Genome-Wide Screen for Salmonella Genes Required for Long-Term Systemic Infection of the Mouse

Abstract

A microarray-based negative selection screen was performed to identify Salmonella enterica serovar Typhimurium (serovar Typhimurium) genes that contribute to long-term systemic infection in 129X1/SvJ (Nramp1^r) mice. A high-complexity transposon-mutagenized library was used to infect mice intraperitoneally, and the selective disappearance of mutants was monitored after 7, 14, 21, and 28 d postinfection. One hundred and eighteen genes were identified to contribute to serovar Typhimurium infection of the spleens of mice by 28 d postinfection. The negatively selected mutants represent many known aspects of Salmonella physiology and pathogenesis, although the majority of the identified genes are of putative or unknown function. Approximately 30% of the negatively selected genes correspond to horizontally acquired regions such as those within Salmonella pathogenicity islands (SPI 1–5), prophages (Gifsy-1 and −2 and remnant), and the pSLT virulence plasmid. In addition, mutations in genes responsible for outer membrane structure and remodeling, such as LPS- and PhoP-regulated and fimbrial genes, were also selected against. Competitive index experiments demonstrated that the secreted SPI2 effectors SseK2 and SseJ as well as the SPI4 locus are attenuated relative to wild-type bacteria during systemic infection. Interestingly, several SPI1-encoded type III secretion system effectors/translocases are required by serovar Typhimurium to establish and, unexpectedly, to persist systemically, challenging the present description of Salmonella pathogenesis. Moreover, we observed a progressive selection against serovar Typhimurium mutants based upon the duration of the infection, suggesting that different classes of genes may be required at distinct stages of infection. Overall, these data indicate that Salmonella long-term systemic infection in the mouse requires a diverse repertoire of virulence factors. This diversity of genes presumably reflects the fact that bacteria sequentially encounter a variety of host environments and that Salmonella has evolved to respond to these selective forces in a way that permits both the bacteria and the host to survive. Bacteria belonging to the genus Salmonella are capable of establishing a long-term systemic infection in a variety of hosts, including humans, rodents, fowl, and cattle. The ability of Salmonella to subvert the active immune response of the host represents millions of years of co-evolution and is the result of specialized virulence factors that promote long-term infection. This study describes a microarray-based genome-wide screen designed to identify genes required by Salmonella enterica serovar Typhimurium (serovar Typhimurium) to persist and replicate in the spleen and liver of mice for up to 28 days. The results demonstrate that serovar Typhimurium utilizes a diverse repertoire of virulence factors, including both known and novel virulence genes, to establish infection and to persist in the host. The authors' data further established a previously unappreciated role for Salmonella pathogenicity island 1 in maintaining a persistent systemic infection. In addition, a progressive selection against serovar Typhimurium mutants based upon the duration of the infection was observed, suggesting that certain classes of genes are required at specific times during infection and providing a foundation to further dissect Salmonella pathogenesis into distinct temporal phases.