Targeting intracellular p-aminobenzoic acid production potentiates the anti-tubercular action of antifolates

Abstract

The ability to revitalize and re-purpose existing drugs offers a powerful approach for novel treatment options against Mycobacterium tuberculosis and other infectious agents. Antifolates are an underutilized drug class in tuberculosis (TB) therapy, capable of disrupting the biosynthesis of tetrahydrofolate, an essential cellular cofactor. Based on the observation that exogenously supplied p-aminobenzoic acid (PABA) can antagonize the action of antifolates that interact with dihydropteroate synthase (DHPS), such as sulfonamides and p-aminosalicylic acid (PAS), we hypothesized that bacterial PABA biosynthesis contributes to intrinsic antifolate resistance. Herein, we demonstrate that disruption of PABA biosynthesis potentiates the anti-tubercular action of DHPS inhibitors and PAS by up to 1000 fold. Disruption of PABA biosynthesis is also demonstrated to lead to loss of viability over time. Further, we demonstrate that this strategy restores the wild type level of PAS susceptibility in a previously characterized PAS resistant strain of M. tuberculosis. Finally, we demonstrate selective inhibition of PABA biosynthesis in M. tuberculosis using the small molecule MAC173979. This study reveals that the M. tuberculosis PABA biosynthetic pathway is responsible for intrinsic resistance to various antifolates and this pathway is a chemically vulnerable target whose disruption could potentiate the tuberculocidal activity of an underutilized class of antimicrobial agents.

Deletion of sigB Causes Increased Sensitivity to para -Aminosalicylic Acid and Sulfamethoxazole in Mycobacterium tuberculosis

“…Previously, Thiede et al reported that disruption of pABA biosynthesis potentiates the antitubercular effect of anti-folates by up to 1,000-fold (33). Here, we showed that SigB regulates the expression of pabB gene in M. tuberculosis and, as a result, deletion of sigB results in decreased expression of pabB, impaired pABA production, and increase susceptibility to antifolates.…”

Mechanism of selectivity reveals novel antifolate drug interactions

“…Since the non-pathogenic mycobacterium, Mycobacterium smegmatis (Ms), is resistant to PAS, we determined if PAS could be utilized in a M. tuberculosis Δ pabB strain expressing in trans folAMtb or folA from M. smegmatis (folAMs). M. tuberculosis Δ pabB strain expressing folAMtb grew only in the presence of PABA, not PAS (Extended Data Figure 2a) 15 . Interestingly, M. tuberculosis Δ pabB expressing folAMs grew in the presence of both PAS and PABA (Extended Data Figure 2).…”

“…Since SMX treatment has previously been shown to increase PABA production in bacteria, we reasoned that SMX is causing an increase in PABA biosynthesis and, thereby, antagonism of PAS 17 . Previous work has shown that strains lacking pabB in M. tuberculosis are PABA auxotrophs and can only grow with exogenously added PABA 15 .…”

Implicancias Estructurales y Fisiológicas de la Célula Bacteriana en los Mecanismos de Resistencia Antibiótica

“…Algunos antibacterianos como las sulfonamidas compiten con el PABA por medio de la inhibición de la enzima dihidrofolato reductasa necesaria para el paso de dihidrofolato a tetrahidrofolato, cofactor necesario en la síntesis de DNA y proteínas. La resistencia bacteriana se presenta por mutación espontánea o transferencia de la misma a través de plásmidos, generando mutación de la dihidroteroato sintetasa, creando una vía metabólica alterna para la sínte-sis del ácido fólico y generando aumento en la capacidad de inactivar o destruir la droga y producción de un antagonista de la droga (Thiede et al, 2016;Gardeweg, 2012). Se destaca, igualmente, la alteración enzimática del sitio objetivo del fármaco, reduciendo la afinidad del antibiótico al sitio blanco; estos mecanismos también son regulados por enzimas eritromicina metilasas ribosomales que otorgan resistencia a macrólidos (Sheldon).…”