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Overexpression of the leptospiral lipoprotein LIC13259 in different avirulent Leptospira strains, and evaluation of their growth fitness in DMCs and immune-protective properties in mice

Grant number: 21/09160-7
Support Opportunities:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): December 13, 2021
Effective date (End): June 12, 2022
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal Investigator:Ana Lucia Tabet Oller do Nascimento
Grantee:Maria Fernanda Cavenague Pereira
Supervisor: Melissa J. Caimano
Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
Research place: University of Connecticut (UCONN), United States  
Associated to the scholarship:18/08131-0 - Heterologous expression of the gene LIC13259 in the saprophyte Leptospira biflexa and their acquisition capacity to evade the human immune system, BP.DR

Abstract

Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira. Different mammal species, particularly rodents, act as asymptomatic carriers, shedding large number of leptospires in urine. Humans are incidental hosts and acquire the spirochetes from direct or indirect contact with contaminated urine of these animals. The availability of the whole-genome sequences for different Leptospira spp. allowed the identification of putative surface exposed proteins, potentially involved in bacterial pathogenesis. Recently, we described the leptospiral protein LIC13259 as a surface lipoprotein that contribute to adhesion, colonization and immune evasion, interacting with host components, such as laminin, plasminogen, vitronectin and the complement components C7, C8 and C9 and inhibits the membrane attack complex (MAC). In the recent years, molecular tools for genetic manipulation of Leptospira spp. have expanded considerably, helping to understand the functions of several leptospiral proteins. Heterologous protein expression in the surrogate L. biflexa is evolving as a useful approach to validate biological activity of leptospiral. This saprophyte Leptospira species has been employed as a model for genetic manipulation and gain-of-function analysis. We analyzed the gain-of-function approach by genetically fusing the lic13259 gene with three different promoters to overexpress LIC13259 in L. biflexa serovar Patoc. However, several difficulties were found during expression of the heterologous protein and translocation into the surface of L. biflexa. Therefore, in this project, we will take advantage of two attenuated LPS mutant strains of L. interogans serovar Manilae available in Dr. Caimano's lab. The strains M895 and M1352, which express a defective LPS will be used to overexpress LIC13259 and validate its function and subcellular localization. Currently, Golden Syrian Hamsters are the most commonly used animal model for virulence test of Leptospira isolates and cultures, investigate aspects of pathogenesis, screen mutant strains for virulence and evaluate vaccine candidates. The C3H/HeJ mice strain is a tlr4-/- KO strain that is more susceptible to leptospiral infection than its wild-type parent strain. Collaborators at UConn Health have developed a C3H/HeJ mice model for leptospiral chronic colonization in which the animals are consistently infected and shed high number of leptospires in urine (up to 108/ml) for a long period of time. Thus, another major aim of this project is to establish the C3H/HeJ mice model of infection as a model for vaccine efficacy evaluation, where the major expected outcome will be preventing leptospirosis shedding in urine after vaccination. (AU)

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