The ESX-1 secretion system senses bacterial contact and prepares mycobacteria for environmental adaptation

The ESX-1 system (6-kDa early secretory antigenic target (ESAT-6) secretion system-1) is essential for Mycobacterium tuberculosis pathogenesis and conjugal transfer in Mycobacterium smegmatis, yet little is known about how its function is regulated. Live-cell fluorescence microscopy showed natively expressed ESX-1 was organized into distinct foci predominantly observed at cell-cell contacts. These foci formed when two cells touched and required a fully assembled ESX-1 system in both bacteria, suggesting the generation of an ESX-1 megacomplex across multiple membranes. The emergence of ESX-1 foci and ESX-1 secretion was environmentally dependent: foci formed in low nitrogen environments in which secretion was suppressed, yet with increasing concentrations of nitrogen, ESX-1 systems diffused along the plasma membrane and secretion was activated. Genome-wide transcriptional profiling revealed ESX-1 dependent induction of genes required for the SOS response and error prone DNA replication in high nitrogen. Based on these findings, we propose a new model of ESX-1 function where ESX-1 localization and secretion are responsive to nitrogen levels and form an integral node in the mycobacterial response to neighboring cells and environmental adaptation.

Early secretory antigenic target 6 and culture filtrate protein 10 as diagnostic indicators in IgA nephropathy associated with renal tuberculosis

Background: This study evaluated the diagnostic value of early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) in immunoglobulin A nephropathy (IgAN) associated with renal tuberculosis (RT).Methods: Between January 2013 and January 2016, 40 patients with IgAN (IgAN group), 32 patients with RT (RT group), and 52 patients with IgAN associated with RT (IgAN + RT group) were selected for this study. A Tuberculin skin test (TST) was conducted, and serum Mycobacterium tuberculosis (MTB) antibody levels were measured. Urine samples were collected to culture MTB. Immunohistochemistry and western blotting were used to determine the expression of ESAT-6 and CFP-10 in renal tissues. Receiver operating characteristic (ROC) analysis was used to evaluate the diagnostic values of ESAT-6 and CFP-10 in IgAN associated with RT.Results: TST, serum MTB antibody, and urine MTB assessments were negative in the IgAN group. The positive rates of the TST and serum MTB antibody and urine MTB testing were higher in the RT group than in the IgAN + RT group. Among the three groups, expression levels of ESAT-6 and CFP-10 were found to be the highest in the IgAN + RT group and were found to be the lowest in the IgAN group. The ROC curves indicated that the area under curve (AUC) value of ESAT-6 protein for IgAN + RT diagnosis was 0.907 with a cut-off of 26.72 as the critical value. Detection by ESAT-6 protein levels achieved 75.0% sensitivity and 94.2% specificity. The AUC value of the CFP-10 protein for diagnosis of IgAN + RT was 0.800, with a cut-off of 25.665 as the critical value. Detection by the protein levels of CFP-10 showed 63.9% sensitivity and 84.6% specificity.Conclusions: Our study provides evidence for the potential of the proteins ESAT-6 and CFP-10 as candidate markers for the diagnosis of IgAN associated with RT.

Differed IL-1 Beta Response between Active TB and LTBI Cases by Ex Vivo Stimulation of Human Monocyte-Derived Macrophage with TB-Specific Antigen

Background. The difference of macrophage-specific interleukin-1 beta (IL-1b) response between latent tuberculosis infection (LTBI) and active tuberculosis (TB) remains less studied. Method. We performed this prospective study and recruited active TB patients, contacts with LTBI, and uninfected contacts. The gene and protein expression of human monocyte-derived macrophage (hMDM) after ex vivo stimulation by early secretory antigenic target-6KD (ESAT-6) and tuberculin purified protein derivatives (PPD) was studied by real-time PCR and flow cytometry. The effect of caspase-1 inhibitor was also studied. Result. The IL-1b gene expression after 6 hr ESAT-6 1 μg/ml stimulation was different among active TB patients (n=12), LTBI cases (n=12), and uninfected contacts (n=23) (log fold change: 0.98±1.26 vs. 2.20±0.96 vs. 2.20±0.96, P=0.013). The IL-1b gene expression at 24 hours was higher than that at 6 hours in LTBI cases (n=4) and uninfected contacts (n=6). After 24 hr ESAT-6 1 μg/ml stimulation, the percentage of IL-1b-expressed hMDM was borderline lower in the active TB patients (n=9) than in the LTBI cases (n=10) (14.0±11.2% vs. 31.6±22.5%, P=0.065). Compared with ESAT-6 1 μg/ml stimulation but without the addition of caspase-1 inhibitor (CasI) (55.6±16.3%), the percentage of IL-1b-positive hMDMs decreased after addition of CasI (50 μg/ml CasI: 49.8±18.2%, P=0.078; 100 μg/ml CasI: 46.6±20.8%, P=0.030; 150 μg/ml CasI: 33.7±15.5%, P=0.016). Conclusions. This study revealed that macrophage-specific IL-1b response differed among different stages of Mycobacterium tuberculosis infection. The role of IL-1b and inflammasome in the process of LTBI progressing to active TB warrants further investigation.

