Study of the Five Rickettsia prowazekii Proteins Annotated as ATP/ADP Translocases (Tlc): Only Tlc1 Transports ATP/ADP, While Tlc4 and Tlc5 Transport Other Ribonucleotides

Abstract

The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a K i similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins.Rickettsia prowazekii is the etiological agent of epidemic typhus in humans and is designated as a select agent based on its potential use as an agent of bioterrorism. R. prowazekii is an obligate intracellular bacterium and grows only within the cytoplasm of a eukaryotic host cell, unbounded by a host-derived membrane vesicle. As a consequence of growth in this nutrient-rich niche, R. prowazekii has evolved several unique transport systems specific for large, charged metabolites, such as UDP-glucose, S-adenosylmethionine, ATP, NAD, UMP, GMP, and AMP (11,12,45,52,54,55). The ability to transport substrates that are present in the host cell cytosol and rarely available in the extracellular milieu has likely contributed to the evolution of the small-sized rickettsial genome (834 open reading frames [ORFs]); many of the de novo biosynthetic pathways characteristic of free-living bacteria are no longer present in rickettsiae (8). The ATP/ADP translocase Tlc1 is the most thoroughly characterized of these transport systems (1-5, 13, 14, 16, 18, 27, 36, 50, 53, 56). Tlc1 is an obligateexchange transport system that catalyzes the equilibration of...

Nucleotide Parasitism by Simkania negevensis ( Chlamydiae )

“…This is consistent with secondarystructure predictions of biochemically characterized nucleotide transport proteins, for which between 11 and 12 transmembrane domains are recognized (7,26,43,63,67,75). The only nucleotide transporter whose topology has been investigated experimentally in some detail is the ATP/ADP translocase from Rickettsia prowazekii, showing 12 transmembrane domains (1,2,6).…”

“…Another possible explanation for why no substrate could be identified for SnNTT4 could be that, unlike other nucleotide transporters characterized in E. coli, SnNTT4 requires posttranslational modifications that the heterologous host cannot perform. It should be noted that two of the five putative nucleotide transporters from R. prowazekii also failed to take up any of the nucleoside mono-, di-, and triphosphates tested, although the presence of mRNA of those transporters suggested that they are required during intracellular growth (7).…”

“…In order to elucidate the biochemical properties of the putative nucleotide transporters from S. negevensis, we performed import studies with E. coli cells expressing the recombinant SnNTT1 to -4, an approach that often has been successfully applied to study the substrate specificities and transport modes of bacterial nucleotide transporters (7,15,26,27,45,63,64,67,75). Here, we initially tested whether the four SnNTTs were able to import ATP, which is accepted as a substrate of several other bacterial NTTs.…”

“…Among bacteria, NTT proteins catalyzing ATP/ADP exchange have been found in Chlamydiae and Rickettsiales, comprising major intracellular pathogens of humans (15,45,48,62,64,67), and recently also in the obligate intracellular veterinary pathogen Lawsonsia intracellularis belonging to the Deltaproteobacteria (63), in the plant pathogen "Candidatus Liberibacter asiaticus" belonging to the Rhizobiales (75), and in the obligate intracellular amoeba symbiont "Candidatus Amoebophilus asiaticus" belonging to the Bacteroidetes (65). In addition to ATP/ADP translocases, some obligate intracellular bacteria encode nucleotide transport isoforms for the import of nucleotides other than ATP or the cofactor NAD ϩ (7,22,26,27), thereby compensating for their inability to synthesize these metabolites de novo (22,33,66,76). Nucleotide transporters are thus important proteins for host cell interaction of obligate intracellular bacteria.…”

Chlamydia trachomatis Transports NAD via the Npt1 ATP/ADP Translocase

“…NTTs may be divided into three different classes, nucleotide antiporters (class I), proton-driven nucleotide symporters (class II), and NAD/ADP antiporters (class III) (7). NTTs appear to be restricted to a limited number of obligate intracellular bacteria other than the members of the order Chlamydiales, including members of the order Rickettsiales, Lawsonia spp., and "Candidatus Liberbacter spp.," as well as plastids, which all possess ATP/ADP translocases (class I NTTs), presumably to acquire energy from their host (8)(9)(10)(11)(12). To date, class II and III NTTs have been characterized only in Chlamydia trachomatis and P. amoebophila (7,10,13).…”