Sequencing, expression, and functional analyses support the candidacy of Ncf2 in susceptibility to Salmonella typhimurium infection in wild-derived mice.
A recessive Salmonella Typhimurium susceptibility locus (immunity to Typhimurium (Ity3) was reported previously on distal mouse chromosome 1 using a cross between C57BL/6J and wild-derived MOLF/Ei mice. This quantitative trait locus is located in a genomic region spanning 84 Mb, rich in candidate genes for which a role in host resistance to Salmonella infection is either known or can be envisioned. In this study, we report the evaluation of neutrophil cytosolic factor 2 (Ncf2) as a candidate Salmonella susceptibility gene for Ity3. Ncf2 encodes p67phox, a subunit of the multiprotein enzyme complex NADPH oxidase, known to be responsible for the generation of superoxides. Congenic mice carrying the Ity3 region from MOLF/Ei, B6.MOLF-Ity/Ity3 were more susceptible to infection compared with control mice heterozygous at Ity3, B6.MOLF-Ity/Ity3(MOLF/B6), confirming the existence of a recessive Salmonella susceptibility locus on distal chromosome 1. Spleen Ncf2 expression levels were lower in infected congenic mice homozygous for the MOLF/Ei allele at Ity3 compared with mice heterozygous at Ity3. C57BL/6J and MOLF/Ei Ncf2 sequence comparisons revealed one nonconservative amino acid change (R394Q) in the functional and highly conserved Phox and Bem1 domain of the protein. Functional analysis revealed that the MOLF/Ei allele had reduced PMA- and Salmonella-induced superoxide induction as compared with their wild-type counterparts ex vivo. The R394Q substitution seems to occur on an amino acid involved in electrostatic interactions with p40phox, crucial in its activation. Moreover, a human mutation in the corresponding R395W, resulting in chronic granulatomous disease, is known to lead to reduced superoxide levels. These results support the candidacy of Ncf2 as the gene underlying Ity3.
Sancho-Shimizu
Vanessa
V
Department of Human Genetics, McGill University, Montreal, Quebec, Canada.
Malo
Danielle
D
D000818:Animals; D027682:Cation Transport Proteins / Q000235:genetics; D005260:Female; D005819:Genetic Markers; D020022:Genetic Predisposition to Disease; D008297:Male; D051379:Mice; D020297:Mice, Congenic; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D010641:Phenotype; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D040641:Quantitative Trait Loci; D012481:Salmonella Infections, Animal / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology; D012486:Salmonella typhimurium / Q000276:immunology*; D017422:Sequence Analysis, DNA; D000099173:Natural Resistance-associated Macrophage Protein 1
D027682:Cation Transport Proteins; D005819:Genetic Markers; D019255:NADPH Oxidases; D010750:Phosphoproteins; D000099173:Natural Resistance-associated Macrophage Protein 1; C079789:neutrophil cytosol factor 67K; C511584:Ncf2 protein, mouse