Mycobacterial DNA-binding protein 1 is critical for BCG survival in stressful environments and simultaneously regulates gene expression.

Survival of the live attenuated Bacillus Calmette-Guérin (BCG) vaccine amidst harsh host environments is key for BCG effectiveness as it allows continuous immune response induction and protection against tuberculosis. Mycobacterial DNA binding protein 1 (MDP1), a nucleoid associated protein, is essential in BCG. However, there is limited knowledge on the extent of MDP1 gene regulation and how this influences BCG survival. Here, we demonstrate that MDP1 conditional knockdown (cKD) BCG grows slower than vector control in vitro, and dies faster upon exposure to antibiotics (bedaquiline) and oxidative stress (H2O2 and menadione). MDP1-cKD BCG also exhibited low infectivity and survival in THP-1 macrophages and mice indicating possible susceptibility to host mediated stress. Consequently, low in vivo survival resulted in reduced cytokine (IFN-gamma and TNF-alpha) production by splenocytes. Temporal transcriptome profiling showed more upregulated (81-240) than downregulated (5-175) genes in response to MDP1 suppression. Pathway analysis showed suppression of biosynthetic pathways that coincide with low in vitro growth. Notable was the deferential expression of genes involved in stress response (sigI), maintenance of DNA integrity (mutT1), REDOX balance (WhiB3), and host interactions (PE/PE_PGRS). Thus, this study shows MDP1's importance in BCG survival and highlights MDP1-dependent gene regulation suggesting its role in growth and stress adaptation.

Antibodies against native proteins of Mycobacterium tuberculosis can detect pulmonary tuberculosis patients.

Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to Mycobacterium tuberculosis proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from M. tuberculosis. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from Escherichia coli (E. coli) couldn't distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from M. tuberculosis. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734-0.890, p < 0.0001) and 0.821 (95% CI 0.752-0.890, p < 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB's POCT by antibody-based diagnosis.

Monitoring IgG against Mycobacterium tuberculosis proteins in an Asian elephant cured of tuberculosis that developed from long-term latency.

Tuberculosis (TB) is fatal in elephants, hence protecting elephants from TB is key not only in the conservation of this endangered animal, but also to prevent TB transmission from elephants to humans. Most human TB cases arise from long-term asymptomatic infections. Significant diagnostic challenges remain in the detection of both infection and disease development from latency in elephants due to their huge bodies. In this study, we assessed cryopreserved sera collected for over 16 years, from the first Japanese treatment case of elephant TB. Semi-quantification of IgG levels to 11 proteins showed high detection levels of 3 proteins, namely ESAT6/CFP10, MPB83 and Ag85B. The level of IgG specific to these 3 antigens was measured longitudinally, revealing high and stable ESAT6/CFP10 IgG levels regardless of onset or treatment. Ag85B-specifc IgG levels were largely responsive to onset or treatment, while those of MPB83 showed intermediate responses. These results suggest that ESAT6/CFP10 is immunodominant in both asymptomatic and symptomatic phases, making it useful in the detection of infection. On the other hand, Ag85B has the potential to be a marker for the prediction of disease onset and in the evaluation of treatment effectiveness in elephants.

Demonstration of Mitochondrial Damage and Mitophagy in Cisplatin-Mediated Nephrotoxicity.

Cisplatin is a chemotherapeutic widely used in the treatment of various types of solid tumors. Acute kidney injury is the most critical dose-limiting factor in cancer patients treated with cisplatin; mitochondrial dysfunction and resultant cell damage by reactive oxygen species released from damaged mitochondria are suspected to be involved in the kidney injury. Pathological features of mitochondrial damage in relation to cisplatin-mediated nephrotoxicity, however, is not fully described. The purpose of this study was to demonstrate mitochondrial damage and clearance of damaged mitochondria by mitophagy in cisplatin-mediated nephrotoxicity. Three groups of rats received a single intraperitoneal injection of cisplatin at 20 mg/kg and were sacrificed at 24, 48 and 72 hours after the treatment. A time-dependent increase in the number of damaged renal tubules and the serum levels of blood urea nitrogen, creatinine, and mitochondrial aspartate transaminase was observed in rats after the treatment. We showed the increased numbers of swollen and fragmented mitochondria, observed by electron microscopy, and of cytochrome c oxidase IV- and 8-nitroguanosine-positive intracytoplasmic granules, detected by immunohistochemistry, in the degenerated renal tubules of the treated animals. Moreover, activated autophagy process was indicated in the degenerated renal epithelial cells, based on the findings of immunohistochemistry of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and lysosomal-associated membrane protein 1 (LAMP-1), a lysosome marker, and swollen and fragmented mitochondria in autophagosomes. These results suggest that mitochondrial damage and clearance of damaged mitochondria by mitophagy is involved in cisplatin-mediated nephrotoxicity.