title: Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA.
abstract: Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI)-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant) AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease) and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses.
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University Columbus, OH, USA.
authors.forename: Ky Van
authors.initials: KV
authors.lastname: Hoang
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University Columbus, OH, USA.
authors.forename: Carolyn G
authors.initials: CG
authors.lastname: Chen
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University Columbus, OH, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Koopman
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University Columbus, OH, USA.
authors.forename: Jasmine
authors.initials: J
authors.lastname: Moshiri
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University Columbus, OH, USA.
authors.forename: Haley E
authors.initials: HE
authors.lastname: Adcox
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University Columbus, OH, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Gunn
keywords: Francisella; acid phosphatase; protein secretion