination on T cell responses in the airway, blood, and tissues (lung, thoracic lymph nodes, and spleen), as well as the expression of cytokines, cytotoxic effectors, and key transcription factors. Our results showed that i.v. BCG induces a robust and sustained immune response, including tissue-resident memory T cells in lungs, polyfunctional CD4+ and CD8αß+ T cells expressing multiple cytokines, and CD8αß+ T cells and NK cells expressing cytotoxic effectors in airways. We also detected higher levels of mycobacteria-specific IgG and IgM in the airways of i.v. BCG-vaccinated MCM. Although i.v. BCG vaccination resulted in an influx of tissue-resident memory T cells in lungs of MCM with controlled SIV replication, MCM with high plasma SIV RNA (>105 copies/ml) typically displayed reduced T cell responses, suggesting that uncontrolled SIV or HIV replication would have a detrimental effect on i.v. BCG-induced protection against M. tuberculosis.

Cutting Edge: Cytosolic Receptor AIM2 Is Induced by Peroxisome Proliferator-activated Receptor γ following Mycobacterium tuberculosis Infection of Human Macrophages but Does Not Contribute to IL-1ß Release.

AIM2 (absent in melanoma 2), an inflammasome component, mediates IL-1ß release in murine macrophages and cell lines. AIM2 and IL-1ß contribute to murine control of Mycobacterium tuberculosis (M.tb) infection, but AIM2's impact in human macrophages, the primary niche for M.tb, remains unclear. We show that M.tb, Mycobacterium bovis bacillus Calmette-Guérin (BCG), and M. smegmatis induce AIM2 expression in primary human macrophages. M.tb-induced AIM2 expression is peroxisome proliferator-activated receptor γ (PPARγ)-dependent and M.tb ESX-1-independent, whereas BCG- and M. smegmatis-induced AIM2 expression is PPARγ-independent. PPARγ and NLRP3, but not AIM2, are important for IL-1ß release in response to M.tb, and NLRP3 colocalizes with M.tb. This is in contrast to the role for AIM2 in inflammasome activation in mice and peritoneal macrophages. Altogether, we show that mycobacteria induce AIM2 expression in primary human macrophages, but AIM2 does not contribute to IL-1ß release during M.tb infection, providing further evidence that AIM2 expression and function are regulated in a cell- and/or species-specific manner.

Assessing vaccine-mediated protection in an ultra-low dose Mycobacterium tuberculosis murine model.

Despite widespread immunization with Bacille-Calmette-Guérin (BCG), the only currently licensed tuberculosis (TB) vaccine, TB remains a leading cause of mortality globally. There are many TB vaccine candidates in the developmental pipeline, but the lack of a robust animal model to assess vaccine efficacy has hindered our ability to prioritize candidates for human clinical trials. Here we use a murine ultra-low dose (ULD) Mycobacterium tuberculosis (Mtb) challenge model to assess protection conferred by BCG vaccination. We show that BCG confers a reduction in lung bacterial burdens that is more durable than that observed after conventional dose challenge, curbs Mtb dissemination to the contralateral lung, and, in a small percentage of mice, prevents detectable infection. These findings are consistent with the ability of human BCG vaccination to mediate protection, particularly against disseminated disease, in specific human populations and clinical settings. Overall, our findings demonstrate that the ultra-low dose Mtb infection model can measure distinct parameters of immune protection that cannot be assessed in conventional dose murine infection models and could provide an improved platform for TB vaccine testing.

BCG vaccination induces cross-protective immunity against pathogenic microorganisms.

Bacillus Calmette-Guérin (BCG) is an attenuated Mycobacterium bovis strain used as a vaccine to prevent Mycobacterium tuberculosis (M. tb) infection. Its ability to potentiate the immune response induced by other vaccines and to promote nonspecific immunomodulatory effects has been described. These effects can be triggered by epigenetic reprogramming and metabolic shifts on innate immune cells, a phenomenon known as trained immunity. The induction of trained immunity may contribute to explain why BCG vaccination effectively decreases disease symptoms caused by pathogens different from M. tb. This article explains the importance of BCG immunization and the possible mechanisms associated with the induction of trained immunity, which might be used as a strategy for rapid activation of the immune system against unrelated pathogens.

Plasma metabolome predicts trained immunity responses after antituberculosis BCG vaccination.

