Rickettsia typhi possesses phospholipase A2 enzymes that are involved in infection of host cells.

The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA2 activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.

Functional characterization of a phospholipase A(2) homolog from Rickettsia typhi.

Phospholipase A(2) (PLA(2)) has long been proposed to be involved in rickettsial entry into host cells, escape from the phagosome to evade destruction by lysosomal exposure, and lysis of the host cells. However, the corresponding rickettsial gene(s) encoding a protein with PLA(2) activity has not been identified or functionally characterized. Here, we report that the Rickettsia typhi genome possesses two genes encoding patatin-like PLA(2) proteins, RT0590 and RT0522. Sequence analysis of RT0522 and RT0590 reveals the presence of the conserved motifs essential for PLA(2) activity. Transcriptional analysis indicates that RT0522, but not RT0590, is transcribed at all stages of intracellular growth of R. typhi in Vero cells. The differential gene expression pattern of RT0522 at various stages of growth suggests its potential role during R. typhi infection of host cells. In silico, RT0522 is predicted to be noncytoplasmic and its gene does not encode a recognizable signal peptide sequence. However, our data indicate that RT0522 is secreted into the host cytoplasm. In addition, we observe that RT0522 protein expression is cytotoxic to both yeast and Vero cells. Importantly, we demonstrate that recombinant RT0522 possesses phospholipase A activity that requires a eukaryotic host cofactor for activation. Both cytotoxicity and phospholipase A activity associated with RT0522 were reduced by PLA(2) inhibitors. Site-directed mutagenesis of predicted catalytic Ser/Asp residues of RT0522 also eliminates cytotoxicity and phospholipase A activity. To our knowledge, RT0522 is the first protein identified from Rickettsia typhi with functional phospholipase A activity.

Rickettsial metK-encoded methionine adenosyltransferase expression in an Escherichia coli metK deletion strain.

The obligate intracellular bacterium Rickettsia prowazekii has recently been shown to transport the essential metabolite S-adenosylmethionine (SAM). The existence of such a transporter would suggest that the metK gene, coding for the enzyme that synthesizes SAM, is unnecessary for rickettsial growth. Genome sequencing has revealed that this is the case for the metK genes of the spotted fever group and the Madrid E strain of R. prowazekii, which contain recognizable inactivating mutations. However, several strains of the typhus group rickettsiae possess metK genes lacking obvious mutations. In order to determine if these genes code for a product that retains MAT function, an Escherichia coli metK deletion mutant was constructed in which individual rickettsial metK genes were tested for the ability to complement the methionine adenosyltransferase deficiency. Both the R. prowazekii Breinl and R. typhi Wilmington metK genes complemented at a level comparable to that of an E. coli metK control, demonstrating that the typhus group rickettsiae have the capability of synthesizing as well as transporting SAM. However, the appearance of mutations that affect the function of the metK gene products (a stop codon in the Madrid E strain and a 6-bp deletion in the Breinl strain) provides experimental support for the hypothesis that these typhus group genes, like the more degenerate spotted fever group orthologs, are in the process of gene degradation.

Molecular and functional analysis of the lepB gene, encoding a type I signal peptidase from Rickettsia rickettsii and Rickettsia typhi.

The type I signal peptidase lepB genes from Rickettsia rickettsii and Rickettsia typhi, the etiologic agents of Rocky Mountain spotted fever and murine typhus, respectively, were cloned and characterized. Sequence analysis of the cloned lepB genes from R. rickettsii and R. typhi shows open reading frames of 801 and 795 nucleotides, respectively. Alignment analysis of the deduced amino acid sequences reveals the presence of highly conserved motifs that are important for the catalytic activity of bacterial type I signal peptidase. Reverse transcription-PCR and Northern blot analysis demonstrated that the lepB gene of R. rickettsii is cotranscribed in a polycistronic message with the putative nuoF (encoding NADH dehydrogenase I chain F), secF (encoding protein export membrane protein), and rnc (encoding RNase III) genes in a secF-nuoF-lepB-rnc cluster. The cloned lepB genes from R. rickettsii and R. typhi have been demonstrated to possess signal peptidase I activity in Escherichia coli preprotein processing in vivo by complementation assay.

Subcellular localization of rickettsial invasion protein, InvA.

