The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation.

20Spores are the major infectious particle of the Gram-positive nosocomial pathogen, 21 Clostridioides (formerly Clostridium) difficile, but the molecular details of how this organism 22 forms these metabolically dormant cells remain poorly characterized. The composition of the 23 spore coat in C. difficile differs markedly from that defined in the well-studied organism, 24 Bacillus subtilis, with only 25% of the ~70 spore coat proteins being conserved between the two 25 organisms, and only 2 of 9 coat assembly (morphogenetic) proteins defined in B. subtilis having 26 homologs in C. difficile. We previously identified SipL as a clostridia-specific coat protein 27 essential for functional spore formation. Heterologous expression analyses in E. coli revealed 28 that SipL directly interacts with C. difficile SpoIVA, a coat morphogenetic protein conserved in 29 all spore-forming organisms, through SipL's C-terminal LysM domain. In this study, we show 30 that SpoIVA-SipL binding is essential for C. difficile spore formation and identify specific 31 residues within the LysM domain that stabilize this interaction. Fluorescence microscopy 32 analyses indicate that binding of SipL's LysM domain to SpoIVA is required for SipL to localize 33 to the forespore, while SpoIVA requires SipL to promote encasement of SpoIVA around the 34 forespore. Since we also show that clostridial LysM domains are functionally interchangeable at 35 least in C. difficile, the basic mechanism for SipL-dependent assembly of clostridial spore coats 36 may be conserved. 37 38 39 3 Importance 40 41The metabolically dormant spore-form of the major nosocomial pathogen, Clostridioides 42 difficile, is its major infectious particle. However, the mechanisms controlling the formation of 43 these resistant cell types are not well understood, particularly with respect to its outermost layer, 44 the spore coat. We previously identified two spore morphogenetic proteins in C. difficile: 45 SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only 46 in the Clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and 47 directly interact through SipL's C-terminal LysM domain. In this study, we demonstrate that the 48 LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the 49 forespore during spore morphogenesis. We further identified residues within the LysM domain 50 that are important for binding SpoIVA and thus functional spore formation. These findings 51 provide important insight into the molecular mechanisms controlling the assembly of infectious 52 C. difficile spores.53 4 Introduction 54 55The Gram-positive pathogen Clostridioides (formerly Clostridium) difficile is a leading 56 cause of antibiotic-associated diarrhea and gastroenteritis in the developed world (1, 2). Since C. 57 difficile is an obligate anaerobe, its major infectious particle is its aerotolerant, metabolically 58 dormant spore form (3, 4). C. difficile spores in the environment ar...

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.

Though the past three decades have led to a renaissance in vaccine design, the development of vaccines that protect against helminth diseases remains elusive. The need for protective vaccines for humans and livestock remains urgent because of the side-effect profiles of anti-helminthic drugs and the growing incidence of antimicrobial resistance and declining efficacy. The “-omics” era has led to renewed interest in vaccine development against helminth diseases, as candidate vaccines can now be designed, evaluated, and refined in a fraction of the time previously required. In this chapter, we describe and review genomic, transcriptomic, and proteomic approaches to the design of vaccines against helminth diseases.