The antituberculosis vaccine Bacillus Calmette-Guérin (BCG) induces nonspecific protection against heterologous infections, at least partly through induction of innate immune memory (trained immunity). The amplitude of the response to BCG is variable, but the factors that influence this response are poorly understood. Metabolites, either released by cells or absorbed from the gut, are known to influence immune responses, but whether they impact BCG responses is not known. We vaccinated 325 healthy individuals with BCG, and collected blood before, 2 weeks and 3 months after vaccination, to assess the influence of circulating metabolites on the immune responses induced by BCG. Circulating metabolite concentrations after BCG vaccination were found to have a more pronounced impact on trained immunity responses, such as the increase in IL-1ß and TNF-α production upon Staphylococcus aureus stimulation, than on specific adaptive immune memory, assessed as IFN-γ production in response to Mycobacterium tuberculosis. Circulating metabolites at baseline were able to predict trained immunity responses at 3 months after vaccination and enrichment analysis based on the metabolites positively associated with trained immunity revealed enrichment of the tricarboxylic acid (TCA) cycle and glutamine metabolism, both of which were previously found to be important for trained immunity. Several new metabolic pathways that influence trained immunity were identified, among which taurine metabolism associated with BCG-induced trained immunity, a finding validated in functional experiments. In conclusion, circulating metabolites are important factors influencing BCG-induced trained immunity in humans. Modulation of metabolic pathways may be a novel strategy to improve vaccine and trained immunity responses.

MicroRNA-99b Regulates Bacillus Calmette-Guerin-Infected Immature Dendritic Cell-Induced CD4+ T Cell Differentiation by Targeting mTOR Signaling.

This study aimed to elucidate the mechanisms by which microRNA-99b (miR-99b) regulates CD4+ T cell differentiation induced by Bacillus Calmette-Guerin (BCG)-infected immature dendritic cells (imDCs). Levels of miR-99b, interferon-gamma (IFN-γ), Foxp3, interleukin (IL)-10, IL-17, IL-23, and ROR-γt were assessed. Effects of miR-99b inhibition and mechanistic target of rapamycin (mTOR) agonist on Th17/Treg cell ratio and cytokine levels (IL-6, IL-17, IL-23) were studied. Expression of mTOR, S6K1, and 4E-BP1 related to miR-99b was analyzed. BCG-infected imDCs led to CD4+ T cell differentiation and altered levels of IFN-γ, Foxp3, IL-10, miR-99b, IL-17, IL-23, and ROR-γt. Inhibition of miR-99b increased the Th17/Treg cell ratio in CD4+ T cells co-cultured with BCG-infected imDCs, and this effect was further enhanced by the mTOR agonist. Additionally, the miR-99b inhibitor elevated the levels of IL-6, IL-17, and IL-23 when CD4+ T cells were co-cultured with BCG-infected imDCs, and the mTOR agonist further amplified this increase. Notably, miR-99b negatively regulated mTOR signaling, as the miR-99b inhibitor upregulated the expression levels of mTOR, S6K1, and 4E-BP1 while decreasing miR-99b. It was concluded that miR-99b modulates CD4+ T cell differentiation via mTOR pathway in response to BCG-infected im-DCs. Inhibiting miR-99b affects Th17/Treg ratio and pro-inflammatory cytokines, potentially impacting tuberculosis immunotherapies.

Wildlife vaccination strategies for eliminating bovine tuberculosis in white-tailed deer populations.

Many pathogens of humans and livestock also infect wildlife that can act as a reservoir and challenge disease control or elimination. Efficient and effective prioritization of research and management actions requires an understanding of the potential for new tools to improve elimination probability with feasible deployment strategies that can be implemented at scale. Wildlife vaccination is gaining interest as a tool for managing several wildlife diseases. To evaluate the effect of vaccinating white-tailed deer (Odocoileus virginianus), in combination with harvest, in reducing and eliminating bovine tuberculosis from deer populations in Michigan, we developed a mechanistic age-structured disease transmission model for bovine tuberculosis with integrated disease management. We evaluated the impact of pulse vaccination across a range of vaccine properties. Pulse vaccination was effective for reducing disease prevalence rapidly with even low (30%) to moderate (60%) vaccine coverage of the susceptible and exposed deer population and was further improved when combined with increased harvest. The impact of increased harvest depended on the relative strength of transmission modes, i.e., direct vs indirect transmission. Vaccine coverage and efficacy were the most important vaccine properties for reducing and eliminating disease from the local population. By fitting the model to the core endemic area of bovine tuberculosis in Michigan, USA, we identified feasible integrated management strategies involving vaccination and increased harvest that reduced disease prevalence in free-ranging deer. Few scenarios led to disease elimination due to the chronic nature of bovine tuberculosis. A long-term commitment to regular vaccination campaigns, and further research on increasing vaccines efficacy and uptake rate in free-ranging deer are important for disease management.

Wildlife Immune Responses to Mycobacterium bovis and to Bacille of Calmette-Guerin.

