Differences in the internalization of self-inactivating VSVG-pseudotyped murine leukemia virus-based vectors in human and murine cells.

Self-inactivating VSVG-pseudotyped murine leukemia virus (SIN-VSVG-MLV) has been widely used to generate stable cell lines and produce gene delivery vectors. Despite the broad cellular tropism of the VSVG-pseudotyped MLV, we observed differential viral transduction efficiency depending on the host cell type used. In order to determine the mechanism underlying these differences, we used a GFP-expressing SIN-VSVG-MLV and analyzed the major steps of viral transduction in different cell lines including human epithelial, T-lymphocytes, monocytes and murine fibroblast cells. We observed the better transduction efficiency in HeLa cells, which was 20-fold higher than THP-1 and NIH/3T3 cells. To quantify viral internalization, we determined genomic RNA content by quantifying the early reverse transcription product. Genomic RNA and transduction levels were correlated with HeLa cells showing the higher amount of early RT product followed by tsA201 cells, while NIH/3T3, Jurkat and THP-1 had the lowest amounts. Similar results were observed when the late reverse transcription product was analyzed. Reverse transcription efficiency was 66-85% in HeLa cells and about 30% in tsA201, NIH/3T3, Jurkat and THP-1 cells. Viral integration, determined by Alu-Nested-qPCR, was higher for HeLa and lowerst for Jurkat and THP-1 cells. Interestingly, we observed that viral entry was correlated with the cellular availability of clathrin-mediated endocytosis, which was higher in HeLa and tsA201 cells, potentially explaining the higher rates of SIN-VSVG-MLV transduction and early RT synthesis observed in these cell lines. In conclusion, the SIN-VSVG-MLV vector showed significantly different rates of infectivity depending on the host cell type, possibly due to differential rates of viral internalization.

Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis.

Infectious pancreatic necrosis virus (IPNV) is a non-enveloped virus belonging to the Birnaviridae family. IPNV produces an acute disease in salmon fingerlings, with high mortality rates and persistent infection in survivors. Although there are reports of IPNV binding to various cells, the viral receptor and entry pathways remain unknown. The aim of this study was to determine the endocytic pathway that allows for IPNV entry. We observed that IPNV stimulated fluid uptake and virus particles co-localysed with the uptake marker dextran in intracellular compartments, suggesting a role for macropinocytosis in viral entry. Consistent with this idea, viral infection was significantly reduced when the Na+/H+ exchanger NHE1 was inhibited with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). Neither chlorpromazine nor filipin complex I affected IPNV infection. To examine the role of macropinocytosis regulators, additional inhibitors were tested. Inhibitors of the EGFR pathway and the effectors Pak1, Rac1 and PKC reduced viral infection. Together, our results indicate that IPNV is mainly internalized into CHSE-214 cells by macropinocytosis.

Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa.

Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CDI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.

Outcome of relapsing Clostridium difficile infections do not correlate with virulence-, spore- and vegetative cell-associated phenotypes.

One of the main clinical challenges of Clostridium difficile infections (CDI) is the high rate of relapse episodes. The main determinants involved in relapse of CDI include the presence of antibiotic-resistant C. difficile spores in the colonic environment and a permanent state of dysbiosis of the microbiota caused by antibiotic therapy. A possible scenario is that phenotypes related to the persistence of C. difficile spores might contribute to relapsing infections. In this study, 8 C. difficile isolates recovered from 4 cases with relapsing infection, and 9 isolates recovered from single infection cases were analyzed for PCR ribotyping and the presence of tcdA, tcdB and cdtAB genes. Factors associated to spore persistence, sporulation, spore adherence and biofilm formation and sporulation during biofilm formation were characterized. We also evaluated motility and cytotoxicity. However, we observed no significant difference in the analyzed phenotypes among the different clinical outcomes, most likely due to the high variability observed among strains within clinical backgrounds in each phenotype and the small sample size. It is noteworthy that C. difficile spores adhered to similar extents to undifferentiated and differentiated Caco-2 cells. By contrast, spores of all clinical isolates tested had increased germination efficiency in presence of taurocholate, while decreased sporulation rate during biofilm development in the presence of glucose. In conclusion, these results show that, at least in this cohort of patients, the described phenotypes are not detrimental in the clinical outcome of the disease.

The Clostridium difficile exosporium cysteine (CdeC)-rich protein is required for exosporium morphogenesis and coat assembly.

Clostridium difficile is an important nosocomial pathogen that has become a major cause of antibiotic-associated diarrhea. There is a general consensus that C. difficile spores play an important role in C. difficile pathogenesis, contributing to infection, persistence, and transmission. Evidence has demonstrated that C. difficile spores have an outermost layer, termed the exosporium, that plays some role in adherence to intestinal epithelial cells. Recently, the protein encoded by CD1067 was shown to be present in trypsin-exosporium extracts of C. difficile 630 spores. In this study, we renamed the CD1067 protein Clostridium difficile exosporium cysteine-rich protein (CdeC) and characterized its role in the structure and properties of C. difficile spores. CdeC is expressed under sporulation conditions and localizes to the C. difficile spore. Through the construction of an ΔcdeC isogenic knockout mutant derivative of C. difficile strain R20291, we demonstrated that (i) the distinctive nap layer is largely missing in ΔcdeC spores; (ii) CdeC is localized in the exosporium-like layer and is accessible to IgGs; (iii) ΔcdeC spores were more sensitive to lysozyme, ethanol, and heat treatment than wild-type spores; and (iv) despite the almost complete absence of the exosporium layer, ΔcdeC spores adhered at higher levels than wild-type spores to intestinal epithelium cell lines (i.e., HT-29 and Caco-2 cells). Collectively, these results indicate that CdeC is essential for exosporium morphogenesis and the correct assembly of the spore coat of C. difficile.