Identification of a novel regulator ofClostridioides difficilecortex formation

Abstract

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. C. difficile infections are transmitted by its metabolically dormant, aerotolerant spore form. Functional spore formation depends on the assembly of two protective layers: a thick layer of modified peptidoglycan known as the cortex layer and a multilayered proteinaceous meshwork known as the coat. We previously identified two spore morphogenetic proteins, SpoIVA and SipL, that are essential for recruiting coat proteins to the developing forespore and making functional spores. While SpoIVA and SipL directly interact, the identities of the proteins they recruit to the forespore remained unknown. We used mass spectrometry-based affinity proteomics to identify proteins that interact with the SpoIVA-SipL complex. These analyses identified the Peptostreptococcaceae family-specific, sporulation-induced bitopic membrane protein CD3457 (renamed SpoVQ) as a protein that interacts with SipL and SpoIVA. Loss of SpoVQ decreased heat-resistant spore formation by ~5-fold and reduced cortex thickness ~2-fold; the thinner cortex layer of ∆spoVQ spores correlated with higher levels of spontaneous germination (i.e., in the absence of germinant). Notably, loss of SpoVQ in either spoIVA or sipL mutants prevented cortex synthesis altogether and greatly impaired the localization of a SipL-mCherry fusion protein around the forespore. Thus, SpoVQ is a novel regulator of C. difficile cortex synthesis that appears to link cortex and coat formation. The identification of SpoVQ as a spore morphogenetic protein further highlights how Peptostreptococcaceae family-specific mechanisms control spore formation in C. difficile.

PrrA modulates Mycobacterium tuberculosis response to multiple environmental cues and is critically regulated by serine/threonine protein kinases

“…Purification of PrrA-FLAG protein was based on a previously described FLAG-tagged protein immunoprecipitation protocol [69]. Thawed pellets were resuspended in 35 ml FLAG immunoprecipitation buffer (FIB) (50 mM Tris, pH 7.5, 150 mM NaCl, and protease inhibitors (Pierce protease inhibitor tablet)).…”