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. 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.

Clostridioides difficile, formerly known as Clostridium difficile, is a spore-forming bacterium considered as the most common cause of nosocomial infections in developed countries. The spore of C. difficile is involved in the transmission of the pathogen and in its first interaction with the host; therefore, a therapeutic approach able to control C. difficile spores would improve the clearance of the infection. The C-terminal (CTD) end of BclA2, a spore surface protein of C. difficile responsible of the interaction with the host intestinal cells, was selected as a putative mucosal antigen. The BclA2 fragment, BclA2_CTD, was purified and used to nasally immunize mice both as a free protein and after adsorption to the spore of Bacillus subtilis, a well-established mucosal delivery vehicle. While the adsorption to spores increased the in vitro stability of BclA2_CTD, in vivo both free and spore-adsorbed BclA2_CTD were able to induce a similar, specific humoral immune response in a murine model. Although in the experimental conditions utilized the immune response was not protective, the induction of specific IgG indicates that free or spore-bound BclA2_CTD could act as a putative mucosal antigen targeting C. difficile spores.

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance an immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets for new therapies. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here, we report that the C-terminal domain of the spore surface protein BclA3, BclA3_CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3_CTD was also exposed on the surface of B. subtilis spores, a mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3_CTD induced antibody production in mice and attenuated some C. difficile infection symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization routes need to be optimized, our results suggest BclA3_CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores.

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance a strong immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets of new therapeutic treatments. In this context, spore surface proteins of C. difficile , are potential antigens for the development of vaccines targeting C. difficile spores. Here we report that the C-terminal domain of the spore surface protein BclA3, BclA3 _CTD , was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3 _CTD was also exposed on the surface of B. subtilis spores, a well-established mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3 _CTD induced antibody production in mice and attenuated some CDI symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization route need to be optimized, our results suggest BclA3 _CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores.

Inflammation is a multifaceted response involving molecular and cellular components triggered by diverse stimuli, including physical, chemical, and biological factors. The connection between these stimuli and inflammatory cellular responses involves numerous interactions among molecular components, resulting in cell outputs that can either help resolve potential harmful outcomes or amplify them, leading to further complications. Notably, heat shock factor 1 (HSF1), a transcription factor primarily associated with cellular responses to elevated temperatures, has also been reported to play a key role in modulating inflammation. Indeed, growing evidence suggests a close relationship between HSF1 and intracellular signaling pathways that regulate the secretion of inflammatory cytokines, impacting both infectious and non-infectious diseases. Furthermore, chaperone proteins known as heat shock proteins (Hsps), which HSF1 transcriptionally regulates, also play important roles during inflammation by modulating numerous host factors involved in this process. Here, we examine the interplay between HSF1 and Hsps in the context of inflammation caused by infectious and non-infectious diseases. We also discuss the potential benefits of targeting these factors as strategies for reducing the effects of exacerbated or damaging inflammation.

Neurotropic viruses can alter neuronal responses in the central nervous system (CNS), significantly affecting viral clearance and disease progression. Herpes simplex virus type 1 (HSV-1) brain infection may lead to life-threatening severe acute encephalitis in untreated patients and neurological sequelae in survivors despite antiviral treatment. Notably, asymptomatic brain infection occurs in an important proportion of healthy individuals (>35%) and is associated with residual chronic neuroinflammatory responses that may lead to neurodegeneration. Therefore, understanding the molecular basis of these detrimental effects and finding and advancing new therapeutic strategies to manage HSV-1 brain infections are needed. Neuropeptides are pleiotropic neuroimmune mediators expressed throughout the CNS that modulate glial activation, cytokine production, and neuronal survival. However, their regulation during HSV-1 brain infections remains largely unexplored. Here, we sought to investigate the expression dynamics of two neuropeptides, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP), in two mouse strains that model human traits of symptomatic and asymptomatic HSV-1 brain infections (BALB/c and C57BL/6, respectively), as well as in the human neuroblastoma cell line SH-SY5Y, to uncover potential differences that could help explain the susceptibility of some individuals to develop severe HSV-1 infection. Our findings provide evidence that HSV-1 brain infection modulates NPY and VIP mRNA expression in a neurovirulence- and host-susceptibility-dependent manner, which may be associated with disease severity and chronic damage, warranting further evaluation.