Expanding the understanding of peptidoglycan turnover in <em>Chlamydiae</em>

Abstract

The obligate intracellular chlamydiae evolved mechanisms to persist inside their host and to evade the host immune system, making them intriguing and highly successful pathogens. This work aimed to asses how these strategies are interlinked with the chlamydial peptidoglycan (PG) turnover process.<br /> First, antimicrobial compounds were used as tools to study the interplay between PG biosynthesis and the chlamydial lifecycle. These experiments included analysis of the host cell, since an antichlamydial effect can be caused by both host cell stress and by inhibition of a bacterial target. In a cell culture based approach combining cytotoxicity analysis and fluorescence microscopy, muraymycin D2 and derivates were verified to induce a persistent state in productive Chlamydia infection. The antichlamydial effect could be attributed to the inhibition of PG precursor synthesis, since no damaging effect on the host cells was observed and biochemical analysis identified Chlamydia MraY as a target. In addition, muraymycin D2 was found to break penicillin-induced persistence in C. trachomatis, suggesting the presence of additional targets.<br /> Degradation of the PG ring during the unique chlamydial cell division poses risks to the organism, as products of this process initiate host immune response. In this work, comparative analyses on cell division amidases from Chlamydia and E. coli corroborated the importance of an autoinhibitory domain in the E. coli enzyme that is absent in the Chlamydia homologs. In line, C. trachomatis AmiA was found to be active by default and to degrade the recognition substrate of immunity receptor NOD2 in vitro. Unlike the dual functioning C. pneumoniae AmiA, C. trachomatis AmiA did not show additional carboxypeptidase activity on PG precursor lipid II, suggesting that different PG degradation machineries exist in Chlamydia. PG-derived peptides generated by amidase activity trigger NOD1 mediated immune response. Here, an ortholog of a Bacillus NlpC/P60 protein was found to be widely conserved among Chlamydia and Chlamydia-related organisms and to cleave the minimal recognition site of NOD1. In silico, biochemical and surrogate host experiments indicate that C. trachomatis YkfC is tailored to recycle PG-derived peptides in the cytoplasm and shows high substrate specificity that prevents interference with PG precursor synthesis. The identification of C. trachomatis YkfC targeting cysteine protease inhibitors provides a basis for future studies on the intersection of energy recovery, cell division and immune evasion in Chlamydia and on the host-pathogen interplay.