Analysis of Pools of Targeted Salmonella Deletion Mutants Identifies Novel Genes Affecting Fitness during Competitive Infection in Mice

Abstract

Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS).

Dual RNA-seq of pathogen and host

“…In contrast, several further host-induced sRNAs identified in the present work did not produce a positive hit in global transposon screens for Salmonella virulence genes (Barquist et al, 2013;Chaudhuri et al, 2013;Santiviago et al, 2009). This means that the mere intracellular induction of a given sRNA cannot be considered strict evidence for it giving rise to a virulence phenotype when deleted.…”

Signature tagged mutagenesis in the functional genetic analysis of gastrointestinal pathogens

“…The P T7 was added to the cassette inserted during mutagenesis and positioned to produce a gene-specific transcript from the genomic sequence adjacent to the insertion. 86 In total there was a pool of over 1,000 gene-targeted Salmonella mutants injected into BALB/C mice and the spleen analyzed 2 d post infection. A threshold of a 2-fold change was designated as having decreased fitness compared with input pool.…”

“…A threshold of a 2-fold change was designated as having decreased fitness compared with input pool. 86 In total 120 mutants were shown to have decreased fitness, among these were mutants associated with TTSS encoded by SPI-2 and associated effector genes, and genes for cell wall biosynthesis all known to play a role during infection. 86 There were 15 mutants previously unknown to have a role during systemic infection in BALB/c mice identified using this method.…”

“…These included all five candidate sRNA, a supernumerary tRNA (LeuX) and nine protein coding genes. 86 Mutations in leuX, encoding tRNA-Leu, were previously known to reduce the expression of Type I fimbriae and reduce bladder epithelial invasion and intracellular proliferation of Uropathogenic E. coli. 87,88 This was the first time a leuX mutant was demonstrated to have a role during Salmonella infection.…”

“…87,88 This was the first time a leuX mutant was demonstrated to have a role during Salmonella infection. 86 One advantage of this method is being able to identify mutants that may enhance survival during infection, exemplified by a mutant in oxyS. OxyS is a member of the OxyR regulon expressed during oxidative stress 86 and encodes a sRNA that regulates over 40 genes in E. coli.…”