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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., Author summary We discovered a mechanism of assembly of the outer most layer of Clostridium difficile spores, the exosporium. While CdeC is conserved in several Peptostreptococcaeace family members, CdeM is unique to C. difficile. We show that two proteins that are rich in cysteine amino acid residues, CdeC and CdeM, are essential for the recruitment of additional spore coat and exosporium proteins. The absence of CdeC, had profound implications in the correct spore coat assembly which were related to decreased spore resistant properties that are relevant for in vivo infection such as lysozyme resistance, macrophage infection. Notably, the absence of either cysteine rich proteins leads to a decrease in spore adherence of C. difficile spores to healthy colonic mucosa; but only the absence of CdeC affected in vivo competitive fitness in a mouse model, recurrence of the disease in a mouse model of recurrent infection. Considering the importance of the outer layers of C. difficile spores in spore-host interactions, our findings have broad implications on the biology of C. difficile spores and to C. difficile pathogenesis.

Smooth muscle-specific transcription is controlled by a multitude of transcriptional regulators that cooperate to drive expression in a temporospatial manner. Previous analysis of the cysteine-rich protein 1 ( CRP1/Csrp) gene revealed an intronic enhancer that is sufficient for expression in arterial smooth muscle cells and requires a serum response factor-binding CArG element for activity. The presence of a CArG box in smooth muscle regulatory regions is practically invariant; however, it stands to reason that additional elements contribute to the modulation of transcription in concert with the CArG. Because of the potential importance of other regulatory elements for expression of the CRP1 gene, we sought to identify additional motifs within the enhancer that are necessary for expression. In this effort, we identified a conserved cAMP response element (CRE) that, when mutated, diminishes the expression of the enhancer in cultured vascular smooth muscle cells. Using transfection and electrophoretic mobility shift assays, we have shown that the CRE binds the cAMP response element-binding protein (CREB) and is activated by Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), but not by CaMKII. Furthermore, our data demonstrate that CaMKIV stimulates CRP1 expression not only through the CRE but also through the CArG box. These findings represent evidence of a functional CRE within a smooth muscle-specific gene and provide support for a mechanism in which CREB functions as a smooth muscle determinant through CaMKIV activation.

The family of cysteine rich proteins (CRP) comprises three closely homologous members that have been reported to interact with α-actinin. Muscular LIM protein (MLP/CRP3), the skeletal muscle variant, was originally discovered as a positive regulator of myogenesis and is suggested to be part of the stretch sensor of the myofibril through its interaction with telethonin (T-Cap). We determined the structure of both LIM domains of human MLP by nuclear magnetic resonance spectroscopy. We confirm by 15N relaxation measurements that both LIM domains act as independent units and that the adjacent linker regions are fully flexible. With the published structures of CRP1 and CRP2, the complete family has now been structurally characterized.

International audience; Plant lipid transfer proteins are small soluble extracellular proteins that are able to bind and transfer a variety of lipids in vitro. Recently, it has been proposed that lipid transfer proteins may play a key role in plant defence mechanisms, especially during the induction of systemic acquired resistance. However, very little is known about the proteins expressed in developing plants and tissues, since almost all the biophysical and structural data available to date on lipid transfer proteins originate from proteins present in storage tissues of monocot cereal seeds. In this paper, we report the structural and functional characteristics of a lipid transfer protein (named LTP1_1) constitutively expressed in young aerial organs of Nicotiana tabacum (common tobacco). The unlabelled and uniformly labelled proteins were produced in the yeast Pichia pastoris, and we determined the three-dimensional (3D) structure of LTP1_1 using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. The global fold of LTP1_1 is very close to the previously published structures of LTP1 extracted from cereal seeds, including an internal cavity. However, the chemical shift variations of several NMR signals upon lipid binding show that tobacco LTP1_1 is able to bind only one LysoMyristoylPhosphatidylCholine (LMPC), while wheat and maize LTPs can bind either one or two. Titration experiments using intrinsic tyrosine fluorescence confirm this result not only with LMPC but also with two fatty acids. These differences can be explained by the presence in tobacco LTP1_1 of a hydrophobic cluster closing the second possible access to the protein cavity. This result suggests that LTP1 lipid binding properties could be modulated by subtle changes in a conserved global structure. The biological significance of this finding is discussed in the light of the signalling properties of the tobacco LTP1_1-jasmonate complex described elsewhere.