Diagnostic Accuracy of Early Secretory Antigenic Target-6–Free Interferon-gamma Release Assay Compared to QuantiFERON-TB Gold In-tube

Background Early secretory antigenic target-6 (ESAT-6) is an immunodominant Mycobacterium tuberculosis (M.tb) antigen included in novel vaccines against tuberculosis (TB) and in interferon-gamma (IFN-γ) release assays (IGRAs). Therefore, the availability of an ESAT-6–free IGRA is essential to determine M.tb infection status following vaccination with ESAT-6–containing vaccines. We aimed to qualify a recently developed ESAT-6–free IGRA and to assess its diagnostic performance in comparison to QuantiFERON-TB Gold In-tube (QFT). Methods Participants with different levels of M.tb exposure and TB disease were enrolled to determine the ESAT-6–free IGRA cutoff, test assay performance in independent cohorts compared to standard QFT, and perform a technical qualification of antigen-coated blood collection tubes. Results ESAT-6–free IGRA antigen recognition was evaluated in QFT-positive and QFT-negative South African adolescents. The ESAT-6–free IGRA cutoff was established at 0.61 IU/mL, based on receiver operating characteristic analysis in M.tb-unexposed controls and microbiologically confirmed pulmonary TB patients. In an independent cohort of healthy adolescents, levels of IFN-γ released in QFT and ESAT-6–free IGRA were highly correlated (P < .0001, r = 0.83) and yielded comparable positivity rates, 41.5% and 43.5%, respectively, with 91% concordance between the tests (kappa = 0.82; 95% confidence interval, 0.74–0.90; McNemar test P = .48). ESAT-6–free IGRA blood collection tubes had acceptable lot-to-lot variability, precision, and stability. Conclusions The novel ESAT-6–free IGRA had diagnostic accuracy comparable to QFT and is suitable for use in clinical trials to assess efficacy of candidate TB vaccines to prevent established M.tb infection.

Retention of EsxA in the Capsule-Like Layer ofMycobacterium tuberculosisIs Associated with Cytotoxicity and Is Counteracted by Lung Surfactant

ABSTRACTMycobacterium tuberculosis, the pathogen that causes tuberculosis, primarily infects macrophages but withstands the host cell’s bactericidal effects. EsxA, also called virulence factor 6-kDa early secretory antigenic target (ESAT-6), is involved in phagosomal rupture and cell death. We provide confocal and electron microscopy data showing thatM. tuberculosisbacteria grown without detergent retain EsxA on their surface. Lung surfactant has detergent-like properties and effectively strips off this surface-associated EsxA, which advocates a novel mechanism of lung surfactant-mediated defense against pathogens. Upon challenge of human macrophages with theseM. tuberculosisbacilli, the amount of surface-associated EsxA rapidly declines in a phagocytosis-independent manner. Furthermore,M. tuberculosisbacteria cultivated under exclusion of detergent exert potent cytotoxic activity associated with bacterial growth. Together, this study suggests that the surface retention of EsxA contributes to the cytotoxicity ofM. tuberculosisand highlights how cultivation conditions affect the experimental outcome.

