Publication Database

Identification and characterization of immuno-active factors of Enterococcus spp. involved in immunomodulatory effects of Probiotic Enterococcus faecium SF68 (2022)

Art

Hochschulschrift

Autor

Ghazisaeedi, Fereshteh (WE 7)

Quelle

Berlin, 2022 — VIII, 158 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/37575

Kontakt

Institut für Mikrobiologie und Tierseuchen

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51843 / 66949
mikrobiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

E. faecium SF68 is a licensed probiotic, which has been shown to ameliorate symptoms of inflammation in enteritis and to reduce the incidence and severity of diarrhea in humans and animals. In vivo trials with weaning piglets performed in our laboratory or in cooperation with our laboratory, yielded controversial observations on the anti-inflammatory effects of E. faecium SF68. Supplementation with the probiotic in animals was reported to contribute to reduced expression of immune-associated genes in intestinal tissues and associated lymphoid organs. Moreover, these animals had lower amounts of serum IgG and fecal IgA, and reduced CD8+ intraepithelial lymphocyte populations. Piglets supplemented with E. faecium SF68 showed higher colonization and greater pathogen shedding in Salmonella infection studies. Since the alterations observed were mainly associated with local innate immune responses, this doctoral study focused mainly on the direct immunomodulatory effects of E. faecium SF68 on intestinal epithelial cells in an in vitro model. NF-κB, one of the key initiators of innate immune responses in intestinal epithelial cells encountering microbiota and invasive enteric pathogens, was selected as a reporter for determination of the effects of E. faecium SF68 on immune response modulation in intestinal epithelial cells. Cell-free, whole bacterial lysates of E. faecium SF68 inhibited NF-κB activation in intestinal epithelial cells from different host species. This inhibitory effect occurred in a reversible manner; after removal of the lysate and addition of fresh culture medium, cells recovered their NF-κB activity. Cells treated with the bacterial lysates ceased to proliferate, based on the results of host cell cycle protein Ki67 staining. Porcine intestinal epithelial cells treated with the probiotic lysate contained lower levels of phosphorylated p65 subunit of NF-κB at serine 536 compared to untreated cells. Phosphorylation at this amino acid is one of the most studied modifications involved in the transcriptional activity of p65 subunit. Moreover, fluorescence microscopy images of porcine intestinal cells suggested a dysregulation in the trafficking of NF-κB between the cytoplasm and nucleus. Ammonium sulfate (AS) fractionation of proteins of the E. faecium SF68 lysates and analysis of the proteins present in different AS-fractions revealed that several enzymes involved in arginine metabolism are present in the active AS-fraction. Other commensal and clinical E. faecium isolates, as well as isolates of other enterococcal species possessing enzymes of arginine deiminase pathway including E. faecalis, E. hirae and E. durans, exhibited the same inhibitory effect on NF-κB activity of IPEC-J2 cells. E. gallinarum and E. casseliflavus species were the two exceptions. These two species are the only motile enterococcal species expressing flagellin which is a strong stimulant of inflammatory pathways such as NF-κB, particularly in our in vitro cell model. Experiments examining pre- and co-incubation of host cells with E. faecium SF68 lysates and different Toll-like receptor (TLR)- and NOD-like receptor (NLR)-ligands showed that cells pre-treated with the E. faecium SF68 bacterial lysates were severely impaired in their NF-κB activation responses to these pathogen-associated molecular pattern (PAMP) signals. Lysates from strains of other enterococcal species with no AD pathway activity (E. avium, E. cecorum and E. raffinosus) did not inhibit the NF-κB activity of treated IPEC-J2 cells. The arcA gene of E. faecium SF68 encoding arginine deiminase was cloned into an E. coli-Enterococcus shuttle vector and introduced into an E. avium strain which showed no inhibitory effects on NF-κB activity. Transformants of E. avium constitutively expressing arginine deiminase of E. faecium SF68 showed the same NF-κB-inhibitory effects on porcine and human intestinal epithelial cell lines as E. faecium SF68. Comparison of results with the Streptococcus suis 10 strain, another bacterial genus possessing AD-pathway enzymes, exhibited the same results. Furthermore, an arcA knockout mutant of S. suis 10 no longer showed inhibition of NF-κB activity of host cells and purified recombinant arginine deiminase of S. suis 10 was shown to have the same inhibitory effects on NF-κB activity as the cell-free, whole bacterial lysates of S. suis 10 and E. faecium SF68. Finally, arginine supplementation of the cell culture medium eliminated the inhibitory effects on NF-κB activity mediated by arginine deiminase or bacterial lysates containing arginine deiminase. In summary, we postulate that the reported anti-inflammatory effects of E. faecium SF68 in vitro and likely in vivo, are associated with arginine deiminase-mediated arginine depletion. Beneficial bacteria in general can have a large number of molecules and metabolites, allowing them to interact with intestinal epithelial and immune cells. Therefore, the immunomodulatory effects elicited by beneficial microbes in the gut may not be limited to a single factor. Additionally, the health condition of the recipients, their age and immune status can determine the outcome of beneficial microbe administration. For instance, neonates with underdeveloped immune systems, not fully shaped microbiota and higher metabolic activity are more sensitive to arginine depletion (Flynn et al. 2002). Moreover, the concurrent presence of an intestinal pathogen such as Salmonella or Giardia, which use arginine depletion as a defensive strategy to hinder the host iNOS production and proliferation of immune cells, can aggravate the clinical condition. These pathogens promote arginine depletion by their own AD-pathway enzymes or induction of arginine consuming enzymes in the host cells. The mechanisms of action of beneficial microbes should be studied well and in detail. More importantly, the assessments and decisions on the administration of a beneficial microbe as therapeutic or preventive measure should be made cautiously and individually for each case to optimize the use of these advantageous biological tools.