Mycobacterium avium complex (MAC) is the leading cause of non-tuberculous mycobacterial pulmonary disease (NTM-PD), a chronic infection with a heterogeneous clinical course. Although murine models of MAC-PD exist, faithfully reproducing the progressive pathology and variable treatment responses of the disease remains challenging. Here, we assessed the virulence of five clinical MAC strains in immunocompetent BALB/c mice, including a newly identified highly virulent isolate, NBRC112750. Two strains, FKJ-1 and NBRC112750, induced progressive pulmonary infection characterized by rising bacterial loads and extensive lung involvement over 25 weeks postinfection. In BALB/c mice, both strains produced necrotizing granulomas resembling those observed in M. tuberculosis-infected C3HeB/FeJ mice, characterized by neutrophilic infiltration, foamy macrophages, and collagen encapsulation. We further established an inhalation-based infection model using FKJ-1, in which low-dose exposure reproducibly generated necrotizing granulomas. Despite demonstrating in vitro drug susceptibility, FKJ-1 responded poorly to standard antimicrobial, therapy indicating strain-dependent variability in treatment efficacy. Together, these findings establish a murine model that accurately reflects the critical pathological and therapeutic features of MAC-PD and provides a valuable platform for studying MAC pathogenesis and evaluating novel therapies.IMPORTANCEThe global incidence of pulmonary disease (PD) caused by non-tuberculous mycobacteria, particularly Mycobacterium avium complex (MAC), is increasing. However, the mechanisms underlying its pathological heterogeneity and variable treatment outcomes remain poorly understood. Here, we establish a murine model that recapitulates the key features of progressive MAC-PD, including necrotizing granuloma formation. We also demonstrate strain-specific differences in treatment responses despite comparable in vitro drug susceptibility. Notably, highly virulent strains induced necrotizing granulomatous lesions similar to those observed in patients with tuberculosis or MAC-PD. This study provides a valuable in vivo platform for investigating host-pathogen interactions, elucidating strain-dependent pathogenesis, and optimizing treatment strategies for MAC-PD.

ABSTRACT Methionine biosynthesis is essential for Mycobacterium tuberculosis, but the in vivo relevance of individual enzymes remains unclear. We performed transposon sequencing in Mycobacterium bovis BCG under methionine‐free conditions and identified metF as a major fitness determinant. To define enzyme‐specific functions, ΔmetE and ΔmetF mutants were generated in M. tuberculosis and evaluated using in vitro growth assays and a murine infection model. The ΔmetE mutant showed vitamin B12‐dependent growth in minimal medium but maintained normal growth in mice, indicating that its in vitro requirement does not translate to an in vivo defect. In contrast, ΔmetF exhibited strict methionine auxotrophy in vitro and completely failed to survive or persist in murine lungs and spleens. Structural modeling showed that MetF is distinct from human homologs, supporting selective inhibition. These findings demonstrate that MetF, but not MetE, is indispensable for M. tuberculosis survival in vivo and identify MetF as a promising metabolic drug target.

Clinical isolates of Mycobacterium avium complex (MAC) differ markedly in their capacity to persist in mouse lungs, complicating strain selection for experimental infection and treatment-response studies. Here, we applied a screening strategy that combined genome-guided pooled infection with whole-genome sequencing to identify mouse-persistent MAC clinical isolates. Thirty-nine MAC clinical isolates, including 13 clarithromycin-resistant isolates, were classified into eight genome-based groups and used for pooled intranasal infection in BALB/c mice. At 8 weeks postinfection, pooled groups that maintained or increased lung bacterial burdens underwent bacterial whole-genome sequencing and deconvolution of isolate proportions based on isolate-specific single-nucleotide variants. Candidate isolates exhibiting increased relative representation after infection were selected for single-isolate validation. MAV22, MI01, and MI05 increased lung bacterial burdens, whereas MAV17 showed a modest decline but retained measurable burdens suitable for downstream assessment. These isolates were subsequently evaluated in a multidrug treatment assay comprising clarithromycin, rifampicin, and ethambutol. Treatment reduced lung bacterial burdens in mice infected with clarithromycin-susceptible isolates, whereas the clarithromycin-resistant isolate MAV22 exhibited a smaller reduction. This screening strategy provides a practical first-pass approach for identifying MAC clinical isolates that maintain measurable lung bacterial burdens and are suitable for CFU-based treatment-response assessment.

