Identification of Salmonella gallinarum virulence genes in a chicken infection model using PCR-based signature-tagged mutagenesis.
Salmonella gallinarum (SG) is a non-motile host-adapted salmonella that causes fowl typhoid, a severe systemic disease responsible for significant economic losses to the poultry industry worldwide. This study describes the application of a PCR-based signature-tagged mutagenesis system to identify in vivo-essential genes of SG. Ninety-six pools representing 1152 SG mutants were screened in a natural-host chicken infection model. Twenty presumptive attenuated mutants were identified and examined further. The identity of the disrupted gene in each mutant was determined by cloning of the DNA sequences adjacent to the transposon, followed by sequencing and comparison with the bacterial genome database. In vitro and in vivo competition indices were determined for each identified mutant and a total of 18 unique, attenuating gene disruptions were identified. These mutations represented six broad genomic classes: Salmonella pathogenicity island-1 (SPI-1), SPI-2, SPI-10, SPI-13, SPI-14 and non-SPI-encoded virulence genes. SPI-13 and SPI-14 are newly identified and designated in this study. Most of the genes identified in this study were not previously believed or known to play a role in the pathogenesis of SG infection in chickens. Each STM identified mutant showed competitiveness and/or virulence defects, confirmed by in vitro and in vivo assays, and challenge tests. This study should contribute to a better understanding of the pathogenic mechanisms involved in progression of disease caused by SG, and identification of novel live vaccine candidates and new potential antibiotic targets.
Shah
Devendra H
DH
Biosafety Research Institute and College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea.
Lee
Mi-Jin
MJ
Biosafety Research Institute and College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea.
Park
Jin-Ho
JH
Biosafety Research Institute and College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea.
Lee
John-Hwa
JH
Biosafety Research Institute and College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea.
Eo
Seong-Kug
SK
Biosafety Research Institute and College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea.
Kwon
Jung-Thek
JT
Halim Inc., 13-14 Euryang-Ri, Samgi-Myeon, Iksan 570-883, Korea.
Chae
Joon-Seok
JS
Biosafety Research Institute and College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea.
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002645:Chickens / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis; D004195:Disease Models, Animal; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012475:Salmonella / Q000235:genetics* / Q000378:metabolism; D012481:Salmonella Infections, Animal / Q000382:microbiology*; D014774:Virulence / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial