Identification of bioactive substances produced by Lactobacillus paracasei 28.4 for the control of oral candidiasis

• Grant number: 17/19219-3
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: April 01, 2018
• End date: July 31, 2020
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Juliana Campos Junqueira
• Grantee: Rodnei Dennis Rossoni
• Host Institution: Instituto de Ciência e Tecnologia (ICT). Universidade Estadual Paulista (UNESP). Campus de São José dos Campos. São José dos Campos , SP, Brazil
• Associated scholarship(s): 18/21239-5 - Galleria mellonella in antimicrobial drug discovery: bioactive substances extracted from probiotic against Candida albicans persisters, BE.EP.PD

Abstract

Currently, the treatment options for fungal infections, such as oral candidosis, present limitations due to the low availability of antifungal drugs and the emergence of drug resistant strains. The use of probiotics is considered promising because they have antimicrobial activity as well as immunomodulatory action, but its clinical application in immunocompromised patients is critical due to the possibility of bacteraemia caused by bacteria of the genus Lactobacillus. In this context, the isolation of the bioactive substances produced by Lactobacillus for the control of oral candidiasis becomes innovative and safe for hospitalized and immunocompromised patients. Therefore, the objective of this study will be to purify and identify the metabolites produced by L. paracasei 28.4 and to evaluate their in vitro effects on C. albicans virulence and its antimicrobial and immunological response in in vivo studies models. The metabolites of L. paracasei 28.4 culture will be extracted with ethyl acetate and subsequently fractionated and identified by High-performance liquid chromatography with diode array detection (CLAE-DAD). Initially, the crude extract and all fractions obtained from the supernatant will be evaluated for cytotoxicity in cell culture of human gingival fibroblasts (FMM-1), mouse macrophages (RAW 264.7) and human skin keratinocytes (HaCaT). Next, the crude extract and supernatant fractions of L. paracasei 28.4 will be tested on 30 clinical strains of C. albicans isolated from the oral cavity of patients with Human Immunodeficiency Virus (HIV) using the Minimum Inhibitory Concentration assay (MIC). The antifungal effect of the crude extract and supernatant fractions of L. paracasei 28.4 will be verified in two C. albicans strains fluconazole resistant using in vitro assays of biofilm formation and filamentation. Also in vitro, the immunomodulatory action of the crude extract and supernatant fractions of L. paracasei 28.4 will be investigated using Candida-activated RAW 264.7 cells for quantification of cytokines using fluorescently labeled beads. Next, the prophylactic and therapeutic effects of the crude extract and fractions of L. paracasei 28.4 supernatant will be investigated in the experimental model of Galleria mellonella by analysis of survival curve, hemocyte count, hemocyte cell characterization by flow cytometry, C. albicans counts of the fatty body and genes expression related to antimicrobial peptides (galiomycin, galleriomycin, cepropin D), phagocytosis (C8-Contig 1910) and the regulation of cytokines and cascade NF-kB (C7 Contig 15362) present on the immune response of this invertebrate model. Therefore, the fraction of L. paracasei 28.4 supernatant with better antifungal and immunomodulatory effect observed in the previous assays will be selected with the purpose of expanding and confirming such effects in the oral candidiasis in immunosuppressed mice. This bioactive substance will be applied on lesions induced by candidiasis in the tongue of animals. The development of oral candidiasis will be monitored recoverying of C. albicans from the oral cavity of the animals, macroscopic evaluation, histological analysis and gene expression of beta defensins 1 and 3. The results will be submitted to an exploratory analysis for selection of the most appropriate statistical test for each experiment in this study. The level of significance considered for each test will be 5%.

Scientific publications

(11)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

GERALDO, BARBARA M. C.; BATALHA, MARIANNA N.; MILHAN, NOALA V. M.; ROSSONI, RODNEI D.; SCORZONI, LILIANA; ANBINDER, ANA LIA. Heat-killed Lactobacillus reuteri and cell-free culture supernatant have similar effects to viable probiotics during interaction with Porphyromonas gingivalis. JOURNAL OF PERIODONTAL RESEARCH, NOV 2019. (16/06946-1, 15/13561-6, 17/19219-3)

ROSSONI, RODNEI DENNIS; RIBEIRO, FELIPE DE CAMARGO; SANTANA DOS SANTOS, HANNA FLAVIA; DOS SANTOS, JESSICA DIANE; OLIVEIRA, NICASSIA DE SOUSA; DOS SANTOS DUTRA, MARIGNES THEOTONIO; BIAZZI DE LAPENA, SIMONE APARECIDA; JUNQUEIRA, JULIANA CAMPOS. Galleria mellonella as an experimental model to study human oral pathogens. ARCHIVES OF ORAL BIOLOGY, v. 101, p. 13-22, MAY 2019. (17/19219-3, 16/25544-1)