v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a candidate gene associated with early tuberculosis onset identified by a genome-wide association study. Here, we investigated the role of Mafb in susceptibility to Mycobacterium tuberculosis (Mtb) infection in myeloid-specific Mafb-knockout (Mafb-cKO) mice. Mtb infection was performed both in vitro using bone marrow-derived macrophages (BMMs) from Mafb-cKO mice and in vivo in Mafb-cKO mice. The absence of Mafb promoted Mtb proliferation in BMMs. RNA sequencing (RNA-seq) revealed activation of the metabolic process and impairment of the response to type I interferons (IFNs) in Mtb-infected BMMs from Mafb-cKO mice, which conforms to our previous findings in Mtb-infected human macrophages with MAFB knockdown. Mafb deficiency increased mortality and bacterial burden in the lungs and spleens during Mtb infection in mice. RNA-seq revealed weakened leukocyte or lymphocyte chemotaxis in Mtb-infected Mafb-cKO mouse lungs. Flow cytometry demonstrated an alteration in the proportion of immune cells in Mtb-infected mouse lungs due to Mafb deficiency. Together, Mafb in myeloid cells is involved not only in the functional antibacterial process of macrophages but also in immune cell recruitment in the lungs, thereby contributing to host defense against Mtb infection.

ABSTRACT Mycobacterium avium complex (MAC) is the leading cause of nontuberculous mycobacterial pulmonary disease (NTM-PD) and mainly comprises M. avium (MAV) and M. intracellulare (MI). Host–pathogen interactions may contribute to the heterogeneous clinical course of MAC pulmonary disease (MAC-PD); however, species- or isolate-associated differences in virulence and host immune responses induced by MAC strains remain poorly understood. Here, we established a panel of MAC clinical isolates exhibiting persistent pulmonary infection in mice and performed transcriptomic analyses to evaluate pulmonary immune responses. Although MAC infections induced broadly shared inflammatory responses, MI infection elicited a robust IL-17/neutrophilic inflammatory signature, whereas MAV infection showed relative enrichment of IFN-γ/cytotoxicity-associated responses, indicating differences in the balance of their immune gene expression programs. The MI-associated IL-17/neutrophilic program remained evident at the isolate level and after adjustment for pulmonary growth phenotype. RT-qPCR and flow cytometric analyses using the representative isolate pair FKJ-1 (MI) and FKJ-8 (MAV) further supported the differential induction of these immune signatures in infected lungs. To define the cellular basis of these immune programs, we performed single-cell RNA sequencing on lungs infected with FKJ-1 and FKJ-8. Infection with the representative MI isolate was associated with increased Il17a -expressing CD4 + T cells and γδ T cells, neutrophilic inflammation, and the expansion of inflammatory macrophages. Taken together, these findings demonstrate that within a selected panel of persistent MAC clinical isolates, MI infection is associated with a robust IL-17/neutrophilic pulmonary immune program, providing a preclinical framework for dissecting species- and isolate-associated host–pathogen interactions in MAC-PD.

Background Impaired mucociliary clearance is associated with nontuberculous mycobacterial lung disease (NTM-LD). While airway epithelial cells (AECs), which are essential for maintaining this defense mechanism, play a central role in NTM-LD pathogenesis, their in vivo gene expression in human NTM-LD remains unexplored. We investigated AEC gene expression using surgical specimens of Mycobacterium avium complex lung disease (MAC-LD) patients. Methods We profiled gene expression of AECs from surgical specimens of age-matched female MAC-LD patients (n=7) and lung cancer patients (n=8), the latter serving as non-infectious controls. Differentially expressed genes identified through weighted gene co-expression network analysis were validated using qRT‒PCR in an independent cohort (MAC-LD: n=19, Lung cancer: n=25). Associations between AEC gene expression and MAC-LD clinical phenotypes were also investigated. Results We identified 54 upregulated and 4 downregulated genes in AECs from MAC-LD patients. In addition to immune-related genes enriched in antigen presentation, complement and coagulation cascades, neutrophil migration, and the IL-17 signaling pathway, SLC26A4 , which encodes the anion exchanger pendrin, was markedly upregulated (log2 fold change=3.0, FDR=0.8×10 -5 ). The increased expression of the selected genes CCL20 , MMP9 , C3 , and SLC26A4 was validated, and their reproducibility was confirmed in the validation cohort. Among the 26 MAC-LD patients, the AEC gene expression level of MMP9 was significantly elevated in patients with cavitary lesions, whereas SLC26A4 expression was correlated with bronchiectasis severity. Conclusions SLC26A4 , an anion exchanger, was markedly upregulated in MAC-LD AECs, alongside various immune-related genes. The associations between gene expression and key disease phenotypes suggest potential targets for novel therapeutics.

