Study of the mechanisms implicated in the internalization of bacteriophage 933W on mammalian cells

• Grant number: 16/14528-5
• Support Opportunities: Research Grants - Visiting Researcher Grant - International
• Start date: February 01, 2017
• End date: March 31, 2017
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Luis Carlos de Souza Ferreira
• Grantee: Luis Carlos de Souza Ferreira
• Visiting researcher: Leticia Verónica Bentancor
• Visiting researcher institution: Universidad Nacional de Quilmes (UNQ), Argentina
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost and physical stability. However, is not clear the mechanism by which the bacteriophage is able to be introduced by eukaryotic cells. The genes encoding for Stx are located in the genome of lamboid bacteriophages. As a result of prophage induction, bacterial host cells lyse and release free phage particles that can infect other bacteria (Cornick et al., 2006). The capacity of mammalian cells to transcribe and translate stx2 genes was reported in vitro (Bentancor-Bilen et al., 2013) and in vivo (Bentancor et al., 2013). Bentancor and collaborators shown that macrophages incubated with a mutant version of bacteriophage933W (in which the encoding sequence for stx was replaced by gfp encoding sequence, fDTOX:GFP) were able to express GFP. During a recently collaboration (between Dr. Luis Carlos de Souza Ferreira and Dr. Leticia Bentancor), we used the non-pathogenic E. coli C600 strain carrying fDTOX:GFP, to track in vivo GFP expression using In Vivo Imaging System (IVIS). We detected fluorescence in liver, kidney, and intestine of mice infected with the recombinant E. coli strain after treatment with ciprofloxacin, which induces the lytic replication and release of bacteriophages. In addition, we showed that chitosan, a linear polysaccharide composed of D-glucosamine residues and with a number of commercial and biomedical uses, had strong anti-bacteriophage effects, as demonstrated at in vitro and in vivo conditions (Amorin et al., 2014). These results let us to continue working on the hypothesis that bacteriophage could have a relevant role on the development of Hemolytic Uremic Syndrome (HUS). On the other hand, the previous results using chitosan as anti-bacteriophage agent, also let us to continue working on the possibility to use the bacteriophage as new target against STEC infections. It is known that lambda bacteriophage is able to be internalized by eukaryotic cells and express protein under eukaryotic promoters (Lankes et al., 2007; Sapinoro et al., 2008; Piersanti et al., 2004; Merril et al., 1971). The mechanism by which the internalization occurs is unknown. There are some hypotheses but there are not confirmed. Analyzing the bacteriophage structure, we found RGD and RGE motifs on the tail fiber protein of bacteriophage 933W. Previous reports had shown that K562-avb3 cells (expressing avb3 integrin on their surface) could be responsible for the entrance of bacteriophage to the cells for the capacity of these integrin to recognize the amino acidic sequence of RGD and RGE. Based on that, we propose to study the relevance of the RGD and RGE motifs in the internalization of the bacteriphage 933W into the eukaryotic cells. (AU)