An ancestral mycobacterial effector promotes dissemination of infection.

The human pathogen Mycobacterium tuberculosis typically causes lung disease but can also disseminate to other tissues. We identified a M. tuberculosis (Mtb) outbreak presenting with unusually high rates of extrapulmonary dissemination and bone disease. We found that the causal strain carried an ancestral full-length version of the type VII-secreted effector EsxM rather than the truncated version present in other modern Mtb lineages. The ancestral EsxM variant exacerbated dissemination through enhancement of macrophage motility, increased egress of macrophages from established granulomas, and alterations in macrophage actin dynamics. Reconstitution of the ancestral version of EsxM in an attenuated modern strain of Mtb altered the migratory mode of infected macrophages, enhancing their motility. In a zebrafish model, full-length EsxM promoted bone disease. The presence of a derived nonsense variant in EsxM throughout the major Mtb lineages 2, 3, and 4 is consistent with a role for EsxM in regulating the extent of dissemination.

A non-canonical type 2 immune response coordinates tuberculous granuloma formation and epithelialization.

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.

Macrophage ACKRobatics: An atypical Cxcr3 keeps macrophages in check.

Discussion on an unusual role for a cxcr3 receptor, in which it antagonizes a paralogous receptor to limit macrophage migration.

Mycobacterial Evolution Intersects With Host Tolerance.

Over the past 200 years, tuberculosis (TB) has caused more deaths than any other infectious disease, likely infecting more people than it has at any other time in human history. Mycobacterium tuberculosis (Mtb), the etiologic agent of TB, is an obligate human pathogen that has evolved through the millennia to become an archetypal human-adapted pathogen. This review focuses on the evolutionary framework by which Mtb emerged as a specialized human pathogen and applies this perspective to the emergence of specific lineages that drive global TB burden. We consider how evolutionary pressures, including transmission dynamics, host tolerance, and human population patterns, may have shaped the evolution of diverse mycobacterial genomes.

Macrophage sphingolipids are essential for the entry of mycobacteria.

Mycobacteria are intracellular pathogens that can invade and survive within host macrophages. Mycobacterial infections remain a major cause of mortality and morbidity worldwide, with serious concerns of emergence of multi and extensively drug-resistant tuberculosis. While significant advances have been made in identifying mycobacterial virulence determinants, the detailed molecular mechanism of internalization of mycobacteria into host cells remains poorly understood. Although several studies have highlighted the crucial role of sphingolipids in mycobacterial growth, persistence and establishment of infection, the role of sphingolipids in the entry of mycobacteria into host cells is not known. In this work, we explored the role of host membrane sphingolipids in the entry of Mycobacterium smegmatis into J774A.1 macrophages. Our results show that metabolic depletion of sphingolipids in host macrophages results in a significant reduction in the entry of M. smegmatis. Importantly, the entry of Escherichia coli into host macrophages under similar conditions remained invariant, implying the specificity of the requirement of sphingolipids in mycobacterial entry. To the best of our knowledge, our results constitute the first report demonstrating the role of host macrophage sphingolipids in the entry of mycobacteria. Our results could help in the development of novel therapeutic strategies targeting sphingolipid-mediated entry of mycobacteria into host cells.

Identification of Mycobacterial Genes Involved in Antibiotic Sensitivity: Implications for the Treatment of Tuberculosis with ß-Lactam-Containing Regimens.

In a Mycobacterium smegmatis mutant library screen, transposon mutants with insertions in fhaA, dprE2, rpsT, and parA displayed hypersusceptibility to antibiotics, including the ß-lactams meropenem, ampicillin, amoxicillin, and cefotaxime. Sub-MIC levels of octoclothepin, a psychotic drug inhibiting ParA, phenocopied the parA insertion and enhanced the bactericidal activity of meropenem against Mycobacterium tuberculosis in combination with clavulanate. Our study identifies novel factors associated with antibiotic resistance, with implications in repurposing ß-lactams for tuberculosis treatment.

Insights into the function of FhaA, a cell division-associated protein in mycobacteria.