Tuberculosis (TB) pathology involves complex immune responses within granulomatous lesions. Using single-cell RNA sequencing, we characterized the cellular compositions of necrotizing granulomatous lesions that developed in the lungs of Mycobacterium tuberculosis-infected C3HeB/FeJ mice. We identified 11 distinct major cell types, including phagocytes such as neutrophils and macrophages, and T cells, natural killer cells, B cells, dendritic cells, and plasmacytoid dendritic cells. Among T cells, particularly, Pdcd1+ γδ T cells were detected in necrotizing granulomatous lesions, suggesting their potential role in the pathogenicity of M. tuberculosis. Within the macrophage populations, we identified a cluster with significantly higher Plin2 expression compared to other clusters, whose transcriptomic profile was consistent with that of foamy macrophages. A subset of the Plin2-expressing macrophages was identified as a major source of Ifnb1 and Cxcl1, suggesting their involvement in type I interferon signaling and neutrophil recruitment. Furthermore, we identified Flrt2, Hyal1, and Mmp13 as novel molecular markers of Plin2-expressing macrophages, which were localized to the peripheral rim regions of necrotizing granulomas. In conclusion, our results provide the immune landscape of necrotizing granulomas and reveal novel functional states of macrophages contributing to TB pathogenesis.

Background:Impaired mucociliary clearance is associated with nontuberculous mycobacterial lung disease (NTM-LD). While airway epithelial cells (AECs), which are essential for maintaining this defence mechanism, play a central role in NTM-LD pathogenesis, their in vivo gene expression in human NTM-LD remains unexplored. We investigated AEC gene expression using surgical specimens of Mycobacterium avium complex lung disease (MAC-LD) patients. Methods:We profiled gene expression of AECs from surgical specimens of age-matched female MAC-LD patients (n=7) and lung cancer patients (n=8), the latter serving as noninfectious controls. Differentially expressed genes identified through weighted gene coexpression network analysis were validated using quantitative reverse transcriptase PCR (qRT‒PCR) in an independent cohort (MAC-LD: n=19, Lung cancer: n=25). Associations between AEC gene expression and MAC-LD clinical phenotypes were also investigated. Results:We identified 54 upregulated and 4 downregulated genes in AECs from MAC-LD patients. In addition to immune-related genes enriched in antigen presentation, complement and coagulation cascades, neutrophil migration and the interleukin-17 signalling pathway, SLC26A4, which encodes the anion exchanger pendrin, was markedly upregulated (log2 fold change of 3.0, false discovery rate of 0.8×10-5). The increased expression of the selected genes CCL20, MMP9, C3 and SLC26A4 was validated, and their reproducibility was confirmed in the validation cohort. Among the 26 MAC-LD patients, the AEC gene expression level of MMP9 was significantly elevated in patients with cavitary lesions, whereas SLC26A4 expression was correlated with bronchiectasis severity. Conclusions:SLC26A4, an anion exchanger, was markedly upregulated in MAC-LD AECs, alongside various immune-related genes. The associations between gene expression and key disease phenotypes suggest potential targets for novel therapeutics.

