Highly Efficient Ex Vivo Correction of COL7A1 through Ribonucleoprotein-Based CRISPR/Cas9 and Homology-Directed Repair to Treat Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of variants in COL7A1 encoding type VII Collagen, the major component of anchoring fibrils that form key attachment structures for dermal-epidermal adherence. In this study, we achieved highly efficient COL7A1 editing in primary RDEB keratinocytes and fibroblasts from 2 patients homozygous for the c.6508C>T (p.Gln2170∗) variant through CRISPR/Cas9-mediated homology-directed repair. Three guide RNAs targeting the c.6508C>T variant or harboring sequences were delivered together with high-fidelity Cas9 as a ribonucleoprotein complex. Among them, one achieved 73% cleavage activity in primary RDEB keratinocytes and RDEB fibroblasts. Then, we treated RDEB keratinocytes and RDEB fibroblasts with this specific ribonucleoprotein complex and the corresponding donor template delivered as single-stranded oligodeoxynucleotide and achieved up to 58% of genetic correction as well as type VII Collagen rescue. Finally, grafting of corrected 3-dimensional skin onto nude mice induced re-expression and normal localization of type VII Collagen as well as anchoring fibril formation at the dermal-epidermal junction 5 and 10 weeks after grafting. With this promising nonviral approach, we achieved therapeutically relevant specific gene editing that could be applicable to all variants in exon 80 of COL7A1 in primary RDEB cells.

Role of Arginine 214 in the Substrate Specificity of OXA-48.

Increasing numbers of variants of the carbapenem-hydrolyzing class D ß-lactamase OXA-48 are identified in Enterobacterales worldwide. Among them, OXA-181 and OXA-232 are of particular interest, as they differ from each other by a single amino acid substitution at position 214 (R in OXA-181 and S in OXA-232) that results in reduced carbapenem-hydrolyzing activity for OXA-232. To investigate the role of amino acid position 214 (AA214), the X-ray structure of OXA-232 was determined and AA214 of OXA-48 and of OXA-232 was replaced by G, L, D, E, S, R, and K using site-directed mutagenesis. These mutants were phenotypically characterized, and three mutants of OXA-232 were purified to study their steady-state kinetic properties. The X-ray structure of OXA-232 along with molecular modeling studies showed that the interaction via a salt bridge between R214 and D159 in OXA-48 is not possible with the G214 or S214 mutation. In contrast, with K214, which is also positively charged, the interaction with D159 is maintained. With the E214 mutant, an alternative binding conformation of imipenem that is not compatible with a nucleophilic attack by S70 was evidenced. Thus, imipenem has a very poor apparent affinity for the E214 mutant because of its nonproductive binding mode. Similarly, we could explain the lack of temocillin hydrolysis by the OXA-232-S214E mutant, which is due to the unfavorable interaction between the negatively charged R1 substituent of temocillin with the E214 residue. Overall, we demonstrate that AA214 in OXA-48-like ß-lactamases is critical for the carbapenemase activity.

Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14.

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14.

Atrophy of primary lymphoid organs induced by Marek's disease virus during early infection is associated with increased apoptosis, inhibition of cell proliferation and a severe B-lymphopenia.

Marek's disease is a multi-faceted highly contagious disease affecting chickens caused by the Marek's disease alphaherpesvirus (MDV). MDV early infection induces a transient immunosuppression, which is associated with thymus and bursa of Fabricius atrophy. Little is known about the cellular processes involved in primary lymphoid organ atrophy. Here, by in situ TUNEL assay, we demonstrate that MDV infection results in a high level of apoptosis in the thymus and bursa of Fabricius, which is concomitant to the MDV lytic cycle. Interestingly, we observed that in the thymus most of the MDV infected cells at 6 days post-infection (dpi) were apoptotic, whereas in the bursa of Fabricius most of the apoptotic cells were uninfected suggesting that MDV triggers apoptosis by two different modes in these two primary lymphoid organs. In addition, a high decrease of cell proliferation was observed from 6 to 14 dpi in the bursa of Fabricius follicles, and not in the thymus. Finally, with an adapted absolute blood lymphocyte count, we demonstrate a major B-lymphopenia during the two 1st weeks of infection, and propose this method as a potent non-invasive tool to diagnose MDV bursa of Fabricius infection and atrophy. Our results demonstrate that the thymus and bursa of Fabricius atrophies are related to different cell mechanisms, with different temporalities, that affect infected and uninfected cells.

Incorporation of the influenza A virus NA segment into virions does not require cognate non-coding sequences.

For each influenza virus genome segment, the coding sequence is flanked by non-coding (NC) regions comprising shared, conserved sequences and specific, non-conserved sequences. The latter and adjacent parts of the coding sequence are involved in genome packaging, but the precise role of the non-conserved NC sequences is still unclear. The aim of this study is to better understand the role of the non-conserved non-coding sequences in the incorporation of the viral segments into virions. The NA-segment NC sequences were systematically replaced by those of the seven other segments. Recombinant viruses harbouring two segments with identical NC sequences were successfully rescued. Virus growth kinetics and serial passages were performed, and incorporation of the viral segments was tested by real-time RT-PCR. An initial virus growth deficiency correlated to a specific defect in NA segment incorporation. Upon serial passages, growth properties were restored. Sequencing revealed that the replacing 5'NC sequence length drove the type of mutations obtained. With sequences longer than the original, point mutations in the coding region with or without substitutions in the 3'NC region were detected. With shorter sequences, insertions were observed in the 5'NC region. Restoration of viral fitness was linked to restoration of the NA segment incorporation.

High hepatitis E virus seroprevalence in forestry workers and in wild boars in France.

Hepatitis E virus (HEV) is a fecally and orally transmitted human pathogen of worldwide distribution. In industrial countries, HEV is observed in an increasing number of autochthonous cases and is considered to be an emerging pathogen. A growing body of evidence suggests that HEV is a zoonotic disease, and pig handlers and pig veterinarians have been reported to be high-risk groups for HEV infection. The aims of the present study were to establish the prevalence of anti-HEV in wild boars in France and to identify whether forestry workers are at a higher risk of HEV infection. Three different anti-HEV tests were used to compare their effectiveness in detecting anti-HEV in the general population. The most sensitive test was then used to investigate HEV seroprevalence in 593 forestry workers and 421 wild boars. Anti-HEV was detected in 31% of the forestry workers and 14% of the wild boars. Detection of anti-HEV in humans was correlated with age, geographical location, and occupational activity and in wild boars was correlated with geographical location. HEV infection is frequent in woodcutters in France, and it varies geographically. Further studies are needed to confirm these findings and to elucidate the transmission route and the exact virus reservoirs.