The origin of SARS-CoV-2 variants of concern remains unclear. Here, we test whether intra-host virus evolution during persistent infections could be a contributing factor by characterizing the long-term SARS-CoV-2 infection dynamics in an immunosuppressed kidney transplant recipient. Applying RT-qPCR and next-generation sequencing (NGS) of sequential respiratory specimens, we identify several mutations in the viral genome late in infection. We demonstrate that a late viral isolate exhibiting genome mutations similar to those found in variants of concern first identified in UK, South Africa, and Brazil, can escape neutralization by COVID-19 antisera. Moreover, infection of susceptible mice with this patient’s escape variant elicits protective immunity against re-infection with either the parental virus and the escape variant, as well as high neutralization titers against the alpha and beta SARS-CoV-2 variants, B.1.1.7 and B.1.351, demonstrating a considerable immune control against such variants of concern. Upon lowering immunosuppressive treatment, the patient generated spike-specific neutralizing antibodies and resolved the infection. Our results suggest that immunocompromised patients could be a source for the emergence of potentially harmful SARS-CoV-2 variants.

Clostridium spiroforme produces the binary actin-ADP-ribosylating toxin CST ( C. spiroforme toxin), which has been proposed to be responsible for diarrhea, enterocolitis, and eventually death, especially in rabbits. Here we report on the recombinant production of the enzyme component (CSTa) and the binding component (CSTb) of C. spiroforme toxin in Bacillus megaterium . By using the recombinant toxin components, we show that CST enters target cells via the lipolysis-stimulated lipoprotein receptor (LSR), which has been recently identified as the host cell receptor of the binary toxins Clostridium difficile transferase (CDT) and Clostridium perfringens iota toxin. Microscopic studies revealed that CST, but not the related Clostridium botulinum C2 toxin, colocalized with LSR during toxin uptake and traffic to endosomal compartments. Our findings indicate that CST shares LSR with C. difficile CDT and C. perfringens iota toxin as a host cell surface receptor.

AIM: To evaluate the effect of oral Escherichia coli (E. coli) Nissle application on the outcome of intestinal-borne dermatoses. METHODS: In a randomized, controlled, non-blinded prospective clinical trial 82 patients with intestinal-borne facial dermatoses characterized by an erythematous papular-pustular rash were screened. At the initiation visit 37 patients entered the experimental arm and 20 patients constituted the control arm. All 57 patients were treated with a vegetarian diet and conventional topical therapy of the dermatoses with ointments containing tetracycline, steroids and retinoids. In the experimental arm patients received a one month therapy with oral E. coli Nissle at a maintenance dose of 2 capsules daily. The experimental group was compared to a non-treatment group only receiving the diet and topical therapy. The primary outcome parameter was improvement of the dermatoses, secondary parameters included life quality and adverse events. In addition the immunological reaction profile (IgA, interleucin-8 and interferon-α) was determined. Furthermore the changes of stool consistency and the microbiota composition over the time of intervention were recorded. RESULTS: Eighty-nine percent of the patients with acne, papular-pustular rosacea and seborrhoic dermatitis responded to E. coli Nissle therapy with significant amelioration or complete recovery in contrast to 56% in the control arm (P < 0.01). Accordingly, in the E. coli Nissle treated patients life quality improved significantly (P < 0.01), and adverse events were not recorded. The clinical improvement was associated with a significant increase of IgA levels to normal values in serum as well as suppression of the proinflammatory cytokine IL-8 (P < 0.01 for both parameters). In the E. coli Nissle treated group a shift towards a protective microbiota with predominance of bifidobacteria and lactobacteria (> 10⁷ CFU/g stool) was observed in 79% and 63% of the patients, respectively (P < 0.01), compared to no change in the control group without E. coli Nissle. Moreover, the detection rate of a pathogenic flora dropped from 73% to 14 % of the patients in the experimental arm (P < 0.01) with no significant change in the control arm (accounting 80% before and 70% after the observation period, P > 0.05). Accordingly, stool consistency, color and smell normalized in the E. coli Nissle treated patients. CONCLUSION: E. coli Nissle protects the mucus barrier by overgrowth of a favorable gut microbiota with less immunoreactive potential which finally leads to clinical improvement of intestinal borne dermatoses.

Clostridium difficile is the leading cause of antibiotics-associated diarrhea and pseudomembranous colitis. Hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT ( C. difficile transferase), in addition to the Rho-glucosylating toxins A and B. We recently identified the lipolysis-stimulated lipoprotein receptor (LSR) as the host receptor that mediates uptake of CDT into target cells. Here we investigated in H1-HeLa cells, which ectopically express LSR, the influence of CDT on the plasma membrane distribution of the receptor. We found by fluorescence microscopy that the binding component of CDT (CDTb) induces clustering of LSR into subcompartments of the plasma membrane. Detergent extraction of cells treated with CDTb, followed by sucrose gradient fractionation, uncovered accumulation of LSR in detergent-resistant membranes (DRMs) that contained typical marker proteins of lipid rafts. Membrane cholesterol depletion with methyl-β-cyclodextrin inhibited the association of LSR with DRMs upon addition of CDTb. The receptor-binding domain of CDTb also triggered LSR clustering into DRMs. CDTb-triggered clustering of LSR into DRMs could be confirmed in Caco-2 cells. Our data suggest that CDT forces its receptor to cluster into lipid rafts and that oligomerization of the B component might enhance but is not essential for this process. IMPORTANCE C. difficile binary toxin CDT is a member of the iota-like, actin ADP-ribosylating toxin family. The mechanism that mediates endocytic uptake of these toxins still remains elusive. Previous studies highlighted the importance of lipid rafts for oligomerization of the binding component of these toxins and for cell entry. Recently, the host cell receptor for this toxin family, namely, the lipolysis-stimulated lipoprotein receptor (LSR), has been identified. Our study now demonstrates that the binding component of CDT (CDTb) induces clustering of LSR into lipid rafts. Importantly, LSR clustering is efficiently induced also by the receptor-binding domain of CDTb, suggesting that oligomerization of the B component of CDT is not the main trigger of this process. The current work extends our knowledge on the cooperative play between iota-like toxins and their receptor.

Purpose: Recent studies point toward a potential benefit of doxycycline use for post-exposure prophylaxis (PEP) or pre-exposure prophylaxis (PrEP) to prevent sexually transmitted infections (STIs). Although prescribing doxycycline in a prophylactic intention is not generally recommended yet, we noticed an increasing number of inquiries from individuals within the LGBTQ community for doxycycline prescriptions. Methods: We conducted an anonymous online survey to evaluate the current extent of doxycycline use for PEP or PrEP within the LGBTQ community using REDCap electronic data capture tools. Participants gained access to the online survey through a QR code on posters in the premises of our STI outpatient department and at LGBTQ community-related events in the south-western region of Germany. Additional access was provided by a direct link shared on social media profiles for men having sex with men (MSM), transgender, and queers. Results: 96 of 99 responses were eligible for analysis. Twenty-two participants (23%) indicated to have already used doxycycline for PEP and six participants (6%) used doxycycline for PrEP. The majority of participants used pills left over from previous doxycycline treatment. Forty percent of indicated modes of access were without a regular prescription, e.g., by provision from acquaintances (with or without healthcare profession) or by ordering online. Conclusion: Our study shows that the concept of doxycycline use for prevention of STIs is already well known and applied in the LGBTQ community. Further analysis, especially modeling studies, are needed to evaluate strategies aiming to reduce doxycycline intake (PEP/PrEP versus repeated targeted therapies) and improve sexual health outcomes within the community.