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

Abstract

Organisms of Chlamydia spp. 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 inhibit Chlamydia growth 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. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal ⌬murA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-toaspartate change in a key residue within the active site of MurA. In E. 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 of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.

Identification and Characterization of a MurA, UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Cariogenic Streptococcus Mutans

“…The expected 20,21) . The sequence analysis demonstrated that there is no replacement in the active site of S. mutans MurA.…”

Fosfomycin and Its Application in the Treatment of Multidrug-Resistant Enterobacteriaceae Infections

“…[85][86][87] Once inside the cell, fosfomycin acts as an analog of phosphoenolpyruvate and inhibits enolpyruvyl transferase (also known as MurA), a cytoplasmic enzyme that allows the first step of synthesis of N-acetylmuramic acid. 86,[88][89][90][91] Because N-acetylmuramic acid is a major component of glycan strands in the cell wall, fosfomycin blocks cell wall synthesis with a lethal effect. 86,92,93 …”

“…The resistance of enolpyruvyl transferase to inactivation by fosfomycin was demonstrated in Mycobacterium tuberculosis, 94 Vibrio fisheri, 95 and Chlamydia trachomatis. 91 In susceptible organisms, several mutations in murA demonstrate a lower affinity of its product to phosphoenolpyruvate and may lead to fosfomycin resistance 96-98 associated with a lower rate of peptidoglycan synthesis. 98 However, resistance to fosfomycin due to overexpression of enolpyruvyl transferase has been also observed.…”

Kinetic Properties of Wild-type and C117D Mutant UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) from Haemophilus influenzae

“…Furthermore, a recombinant E. coli strain where the MurA gene was replaced with the C. trachomatis MurA gene was resistance to fosfomycin, 12 suggesting that the aspartate substitution is highly important to fosfomycin resistance. The structure or kinetic properties of MurAs from C. trachomatis or M. tuberculosis have not yet been reported due to the difficulties associated with expressing recombinant proteins, even though inhibitors of these MurAs would be valuable for the development of antibiotics against C. trachomatis or M. tuberculosis.…”

“…12,13 The fosfomysin resistance of these bacteria is probably due to the resistance of MurA, since MurA of M. tuberculosis or C. trachomatis contains an aspartate residue at the active site loop instead of cysteine. Therefore, we tested whether C117D substitution affected the sensitivity of Hi MurA to fosfomycin.…”