To understand further the molecular basis of rickettsial host cell invasion, Rickettsia prowazekii invasion gene homolog (invA) has been characterized. Our previous experiments have shown that InvA is an Ap5A pyrophosphatase, a member of the Nudix hydrolase family, which is up-regulated during the internalization, early growth phase, and exit steps during rickettsial mammalian cell infection. In addition to the molecular characterization, subcellular localization of InvA was investigated. InvA-specific antibodies were raised in mice and used for immunoelectron microscopy. The generated antibodies were shown to recognize InvA and by immunogold labeling showed InvA in the cytoplasm of rickettsiae. A cytoplasmic location for InvA would allow for a rapid response to any internal substance and efficient functioning in hydrolysis of toxic metabolic by-products that are accumulated in the rickettsial cytoplasm during host cell invasion. Protecting bacteria from a hazardous environment could enhance their viability and allow them to remain metabolically active, which is a necessary step for the rickettsial obligate intracellular lifestyle.

Identification of a natural cycle involving Rickettsia typhi infection of Monopsyllus sciurorum sciurorum fleas from the nests of the fat dormouse (Glis glis).

In a mountainous area in the Dinaric Beech-Fir Forest of southern Slovenia, summer nests of the European fat dormouse (Glis glis) were collected. From these dormouse nests, 180 Monopsyllus sciurorum sciurorum fleas were examined by polymerase chain reaction with primers for the Rickettsia citrate synthase gene. Samples from one nest yielded the expected 381 base pair DNA product. The origin of the DNA product was identified as Rickettsia typhi by AluI restriction fragment length polymorphism analysis. Inoculation of the triturated positive fleas into Vero cell culture resulted in the cultivation of a rickettsia which reacted with polyclonal and species-specific monoclonal antibodies for R. typhi. The widespread distribution of this sylvatic flea species in nearly all of Europe as well as in the Middle East and its presence on other mammalian and avian hosts suggests that R. typhi might exist in unrecognised enzootic cycles. Further investigations are needed to determine the extent of these cycles in Europe and the potential occurrence of human infections.

Enzymatic activities leading to pyrimidine nucleotide biosynthesis from cell-free extracts of Rickettsia typhi.

Cell-free extracts from Rickettsia typhi were examined for the presence or absence of pyrimidine phosphotransferase enzymes and compared with the enzymes of mouse L cells and Salmonella typhimurium. The organisms were grown in mouse L cells and in the yolk sacs of chicken embryos, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The enzymes for the reutilization of uridine and thymidine, uridine kinase (EC 2.7.1.48) and thymidine kinase (EC 2.7.1.21), were not detected in R. typhi extracts with the phosphate donors effective for control enzymes. The following enzyme activities were demonstrated in R. typhi: uridine-5'-monophosphate kinase (UMPK, EC 2.7.4.4), deoxythymidine-5'-monophosphate kinase (dTMPK, EC 2.7.4.9), and nucleosidediphosphate kinase (NDPK, EC 2.7.4.6). Physicochemical and enzymatic analyses demonstrated that the pyrimidine nucleotide kinases of R. typhi were not of host origin and that the source (yolk sac and mouse L cells) did not influence the relative enzymatic activities. The specific activities of UMPK and dTMPK were higher when the rickettsiae were harvested before embryo death, whereas NDPK levels were slightly decreased. The specific activities of UMPK, dTMPK, and NDPK were comparable to those of S. typhimurium, and consequently the rickettsiae have potential for the anabolism of monophosphates, as do the host-independent bacteria. These results suggest that R. typhi cannot utilize host uridine or thymidine pools directly but must rely on themonophosphorylated molecules of the host cell or must synthesize the monophosphates de novo.

Enzymatic activities of cell-free extracts of Rickettsia typhi.

Cell-free extracts of Rickettsia typhi were tested for activities of enzymes of the tricarboxylic acid cycle, of glutamate catabolism, and of glycolysis. The organisms were grown in the yolk sacs of chicken embryos, harvested shortly before the time of embryo death, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The following enzymatic activities were demonstrated: high levels of malate dehydrogenase (MDH), moderate levels of glutamate-oxaloacetate transaminase, glutamate, succinate, and isocitrate dehydrogenases, and citrate synthase, and low levels of glutamate-pyruvate transaminase. The specific activities of some of these enzymes were higher when the rickettsiae were harvested at a time of active proliferation, 3 to 4 days prior to embryo death. Rickettsial MDH was differentiated from host MDH by its migration pattern on polyacrylamide gel electrophoresis. The activities of MDH and two other dehydrogenases, demonstrable after the cells had been disrupted, were absent from purified, intact rickettsial preparations. No activity was detected for glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, lactate dehydrogenases, phosphoglucose isomerase, fructoaldolase, or pyruvate kinase. Our results suggest that extracts of R. typhi that contain demonstrable enzymes involved in the catabolism of glutamate and tricarboxylic acid cycle intermediates, unlike Coxiella burnetti, lack detectable glycolytic activity.