Silencing of VAMP3 expression does not affect Brucella melitensis infection in mouse macrophages

Abstract

It has been proposed that intracellular pathogens may interfere with expression or function of proteins that mediate vesicular traffic in order to survive inside cells. Brucella melitensis is an intracellular pathogen that evades phagosome-lysosome fusion, surviving in the so-called Brucella-containing vacuoles (BCV). Vesicle-associated membrane protein 3 (VAMP3) is a v-SNARE protein that promotes the exocytosis of the proinflammatory cytokine TNF at the phagocytic cup when docking to its cognate t-SNARE proteins syntaxin-4 and SNAP-23 at the plasma membrane. We determined the expression level of VAMP3 in J774.1 murine macrophages stimulated with B. melitensis lipopolysaccharide (LPS) and detected a transitory increase of VAMP3 mRNA expression at 30 min. A similar result was obtained when cells were incubated in the presence of LPS from Salmonella enterica serovar Minnesota (SeM). This increase of VAMP3 mRNA was also observed on infected cells with B. melitensis even after one hour. In contrast, infection with Salmonella enterica serovar Enteritidis (SeE) did not cause such increase, suggesting that membrane components other than LPS modulate VAMP3 expression differently. To determine the effect of VAMP3 inhibition on macrophages infection, the expression of VAMP3 in J774.A1 cells was silenced and then infected with wild-type B. melitensis. Although a slight decrease in the rate of recovery of surviving bacteria was observed between 12 h and 36 h post-infection with B. melitensis, this was not significant indicating that VAMP3 is not involved in Brucella survival.

Keywords: :

• Brucella
• intracellular
• pathogen
• phagocytosis
• macropinocytosis
• SNARE
• siRNA

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by PAPIIT IN-222907 and PAPIIT IN-212610, UNAM. The authors acknowledge Daniel Martínez for his helpful discussions, Raymundo Iturbe for his technical support, Ma. Teresa Ramírez for her support in the translation of this paper and Mrs. Francisca Muños for her administrative support.