Faculty Collaboration Database

Modified needle-tip PcrV proteins reveal distinct phenotypes relevant to the control of type III secretion and intoxication by Pseudomonas aeruginosa. PLoS One 2011 Mar 29;6(3):e18356

Date

04/12/2011

Pubmed ID

21479247

Pubmed Central ID

PMC3066235

DOI

10.1371/journal.pone.0018356

Scopus ID

2-s2.0-79953167847 (requires institutional sign-in at Scopus site)   24 Citations

Abstract

The type III secretion system (T3SS) is employed to deliver effector proteins to the cytosol of eukaryotic hosts by multiple species of Gram-negative bacteria, including Pseudomonas aeruginosa. Translocation of effectors is dependent on the proteins encoded by the pcrGVHpopBD operon. These proteins form a T3S translocator complex, composed of a needle-tip complex (PcrV), translocons (PopB and PopD), and chaperones (PcrG and PcrH). PcrV mediates the folding and insertion of PopB/PopD in host plasmic membranes, where assembled translocons form a translocation channel. Assembly of this complex and delivery of effectors through this machinery is tightly controlled by PcrV, yet the multifunctional aspects of this molecule have not been defined. In addition, PcrV is a protective antigen for P. aeruginosa infection as is the ortholog, LcrV, for Yersinia. We constructed PcrV derivatives containing in-frame linker insertions and site-specific mutations. The expression of these derivatives was regulated by a T3S-specific promoter in a pcrV-null mutant of PA103. Nine derivatives disrupted the regulation of effector secretion and constitutively released an effector protein into growth medium. Three of these regulatory mutants, in which the linker was inserted in the N-terminal globular domain, were competent for the translocation of a cytotoxin, ExoU, into eukaryotic host cells. We also isolated strains expressing a delayed-toxicity phenotype, which secrete translocators slowly despite the normal level of effector secretion. Most of the cytotoxic translocation-competent strains retained the protective epitope of PcrV derivatives, and Mab166 was able to protect erythrocytes during infection with these strains. The use of defined PcrV derivatives possessing distinct phenotypes may lead to a better understanding of the functional aspects of T3 needle-tip proteins and the development of therapeutic agents or vaccines targeting T3SS-mediated intoxication.

Author List

Sato H, Hunt ML, Weiner JJ, Hansen AT, Frank DW

Author

Dara W. Frank PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Animals
Antigens, Bacterial
Bacterial Proteins
Bacterial Toxins
Cell Death
Epitopes
HeLa Cells
Humans
Models, Molecular
Molecular Sequence Data
Mutagenesis, Insertional
Mutation
Phenotype
Pore Forming Cytotoxic Proteins
Protein Structure, Tertiary
Pseudomonas Infections
Pseudomonas aeruginosa
Recombinant Fusion Proteins
Sheep
Structural Homology, Protein