Understanding the functions of human transcriptional regulatory genes SP110 and SP140 during Mycobacterium tuberculosis infection is crucial; in a mouse model, homologous genes Sp110 and Sp140 have been shown to negatively regulate inflammatory response genes, including the type I interferon (IFN) response. The reduction of these genes in mice is associated with susceptibility to M. tuberculosis infection and the development of necrotizing granulomatous lesions. To investigate the involvement of SP110 and SP140 in human inflammatory response, we analyzed their regulatory manner in THP-1 macrophages infected with M. tuberculosis. Genome-wide transcriptional profiling revealed that the depletion of SP110 and/or SP140 impaired the induction of gene expression associated with inflammatory responses, including IFN response genes, although it had little effect on the intracellular proliferation of M. tuberculosis. By contrast, genes related to phosphorylation were upregulated in infected macrophages with SP110 and/or SP140 knockdown, but downregulated in infected control macrophages without their knockdown. Reverse transcription-quantitative PCR and ELISA further confirmed the impairment of the induction of IFN response genes by the depletion of SP110 and/or SP140 in M. tuberculosis-infected macrophages. These findings suggest that human SP110 and SP140 act as positive regulators for genes associated with inflammatory responses in M. tuberculosis-infected macrophages. IMPORTANCE Tuberculosis (TB) is one of the most serious infectious diseases, with high morbidity and mortality worldwide. C3HeB/FeJ mice are widely utilized for evaluating anti-TB drugs because their drug sensitivity and pathology during M. tuberculosis infection resemble those of human TB, including the development of necrotizing granulomas. Downregulation of the transcriptional regulatory genes Sp110 and Sp140 in C3HeB/FeJ mice has been demonstrated to activate gene expression associated with inflammatory responses during M. tuberculosis infection, resulting in susceptibility to the infection. Here, we examined the regulatory manner of SP110 and SP140 using transcriptomic analysis in M. tuberculosis-infected human macrophages. Depletion of SP110 and/or SP140 in M. tuberculosis-infected THP-1 macrophages impaired the induction of gene expression associated with inflammatory responses, including interferon response genes, compared with that in control macrophages. These results suggest that human SP110 and SP140 act as positive regulators for genes associated with inflammatory responses upon M. tuberculosis infection.

IntroductionIt is assumed that host defense systems eliminating the pathogen and regulating tissue damage make a strong impact on the outcome of tuberculosis (TB) disease and that these processes are affected by rifampicin (RIF) resistance–conferring mutations of Mycobacterium tuberculosis (Mtb). However, the host responses to the pathogen harboring different mutations have not been studied comprehensively in clinical settings. We analyzed clinico-epidemiological factors and blood transcriptomic signatures associated with major rpoB mutations conferring RIF resistance in a cohort study.MethodsDemographic data were collected from 295 active pulmonary TB patients with treatment history in Hanoi, Vietnam. When recruited, drug resistance–conferring mutations and lineage-specific variations were identified using whole-genome sequencing of clinical Mtb isolates. Before starting retreatment, total RNA was extracted from the whole blood of HIV-negative patients infected with Mtb that carried either the rpoB H445Y or rpoB S450L mutation, and the total RNA was subjected to RNA sequencing after age-gender matching. The individual RNA expression levels in the blood sample set were also measured using real-time RT-PCR. Logistic and linear regression models were used to assess possible associations.ResultsIn our cohort, rpoB S450L and rpoB H445Y were major RIF resistance–conferring mutations [32/87 (36.8%) and 15/87 (17.2%), respectively]. H445Y was enriched in the ancient Beijing genotype and was associated with nonsynonymous mutations of Rv1830 that has been reported to regulate antibiotic resilience. H445Y was also more frequently observed in genetically clustered strains and in samples from patients who had received more than one TB treatment episode. According to the RNA sequencing, gene sets involved in the interferon-γ and-α pathways were downregulated in H445Y compared with S450L. The qRT-PCR analysis also confirmed the low expression levels of interferon-inducible genes, including BATF2 and SERPING1, in the H445Y group, particularly in patients with extensive lesions on chest X-ray.DiscussionOur study results showed that rpoB mutations as well as Mtb sublineage with additional genetic variants may have significant effects on host response. These findings strengthen the rationale for investigation of host-pathogen interactions to develop countermeasures against epidemics of drug-resistant TB.

