Characterization of the chlamydial machinery for peptidoglycan remodelling

• Applicant: Dr. Beate Henrichfreise
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 390536577

Obligate intracellular bacteria from the phylum Chlamydiae play a major role in global human health. Although the cell wall (peptidoglycan) of free-living bacteria is important for osmoprotection and cell division and serves as major antibiotic target, research on the chlamydial cell wall is still in its infancy. It is now established that biosynthesis of peptidoglycan (PG)-like cell wall material promotes cell division in Chlamydiae. Because an unknown chemical modification of PG in Protochlamydia amoebophila and a transient accumulation of fluorescently-labeled PG precursors at the division septum of Chlamydia trachomatis was recently reported, it is imperative to i) identify chlamydial PG-remodelling enzymes, ii) define their enzymatic properties and substrate specificities, and iii) characterize their subcellular localization. To this end, we will use a combination of forward genetic (structure-function) studies, biochemical assays and in situ/in vivo localization experiments.This proposal builds on our functional analyses of two uncharacterized chlamydial cell wall remodelling enzymes: the PG amidase AmiA and the LysM-domain protein mis-annotated as NlpD. While AmiA is a bifunctional amidase/penicillin-sensitive carboxypeptidase cleaving PG and/or its precursors, 'NlpD' binds PG with the LysM domain and seems to act on peptide side chains of PG (precursors). We will extend this published work by conducting structure-function studies on chlamydial AmiA, assessing if AmiA is by default an active enzyme and whether its activity can be regulated by auto-inhibition as for the E. coli amidases (Aim 1). We will also assess the role of the LysM-domain(s) of 'NlpD' for binding and/or cleaving PG (precursors) in vitro and in vivo by comparing the performance of wild-type against mutants harboring single or multiple substitutions in the LysM domain, as well as chimeric variants in which the 'NlpD' LysM domain has been swapped with that from an uncharacterized chlamydial LysM protein (Aim 2). Next, we will exploit the recently established C. trachomatis transformation system to determine if AmiA, 'NlpD' and the LysM protein are localized to the division plane of live cells and if their overexpression impairs constriction or alters the architecture of septal PG (Aim 3). Together our work will reveal which PG remodelling steps operate in obligate intracellular pathogens such as chlamydiae, while also unearthing important deviations from the canonical cell division and septal PG control mechanisms of free-living (model) bacteria.

• DFG Programme: Research Grants