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.

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.

Extracellular DNA of slow growers of mycobacteria and its contribution to biofilm formation and drug tolerance.

DNA is basically an intracellular molecule that stores genetic information and carries instructions for growth and reproduction in all cellular organisms. However, in some bacteria, DNA has additional roles outside the cells as extracellular DNA (eDNA), which is an essential component of biofilm formation and hence antibiotic tolerance. Mycobacteria include life-threating human pathogens, most of which are slow growers. However, little is known about the nature of pathogenic mycobacteria's eDNA. Here we found that eDNA is present in slow-growing mycobacterial pathogens, such as Mycobacterium tuberculosis, M. intracellulare, and M. avium at exponential growth phase. In contrast, eDNA is little in all tested rapid-growing mycobacteria. The physiological impact of disrupted eDNA on slow-growing mycobacteria include reduced pellicle formation, floating biofilm, and enhanced susceptibility to isoniazid and amikacin. Isolation and sequencing of eDNA revealed that it is identical to the genomic DNA in M. tuberculosis and M. intracellulare. In contrast, accumulation of phage DNA in eDNA of M. avium, suggests that the DNA released differs among mycobacterial species. Our data show important functions of eDNA necessary for biofilm formation and drug tolerance in slow-growing mycobacteria.

Higher genome mutation rates of Beijing lineage of Mycobacterium tuberculosis during human infection.

Mycobacterium tuberculosis (Mtb) strains of Beijing lineage have caused great concern because of their rapid emergence of drug resistance and worldwide spread. DNA mutation rates that reflect evolutional adaptation to host responses and the appearance of drug resistance have not been elucidated in human-infected Beijing strains. We tracked and obtained an original Mtb isolate of Beijing lineage from the 1999 tuberculosis outbreak in Japan, as well as five other isolates that spread in humans, and two isolates from the patient caused recurrence. Three isolates were from patients who developed TB within one year after infection (rapid-progressor, RP), and the other three isolates were from those who developed TB more than one year after infection (slow-progressor, SP). We sequenced genomes of these isolates and analyzed the propensity and rate of genomic mutations. Generation time versus mutation rate curves were significantly higher for RP. The ratio of oxidative versus non-oxidation damages induced mutations was higher in SP than RP, suggesting that persistent Mtb are exposed to oxidative stress in the latent state. Our data thus demonstrates that higher mutation rates of Mtb Beijing strains during human infection is likely to account for the higher adaptability and an emergence ratio of drug resistance.