Characterization of the Cell Division-Associated Peptidoglycan Amidase AmiA of Chlamydia trachomatis

Dannenberg, J.; Lee, J.; Liechti, G. W.; Otten, C.; Loeckener, I.; Reuter, J.; Klockner, A.; Krannich, S.; Schneider, T.; Ouellette, S. P.; Henrichfreise, B.

doi:10.1101/2025.10.06.680639

bioRxiv · 10.1101/2025.10.06.680639

Characterization of the Cell Division-Associated Peptidoglycan Amidase AmiA of Chlamydia trachomatis

Dannenberg, J.·Lee, J.·Liechti, G. W.·Otten, C.·Loeckener, I.·Reuter, J.·Klockner, A.·Krannich, S.·Schneider, T.·Ouellette, S. P.·Henrichfreise, B.

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Abstract

Chlamydia is an obligate intracellular bacterium that differentiates between infectious, non-dividing EBs and non-infectious, dividing RBs. Pathogenic Chlamydia species are unusual in lacking a peptidoglycan sacculus, yet they do synthesize a transient and localized peptidoglycan structure at the divisome of the RB during their polarized division process. Although several studies have described the components of the chlamydial divisome necessary to generate peptidoglycan at a specific site on the membrane, less is understood about how the peptidoglycan structure is degraded to allow for the daughter cell to form and the division process to complete. Amidases are key components of the cell wall in model system bacteria as they catalyze the degradation and remodeling of peptidoglycan, including in the division septum. Here, we characterized the cell division-associated amidase, AmiA_Ct, of Chlamydia trachomatis both in vitro and in vivo. Our in vitro data show that AmiA_Ct is a bona fide, metal-dependent amidase capable of cleaving peptidoglycan. AmiA_Ct complemented an E. coli amidase deficient strain and supported the growth and separation of daughter cells. To assess the function of AmiA_Ct in C. trachomatis, we generated a transformant strain carrying an inducible CRISPR interference system targeting the amiA gene. Knocking down expression of amiA resulted in altered bacterial morphology, a reduction in infectious EBs, and the accumulation of peptidoglycan in the organisms. These data indicate a critical function for AmiA_Ct in the unique cell division process of Chlamydia. ImportancePeptidoglycan is an important structural cell wall polymer that serves to give bacteria their shape and resistance to changes in extracellular solute concentrations. For Chlamydia trachomatis, an obligate intracellular pathogen that divides within a host cell, peptidoglycan is only used for cell division and is not a component of its cell wall. In this study, we characterize the function of a chlamydial amidase that helps degrade peptidoglycan during cell division. We show a critical function for amidase activity in facilitating changes to the peptidoglycan structure during chlamydial cell division that support normal growth and development of this pathogenic bacterium.

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Dannenberg, J., Lee, J., Liechti, G. W., Otten, C., Loeckener, I., Reuter, J., Klockner, A., Krannich, S., Schneider, T., Ouellette, S. P., Henrichfreise, B.. 2025-10-07. Characterization of the Cell Division-Associated Peptidoglycan Amidase AmiA of Chlamydia trachomatis. https://doi.org/10.1101/2025.10.06.680639

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