FhaA is a forkhead-associated domain-containing protein, the depletion of which leads to accumulation of peptidoglycan (PG) precursors at the septum and poles in Mycobacterium smegmatis (M. smegmatis), by a mechanism undefined thus far. To elucidate its function, we constructed an fhaA (MSMEG_0035) knockout (ΔfhaA) strain in M. smegmatis and demonstrated that this gene is dispensable for in vitro growth. The mutant showed a short cell length phenotype due to a probable defect in cell elongation/cell wall synthesis, which was reversed by complementation with both M. smegmatis and Mycobacterium tuberculosis (M. tb) fhaA (Rv0020c), confirming their association with the observed phenotype. The identification of penicillin binding protein A (PbpA), a PG biosynthesis enzyme as an interacting partner for mycobacterial FhaA, provided a hint into the functioning of FhaA. A drastic reduction in the levels of ectopically expressed PbpA in the ΔfhaA mutant vs wild-type M. smegmatis suggested that FhaA interacts with and stabilises PbpA. In addition, the fhaA deletion mutant was sensitive to multiple classes of antibiotics pointing to a general permeability defect. Our findings uncover a role for FhaA in PG biosynthesis and suggest its involvement in the maintenance of mycobacterial cell envelope integrity.

Identification of novel loci associated with mycobacterial isoniazid resistance.

Despite the known association of several genes to clinical Isoniazid (INH) resistance, its molecular basis remains unknown in ~16% of clinical isolates of Mycobacterium tuberculosis (M. tb). While screening a set of Mycobacterium smegmatis (M. smegmatis) transposon mutants with altered colony morphology for differential susceptibility to INH, we found six resistant mutants and mapped their transposon insertion sites. The disrupted genes in six INH resistant mutants were homologs of M. tb ctaE, rplY, tatA, csd and tatB with one insertion mapping to the promoter region of M. smegmatis ctaE. MIC measurements indicated a wide spectrum of INH resistance in these mutants, with complementation analyses of four selected mutants with the cognate M. smegmatis genes and their M. tb homologs confirming the association of the disrupted genes with INH resistance. Our discovery of novel genes associated with INH resistance could lead to the identification of novel INH resistance mechanisms and possibly new diagnostic modalities as well.

Identifying novel mycobacterial stress associated genes using a random mutagenesis screen in Mycobacterium smegmatis.

Cell envelope associated components of Mycobacterium tuberculosis (M.tb) have been implicated in stress response, immune modulation and in vivo survival of the pathogen. Although many such factors have been identified, there is a large disparity between the number of genes predicted to be involved in functions linked to the envelope and those described in the literature. To identify and characterise novel stress related factors associated with the mycobacterial cell envelope, we isolated colony morphotype mutants of Mycobacterium smegmatis (M. smegmatis), based on the hypothesis that mutants with unusual colony morphology may have defects in the biosynthesis of cell envelope components. On testing their susceptibility to stress conditions relevant to M.tb physiology, multiple mutants were found to be sensitive to Isoniazid, Diamide and H2O2, indicative of altered permeability due to changes in cell envelope composition. Two mutants showed defects in biofilm formation implying possible roles for the target genes in antibiotic tolerance and/or virulence. These assays identified novel stress associated roles for several mycobacterial genes including sahH, tatB and aceE. Complementation analysis of selected mutants with the M. smegmatis genes and their M.tb homologues showed phenotypic restoration, validating their link to the observed phenotypes. A mutant carrying an insertion in fhaA encoding a forkhead associated domain containing protein, showed reduced survival in THP-1 macrophages, providing in vivo validation to this screen. Taken together, these results suggest that the M.tb homologues of a majority of the identified genes may play significant roles in the pathogenesis of tuberculosis.

Dissecting the membrane cholesterol requirement for mycobacterial entry into host cells.

Mycobacteria are intracellular pathogens that can invade and survive within host macrophages, and are a major cause of mortality and morbidity worldwide. The molecular mechanism involved in the internalization of mycobacteria is poorly understood. In this work, we have explored the role of host membrane cholesterol in the entry of the avirulent surrogate mycobacterial strain Mycobacterium smegmatis into THP-1 macrophages. Our results show that depletion of host membrane cholesterol using methyl-ß-cyclodextrin results in a significant reduction in the entry of M. smegmatis into host cells. More importantly, we show that the inhibition in the ability of M. smegmatis to enter host macrophages could be reversed upon replenishment of membrane cholesterol. To the best of our knowledge, these results constitute the first report showing that membrane cholesterol replenishment can reverse the inhibition in the entry of mycobacteria into host cells. In addition, we demonstrate that cholesterol complexation using amphotericin B (without physical depletion) is sufficient to inhibit mycobacterial entry. Importantly, we observed a significant reduction in mycobacterial entry upon enrichment of host membrane cholesterol. Taken together, our results demonstrate, for the first time, that an optimum host plasma membrane cholesterol is necessary for the entry of mycobacteria. These results assume relevance in the context of developing novel therapeutic strategies targeting cholesterol-mediated mycobacterial host cell entry.