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The analysis of cell electrophysiology for pathogenic samples at BSL3 can be problematic. It is virtually impossible to isolate infected from uninfected without a label, for example green fluorescent protein, which can potentially alter the cell electrical properties. Furthermore, the measurement of highly pathogenic organisms often requires equipment dedicated only for use with these organisms due to safety considerations. To address this, we have used dielectrophoresis to study the electrical properties of the human THP-1 cell line and monocyte-derived macrophages before and after infection with non-labelled Mycobacterium tuberculosis. Infection with these highly pathogenic bacilli resulted in changes including a raised surface conductance (associated with reduced zeta potential) and increased capacitance, suggesting an increase in surface roughness. We have also investigated the effect of fixation on THP-1 cells as a means to enable study on fixed samples in BSL1 or 2 laboratories, which suggests that the properties of these cells are largely unaffected by the fixation process. This advance results in a novel technique enabling the isolation of infected and non-infected cells in a sample without labelling., (© 2024 The Authors. ELECTROPHORESIS published by Wiley‐VCH GmbH.)

In low-resource settings with high tuberculosis (TB) burdens, lack of rapid diagnostic methods for detection and differentiation of Mycobacterium tuberculosis complex (MTBC) is a major challenge affecting TB management. This study utilized comparative genomic analyses of MTBC lineages; M. tuberculosis , M. africanum Lineages 5/6 and M. bovis to identify lineage-specific genes. Primers were designed for the development of a Multiplex PCR assay which was successful in differentiating the MTBC lineages. There was no cross-reaction with other respiratory pathogens tested. Validation of the assay using clinical samples was performed with sputum DNA extracts from 341 clinically confirmed active TB patients. It was observed that 24.9% of cases were caused by M. tuberculosis , while M. africanum L5 & L6 reported 9.0% and 14.4%, respectively. M. bovis infection was the least frequently detected lineage with 1.8%. Also, 27.0% and 17.0% of the cases were PCR negative and unspeciated, respectively. However, mixed-lineage TB infections were recorded at a surprising 5.9%. This multiplex PCR assay will allow speciation of MTBC lineages in low-resource regions, providing rapid differentiation of TB infections to select appropriate medication at the earliest possible time point. It will also be useful in epidemiological surveillance studies providing reliable information on the prevalence of TB lineages as well as identifying difficult to treat cases of mixed-lineage tuberculosis infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Owusu, van Vliet, Riddell, Stewart, Akwani, Aryeetey, Arthur, Sylverken and Hingley-Wilson.)

Mycobacterium tuberculosis-induced cellular aggregation is essential for granuloma formation and may assist establishment and early spread of M. tuberculosis infection. The M. tuberculosis ESX1 mutant, which has a non-functional type VII secretion system, induced significantly less production of the host macrophage-derived chemokine fractalkine (CX3CL1). Upon infection of human macrophages ESX1-dependent fractalkine production mediated selective recruitment of CD11b+ monocytic cells and increased infection of neighbouring cells consistent with early local spread of infection. Fractalkine levels were raised in vivo at tuberculous disease sites in humans and were significantly associated with increased CD11b+ monocytic cellular recruitment and extent of granulomatous disease. These findings suggest a novel fractalkine-dependent ESX1-mediated mechanism in early tuberculous disease pathogenesis in humans. Modulation of M. tuberculosis-mediated fractalkine induction may represent a potential treatment option in the future, perhaps allowing us to switch off a key mechanism required by the pathogen to spread between cells., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance.

The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 10-kDa culture filtrate antigen (CFP-10), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv3615c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-10 and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospot- and flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus Calmette-Guérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-10 in both active and latent TB infection. EspC contained broadly recognized CD4(+) and CD8(+) epitopes, inducing a predominantly CD4(+) T-cell response that comprised functional T-cell subsets secreting both IFN-γ and IL-2 as well as functional T-cell subsets secreting only IFN-γ. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-10, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-10, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis.

