RESUMEN
Objective: Gastrointestinal [GI] tract, like other mucosal surface, is colonized with a microbial population known as gut microbiota. Outer membrane vesicles [OMVs] which are produced by gram negative bacteria could be sensed by Toll like receptors [TLRs]. The interaction between gut microbiota and TLRs affects homeostasis and immune responses. In this study, we evaluated TLR2, TLR4 genes expression and cytokines concentration in Caco-2 cell line treated with Bacteroides fragilis [B. fragilis] and its OMVs
Materials and Methods: In this experimental study, OMVs were extracted using sequential centrifugation and their physicochemical properties were evaluated as part of quality control assessment. Caco-2 cells were treated with B. fragilis and its OMVs [180 and 350 microg/ml]. Quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR] was performed to assess TLR2 and TLR4 mRNA expression levels. Pro-inflammatory [IFN?] and anti-inflammatory [IL- 4 and IL-10] cytokines were evaluated by ELISA
Results: B. fragilis significantly decreased TLR2 and slightly increased TLR4 mRNA levels in Caco-2 cell line. The TLR2 mRNA level was slightly increased at 180 and 350 microg/ml of OMVs. Conversely, the TLR4 mRNA level was decreased at 180 microg/ml of OMVs, while it was significantly increased at 350 microg/ml of OMVs. Furthermore, B. fragilis and its OMVs significantly increased and decreased IFN? concentration, respectively. Anti-inflammatory cytokines were increased by B. fragilis and its OMVs
Conclusion: B. fragilis and its OMVs have pivotal role in the cross talk between gut microbiota and the host especially in the modulation of the immune system. Based on the last studies on immunomodulatory effect of B. fragilis derived OMVs on immune cells and our results, we postulate that B. fragilis derived OMVs could be possible candidates for the reduction of immune responses
RESUMEN
Background: Diarrhea, caused by enteroaggregative Escherichia coli [EAEC], is an important infection leading toillness and death. Numerous virulent factors have been described in EAEC. However, their prevalence was highly variable among EAECs of distinct geographic locations. Studies have shown that dispersin [antiaggregation protein, aap] is one of the important and abundant virulent factors in EAEC. In this study, we aimed to determine the presence, conservation, and immunogenicity of aap gene in EAEC isolated from Iranian patients
Methods: PCR amplification of aap gene in the EAEC isolates was performed, and the aap gene was cloned in pBAD-gIIIA vector. The sequence of aap gene was analyzed using the ExPASy and BLAST tools. The expression of aap gene was performed in E. coli Top10, and expression confirmation was carried out by SDS-PAGE and Western-blot techniques. Rabbits were immunized with purified dispersin protein emulsified with Freund's adjuvant. Sera were collected and examined for antibody response. Finally, in vitro efficacy of dispersin and anti-dispersin was evaluated
Results: The results of PCR showed the presence of aap gene in all of the EAEC isolates with significant homology. Finally, the significant difference between the levels of IgG response in dispersin-injected rabbits and control group was observed
Conclusion: Our results were in accordance with other studies that reported the presence of dispersin in the EAEC isolates with high conservation and immunogenicity. Hence, dispersin could be a promising candidate for any probable prevention against EAEC infections