Bovine tuberculosis (bTB) is a zoonotic bacterial disease presenting public health, veterinary, and economic threats around the globe. Although cattle producers rely on regular testing and management practices to minimize domestic herd exposure, wildlife species around the world continue to be the main reservoirs for disease. Wildlife reservoirs for bTB include the Eurasian badger (Meles meles) in Great Britain and Ireland, the brushtail possum (Trichosurus vulpecula) in New Zealand, wild boar (Sus scrofa) in Spain, as well as white-tailed deer (Odocoileus virginianus) in the United States and red deer (Cervus elaphus) in Spain. Although all reservoir species share the ability to infect cattle, they differ in transmission capability, disease pathogenesis, diagnostic detection, and vaccination strategies. In this review, bTB interactions with these wildlife reservoirs are discussed, illustrating the need to address bTB disease in wildlife hosts to achieve eradication in domestic livestock.

Recombinant Bacillus Calmette-Guérin Expressing SARS-CoV-2 Chimeric Protein Protects K18-hACE2 Mice against Viral Challenge.

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette-Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

Dual-specificity phosphatase 5-mediated fatty acid oxidation promotes Mycobacterium bovis BCG -induced inflammatory responses.

Mycobacterium tuberculosis (Mtb) reprograms FAs metabolism of macrophages during infection and affects inflammatory reaction eventually, however, the mechanism remains poorly understood. Here we show that Mycobacterium bovis (BCG) induces DUSP5 expression through TLR2-MAPKs signaling pathway and promotes fatty acid oxidation (FAO). Silencing DUSP5 by adeno-associated virus vector (AAV) ameliorates lung injury and DUSP5 knockdown reduces the expression of IL-1ß, IL-6 and inactivated NF-κB signaling in BCG-infected macrophages. Of note, DUSP5 specific siRNA increases the content of free fatty acids (FFAs) and triglyceride (TG), but represses the expression of FAO associated enzymes such as CPT1A and PPARα, suggesting DUSP5 mediated FAO during BCG infection. Moreover, Inhibiting FAO by pharmacological manner suppresses IL-1ß, IL-6, TNF-α expression and relieves lung damage. Taken together, our data indicates DUSP5 mediates FAO reprogramming and promotes inflammatory response to BCG infection.

Enhancement of mycobacterial pathogenesis by host interferon-γ.

The cytokine IFNγ is a principal effector of macrophage activation and immune resistance to mycobacterial infection; however, pathogenic mycobacteria are capable of surviving in IFNγ-activated macrophages by largely unknown mechanisms. In this study, we find that pathogenic mycobacteria, including M. bovis BCG and M. tuberculosis can sense IFNγ to promote their proliferative activity and virulence phenotype. Moreover, interaction with the host intracellular environment increases the susceptibility of mycobacteria to IFNγ through upregulating expression of mmpL10, a mycobacterial IFNγ receptor, thereby facilitating IFNγ-dependent survival and growth of mycobacteria in macrophages. Transmission electron microscopy analysis reveals that IFNγ triggers the secretion of extracellular vesicles, an essential virulence strategy of intracellular mycobacteria, while proteomics identifies numerous pivotal IFNγ-induced effectors required for mycobacterial infection in macrophages. Our study suggests that sensing host IFNγ is a crucial virulence mechanism used by pathogenic mycobacteria to survive and proliferate inside macrophages.

Epidemiology of Mycobacterium bovis infection in free-ranging rhinoceros in Kruger National Park, South Africa.

Mycobacterium bovis infection, which is a prominent cause of bovine tuberculosis, has been confirmed by mycobacterial culture in African rhinoceros species in Kruger National Park (KNP), South Africa. In this population-based study of the epidemiology of M. bovis in 437 African rhinoceros (Diceros bicornis, Ceratotherium simum), we report an estimated prevalence of 15.4% (95% CI: 10.4 to 21.0%), based on results from mycobacterial culture and an antigen-specific interferon gamma release assay from animals sampled between 2016 and 2020. A significant spatial cluster of cases was detected near the southwestern park border, although infection was widely distributed. Multivariable logistic regression models, including demographic and spatiotemporal variables, showed a significant, increasing probability of M. bovis infection in white rhinoceros based on increased numbers of African buffalo (Syncerus caffer) herds in the vicinity of the rhinoceros sampling location. Since African buffaloes are important maintenance hosts for M. bovis in KNP, spillover of infection from these hosts to white rhinoceros sharing the environment is suspected. There was also a significantly higher proportion of M. bovis infection in black rhinoceros in the early years of the study (2016­2018) than in 2019 and 2020, which coincided with periods of intense drought, although other temporal factors could be implicated. Species of rhinoceros, age, and sex were not identified as risk factors for M. bovis infection. These study findings provide a foundation for further epidemiological investigation of M. bovis, a multihost pathogen, in a complex ecosystem that includes susceptible species that are threatened and endangered.

