Amino acid and C1 carbon source metabolism in the Salmonella typhimurium phagosome: Role of amino acids and glycine conversion in intracellular growth

我们构建了一组独特的鼠伤寒沙门氏菌突变体，它们在氨基酸生物合成和摄取、肽和寡肽摄取以及氨基酸分解代谢方面存在缺陷，并测定了它们在肠上皮细胞中的细胞内生长特征。我们的研究表明，胞内沙门氏菌不仅可以获得宿主衍生的氨基酸库，而且还能够在缺乏生物合成和摄取系统的情况下使用肽来满足氨基酸需求。一个例外是glyA突变的菌株，编码丝氨酸羟甲基转移酶（SHMT），负责丝氨酸到甘氨酸的可逆转化，显示出严重的细胞内生长缺陷。在培养中，甘氨酸可以纠正glyA突变体在葡萄糖极少培养基中的生长缺陷；然而，在宿主细胞中，glyA突变体表现出严重的生长缺陷。此外，我们发现沙门氏菌aroA突变株的衰减不是由于芳香氨基酸的生物合成缺陷，而是可能由于对氨基苯甲酸的限制，对氨基苯甲酸也来源于相同的前体，chorismic酸。所有三种芳香氨基酸生物合成缺陷突变体在细胞内没有生长缺陷，而pabA、pabB和/或pabC突变体在宿主细胞内表现出严重的生长缺陷。最后，一个以前未被发现的基因ltaA，通过调节苏氨酸进入甘氨酸或支链氨基酸途径的流动，在维持细胞内生长中发挥核心作用。结果表明，胞内沙门氏菌优先从宿主那里获取氨基酸以最大化胞内生长，并且高度依赖于参与C1碳单元生物合成的氨基酸库的维持。

We constructed a unique collection of mutants of Salmonella typhimurium defective in amino acid biosynthesis and uptake, peptide and oligopeptide uptake and amino acid catabolism and determined their intracellular growth characteristics in intestinal epithelial cells. Our studies showed that intracellular Salmonella not only have access to host-derived amino acid pools but are also capable of using peptides to satisfy amino acid requirements in the absence of biosynthetic and uptake systems. One exception is a strain with a mutation in glyA, encoding serine hydroxymethyltransferase (SHMT), responsible for the reversible conversion of serine to glycine, which shows a severe intracellular growth defect. In culture, glycine can correct the growth defect of a glyA mutant in glucose minimal medium; however, within host cells, a glyA mutant shows a severe growth defect. In addition, we found that the attenuation of aroA mutant strains of Salmonella is not due to defects in aromatic amino acid biosynthesis, but likely limitations in para-aminobenzoic acid, also derived from the same precursor, chorismic acid. Mutants defective in biosynthesis of all three aromatic amino acids show no intracellular growth defects, whereas mutants in pabA, pabB and/or pabC show severe growth defects within host cells. Finally, a previously uncharacterized gene, ltaA, was found to play a central role in maintaining intracellular growth by regulating the flow of threonine into either the glycine or branched chain amino acid pathways. The results indicate that intracellular Salmonella preferentially acquires amino acids from the host to maximize intracellular growth, and shows a high dependence on maintenance of amino acid pools involved in C1 carbon unit biosynthesis.