Microbial Responses to Antibiotics – Stability of Resistance and Extended Potential of Targeting the Folate Synthesis

Sammanfattning: Resistance to antimicrobials is an increasing problem in the world of today, and develops faster than man can counter. It is therefore of importance to study metabolic pathways in order to develop new antibiotics, but also to understand how resistance spreads and stabilizes in microbial populations.The commensal flora could be an important factor in the spread of antimicrobial resistance, as drugs aimed at other targets also hit the harmless commensal bacteria. If stable resistance develops in such a population, it could seriously impair a later treatment with the same drug. After a treatment with the macrolide clarithromycin, resistance to this antibiotic increased markedly in the untargeted throat flora, and resistance levels did not recede until at least one year later. Another example of stable resistance can also be seen in sulfonamide resistant Streptococcus pyogenes. Sequence determinations of the dihydropteroate synthase (dhps) gene conferring this resistance revealed a mosaic organisation implying that the it had been brought there by horizontal transfer. Molecular characterization of this gene showed that the sulfonamide resistance was due to mutations of structurally important amino acids in position 65 and 213.The folate synthesis pathway has potential for being exploited further as a drug target. One possible new drug target is hydroxymethyl-dihydropterin pyrophosphokinase (hppk). In the malaria parasite Plasmodium falciparum this enzyme is part of a polyfunctional entity, also encoding dhps. The HPPK part can be separated from DHPS, but that the opposite is not possible. The PfHPPK has two insertions: one also present in other plasmodia, and one apparently unique to P. falciparum. Both are crucial for enzyme activity.To further characterize HPPK, we developed a spectrophotometric activity assay and a method to measure substrate channelling of hydroxymethyl-dihydropterin diphosphate.