In male mice, defeat in social encounters is associated with an acute non-opioid analgesia, a reaction that may also be seen in response to the scent of a territorial conspecific. As this form of pain inhibition is blocked by diazepam and Ro15-1788, benzodiazepine receptor mediation has been proposed. To further test this hypothesis, the effects of a novel benzodiazepine receptor antagonist (Ro15-3505; 0.625–20 mg/kg) on basal nociception and defeat analgesia have been examined. Results show that, although devoid of intrinsic activity on the mouse tail-flick assay, Ro15-3505 totally blocks the analgesic consequences of defeat at doses above 1.25 mg/kg. Despite certain inconsistencies in the literature, present data provide further support for benzodiazepine receptor mediation of this ecologically-relevant form of pain inhibition.

MAFB, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B, has been identified as a candidate gene for early tuberculosis (TB) onset in Thai and Japanese populations. Here, we investigated the genome-wide transcriptional profiles of MAFB-knockdown (KD) macrophages infected with Mycobacterium tuberculosis (Mtb) to highlight the potential role of MAFB in host immunity against TB. Gene expression analysis revealed impaired type I and type II interferon (IFN) responses and enhanced oxidative phosphorylation in MAFB-KD macrophages infected with Mtb. The expression of inflammatory chemokines, including IFN-γ-inducible genes, was confirmed to be significantly reduced by knockdown of MAFB during Mtb infection. A similar effect of MAFB knockdown on type I and type II IFN responses and oxidative phosphorylation was also observed when Mtb-infected macrophages were activated by IFN-γ. Taken together, our results demonstrate that MAFB is involved in the immune response and metabolism in Mtb-infected macrophages, providing new insight into MAFB as a candidate gene to guide further study to control TB.

Infection with Mycobacterium tuberculosis leads to the development of tuberculosis (TB) with the formation of granulomatous lesions. Foamy macrophages (FM) are a hallmark of TB granulomas, because they provide the primary platform of M. tuberculosis proliferation and the main source of caseous necrosis. In this study, we applied spatial multiomic profiling to identify the signatures of FM within the necrotic granulomas developed in a mouse model resembling human TB histopathology. C3HeB/FeJ mice were infected with M. tuberculosis to induce the formation of necrotic granulomas in the lungs. Using laser microdissection, necrotic granulomas were fractionated into three distinct regions, including the central caseous necrosis, the rim containing FM, and the peripheral layer of macrophages and lymphocytes, and subjected to proteomic and transcriptomic analyses. Comparison of proteomic and transcriptomic analyses of three distinct granulomatous regions revealed that four proteins/genes are commonly enriched in the rim region. Immunohistochemistry confirmed the localization of identified signatures to the rim of necrotic granulomas. We also investigated the localization of the representative markers for M1 macrophages in granulomas because the signatures of the rim included M2 macrophage markers. The localization of both macrophage markers suggests that FM in necrotic granulomas possessed the features of M1 or M2 macrophages. Gene set enrichment analysis of transcriptomic profiling revealed the upregulation of genes related to M2 macrophage activation and mTORC1 signaling in the rim. These results will provide new insights into the process of FM biogenesis, leading to further understanding of the pathophysiology of TB granulomas.

Mycobacterium tuberculosis (Mtb) has different features depending on different geographic areas. We collected Mtb strains from patients with smear-positive pulmonary tuberculosis in Da Nang, central Vietnam. Using a whole genome sequencing platform, including genome assembly complemented by long-read-sequencing data, genomic characteristics were studied. Of 181 Mtb isolates, predominant Vietnamese EAI4_VNM and EAI4-like spoligotypes (31.5%), ZERO strains (5.0%), and part of EAI5 (11.1%) were included in a lineage-1 (L1) sublineage, i.e., L1.1.1.1. These strains were found less often in younger people, and they genetically clustered less frequently than other modern strains. Patients infected with ZERO strains demonstrated less lung infiltration. A region in RD2bcg spanning six loci, i.e., PE_PGRS35, cfp21, Rv1985c, Rv1986, Rv1987, and erm(37), was deleted in EAI4_VNM, EAI4-like, and ZERO strains, whereas another 118 bp deletion in furA was specific only to ZERO strains. L1.1.1.1-sublineage-specific deletions in PE_PGRS4 and PE_PGRS22 were also identified. RD900, seen in ancestral lineages, was present in majority of the L1 members. All strains without IS6110 (5.0%) had the ZERO spoligo-pattern. Distinctive features of the ancestral L1 strains provide a basis for investigation of the modern versus ancestral Mtb lineages and allow consideration of countermeasures against this heterogeneous pathogen.

