TY - JOUR
T1 - Clostridium difficile exosporium cysteine-rich proteins are essential for the morphogenesis of the exosporium layer, spore resistance, and affect C. difficile pathogenesis
AU - Calderón-Romero, Paulina
AU - Castro-Córdova, Pablo
AU - Reyes-Ramírez, Rodrigo
AU - Milano-Céspedes, Mauro
AU - Guerrero-Araya, Enzo
AU - Pizarro-Guajardo, Marjorie
AU - Olguín-Araneda, Valeria
AU - Gil, Fernando
AU - Paredes-Sabja, Daniel
N1 - Funding Information:
Comisión Nacional de Ciencia y Tecnología de Chile supported this work by Fondecyt Grant 1151025 to D.P-S., and doctoral fellowships 21151202, 21140380 and 211611395 to MP-G, PC-R., and PC-C., respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are thankful to Dr. Aimee Shen from Tufts University for the gift of anti-SpoIVA antibodies and Dr. Adriano O. Henriques from Universidade Nova de Lisboa for the gift of anti-CdeM antibody.
Publisher Copyright:
© 2018 Calderón-Romero et al. http://creativecommons.org/licenses/by/4.0/.
PY - 2018/8/1
Y1 - 2018/8/1
N2 - Clostridium difficile is a Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea that can culminate in fatal colitis. During the infection, C. difficile produces metabolically dormant spores, which persist in the host and can cause recurrence of the infection. The surface of C. difficile spores seems to be the key in spore-host interactions and persistence. The proteome of the outermost exosporium layer of C. difficile spores has been determined, identifying two cysteine-rich exosporium proteins, CdeC and CdeM. In this work, we explore the contribution of both cysteine-rich proteins in exosporium integrity, spore biology and pathogenesis. Using targeted mutagenesis coupled with transmission electron microscopy we demonstrate that both cysteine rich proteins, CdeC and CdeM, are morphogenetic factors of the exosporium layer of C. difficile spores. Notably, cdeC, but not cdeM spores, exhibited defective spore coat, and were more sensitive to ethanol, heat and phagocytic cells. In a healthy colonic mucosa (mouse ileal loop assay), cdeC and cdeM spore adherence was lower than that of wild-type spores; while in a mouse model of recurrence of the disease, cdeC mutant exhibited an increased infection and persistence during recurrence. In a competitive infection mouse model, cdeC mutant had increased fitness over wild-type. Through complementation analysis with FLAG fusion of known exosporium and coat proteins, we demonstrate that CdeC and CdeM are required for the recruitment of several exosporium proteins to the surface of C. difficile spores. CdeC appears to be conserved exclusively in related Peptostreptococcaeace family members, while CdeM is unique to C. difficile. Our results sheds light on how CdeC and CdeM affect the biology of C. difficile spores and the assembly of the exosporium layer and, demonstrate that CdeC affect C. difficile pathogenesis.
AB - Clostridium difficile is a Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea that can culminate in fatal colitis. During the infection, C. difficile produces metabolically dormant spores, which persist in the host and can cause recurrence of the infection. The surface of C. difficile spores seems to be the key in spore-host interactions and persistence. The proteome of the outermost exosporium layer of C. difficile spores has been determined, identifying two cysteine-rich exosporium proteins, CdeC and CdeM. In this work, we explore the contribution of both cysteine-rich proteins in exosporium integrity, spore biology and pathogenesis. Using targeted mutagenesis coupled with transmission electron microscopy we demonstrate that both cysteine rich proteins, CdeC and CdeM, are morphogenetic factors of the exosporium layer of C. difficile spores. Notably, cdeC, but not cdeM spores, exhibited defective spore coat, and were more sensitive to ethanol, heat and phagocytic cells. In a healthy colonic mucosa (mouse ileal loop assay), cdeC and cdeM spore adherence was lower than that of wild-type spores; while in a mouse model of recurrence of the disease, cdeC mutant exhibited an increased infection and persistence during recurrence. In a competitive infection mouse model, cdeC mutant had increased fitness over wild-type. Through complementation analysis with FLAG fusion of known exosporium and coat proteins, we demonstrate that CdeC and CdeM are required for the recruitment of several exosporium proteins to the surface of C. difficile spores. CdeC appears to be conserved exclusively in related Peptostreptococcaeace family members, while CdeM is unique to C. difficile. Our results sheds light on how CdeC and CdeM affect the biology of C. difficile spores and the assembly of the exosporium layer and, demonstrate that CdeC affect C. difficile pathogenesis.
UR - http://www.scopus.com/inward/record.url?scp=85053075832&partnerID=8YFLogxK
U2 - 10.1371/journal.ppat.1007199
DO - 10.1371/journal.ppat.1007199
M3 - Article
AN - SCOPUS:85053075832
SN - 1553-7366
VL - 14
JO - PLoS Pathogens
JF - PLoS Pathogens
IS - 8
M1 - e1007199
ER -