The mechanistic basis of susceptibility and resistance to the antitubercular drug para-aminosalicylic acid

Abstract

Tuberculosis (TB) is responsible for the deaths of 1.6 million people worldwide each year and is the leading cause of death by the pathogen Mycobacterium tuberculosis. The treatment regimen for M. tuberculosis involves lengthy, intensive drug therapy that causes severe side-effects. The antimicrobial para-aminosalicylic acid (PAS) is used to treat drug-resistant M. tuberculosis infections. Despite the use of PAS to treat M. tuberculosis for over 70 years, the biochemical mechanisms which govern PAS susceptibility and resistance in M. tuberculosis are incomplete. The focus of this dissertation is to determine these mechanisms and can be summarized into three interrelated studies: 1) The mechanism of action of PAS on the M. tuberculosis folate metabolic pathway; 2) Understanding the mechanisms of PAS resistance; and 3) Examining the drug-drug interactions between PAS and other anti-folate drugs that are used to treat opportunistic infections in patients with HIV-M. tuberculosis co-infections. Chapter 2 shows that PAS is a pro-drug and is converted, via the M. tuberculosis folate biosynthetic pathway, to hydroxy-dihydrofolate. The folate biosynthesis pathway is an essential metabolic pathway used maintain the production of DNA, RNA, and proteins. Results showed that hydroxy-dihydrofolate acted as a potent inhibitor for dihydrofolate reductase and confirm the biochemical mode of action of PAS. Although PAS was originally found to be effective at inhibiting M. tuberculosis and showed no activity against other bacterial species. PAS activity was tested against other bacterial pathogens. While M. tuberculosis was extraordinarily sensitive to PAS, other bacteria resisted PAS-mediated killing. Chapter 2 found these bacterial species could utilize PAS as a fully functional folate analog into one-carbon metabolism. The folate biosynthesis precursor, para-aminobenzoic acid (PABA), is a known antagonist of antifolates, namely sulfonamides. Since PAS was shown to act similarly to PABA in folate biosynthesis, PAS-sulfonamide interactions were tested against a panel of bacterial pathogens. PAS could antagonize sulfonamides in all of these organisms. HIV-infected individuals are given numerous drugs to both treat the HIV infection and prophylactically treat and prevent opportunistic infections. The most commonly prescribed prophylactic drugs are the sulfonamides. These findings strongly support that PAS and sulfonamides should cease to be used in combination in individuals with HIV-M. tuberculosis co-infections. Many patients discontinue treatment of PAS because of the side effects, namely, gastrointestinal distress. Bacteria must make their own folates and many bacteria in the human colon excrete folate to supply human enterocytes with folates. All rapidly dividing human cells require folates for the synthesis of new DNA, RNA, and proteins. All human cells contain folate receptors which recognize the metabolites dihydrofolate or folate. Purified human dihydrofolate reductase enzyme and could use hydroxy-dihydrofolate but much less effectively than the native substrate. Since enterocytes require a large amount of folates for rapid cell growth, enterocytes using hydroxy-dihydrofolate as a source of folates may not be able to grow as fast leading to severe gastrointestinal distress. Indeed, hydroxy-dihydrofolate, not PAS, was cytotoxic to enterocytes and hepatocytes. These findings will allow us to ultimately to design better ways to administer PAS to prevent patients from discontinuing PAS treatment. In Chapter 3, we hypothesized mice could be co-treated with sulfonamides to prevent PAS bioactivation and resulting PAS toxicity. Surprisingly, sulfonamides antagonized the anti-mycobacterial action of PAS in mice, resulting in the unrestricted growth of M. tuberculosis in the lungs and dissemination of M. tuberculosis into the liver and spleen. Taken together, these data indicate that combining PAS with sulfonamide in the clinic would not be useful, could be detrimental to patient outcome, and further highlights the need for mechanistic studies of drug-drug interactions. . Chapter 4 established that PAS resistance in M. tuberculosis primarily mapped to the folate biosynthetic pathway. The most prevalent mutations mapped to thyA and folC, a thymidylate synthase and dihydrofolate synthase, respectively. We hypothesized that folC mediated resistance occurred through an increase in PABA biosynthesis. Indeed PABA biosynthesis genes were upregulated in folC resistant mutants but not in thyA resistant mutants. The folC mutants had restored susceptibility to PAS when PABA biosynthesis was disrupted. Furthermore, it was found that disruptions in PABA biosynthesis were bactericidal in M. tuberculosis. These data represent a novel intrinsic mechanism of resistance to PAS and highlights a novel drug target in M. tuberculosis. This dissertation is the first to determine that PAS selectively inhibits M. tuberculosis dihydrofolate reductase enzyme and subsequently, the folate biosynthetic pathway. The work presented in this dissertation found that using PAS and sulfonamides together prevented both drugs from working correctly in M. tuberculosis and in other bacterial pathogens. The results generated from this dissertation will be used to inform the current clinical practices in combination therapy and foster a paradigm shift in the treatment regimen administered to HIV-M. tuberculosis co-infected patients, leading to decreased mortality rates among this population.