SpoIVA-SipL Complex Formation Is Essential for Clostridioides difficile Spore Assembly

Abstract

Spores are the major infectious particle of the Gram-positive nosocomial pathogen Clostridioides difficile (formerly Clostridium difficile), but the molecular details of how this organism forms these metabolically dormant cells remain poorly characterized. The composition of the spore coat in C. difficile differs markedly from that defined in the well-studied organism Bacillus subtilis, with only 25% of the ∼70 spore coat proteins being conserved between the two organisms and with only 2 of 9 coat assembly (morphogenetic) proteins defined in B. subtilis having homologs in C. difficile. We previously identified SipL as a clostridium-specific coat protein essential for functional spore formation. Heterologous expression analyses in Escherichia coli revealed that SipL directly interacts with C. difficile SpoIVA, a coat-morphogenetic protein conserved in all spore-forming organisms, through SipL’s C-terminal LysM domain. In this study, we show that SpoIVA-SipL binding is essential for C. difficile spore formation and identify specific residues within the LysM domain that stabilize this interaction. Fluorescence microscopy analyses indicate that binding of SipL’s LysM domain to SpoIVA is required for SipL to localize to the forespore while SpoIVA requires SipL to promote encasement of SpoIVA around the forespore. Since we also show that clostridial LysM domains are functionally interchangeable at least in C. difficile, the basic mechanism for SipL-dependent assembly of clostridial spore coats may be conserved. IMPORTANCE The metabolically dormant spore form of the major nosocomial pathogen Clostridioides difficile is its major infectious particle. However, the mechanisms controlling the formation of this resistant cell type are not well understood, particularly with respect to its outermost layer, the spore coat. We previously identified two spore-morphogenetic proteins in C. difficile: SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only in the clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and directly interact through SipL’s C-terminal LysM domain. In this study, we demonstrate that the LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the forespore during spore morphogenesis. We further identified residues within the LysM domain that are important for binding SpoIVA and, thus, functional spore formation. These findings provide important insight into the molecular mechanisms controlling the assembly of infectious C. difficile spores.

A lipoprotein allosterically activates the CwlD amidase during Clostridioides difficile spore formation

“…Immunoprecipitations were performed on lysates prepared from cultures induced to sporulate on 70:30 plates for 22 h. The samples were processed as previously described [ 49 ]. Cultures from three 70:30 plates per strain were scraped into 3 x 1 mL of FLAG IP buffer (FIB: 50 mM Tris pH 7.5, 150 mM NaCl, 0.02% sodium azide, 1X Halt protease inhibitor (ThermoScientific).…”

Grad-seq identifies KhpB as a global RNA-binding protein inClostridioides difficilethat regulates toxin production

“… 2019 ; Touchette et al . 2019 ; Unal et al . 2019 ), and given the bacterium's limited genetic tractability, often based on heterologous protein expression in E. coli or B. subtilis (Aboulnaga et al .…”

SpoIVA is an essential morphogenetic protein for the formation of heat- and lysozyme-resistant spores in Clostridium sporogenes NBRC 14293

“…The SpoVM-binding domain composed of 10 amino acids is also completely conserved among these four C. sporogenes strains ( Figure 1 ). Other partners of SpoIVA in the basement layer of the coat, which can be either SafA and SpoVID in B. subtilis or SipL in C. difficile ( Touchette et al, 2019 ). These proteins contain a LysM domain ( Touchette et al, 2019 ).…”

“…Additionally, it interacts with other morphogenetic proteins such as SafA and CotE ( Driks, 1999 ). In C. difficile , SpoIVA and SipL form a complex that is essential for the correct assembly of the spore coat ( Touchette et al, 2019 ). In a B. subtilis spoIVA mutant, both cortex and spore coat failed to fully develop, resulting in the formation of abnormal structures resembling swirls in the MC when observed using transmission electron microscopy (TEM) ( Roels et al, 1992 ).…”