Clinical Evaluation of a Blood Assay to Diagnose Paucibacillary Tuberculosis via Bacterial Antigens

BACKGROUND The diagnosis of active tuberculosis (TB) cases primarily relies on methods that detect Mycobacterium tuberculosis (Mtb) bacilli or their DNA in patient samples (e.g., mycobacterial culture and Xpert MTB/RIF assays), but these tests have low clinical sensitivity for patients with paucibacillary TB disease. Our goal was to evaluate the clinical performance of a newly developed assay that can rapidly diagnose active TB cases by direct detection of Mtb-derived antigens in patients' blood samples. METHODS Nanoparticle (NanoDisk)-enriched peptides derived from the Mtb virulence factors CFP-10 (10-kDa culture factor protein) and ESAT-6 (6-kDa early secretory antigenic target) were analyzed by high-throughput mass spectrometry (MS). Serum from 294 prospectively enrolled Chinese adults were analyzed with this NanoDisk-MS method to evaluate the performance of direct serum Mtb antigen measurement as a means for rapid diagnosis of active TB cases. RESULTS NanoDisk-MS diagnosed 174 (88.3%) of the study's TB cases, with 95.8% clinical specificity, and with 91.6% and 85.3% clinical sensitivity for culture-positive and culture-negative TB cases, respectively. NanoDisk-MS also exhibited 88% clinical sensitivity for pulmonary and 90% for extrapulmonary TB, exceeding the diagnostic performance of mycobacterial culture for these cases. CONCLUSIONS Direct detection and quantification of serum Mtb antigens by NanoDisk-MS can rapidly and accurately diagnose active TB in adults, independent of disease site or culture status, and outperform Mycobacterium-based TB diagnostics.

Rapid diagnosis of pulmonary tuberculosis by combined molecular and immunological methods

Diagnosing pulmonary tuberculosis (TB) may be delayed until culture results become available.We ascertained the accuracy of a stepwise diagnostic algorithm for the rapid diagnosis of pulmonary TB by GeneXpert from sputum and/or bronchoalveolar lavage (BAL) followed by aMycobacterium tuberculosis-specific BAL ELISPOT assay in patients with a suspected diagnosis of pulmonary TB at a clinical referral centre in Germany.Among 166 patients with a presumptive diagnosis of pulmonary TB, 81 cases were confirmed byM. tuberculosisculture from sputum and/or BAL. In 66 out of 81 (81.5%) cases, patients initially hadM. tuberculosisdetected by GeneXpert from sputum; in addition, six out of 81 (7.4%) cases were diagnosed by GeneXpert on BAL fluid (together 72 out of 81 (88.9%) patients). Out of the remaining nine patients with negative GeneXpert results from sputum and BAL, BAL ELISPOT identified eight patients with culture-confirmed TB correctly (median time to culture positivity 26 days). At a cut-off of >4000 early secretory antigenic target-6- or culture filtrate protein-10-specific interferon-γ-producing lymphocytes per 1 000 0000 lymphocytes, the specificity of the BAL ELISPOT for active TB was 97%.In low TB incidence countries, nearly all patients with active pulmonary TB can be identified within the first few days of clinical presentation using a stepwise strategy with GeneXpert and BAL ELISPOT.

Adaptación de Mycobacterium smegmatis ante el agotamiento de nutrientes y su efecto en la expresión de esat-6

Cuando los recursos son escasos, las micobacterias detienen su crecimiento para dar paso a los genes de la adaptación. Contrariamente, cuando el crecimiento continúa bajo condiciones de estrés, se activan genes específicos de redes metabólicas para su protección. En este sentido, la proteína codificada por esat-6 (por sus siglas en inglés: early secretory antigenic target, 6 kDa) en Mycobacterium tuberculosis, actúa en la lisis del epitelio alveolar y membranas de los macrófagos para escapar e invadir otras células. Pero puede tener otras funciones, tales como interferir en el contacto célula-célula y transferir su ADN. En M. smegmatis, el sistema ESX-1 (por sus siglas en inglés: Secretion Ejectosoma BOX) facilita la secreción de la proteína ESAT-6, probablemente es sensible a uno o más nutrientes del medio de cultivo. Por lo que en el presente estudio se evalúan las condiciones de cultivo limitantes en nutrientes para el crecimiento de M. smegmatis y su relación con la expresión del gen esat-6. Los medios de cultivos probados fueron Hartmans de Bond medio mínimo (HdB), limitado en carbono (HdB<C), nitrógeno (HdB<N) y fosfato inorgánico (HdB<Pi). M. smegmatis se adapta a HdB medio mínimo, HdB<C y HdB<N y reanuda su actividad metabólica en medio fresco, pero no se expresa esat-6. En HdB<Pi M. smegmatis pierde su capacidad metabólica respecto a la resistencia alcohol-ácido y expresa esat-6. Por lo tanto, se proponen los medios de cultivo probados como modelo para la expresión génica bajo limitación por nutrientes.