Glutathione and Nitrosoglutathione in Macrophage Defense againstMycobacterium tuberculosis

Abstract

We demonstrate that Mycobacterium tuberculosis grown in vitro is sensitive to glutathione and its derivative S-nitrosoglutathione. Furthermore, our infection studies with J774.1 macrophages indicate that glutathione is essential for the control of the intracellular growth of M. tuberculosis. This study indicates the important role of glutathione in the control of macrophages by M. tuberculosis.Tuberculosis is one of the most prevalent infectious diseases in the world (3). The situation is exacerbated by the emergence of mutlidrug-resistant strains and an ever-growing number of highly susceptible immunocompromised individuals arising from the AIDS pandemic (3). The production of reactive nitrogen intermediates by murine macrophages is considered to be a relatively effective host defense mechanism against Mycobacterium tuberculosis (1,4,7,8,9,13,16).In this study, we show that a virulent laboratory strain of M. tuberculosis, H37Rv, grown in vitro is sensitive to glutathione (GSH) and nitrosoglutathione (GSNO) at physiological concentrations. While GSH at a 5 mM concentration is bacteriostatic to H37Rv ( Fig. 1; Table 1), GSNO at a 5 mM concentration is bactericidal ( Fig. 1; Table 1), as was observed by determining optical density (OD) and numbers of CFU. H37Rv was grown in 7H9 containing albumin dextrose complex (ADC) and Tween 80 to mid-log phase and then diluted to an OD of approximately 0.1. The bacterial suspensions were then treated with 5 mM GSH (Sigma) or 5 mM GSNO (Sigma) or left untreated. The OD was measured every day for 5 days ( Fig. 1). Each day, an aliquot was taken from every sample, diluted 100-fold, and plated on 7H11 plates containing ADC for determination of numbers of CFU (Table 1). All experiments were performed three times in triplicate.The mechanism of action of the antimycobacterial activity of GSH is not certain. One possibility is that the presence of a high concentration of GSH may result in an imbalance in a bacterium containing an alternative thiol for regulating reduction or oxidation activity (i.e., mycothiol). We also consider the hypothesis of Spallholz in this connection (14). According to Spallholz, "GSH is structurally similar to the precursor of the antibiotics produced in fungi in the genera Penicillium and Cephalasporium." Its potential conversion to the penicillinlike derivative glutacillin, a ␤-lactam form of GSH, raises the intriguing question of whether GSH was once a universal penem-like precursor of antibiotics in cells of many life forms (14).

Hsp 70/Hsp 90 organizing protein as a nitrosylation target in cystic fibrosis therapy

“…(iii) It has been used safely in human trials (9,31). (iv) It has CFTR-independent beneficial effects that include relaxing airways smooth muscle, augmenting ventilation/ perfusion matching, increasing ciliary beat frequency, inhibiting amiloride-sensitive Na + transport, promoting inflammatory cell apoptosis, and antimicrobial effects (8)(9)(10)(11)(12)(13)32). (v) It can increase wild-type CFTR function (17).…”

“…S-nitrosoglutathione (GSNO) is an endogenous signaling molecule that relaxes airway smooth muscle (8)(9)(10), enhances ventilation-perfusion matching (9,10), increases ciliary beat frequency (11), has antimicrobial effects (12,13), and is one of a class of S-nitrosylating agents that increase expression, maturation, and function of wild-type (WT) and ΔF508 CFTR in primary nasal and monolayer cultures of epithelial cells (7,(14)(15)(16)(17)(18)(19). The GSNO effect on CFTR is partly transcriptional (16), but is primarily posttranscriptional (17,18).…”

Avaliação da virulência micobacteriana e modulação da resposta imune durante a infecção por isolados clínicos de Mycobacterium bovis e Mycobacterium tuberculosis.

“…A produção de intermediários reativos de nitrogênio e oxigênio por macrófagos murinos é considerada um mecanismo relativamente efetivo do hospedeiro contra M. tuberculosis, promovendo a inibição do crescimento do bacilo (Adams et al, 1997;Yu et al, 1999;Venketaraman et al, 2005). M. tuberculosis utiliza vários mecanismos para combater o estresse oxidativo, os quais incluem a enzima catalase/peroxidase (KatG), superóxido dismutase e a resposta de detoxificação baseada em tiol (micotiol) (Miller et al, 2007;Attarian et al, 2009).…”

“…Este dipeptídeo, então, é transportado para dentro da célula bacteriana pelo complexo multicomponente ABC transporter dipeptídeo permease, carreando, desta forma, a molécula de NO, levando à morte da bactéria. Estudos recentes mostraram que a ausência da enzima -glutamil transpeptidase leva à resistência da M. tuberculosis à ação do NO, sendo isto mostrado através de comparações in vitro utilizando cepas mutantes para esta enzima (Green et al, 2000;Venketaraman et al, 2005;Dayaram et al, 2006).…”

“…Para tanto, as bactérias foram plaqueadas em meio de cultura 7H9 suplementado com ADC, na D.O de 0,100 e acrescentamos concentrações crescentes do doador de NO, GSNO, sendo já descrito a concentração de 5 mM bactericida para a cepa H37Rv (Venketaraman et al, 2005). Porém, em ensaios prévios vimos que, a concentração de 1mM foi capaz de exercer atividade bactericida para o isolado H37Rv e outros isolados de M. tuberculosis, assim utilizamos concentrações partindo de 0M à 1mM de GSNO.…”