Characterization of Mycobacterium orygis, Mycobacterium bovis, and Mycobacterium caprae Infections in Humans in Western Canada.

Epidemiologic research on zoonotic tuberculosis historically used Mycobacterium bovis as a surrogate measure; however, increased reports of human tuberculosis caused by other animal-associated Mycobacterium tuberculosis complex members like Mycobacterium orygis necessitates their inclusion. We performed a retrospective cohort study including persons infected with any animal-lineage M tuberculosis complex species in Alberta, Canada, from January 1995 to July 2021, identifying 42 patients (20 M bovis, 21 M orygis, 1 M caprae). Demographic, epidemiologic, and clinical characteristics were compared against persons with culture-confirmed M tuberculosis infection. The proportion of culture-positive infections caused by M orygis increased continuously from 2016 to 2020. Significantly more females at a higher median age were impacted by M orygis, with all patients originating from South Asia. Mycobacterium bovis caused significantly more extrapulmonary disease and disproportionately impacted young females, particularly those pregnant or postpartum. All infections were acquired abroad. These findings can aid in developing targeted public health interventions.

Heparin-Binding Hemagglutinin of Mycobacterium tuberculosis Inhibits Autophagy via Toll-like Receptor 4 and Drives M2 Polarization in Macrophages.

BACKGROUND: Tuberculosis (TB), predominantly caused by Mycobacterium tuberculosis (MTB) infection, remains a prominent global health challenge. Macrophages are the frontline defense against MTB, relying on autophagy for intracellular bacterial clearance. However, MTB can combat and evade autophagy, and it influences macrophage polarization, facilitating immune evasion and promoting infection. We previously found that heparin-binding hemagglutinin (HBHA) inhibits autophagy in A549 cells; however, its role in macrophage autophagy and polarization remains unclear. METHODS: Bacterial cultures, cell cultures, Western blotting, immunofluorescence, macrophage infection assays, siRNA knockdown, and enzyme-linked immunosorbent assay were used to investigate HBHA's impact on macrophages and its relevance in Mycobacterium infection. RESULTS: HBHA inhibited macrophage autophagy. Expression of recombinant HBHA in Mycobacterium smegmatis (rMS-HBHA) inhibited autophagy, promoting bacterial survival within macrophages. Conversely, HBHA knockout in the Mycobacterium bovis bacillus Calmette-Guérin (BCG) mutant (BCG-ΔHBHA) activated autophagy and reduced bacterial survival. Mechanistic investigations revealed that HBHA may inhibit macrophage autophagy through the Toll-like receptor 4-dependent PI3K-AKT-mTOR signaling pathway. Furthermore, HBHA induced macrophage M2 polarization. CONCLUSIONS: Mycobacterium may exploit HBHA to suppress the antimicrobial immune response in macrophages, facilitating intracellular survival and immune evasion through autophagy inhibition and M2 polarization induction. Our findings may help identify novel therapeutic targets and develop more effective treatments against MTB infection.

The SapM phosphatase can arrest phagosome maturation in an ESX-1 independent manner in Mycobacterium tuberculosis and BCG.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that survives and grows in macrophages. A mechanism used by Mtb to achieve intracellular survival is to secrete effector molecules that arrest the normal process of phagosome maturation. Through phagosome maturation arrest (PMA), Mtb remains in an early phagosome and avoids delivery to degradative phagolysosomes. One PMA effector of Mtb is the secreted SapM phosphatase. Because the host target of SapM, phosphatidylinositol-3-phosphate (PI3P), is located on the cytosolic face of the phagosome, SapM needs to not only be released by the mycobacteria but also travel out of the phagosome to carry out its function. To date, the only mechanism known for Mtb molecules to leave the phagosome is phagosome permeabilization by the ESX-1 secretion system. To understand this step of SapM function in PMA, we generated identical in-frame sapM mutants in both the attenuated Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine strain, which lacks the ESX-1 system, and Mtb. Characterization of these mutants demonstrated that SapM is required for PMA in BCG and Mtb. Further, by establishing a role for SapM in PMA in BCG, and subsequently in a Mtb mutant lacking the ESX-1 system, we demonstrated that the role of SapM does not require ESX-1. We further determined that ESX-2 or ESX-4 is also not required for SapM to function in PMA. These results indicate that SapM is a secreted effector of PMA in both BCG and Mtb, and that it can function independent of the known mechanism for Mtb molecules to leave the phagosome.