An Untargeted Metabolomic Analysis of Lacticaseibacillus (L.) rhamnosus, Lactobacillus (L.) acidophilus, Lactiplantibacillus (L.) plantarum and Limosilactobacillus (L.) reuteri Reveals an Upregulated Production of Inosine from L. rhamnosus

Lactic acid bacteria are considered an inexhaustible source of bioactive compounds; indeed, products from their metabolism are known to have immunomodulatory and anti-inflammatory activity. Recently, we demonstrated that Cell-Free Supernatants (CFS) obtained from Lactobacillus (L.) acidophilus, Lactiplantibacillus (L.) plantarum, Lacticaseibacillus (L.) rhamnosus, and Limosilactobacillus (L.) reuteri can impair Candida pathogenic potential in an in vitro model of epithelial vaginal infection. This effect could be ascribed to a direct effect of living lactic acid bacteria on Candida virulence and to the production of metabolites that are able to impair fungal virulence. In the present work, stemming from these data, we deepened our knowledge of CFS from these four lactic acid bacteria by performing a metabolomic analysis to better characterize their composition. By using an untargeted metabolomic approach, we detected consistent differences in the metabolites produced by these four different lactic acid bacteria. Interestingly, L. rhamnosus and L. acidophilus showed the most peculiar metabolic profiles. Specifically, after a hierarchical clustering analysis, L. rhamnosus and L. acidophilus showed specific areas of significantly overexpressed metabolites that strongly differed from the same areas in other lactic acid bacteria. From the overexpressed compounds in these areas, inosine from L. rhamnosus returned with the best identification profile. This molecule has been described as having antioxidant, anti-inflammatory, anti-infective, and neuroprotective properties. The biological significance of its overproduction by L. rhamnosus might be important in its probiotic and/or postbiotic activity

C5aR2 Deficiency Ameliorates Inflammation in Murine Epidermolysis Bullosa Acquisita by Regulating Fcγ Receptor Expression on Neutrophils

Epidermolysis bullosa acquisita (EBA) is a rare blistering skin disease induced by autoantibodies directed against type VII collagen. The transfer of antibodies against murine type VII collagen into mice mimics the effector phase of EBA and results in a subepidermal blistering phenotype. Activation of the complement system, and especially the C5a/C5aR1 axis driving neutrophil activation, is critical for EBA pathogenesis. However, the role of the alternative C5a receptor, C5aR2, which is commonly thought to be more immunosuppressive, in the pathogenesis of EBA is still elusive. Therefore, we sought to delineate the functional relevance of C5aR2 during the effector phase of EBA. Interestingly, C5ar2-/- mice showed an attenuated disease phenotype, suggesting a pathogenic contribution of C5aR2 in disease progression. In vitro, C5ar2-/- neutrophils exhibited significantly reduced intracellular calcium flux, ROS release, and migratory capacity when activated with immune complexes or exposed to C5a. These functions were completely absent when C5ar1-/- neutrophils were activated. Moreover, C5aR2 deficiency lowered the ratio of activating and inhibitory FcγRs, impeding the sustainment of inflammation. Collectively, we show here a proinflammatory contribution of C5aR2 in the pathogenesis of antibody-induced tissue damage in experimental EBA

Candida albicans Factor H Binding Molecule Hgt1p – A Low Glucose-Induced Transmembrane Protein Is Trafficked to the Cell Wall and Impairs Phagocytosis and Killing by Human Neutrophils

Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. Candida albicans is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein “High affinity glucose transporter 1” (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased in vitro phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of C. albicans

Candida albicans factor H binding molecule Hgt1p as a dynamic complement evasion molecule

Komplement ist ein stark kontrolliertes System, das in der Lage ist, Krankheitserreger während der Phase im Blutverteilung zu töten. Faktor H (FH) ist der Hauptregulator des alternativen Pathways, und das Einfangen von FH aus dem Serum durch Krankheitserreger bietet ihnen einen gewissen Schutz vor komplement. Candida albicans ist klinisch ein wichtiger opportunistischer Pilz. Er hat auf seiner Zelloberfläche verschiedene FH-Bindungsmoleküle entwickelt, die zur herunterregulirung von Komplement dienen. Das Transmembranprotein „hoher Affinitäts Glucose-Transporter 1“ (Hgt1p), das am Glucosestoffwechsel beteiligt ist, ist einer von ihnen. In der Dissertation wird Glucose-Abhängigkeit des Hgt1p-Abwelsmechanismus, gezeigt, ein Weg, um die C. albicans-Infektion und die Komplementinhibition besser zu verstehen. Dazu was es notwendig eine physiologische Glukose konzentrationin den Medien zu verwenden. Es wird detailliertdass C. albicans Hgt1p durch Vesikeltransport in die Zellwand exportiert wird und die freigesetzten Exosomen integriertes Hgt1p enthalten. An der Zellwand wird das Komplementsystem insbesondere über die FH-Bindung herunterreguliert, gefolgt von einer gestörten C3b / iC3b umwandlung und phagozytose. Die Blockierung von Hgt1p durch spezifische Blocker zeigte eine Verringerung des abgeschiedenen FH und eine parallele signifikante Zunahme der C3b/iC3b-Ablagerung und der Phagozytose durch humane neutrophile Granulozyten. Diese in vitro Daten wurden durch einen in vivo Test unter Verwendung des Galleria Mellonella modells bestätigt. Zusammenfassend weist Hgt1p auch nichtkanonische Funktionen auf, wie die Bindung von FH nach dem Export an die Zellwand. Das Blockieren von Hgt1p-FH Wechselwirkungen kann ein Mittel darstellen, um die Komplementinhibierung zu begrenzen und somit die Aktivierung und Phagozytose während der Blutdisseminationsphase von C. albicans zu verstärken.Complement is a tightly controlled system, capable of killing pathogens during their blood dissemination phase in the host. Factor H (FH) is the main regulator of the alternative pathway and hijacking of FH from serum by pathogens provides them with some protection. Candida albicans is clinically an important opportunistic yeast and has evolved different FH binding molecules on its cell surface which can be employed to evade complement. The transmembrane protein “High affinity glucose transporter 1” (Hgt1p), involved in glucose metabolism, represents one of them. In this dissertation a glucose dependence of the Hgt1p evasion mechanism was shown and, in order to get a better understanding of C. albicans infection and complement inhibition, a physiological glucose condition was used in the media. It was demonstrated that C. albicans exports Hgt1p to the cell wall by vesicle trafficking and that its released exosomes contain integrated Hgt1p. On the cell wall the complement system is downregulated, especially via FH binding, followed by an impaired C3b/iC3b conversion and phagocytosis. Blocking of Hgt1p by specific blockers showed a reduction of bound FH and a parallel significant increase of C3b/iC3b deposition and phagocytosis by human neutrophil granulocytes. These in vitro data were corroborated by an in vivo assay using the Galleria mellonella model. In conclusion, Hgt1p also exhibits non-canonical functions, as the binding of FH after export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to limit complement inhibition and thus enhance activation and phagocytosis during C. albicans blood dissemination.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftInnsbruck, Med. Univ., Diss., 2019(VLID)336473

