Table_4_Transcriptome Analyses of Chicken Primary Macrophages Infected With Attenuated Salmonella Typhimurium Mutants.XLSX

Salmonella enterica is one of the most common foodborne illnesses in the United States and worldwide, with nearly one-third of the cases attributed to contaminated eggs and poultry products. Vaccination has proven to be an effective strategy to reduce Salmonella load in poultry. The Salmonella Typhimurium Δcrp-cya (MeganVac1) strain is the most commonly used vaccine in the United States; however, the mechanisms of virulence attenuation and host response to this vaccine strain are poorly understood. Here, we profiled the invasion and intracellular survival phenotypes of Δcrp-cya and its derivatives (lacking key genes required for intra-macrophage survival) in HD11 macrophages and the transcriptome response in primary chicken macrophages using RNA-seq. Compared to the parent strain UK1, all the mutant strains were highly defective in metabolizing carbon sources related to the TCA cycle and had greater doubling times in macrophage-simulating conditions. Compared to UK1, the majority of the mutants were attenuated for invasion and intra-macrophage survival. Compared to Δcrp-cya, while derivatives lacking phoPQ, ompR-envZ, feoABC and sifA were highly attenuated for invasion and intracellular survival within macrophages, derivatives lacking ssrAB, SPI13, SPI2, mgtRBC, sitABCD, sopF, sseJ and sspH2 showed increased ability to invade and survive within macrophages. Transcriptome analyses of macrophages infected with UK1, Δcrp-cya and its derivatives lacking phoPQ, sifA and sopF demonstrated that, compared to uninfected macrophages, 138, 148, 153, 155 and 142 genes were differentially expressed in these strains, respectively. Similar changes in gene expression were observed in macrophages infected with these strains; the upregulated genes belonged to innate immune response and host defense and the downregulated genes belonged to various metabolic pathways. Together, these data provide novel insights on the relative phenotypes and early response of macrophages to the vaccine strain and its derivatives. The Δcrp-cya derivatives could facilitate development of next-generation vaccines with improved safety.

沙门氏菌（Salmonella enterica）是美国及全球范围内最常见的食源性病原体之一，其中近三分之一病例归因于受污染的鸡蛋和家禽产品。疫苗接种已被证实为降低家禽沙门氏菌载量的有效策略。沙门氏菌鼠伤寒亚种Δcrp-cya（MeganVac1）菌株是美国最常用的疫苗菌株；然而，关于其致病力减弱机制和宿主对这种疫苗菌株的免疫反应仍知之甚少。在本研究中，我们利用RNA测序技术，对Δcrp-cya及其衍生菌株（缺乏在巨噬细胞内生存所需的关键基因）在HD11巨噬细胞中的侵袭和细胞内存活表型，以及原代鸡巨噬细胞中的转录组反应进行了分析。与亲本菌株UK1相比，所有突变菌株在巨噬细胞模拟条件下对与三羧酸循环相关的碳源的代谢能力均显著受损，且其倍增时间显著延长。与UK1相比，大多数突变菌株在侵袭和巨噬细胞内生存方面均表现出减弱。与Δcrp-cya相比，缺乏phoPQ、ompR-envZ、feoABC和sifA的衍生菌株在巨噬细胞内的侵袭和细胞内存活方面表现出高度减弱，而缺乏ssrAB、SPI13、SPI2、mgtRBC、sitABCD、sopF、sseJ和sspH2的衍生菌株则显示出在巨噬细胞内侵袭和存活能力增强。对感染UK1、Δcrp-cya及其缺乏phoPQ、sifA和sopF的衍生菌株的巨噬细胞进行的转录组分析表明，与未感染巨噬细胞相比，这些菌株分别有138、148、153、155和142个基因表达发生差异。在这些菌株感染巨噬细胞的过程中，也观察到了类似的基因表达变化；上调的基因主要涉及先天免疫反应和宿主防御，而下调的基因则涉及多种代谢途径。综上所述，这些数据为疫苗菌株及其衍生菌株的相对表型和巨噬细胞的早期反应提供了新的见解。Δcrp-cya的衍生菌株可能有助于开发安全性更优的下一代疫苗。