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Projeto de investigação
Microbiology Advancing Health: Microbiome-Autoimmunity Interplay and the Artificial Intelligence of Infection Prevention and Control
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Influence of commensal bacteria on the proteolytic and antigenic profiles of INFOGEST-like digested wheat gliadin
Publication . Pereira-Costa, Flávio; Domingues, Vanessa S.; Roque, Ana; Almeida, Zaida L.; Cruz, Pedro F.; Cordeiro, Rachel; Trindade, Daniela; Moura, Carla; Melo, Joana B.; Pereira, Sónia G.; Vaz, Daniela C.
Introduction: Celiac disease (CeD) is a chronic autoimmune enteropathy developed by genetically predisposed individuals when exposed to gluten. Gluten gliadins, along with gut microbiota, may influence CeD onset and progression through mechanisms that remain unclear. Methods: Gliadin-degrading bacterial isolates obtained from CeD patients, and their 1st-degree relatives’ stool and blood were identified (Bacillus tropicus, Enterococcus faecalis, Micrococcus sp., Cronobacter sakazakii, Pseudomonas aeruginosa, and Serratia marcescens) and used in an INFOGEST-like protocol to simulate gliadin digestion after 4 h (digested gliadin, d-gliadin). The d-gliadin digesta were analyzed by fast protein liquid chromatography (FPLC), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), fluorescence spectroscopy, and polyclonal and monoclonal (R5 and G12) enzyme-linked immunosorbent assays (ELISA). Results and discussion: In the absence of the bacterial isolates, gliadin is poorly digested and self-assembles within 1 day into intermediate and large protein oligomers/aggregates, enriched in β-sheet structure (FTIR amide I band between 1,600 and 1,700 cm−1) and able to bind thioflavin T and Congo red. Conversely, in the presence of the bacterial isolates, gliadin is further digested, leading to an increase in protein fragments. After 4 h, the P. aeruginosa, C. sakazakii, and B. tropicus d-gliadin digesta presented a mixture of d-gliadin peptides and aggregates that showed higher antigenicity (associated with the exposure of the 5-amino acid QQPFP and 6-amino acid QPQLPY epitopes, present in the 25-mer and 33-mer, respectively) than control digestions (without bacteria), while E. faecalis led to lower antigenicity. In turn, within 24 h of incubation, all bacterial isolates led to the formation of undigested material with lower antigenicity, either due to fewer 33-mers and 25-mers in solution, or to fragment aggregation into amorphous material, not exposing antigenic sequences. Hence, intestinal flora may enhance or diminish the antigenicity of gliadin, thereby modulating the immunogenic response to gliadin/gluten.
Neuropeptides neurotensin and substance P accelerate diabetic wound healing by modulating immunity and the skin microbiome
Publication . Maranha, Ana; Leal, Ermelindo C.; Alarico, Susana; Tiago, Igor; Pereira, Sónia G.; Empadinhas, Nuno; Carvalho, Eugénia
Diabetic foot ulcers (DFUs) are a common and debilitating complication of diabetes mellitus, typified by impaired healing, persistent inflammation, and skin microbiota dysbiosis. Although some neuropeptides are immunomodulatory, their effects on skin microbiota and wound resolution remain largely unexplored. We examined the therapeutic potential of the neuropeptides neurotensin (NT) and substance P (SP) in reshaping inflammatory and microbial dynamics and accelerating wound closure in a healthy and streptozotocin-induced diabetic mice model. Full-thickness cutaneous wounds were induced on both groups, followed by daily topical applications of saline (CT), NT, or SP. Wound closure was monitored and changes in skin bacterial communities characterized using next-generation sequencing. Wound tissue samples were collected for immunohistochemical analysis. Topical application of NT or SP significantly accelerated wound healing, with the most pronounced effects in diabetic animals. Both neuropeptides reduced the number of pro-inflammatory cells, lowering M1 macrophages in diabetic wounds from 16.4 ± 4 to 12.1 ± 3 with NT and 10.9 ± 3 with SP, while increasing M2 macrophages from 11.6 ± 4 to 18.1 ± 4 (NT) and 21.6 ± 5 (SP). SP exerted the most pronounced immunomodulatory effect, normalizing the M1/M2 ratio and reducing the number of CD3⁺ T cells (12.4 ± 3) and neutrophils (12.1 ± 3) to levels comparable to non-diabetic skin. Concurrently, SP treatment induced substantial microbiota remodeling in diabetic wounds, reducing Staphylococcus by circa 34%, Aerococcaceae_unclassified by 6.8%, Aerococcus by 1.5% and nearly eliminating Weissella. These reductions were accompanied by an over twofold increase in commensal genera (Lactobacillus, Micrococcaceae_unclassified, Actinobacteria_unclassified), shifting the microbial community toward a more non-diabetic profile. Our findings suggest that NT and especially SP, enhance diabetic wound healing through dual local immunomodulation and microbiota restructuring.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia, I.P.
Programa de financiamento
CEEC INST 2018
Número da atribuição
CEECINST/00051/2018/CP1566/CT0004