Studying defined mutants of Mycobacterium tuberculosis in the mouse model of infection has led to the discovery of attenuated mutants that fall into several phenotypic classes. These mutants are categorized by their growth characteristics compared with those of wild-type M. tuberculosis, and include severe growth in vivo mutants, growth in vivo mutants, persistence mutants, pathology mutants and dissemination mutants. Here, examples of each of these mutant phenotypes are described and classified accordingly. Defining the importance of mycobacterial gene products responsible for in vivo growth, persistence and the induction of immunopathology will lead to a greater understanding of the host-pathogen interaction and potentially to new antimycobacterial treatment options.

Mcl-1 protein expression was found to be up-regulated during infection with virulent Mycobacterium tuberculosis strain H37Rv. Mcl-1 induction in THP-1 cells was optimal at a multiplicity of infection of 0.8-1.2 bacilli per macrophage and was independent of opsonin coating of the bacteria. Mcl-1 expression was elevated as early as 4 h, peaked at 5.8-fold above control cells at 24 h, and remained elevated at 48 h after infection. In THP-1 cells, mMcl-1 mRNA was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. In THP-1 cells and monocyte-derived macrophages (MDMs), Mcl-1 protein was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. Treatment of uninfected, H37Ra-infected, and H37Rv-infected THP-1 cells and MDMs with antisense oligonucleotides to mcl-1 reduced Mcl-1 expression by >84%. This resulted in an increase in apoptosis of both MDMs and THP-1 cells that were infected with H37Rv, but not cells that were uninfected or infected with H37Ra. Increased apoptosis correlated with a decrease in M. tuberculosis CFUs recovered from antisense-treated, H37Rv-infected cells at 4 and 7 days after infection. In contrast, CFU recoveries from sense-treated, H37Rv-infected cells or from antisense- or sense-treated, H37Ra-infected cells were unchanged from controls. Thus, the antiapoptotic effect of the induction of Mcl-1 expression in H37Rv-infected macrophages promotes the survival of virulent M. tuberculosis.

The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 1O-kDa culture filtrate antigen (CFP-1O), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv361 5c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-1O and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospotand flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus CalmetteGuérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-1O in both active and latent TB infection. EspC contained broadly recognized CD4 and CD8 epitopes, inducing a predominantly CD4* T-cell response that comprised functional T-cell subsets secreting both IFN-y and lL-2 as well as functional T-cell subsets secreting only IFN-y. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-1O, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-1O, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis. [ABSTRACT FROM AUTHOR]

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting δRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis δRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the δRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We ,conclude that the primary attenuating mechanism of bacillus Calmette-Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness. [ABSTRACT FROM AUTHOR]

It is thought that during latent infection, Mycobacterium tuberculosis bacilli are retained within granulomas in a lowoxygen environment. The dormancy survival (Dos) regulon, regulated by the response regulator DosR, appears to be essential for hypoxic survival in M. tuberculosis, but it is not known how the regulon promotes survival. Here we report that mycobacteria, in contrast to enteric bacteria, do not form higher-order structures (e.g. ribosomal dimers) upon entry into stasis. Instead, ribosomes are stabilized in the associated form (70S). Using a strategy incorporating microfluidic, proteomic, and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during hypoxic stasis, we show that the dormancy regulator DosR is required for optimal ribosome stabilization. We present evidence that the majority of this effect is mediated by the DosR-regulated protein MSMEG_3935 (a S30AE domain protein), which is associated with the ribosome under hypoxic conditions. A Δ3935 mutant phenocopies the ΔdosR mutant during hypoxia, and complementation of ΔdosR with the MSMEG_3935 gene leads to complete recovery of dosR mutant phenotypes during hypoxia. We suggest that this protein is named ribosome-associated factor under hypoxia (RafH) and that it is the major factor responsible for DosR-mediated hypoxic survival in mycobacteria. [ABSTRACT FROM AUTHOR]