Biology subjects

Candela, T.

Publications and source records attributed to Candela, T..

2 recordsLinked to original sources

bioRxiv2023

A streamlined method to obtain biologically active TcdA and TcdB toxins from Clostridioides difficile

The major virulence factors of Clostridioides difficile (C. difficile) are enterotoxin A (TcdA) and cytotoxin B (TcdB). The study of toxins is a crucial step in exploring the virulence of this pathogen. Currently, the toxin purification process is either laborious and time-consuming in C. difficile or performed in heterologous hosts. Therefore, we propose a streamlined method to obtain functional toxins in C. difficile. Two C. difficile strains were generated each harboring a sequence encoding a His-tag at the 3 end of C. difficile 630{Delta}erm tcdA or tcdB genes. Each toxin gene is expressed using the Ptet promoter inducible by anhydro-tetracycline. The purification yields were estimated to be 0.28 mg per liter and 0.1 mg per liter for rTcdA and rTcdB, respectively. In this study, we successfully developed a simple routine method that allows the production and purification of biologically rTcdA and rTcdB active toxins with similar activities compared to native toxins.

microbiology↗

bioRxiv2020

Ser/Thr kinase-dependent phosphorylation of the peptidoglycan hydrolase CwlA controls its export and modulates cell division in Clostridioides difficile

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 serine/threonine kinases (STKs) of the Hanks family control cell division and envelope homeostasis, but only a few kinase substrates and associated molecular mechanisms have been identified. In this work, we identified CwlA as the first STK-PrkC substrate in the human pathogen Clostridiodes difficile and showed that CwlA is an endopeptidase involved in daughter cell separation. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, therefore controlling the hydrolytic activity in the cell wall. High level of CwlA at the cell surface led to cell elongation, whereas low level caused cell separation defects. We thus provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division.

microbiology↗