In Vitro and In Vivo Functional Activity ofChlamydiaMurA, a UDP-N-Acetylglucosamine Enolpyruvyl Transferase Involved in Peptidoglycan Synthesis and Fosfomycin Resistance

Publisher Website
Google Scholar
Abstract
Organisms ofChlamydiaspp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibitChlamydiagrowth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. ThemurAgene fromChlamydiatrachomatisserovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressiblearapromoter and transformed intoEscherichia coli. After transduction of a lethal ΔmurAmutation into the strain, viability of theE. colistrain became dependent upon expression of theC. trachomatis murA.DNA sequence analysis ofmurAfromC. trachomatispredicted a cysteine-to-aspartate change in a key residue within the active site of MurA. InE. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth ofC. trachomatiswas also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to theE. colistrain expressing the chlamydialmurA. Conversion ofC. trachomatiselementary bodies to reticulate bodies and cell division are correlated with expression ofmurAmRNA. mRNA frommurB, the second enzymatic reaction in the PG pathway, was also detected duringC. trachomatisinfection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists inChlamydiaspp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG inChlamydiaspp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.