A Yeast VPS Screening to Identify Brucella Proteins that Interfere with Membrane Trafficking

MARÍA EUGENIA CORTINA; MARCHESINI, M. I.; GUIDOLIN L. S.; GIMENEZ A.; COMERCI D. J; CIOCCHINI AE

Rosario

Congreso; L Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Brucella deliver effector proteins into host cells to interfere with cellular processes and promote infection. Identification and characterization of such proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. In this work, a pathogen effector protein screening in yeast was carried out identifying three B. abortus DNA fragments that produced vacuole protein sorting (VPS) defects in yeast. Cloning of the complete genes codified by these fragments independently demonstrated that htpX was responsible for the VPS defects in yeast. HtpX is a zinc metalloprotease homologue to S. cerevisiae STE24 involved in NH2- and COOH-terminal CAAX processing events of a-factor maturation. An htpX deletion mutant in B. abortus was constructed showing a reduce virulence in mice in the chronic infection phase. Overexpression of HtpX in HeLa cells demonstrated that HtpX target the endoplasmic reticulum. Additionally, expression of a Myc-tagged HtpX protein in HEK293T cells led to a significant decrease in the secretion of the reporter protein embryonic alkaline phosphatase indicating that HtpX, as in yeast, also interferes with the host secretory pathway. The use of the VPS yeast screening allowed us to identify HtpX as a potential new Brucella effector protein that interferes with host cell membrane trafficking.