The Study of stress response mechanism of Mycobacterium tuberculosis induced by isoniazid treatment

Abstract

Tuberculosis (TB) is a contagious bacterial infection caused by Mycobacterium tuberculosis (MTB). Transmission occurs through airborne droplets, primarily affecting the lungs and causing pulmonary disease. However, the infection can also occur in other parts of the body, such as the kidneys, spine, and brain. Even though several anti-TB drugs are available, treating TB is challenging due to the development of drug resistance. The characteristics of the mycobacterial cell wall, which act as barriers and contribute to virulence, provide mycobacteria with high natural resistance. Mannose-capped lipoarabinomannan (ManLAM) is an essential component of the mycobacterial cell wall that modulates the host's immune response, allowing MTB to persist in the host. ManLAM is expressed in Mycobacterium tuberculosis complex and other pathogenic strains, with varying levels between rapid and stasis growth. Changes in the structure of ManLAM affect cell wall integrity, antibiotic sensitivity, and pathogenesis. This demonstrates the importance of controlling ManLAM biosynthesis in the virulence and drug resistance of mycobacteria. Furthermore, the adaptation of MTB in response to various stress conditions, including the presence of antibiotics, makes it a successful pathogen during infection in the host. The first-line drug, isoniazid (INH), is widely used for TB prevention and treatment. However, drug resistance can emerge within the host during treatment, leading to poor clinical outcomes and additional resistance to other drugs. Therefore, understanding mycobacterial stress response mechanisms induced by INH in the host environment is essential and needs to be explored. In this study, MTB strains with different drug-resistant profiles, including H37Rv (drug-sensitive strain; DS), isoniazid-resistant (INH-R), rifampicin-resistant (RIF-R), and multi-drug resistant (MDR) strains, were cultivated in two different culture models; 1) The nutrient-rich (NR) model, which provides the nutrients and supplements required for MTB growth and MTB were cultured in aerobic condition; and 2) The multi-stress (MS) model which simulates the major stresses encountered by MTB in host macrophages, including acidic pH, nutrient starvation, nitric oxide, reactive nitrogen intermediates, and MTB were cultured in anaerobic condition. MTB strains were cultivated in the NR and MS models, and then treated with or without INH. This study analyzed the expression levels of stress-related genes reported to be associated with stress response (hspX, tgs1, sigE, and icl1) and ManLAM-related genes involved in the biosynthesis of ManLAM (pimB, mptA, mptB, dprE1, dprE2, and embC) in four strains that respond to isoniazid under nutrient-rich (NR) and stress-mimicking (MS) conditions. Results show that INH influences the expression levels of stress-related genes and ManLAM-related genes, which present a distinct pattern among strains and between NR versus MS models. In NR conditions, mptA, which is responsible for the elongation of the mannan backbone, is upregulated in all strains following INH treatment, and almost all ManLAM-related genes were upregulated in drug-resistant strains (DR). Interestingly, the expression level of tgs1, which is responsible for triacylglycerol synthesis, was significantly upregulated in MDR, while sigE, one of the main master regulators of MTB in response to stress, was significantly upregulated in DS. These findings indicate that the mechanisms utilized by MTB in response to INH vary between strains. In the MS model, without INH treatment, stress-related genes and ManLAM-related genes were up-regulated in DR, while none of these genes were up-regulated in DS. The findings demonstrate a clear relationship between drug resistance and bacterial adaptation, highlighting distinct differences between DR and DS. Additionally, the upregulation of icl1 and dprE1 in DR further supports the potential use of these genes as indicators of virulence and as targets for future drug treatments. In MS with INH, stress-related genes including hspX, tgs1, and sigE were clearly up-regulated in INH-R and RIF-R strains, which are mono-resistant strains, while icl1 was significantly up-regulated in DS and slightly up-regulated in INH-R and MDR strains. Since the stress response system is crucial for MTB, enabling it to withstand stress and become antibiotic-tolerant, the results indicate that INH is a stressor induces mycobacterial adaptation and may be an initial step toward additional antibiotic resistance. Surprisingly, ManLAM-related genes in DS were strongly up-regulated in response to INH treatment, suggesting that ManLAM regulation may be required for DS to survive in the presence of INH during infection in the host. This study provides new insights into the stress response mechanisms of MTB through the regulation of gene expression involved in ManLAM production, which may be one of the adaptive strategies during isoniazid treatment and could be related to the development of drug resistance. Additionally, the mechanisms related to mycobacterial adaptation identified in this study can be used to develop drug targets, enhancing the effectiveness of future treatments.