Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile

Abstract

Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The synthesis or activity of several key enzymes along the PG biosynthetic pathway is controlled by the Hanks-type serine/threonine kinases (STKs). In Gram-positive bacteria, inactivation of genes encoding STKs is associated with a range of phenotypes, including cell division defects and changes in cell wall metabolism, but only a few kinase substrates and associated mechanisms have been identified. We previously demonstrated that STK-PrkC plays an important role in cell division, cell wall metabolism, and resistance to antimicrobial compounds in the human enteropathogen Clostridioides difficile. In this work, we characterized a PG hydrolase, CwlA, which belongs to the NlpC/P60 family of endopeptidases and hydrolyses cross-linked PG between daughter cells to allow cell separation. We identified CwlA as the first PrkC substrate in C. difficile. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, thereby controlling hydrolytic activity in the cell wall. High levels of CwlA at the cell surface led to cell elongation, whereas low levels caused cell separation defects. Thus, we provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division. IMPORTANCE Bacterial cells are encased in a PG exoskeleton that helps to maintain cell shape and confers physical protection. To allow bacterial growth and cell separation, PG needs to be continuously remodeled by hydrolytic enzymes that cleave PG at critical sites. How these enzymes are regulated remains poorly understood. We identify a new PG hydrolase involved in cell division, CwlA, in the enteropathogen C. difficile. Lack or accumulation of CwlA at the bacterial surface is responsible for a division defect, while its accumulation in the absence of PrkC also increases susceptibility to antimicrobial compounds targeting the cell wall. CwlA is a substrate of the kinase PrkC in C. difficile. PrkC-dependent phosphorylation controls the export of CwlA, modulating its levels and, consequently, its activity in the cell wall. This work provides a novel regulatory mechanism by STK in tightly controlling protein export.

Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291

“…Notably, the homologue protein CD1135 (CwlA) in C . difficile 630 was recently characterized as a γ‐ d ‐Glu‐mDAP‐endopeptidase by using RP‐HPLC (Garcia‐Garcia et al ., 2020).…”

“…Recently, one study published on bioRxiv by Garcia‐Garcia et al . (2020) showed that CwlA (the homologue of Cwl0971 in CD630) could modulate C . difficile cell division through serine/threonine kinase (PrkC)‐dependent phosphorylation, suggesting a novel regulation mechanism for cell wall hydrolysis in C .…”

“…Other endopeptidases, CwlK and LytH, were also reported to be involved in bacterial physiology (Horsburgh et al, 2003;Fukushima et al, 2007). Recently, one study published on bioRxiv by Garcia-Garcia et al (2020) showed that CwlA (the homologue of Cwl0971 in CD630) could modulate C. difficile cell division through serine/threonine kinase (PrkC)-dependent phosphorylation, suggesting a novel regulation mechanism for cell wall hydrolysis in C. difficile 630. Though the cell length of R20291Δ0971 was significantly longer than that of R20291 (Fig.…”

Design and characterization of a novel lytic protein against Clostridium difficile

“…In recent decades, increasing amounts of novel C. difficile autolysins were discovered. Recently, Cwp19 was characterized as a lytic transglycosylase, which can contribute to toxin release through bacteriolysis (Bradshaw et al 2017;El Meouche and Peltier 2018;Wydau-Dematteis et al 2018); Cwl0971, involved in cell wall lysis and cell viability, affects toxin release, sporulation, germination, and pathogenicity of C. difficile (Zhu et al 2021); CwlA, which was the first substrate of PrkC in C. difficile, controls hydrolytic activity in the cell wall (Cuenot et al 2019;Garcia-Garcia et al 2021); Acd24020 exhibited C. difficile-specific lytic activity with an hourglass-shaped substrate-binding groove across the molecule, which is responsible for recognizing the cross-linking structure unique to C. difficile peptidoglycan (Sekiya et al 2021) . However, all these autolysins are not directly used for the treatment of CDI because of their weak lytic activity or low cellular specificity.…”

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

“…However, CwlA, which is an endopeptidase-hydrolyzing peptidoglycan (PG), is the sole PrkC substrate identified so far. The PrkC-dependent phosphorylation of CwlA controls its localization at the cell wall and modulates cell division ( 27 ). CD2148 is a kinase lacking extracellular PASTA domains that is anchored in the cell wall and enriched at the septum of the cells.…”