Elucidating the immunological function of the Cryptococcus neoformans capsule

The encapsulated fungal pathogen Cryptococcus neoformans represents a significant agent of life-threatening infections in immunocompromised subjects. A unique characteristic of Cryptococcus species is the presence of a polysaccharide capsule, which is essential for virulence and endows Cryptococcus with potent immunoregulatory properties. This review provides an overview of the immunological properties of the principal components of C. neoformans capsule

A novel bioluminescence mouse model for monitoring oropharyngeal candidiasis in mice.

International audienceOropharyngeal Candida albicans (C. albicans) infection usually occurs in patients with altered cell-mediated immune response. Many animal models have been developed for studying the pathogenesis of disease. Here we describe a new model for real-time monitoring of oral candidiasis. Mice were rendered susceptible to oral candidiasis by injection with cortisone acetate. Oral infection was performed by placing a swab saturated with genetically engineered bioluminescent strain of C. albicans sublingually. An in vivo imaging technique, exploiting stably trasformed C. albicans that costitutively express luciferase, was adopted. This novel longitudinal study represents a powerful tool to: (1) test real-time progression of infection, (2) identify the target site of C. albicans in specific organs, (3) evaluate the efficacy of antifungal therapies and (4) explore the spread of C. albicans from the local to systemic compartment in a new way

Shiga Toxin 2a Binds to Complement Components C3b and C5 and Upregulates Their Gene Expression in Human Cell Lines

Enterohemorrhagic Escherichia coli (EHEC) infections can cause EHEC-associated hemolytic uremic syndrome (eHUS) via its main virulent factor, Shiga toxins (Stxs). Complement has been reported to be involved in the progression of eHUS. The aim of this study was to investigate the interactions of the most effective subtype of the toxin, Stx2a, with pivotal complement proteins C3b and C5. The study further examined the effect of Stx2a stimulation on the transcription and synthesis of these complement proteins in human target cell lines. Binding of Stx2a to C3b and C5 was evaluated by ELISA. Kidney and gut cell lines (HK-2 and HCT-8) were stimulated with varied concentrations of Stx2a. Subsequent evaluation of complement gene transcription was studied by real-time PCR (qPCR), and ELISAs and Western blots were performed to examine protein synthesis of C3 and C5 in supernatants and lysates of stimulated HK-2 cells. Stx2a showed a specific binding to C3b and C5. Gene transcription of C3 and C5 was upregulated with increasing concentrations of Stx2a in both cell lines, but protein synthesis was not. This study demonstrates the binding of Stx2a to complement proteins C3b and C5, which could potentially be involved in regulating complement during eHUS infection, supporting further investigations into elucidating the role of complement in eHUS pathogenesis

Influence of Glucose on Candida albicans and the Relevance of the Complement FH-Binding Molecule Hgt1 in a Murine Model of Candidiasis

Candidiasis is common in diabetic patients. Complement evasion is facilitated by binding complement factor H (FH). Since the expression of high-affinity glucose transporter 1 (Hgt1), a FH-binding molecule, is glucose-dependent, we aimed to study its relevance to the pathogenesis of Candida albicans. Euglycemic and diabetic mice were intravenously challenged with either Candida albicans lacking Hgt1 (hgt1-/-) or its parental strain (SN152). Survival and clinical status were monitored over 14 days. In vitro, Candida albicans strains were grown at different glucose concentrations, opsonized with human serum, and checked for C3b/iC3b and FH deposition. Phagocytosis was studied by fluorescein isothiocyanate-labeled opsonized yeast cells incubated with granulocytes. The murine model demonstrated a significantly higher virulence of SN152 in diabetic mice and an overall increased lethality of mice challenged with hgt1-/-. In vitro lower phagocytosis and C3b/iC3b deposition and higher FH deposition were demonstrated for SN152 incubated at higher glucose concentrations, while there was no difference on hgt1-/- at physiological glucose concentrations. Despite C3b/iC3b and FH deposition being glucose-dependent, this effect has a minor influence on phagocytosis. The absence of Hgt1 is diminishing this dependency on complement deposition, but it cannot be attributed to being beneficial in a murine model