Department of Molecular Microbiology and Immunology, L220, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
Infect Immun. 2011 Jan;79(1):360-8. doi: 10.1128/IAI.00873-10. Epub 2010 Nov 1.
Salmonella enterica serovar Typhimurium is an intracellular pathogen and a main cause of food-borne illness. In this study, a quantitative PCR (qPCR)-based competitive index (CI) method was developed to simultaneously compare the growth of multiple Salmonella strains. This method was applied to a mixture of 17 Salmonella mutants lacking regulator genes, and their survival ratios were compared based on expression of natural resistance-associated macrophage protein 1 (Nramp1). Nramp1, as a major host innate immune component, controls the intracellular replication of pathogens. Deletion strains containing unique DNA barcodes in place of regulator genes were mixed with the parental control, and the bacteria were inoculated into congenic mice differing only at Nramp1. Most of the deletion strains were outcompeted by wild-type bacteria in either mouse strain, and the lack of Nramp1 didn't increase the tested strain/parent control replication ratios. When the same collection of mutants was tested in congenic mouse-derived primary macrophages, a major Nramp1-expressing cell type, six strains (ΔhimD, ΔphoP/phoQ, ΔrpoE, ΔrpoS, ΔompR/envZ, and Δhfq strains) grew better in Nramp1(-/-) than in Nramp1(+/+) macrophages, suggesting that these six regulators may play roles in overcoming Nramp1-mediated bactericidal activity in primary macrophages. The discrepancy in survival of macrophages and that of mice suggests either that there are differences in macrophage populations or that other cell types expressing Nramp1 control Salmonella proliferation in the host. The method described allows competitive infection analysis to be carried out on complex mixtures of bacteria and provides high reproducibility from independent biological replicates.
肠炎沙门氏菌血清型鼠伤寒是一种细胞内病原体，也是食源性疾病的主要原因。在本研究中，开发了一种基于定量 PCR（qPCR）的竞争指数（CI）方法，用于同时比较多种沙门氏菌菌株的生长情况。该方法应用于缺乏调控基因的 17 种沙门氏菌突变体的混合物中，并基于天然抗性相关巨噬细胞蛋白 1（Nramp1）的表达来比较它们的存活率。Nramp1 作为主要的宿主先天免疫成分，控制病原体的细胞内复制。用含有独特 DNA 条码的缺失株取代调控基因，与亲本对照混合，并将细菌接种到仅在 Nramp1 上不同的同基因小鼠中。大多数缺失株在两种小鼠品系中均被野生型细菌竞争淘汰，而缺乏 Nramp1 并未增加测试菌株/亲本对照的复制比值。当同一批突变体在同基因小鼠衍生的原代巨噬细胞中进行测试时，Nramp1 是主要表达的细胞类型之一，六种菌株（ΔhimD、ΔphoP/phoQ、ΔrpoE、ΔrpoS、ΔompR/envZ 和 Δhfq 菌株）在 Nramp1(-/-)中的生长情况优于 Nramp1(+/+)巨噬细胞，表明这六种调控因子可能在克服原代巨噬细胞中 Nramp1 介导的杀菌活性方面发挥作用。巨噬细胞和小鼠的存活差异表明，巨噬细胞群体存在差异，或者其他表达 Nramp1 的细胞类型在宿主中控制沙门氏菌的增殖。所描述的方法允许对复杂的细菌混合物进行竞争感染分析，并从独立的生物学重复中提供高度可重复性。