PEREIRA, THAIS CRISTINE; DE BARROS, PATRICIA PIMENTEL; DE OLIVEIRA FUGISAKI, LUCIANA RUANO; ROSSONI, RODNEI DENNIS; RIBEIRO, FELIPE DE CAMARGO; DE MENEZES, RAQUEL TELES; JUNQUEIRA, JULIANA CAMPOS; SCORZONI, LILIANA. Recent Advances in the Use of Galleria mellonella Model to Study Immune Responses against Human Pathogens. JOURNAL OF FUNGI, v. 4, n. 4, DEC 2018. (17/19219-3, 16/25544-1, 17/02652-6)

GERALDO, BARBARA M. C.; BATALHA, MARIANNA N.; MILHAN, NOALA V. M.; ROSSONI, RODNEI D.; SCORZONI, LILIANA; ANBINDER, ANA LIA. Heat-killed Lactobacillus reuteri and cell-free culture supernatant have similar effects to viable probiotics during interaction with Porphyromonas gingivalis. JOURNAL OF PERIODONTAL RESEARCH, v. 55, n. 2, p. 6-pg., 2019-11-06. (15/13561-6, 16/06946-1, 17/19219-3)

ROSSONI, RODNEI DENNIS; DE BARROS, PATRICIA PIMENTEL; DAS CHAGAS LOPES, LUCAS ALEXANDRE; RIBEIRO, FELIPE CAMARGO; NAKATSUKA, TOSHIYUKI; KASABA, HIDETO; JUNQUEIRA, JULIANA CAMPOS. Effects of surface pre-reacted glass-ionomer (S-PRG) eluate on Candida spp.: antifungal activity, anti-biofilm properties, and protective effects on Galleria mellonella against C. albicans infection. BIOFOULING, NOV 2019. (18/21239-5, 17/02652-6, 17/19219-3, 19/05664-0)

DE BARROS, PATRICIA PIMENTEL; ROSSONI, RODNEI DENNIS; DE SOUZA, CHEYENNE MARCAL; SCORZONI, LILIANA; FENLEY, JULIANA DE CAMARGO; JUNQUEIRA, JULIANA CAMPOS. CandidaBiofilms: An Update on Developmental Mechanisms and Therapeutic Challenges. Mycopathologia, v. 185, n. 3, p. 415-424, JUN 2020. (19/05664-0, 17/19219-3, 17/02652-6)

ROSSONI, RODNEI DENNIS; DE BARROS, PATRICIA PIMENTEL; MENDONCA, IATA DO CARMO; MEDINA, REBECA PREVIATE; SIQUEIRA SILVA, DULCE HELENA; FUCHS, BETH BURGWYN; JUNQUEIRA, JULIANA CAMPOS; MYLONAKIS, ELEFTHERIOS. The Postbiotic Activity of Lactobacillus paracasei 28.4 Against Candida auris. FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, v. 10, AUG 4 2020. (18/21239-5, 17/19219-3)

RIBEIRO, F. C.; ROSSONI, R. D.; DE BARROS, P. P.; SANTOS, J. D.; FUGISAKI, L. R. O.; LEAO, M. P. V.; JUNQUEIRA, J. C.. Action mechanisms of probiotics on Candida spp. and candidiasis prevention: an update. Journal of Applied Microbiology, v. 129, n. 2, NOV 2019. (17/02652-6, 17/19219-3, 16/25544-1)

DE BARROS, PATRICIA PIMENTEL; ROSSONI, RODNEI DENNIS; RIBEIRO, FELIPE DE CAMARGO; SILVA, MICHELLE PENELUPPI; DE SOUZA, CHEYENNE MARCAL; CARDOSO JORGE, ANTONIO OLAVO; JUNQUEIRA, JULIANA CAMPOS. Two sporulated Bacillus enhance immunity in Galleria mellonella protecting against Candida albicans. Microbial Pathogenesis, v. 132, p. 335-342, JUL 2019. (17/19219-3, 17/02652-6)

ROSSONI, RODNEI DENNIS; VELLOSO, MARISOL DOS SANTOS; DE BARROS, PATRICIA PIMENTEL; DE ALVARENGA, JANAINA ARAUJO; DOS SANTOS, JESSICA DIANE; CHIPOLETTI DOS SANTOS PRADO, ANA CAROLINA; RIBEIRO, FELIPE DE CAMARGO; ANBINDER, ANA LIA; JUNQUEIRA, JULIANA CAMPOS. Inhibitory effect of probiotic Lactobacillus supernatants from the oral cavity on Streptococcus mutans biofilms. Microbial Pathogenesis, v. 123, p. 361-367, OCT 2018. (14/11841-9, 13/25181-8, 17/19219-3, 15/09770-9)

ROSSONI, RODNEI DENNIS; DE BARROS, PATRICIA PIMENTEL; DAS CHAGAS LOPES, LUCAS ALEXANDRE; RIBEIRO, FELIPE CAMARGO; NAKATSUKA, TOSHIYUKI; KASABA, HIDETO; JUNQUEIRA, JULIANA CAMPOS. Effects of surface pre-reacted glass-ionomer (S-PRG) eluate on Candida spp.: antifungal activity, anti-biofilm properties, and protective effects on Galleria mellonella against C. albicans infection. BIOFOULING, v. 35, n. 9, p. 10-pg., 2019-11-08. (17/02652-6, 17/19219-3, 18/21239-5, 19/05664-0)