The intracellular bacterial pathogen Legionella pneumophila uses many effector proteins delivered by the bacterial type IV secretion system (T4SS) to hijack the early secretory pathway to establish its replicative niche, known as the Legione lla-containing vacuole (LCV), On LCV biogenesis, the endoplasmic reticulum (ER) vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNARE) Sec22b is recruited to the bacterial phagosome and forms non-canonical pairings with target membrane SNAREs (t-SNAREs) from the plasma membrane. Here, we identify a Legionella deubiquitinase (DUB), LotB, that can modulate the early secretory pathway by interacting with coatomer protein complex I (COPI) vesicles when ectopically expressed. We show that Sec22b is ubiquitinated upon L. pneumophila infection in a T4SS-dependent manner and that, subsequently, LotB deconjugates K63-linked ubiquitins from Sec22b. The DUB activity of LotB stimulates dissociation of the t-SNARE syntaxin 3 (Stx3) from Sec22b, which resides on the LCV. Our study highlights a bacterial strategy manipulating the dynamics of infection-induced SNARE pairing using a bacterial DUB.

Tuberculosis (TB) and Mycobacterium avium complex lung disease (MAC-LD) are both characterized pathologically by granuloma lesions, which are typically composed of a necrotic caseum at the center surrounded by fibrotic cells and lymphocytes. Although the histological characterization of TB and MAC-LD granulomas has been well-documented, their molecular signatures have not been fully evaluated. In this research we applied mass spectrometry-based proteomics combined with laser microdissection to investigate the unique protein markers in human mycobacterial granulomatous lesions. Comparing the protein abundance between caseous and cellular sub-compartments of mycobacterial granulomas, we found distinct differences. Proteins involved in cellular metabolism in transcription and translation were abundant in cellular regions, while in caseous regions proteins related to antimicrobial response accumulated. To investigate the determinants of their heterogeneity, we compared the protein abundance in caseous regions between TB and MAC-LD granulomas. We found that several proteins were significantly abundant in the MAC-LD caseum of which proteomic profiles were different from those of the TB caseum. Immunohistochemistry demonstrated that one of these proteins, Angiogenin, specifically localized to the caseous regions of selected MAC-LD granulomas. We also detected peptides derived from mycobacterial proteins in the granulomas of both diseases. This study provides new insights into the architecture of granulomatous lesions in TB and MAC-LD.

Background: Isoniazid (INH)-resistant tuberculosis (TB) could seriously affect the fight against TB. We investigated genomic factors that may be associated with katG-S315T, a major INH-resistance conferring mutation, in two large cities of Vietnam. Methods:Mycobacterium tuberculosis (Mtb) isolates were collected from patients with smear-positive pulmonary TB living in two different geographical areas of Vietnam; one in the central and the other in the northern area. Whole genome sequencing was performed using an Illumina MiSeq platform. Major drug resistance-conferring mutations were extracted with the TB-Profiler tool. Using a bacterial genome-wide approach based on linear mixed models, we investigated associations between 31-bp k-mers and Mtb isolates harboring katG-S315T in each cohort, and then compared them. Results: Genome sequences were obtained from 181 Mtb isolates in the central and 322 in the northern area. Isolates with katG-S315T accounted for 12.7% and 26.2% respectively. Ten genes harboring k-mers significantly associated with the katG-S315T phenotype were shared by the two cohorts. Genomic variants responsible for these k-mers of all these genes also showed significant associations with the S315T mutation, when a variant-based approach was conducted. A deletion-based analysis also identified several significant genes, including esxD with a 324-bp deletion and PE_PGRS30 with 109-bp deletion in the central-area cohort. Conclusions: We demonstrated that several genomic variations of the pathogen are associated with katG-S315T in common, and these genomic factors may be related to the low fitness cost of INH-resistant strains in Vietnam.