Research Article: Clostridium difficile exosporium cysteine-rich proteins are essential for the morphogenesis of the exosporium layer, spore resistance, and affect C. difficile pathogenesis

Date Published: August 8, 2018

Publisher: Public Library of Science

Author(s): Paulina Calderón-Romero, Pablo Castro-Córdova, Rodrigo Reyes-Ramírez, Mauro Milano-Céspedes, Enzo Guerrero-Araya, Marjorie Pizarro-Guajardo, Valeria Olguín-Araneda, Fernando Gil, Daniel Paredes-Sabja, Theresa M. Koehler.


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.

Partial Text

Clostridium difficile [1], first reclassified as Peptoclostridium difficile [1] and more recently re-classified as Clostridioides difficile [2], is a Gram-positive, sporogenic anaerobic bacterium that is the most common cause of antibiotic-associated diarrhea within healthcare systems of the developed world [3, 4]. The clinical manifestation of the infection is diarrhea and in severe cases can produce pseudomembranous colitis, toxic megacolon and death [5]. Mortality of C. difficile infections (CDI) may reach up to 5% of CDI cases, but in several outbreaks, it has increased up to 20% [3]. Conventional metronidazole and/or vancomycin treatment (depending on the severity of the symptoms) although resolve single episodes of CDI, exhibit high rates of recurrence of the infection after a first episode. The rate of recurrence of CDI of a first, second and third episode may reach up to 20%, 40% and 60%, respectively [6, 7].

C. difficile spores exhibit an outermost exosporium layer that provides the first site of interaction with the host. Recent studies on the outermost exosporium layer of C. difficile spores have uncovered the ultrastructural variability, composition and functional properties of this layer [14, 20, 23, 38–40]. Extensive studies have demonstrated that cysteine-rich proteins have been involved in the assembly of the exosporium layer of spores of members of the B. cereus group and in the outer crust layer of B. subtilis spores [18, 24–26]. In C. difficile, there are three cysteine-rich proteins identified in the exosporium layer of C. difficile spores, CdeC, CdeM and CdeA [23]. Previously, we demonstrated that CdeC is an exosporium morphogenetic protein in epidemic C. difficile strain R20291 by performing functional analysis of a cdeC mutant strain [13]. In this work, we have used the laboratory strain 630erm due to its genetic ease, to investigate how two exosporium cysteine-rich proteins, CdeC and CdeM, contribute differentially to the spore biology and pathogenesis of C. difficile: CdeC and CdeM are both required for the correct formation of the exosporium layer. Whereas cdeC mutant exhibit defective spore coat assembly (Fig 2A and 2B) and permeability to lysozyme (Fig 4A and 4B), increased susceptibility to ethanol, heat- and macrophage-inactivation (Fig 5A, 5B and 5C), cdeM spores behaved as wild-type spores. Notably, CdeC is specific to C. difficile and related Peptostreptococcaceae family members, while CdeM is specific to C. difficile (Fig 1). In a healthy colonic mucosa, spore adherence of cdeC and cdeM spores was lower than wild-type spores (Fig 6); while during infection cdeC mutant, but not cdeM, exhibited higher diarrhea score, and persistence during recurrence of infection (Fig 7). In concordance, cdeC mutant, but not cdeM mutant, exhibited increased fitness in a competitive infection mouse model. Thus, this work contributes to our understanding on the mechanisms underlying exosporium assembly, and how this impacts C. difficile spore biology and pathogenesis.