Genetic dissection of the ity3 locus identifies a role for ncf2 co-expression modules and suggests selp as a candidate gene underlying the ity3.2 locus

Abstract

Typhoid fever and salmonellosis, which are caused by Salmonella typhi and typhimurium, respectively, are responsible for significant morbidity and mortality in both developed and developing countries. We model typhoid fever using mice infected with Salmonella typhimurium, which results in a systemic disease, whereby the outcome of infection is variable in different inbred strains of mice. This model recapitulates several clinical aspects of the human disease and allows the study of the host response to Salmonella typhimurium infection in vivo. Previous work in our laboratory has identified three loci (Ity, Ity2, and Ity3) in the wild-derived MOLF/Ei mice influencing survival after infection with Salmonella typhimurium. Fine mapping of the Ity3 locus indicated that two sub-loci contribute collectively to the susceptibility of B6.MOLF-Ity/Ity3 congenic mice to Salmonella infection. In the current paper, we provided further evidence supporting a role for Ncf2 (neutrophil cytosolic factor 2 a subunit of NADPH oxidase) as the gene underlying the Ity3.1 sub-locus. Gene expression profiling indicated that the Ity3.1 sub-locus defined a global gene expression signature with networks articulated around Ncf2. Furthermore, based on differential expression and complementation analysis using Selp (selectin-P) knock-out mice, Selp was identified as a strong candidate gene for the Ity3.2 sub-locus.

Keywords: Ity3; Ncf2; Salmonella; innate immunity; murine model for typhoid; selectin P.

Figure 1

Figure illustrating the two approaches used to study the changes in gene expression during infection of congenic and sub-congenic mice. (A) A representation of an initial approach used for studying the change in gene expression over time. The gene expression at day 3 post-infection was compared to day 0, and the final number of genes differentially regulated is stated on the right. (B) The second approach used, where gene expression in each strain was compared to the control Ity strain, at both day 0 and day 3. The final number of genes within each category is shown at the bottom of the panel.

Figure 2

Pathway and process analyses of genes differentially regulated in the spleen of Ity, Ity3, Ity3.RecG, and Ity3.RecN mice during infection. Gene ontology classifications generated by clustering the genes that are differentially regulated in each strain upon infection. (A) Gene ontology (GO) molecular pathways and (B) Gene ontology (GO) processes (C) pathways (D) Process networks that are enriched in the four mouse strains. The sub-congenic strain Ity3.RecG shows a lower −log(p-value) for chemokine and cytokine receptor activity within the GO molecular pathways as well as lower −log(p-values) for response to bacteria in the GO processes. This is consistent with previous data suggesting that the Ity3.RecG mice have a diminished inflammatory response following infection compared to parental strains (16).

Figure 3

Genes that are under the control of the Ity3.1 locus. Sample box plots of the gene lists provided in Table S2 in Supplementary Material are shown. (A) Represents genes, which do not show any changes in expression during infection and show a similar expression pattern in Ity3 and Ity3.RecG. (B,C) Represents a sample box plot of gene expression, which show a similar regulation pattern in Ity3 and Ity3.RecG. (D–G) Also illustrate genes within Tables S2D,G in Supplementary Material, which show similar expression patterns in Ity3 and Ity3.RecG, but different from Ity and Ity3.RecN. These genes are likely under the control of the Ity3.1 locus. Csrp1 (cysteine and glycine-rich protein 1), Chi3l1 (chitinase-like 1), (Plekhm3) Pleckstrin homology domain containing, family M, member 3), Sdsl (serine dehydratase-like), Cd59a (CD59a antigen), Lpxn (leupaxin), Asprv1 (aspartic peptidase, retroviral-like 1).

Figure 4

Genes that are differentially expressed in all susceptible and intermediate strains are under the influence of Ity3.1 sub-locus. Box plots of the expression pattern of two genes (A) Tor3a (torsin family 3, member A) and (B) Fam20b (family with sequence similarity 20, member B) are shown as examples to illustrate the expression pattern seen in the gene list provided in Table S2I in Supplementary Material. This list of genes show a similar expression in Ity3, Ity3.RecG, and Ity3.RecN and highlights the complex nature of the Ity3 locus as there are multiple genes in which expression is influenced by the combination of the two sub-loci. The genes that show a similar expression pattern in Ity3, Ity3.RecG, and Ity3.RecN were studied using GeneMania and the results are shown (C,D), with the query genes being highlighted in black. Genes, which are known to be co-expressed, co-localized, have shared domains or predicted interactions with the list of genes in Table S2I in Supplementary Material are shown. The functional categories, which are enriched within this gene list, are inflammatory response, angiogenesis, and integrin mediated signaling pathways shown in yellow, blue, and red, respectively. Genes that were not differentially expressed but important in these pathways are shown in gray. (D) Co-expression of query genes, as well as other genes within these pathways, with Ncf2 is shown.

Figure 5

Genes under the influence of Ity3.2. List of genes showing a similar expression pattern in Ity3 and Ity3.RecN, and different from Ity and Ity3.RecG. Ints7 (integrator complex subunit 7), F5 (coagulation factor V), Pbx1 (pre B cell leukemia homeobox 1), Cacybp (calcyclin binding protein), Nenf (neuron derived neurotrophic factor), Angel2 [angel homolog 2 (Drosophila)], Cox6a2 (cytochrome c oxidase subunit VIa polypeptide 2), Selp (selectin, platelet), Vamp4 (vesicle-associated membrane protein 4), Sccpdh [saccharopine dehydrogenase (putative)], Ctsw (cathepsin W), Fh1 (fumarate hydratase 1).

Figure 6

Allelic complementation between Ity3 congenic mouse and Selp deficient mice. In order to assess the impact of the MOLF/Ei Selp locus, Selp^−/− mice were crossed with Ity and Ity3 mice to generate mice carrying the knock-out allele complemented by the MOLF/Ei allele at Selp or C57BL/6J allele at the Ity3 locus. (A) Survival curves of the congenic Ity (n = 7), Ity3 (n = 7), knock-out Selp^−/− (n = 21), and Selp^−/MOLF (n = 36), Selp^−/B6 (n = 56) mice after infection with Salmonella typhimurium. The Selp^−/− and Selp^−/MOLF show a similar curve after infection with Salmonella typhimurium, both of which are more susceptible than the control Ity congenic mice and Selp^−/B6, but more resistant than the mice carrying the entire Ity3 locus (Ity3). (B) Bacterial burden in the spleen of congenic Ity (n = 8) and Ity3 (n = 6), sub-congenic Ity3.RecG (n = 8), and Ity3.RecN (n = 8) and Selp^−/− (n = 6) and compound heterozygous (n = 7 for Selp^−/MOLF and n = 10 for Selp^−/B6 mice after infection).