Fonseca Maris V, Sauer John-Demian, Crepin Sebastien, Byrne Brenda, Swanson Michele S
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
Infect Immun. 2014 Feb;82(2):720-30. doi: 10.1128/IAI.01043-13. Epub 2013 Dec 2.
The phagosomal transporter (Pht) family of the major facilitator superfamily (MFS) is encoded by phylogenetically related intracellular gammaproteobacteria, including the opportunistic pathogen Legionella pneumophila. The location of the pht genes between the putative thymidine kinase (tdk) and phosphopentomutase (deoB) genes suggested that the phtC and phtD loci contribute to thymidine salvage in L. pneumophila. Indeed, a phtC(+) allele in trans restored pyrimidine uptake to an Escherichia coli mutant that lacked all known nucleoside transporters, whereas a phtD(+) allele did not. The results of phenotypic analyses of L. pneumophila strains lacking phtC or phtD strongly indicate that L. pneumophila requires PhtC and PhtD function under conditions where sustained dTMP synthesis is compromised. First, in broth cultures that mimicked thymidine limitation or starvation, L. pneumophila exhibited a marked requirement for PhtC function. Conversely, mutation of phtD conferred a survival advantage. Second, in medium that lacked thymidine, multicopy phtC(+) or phtD(+) alleles enhanced the survival of L. pneumophila thymidylate synthase (thyA)-deficient strains, which cannot synthesize dTMP endogenously. Third, under conditions in which transport of the pyrimidine nucleoside analog 5-fluorodeoxyuridine (FUdR) would inhibit growth, PhtC and PhtD conferred a growth advantage to L. pneumophila thyA(+) strains. Finally, when cultured in macrophages, L. pneumophila required the phtC-phtD locus to replicate. Accordingly, we propose that PhtC and PhtD contribute to protect L. pneumophila from dTMP starvation during its intracellular life cycle.
主要易化子超家族（MFS）的吞噬体转运蛋白（Pht）家族由系统发育相关的胞内γ-变形菌编码，包括机会致病菌嗜肺军团菌。pht基因位于假定的胸苷激酶（tdk）和磷酸戊糖变位酶（deoB）基因之间，这表明phtC和phtD位点有助于嗜肺军团菌的胸苷补救。实际上，反式的phtC(+)等位基因可恢复缺乏所有已知核苷转运蛋白的大肠杆菌突变体的嘧啶摄取，而phtD(+)等位基因则不能。对缺乏phtC或phtD的嗜肺军团菌菌株进行的表型分析结果强烈表明，在持续的dTMP合成受损的条件下，嗜肺军团菌需要PhtC和PhtD的功能。首先，在模拟胸苷限制或饥饿的肉汤培养中，嗜肺军团菌对PhtC功能有明显需求。相反，phtD突变赋予了生存优势。其次，在缺乏胸苷的培养基中，多拷贝的phtC(+)或phtD(+)等位基因可提高嗜肺军团菌胸苷酸合成酶（thyA）缺陷菌株的存活率，这些菌株无法内源性合成dTMP。第三，在嘧啶核苷类似物5-氟脱氧尿苷（FUdR）的转运抑制生长的条件下，PhtC和PhtD赋予嗜肺军团菌thyA(+)菌株生长优势。最后，当在巨噬细胞中培养时，嗜肺军团菌需要phtC-phtD位点进行复制。因此我们提出，PhtC和PhtD有助于保护嗜肺军团菌在其细胞内生命周期中免受dTMP饥饿的影响。
