The Rickettsia prowazekii Invasion Gene Homolog (invA) Encodes a Nudix Hydrolase Active on Adenosine (5′)-pentaphospho-(5′)-adenosine

The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA). The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family. To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli. The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives. The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (NpnN; n ≥ 5), a group of cellular signaling molecules. At optimal pH 8.5, the enzyme actively degrades adenosine (5′)-pentaphospho-(5′)-adenosine into ATP and ADP with a Km of 0.1 mm and kcat of 1.9 s−1. Guanosine (5′)-pentaphospho-(5′)-guanosine and adenosine-(5′)-hexaphospho (5′)-adenosine are also substrates. Similar to other Nudix hydrolases, InvA requires a divalent metal cation, Mg2+ or Zn2+, for optimal activity. These data suggest that the rickettsial invasion protein likely plays a role in controlling the concentration of stress-induced dinucleoside oligophosphates following bacterial invasion. The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA). The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family. To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli. The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives. The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (NpnN; n ≥ 5), a group of cellular signaling molecules. At optimal pH 8.5, the enzyme actively degrades adenosine (5′)-pentaphospho-(5′)-adenosine into ATP and ADP with a Km of 0.1 mm and kcat of 1.9 s−1. Guanosine (5′)-pentaphospho-(5′)-guanosine and adenosine-(5′)-hexaphospho (5′)-adenosine are also substrates. Similar to other Nudix hydrolases, InvA requires a divalent metal cation, Mg2+ or Zn2+, for optimal activity. These data suggest that the rickettsial invasion protein likely plays a role in controlling the concentration of stress-induced dinucleoside oligophosphates following bacterial invasion. Rickettsia prowazekii is the etiologic agent of epidemic typhus and Brill-Zinsser disease. These illnesses are louse-borne rickettsioses that are reemerging worldwide (1.Azad A.F. Beard C.B. Rickettsial pathogens and their arthropod vectors.Emerg. Infect. Dis. 1998; 2: 179-186Google Scholar, 2.Raoult D. Ndihokubwayo J.B. Tissot-Dupont H. Roux V. Faugere B. Abegbinni R. Birtles R.J. Outbreak of epidemic typhus associated with trench fever in Burundi.Lancet. 1998; 352: 353-358Google Scholar). The organism is an obligate intracellular Gram-negative bacterium growing only within the eukaryotic host cell cytoplasm (3.Winkler H.H. Rickettsia species (as organisms).Annu. Rev. Microbiol. 1990; 44: 131-153Google Scholar). Humans are exposed to R. prowazekii through direct contact with contaminated body louse feces. The bacterium begins its life cycle in the human host by invading the epithelial cells via the process of induced phagocytosis (3.Winkler H.H. Rickettsia species (as organisms).Annu. Rev. Microbiol. 1990; 44: 131-153Google Scholar, 4.Walker T.S. Winkler H.H. Penetration of cultured mouse fibroblasts (L cells) by Rickettsia prowazekii.Infect. Immun. 1978; 22: 200-208Google Scholar). Then, it rapidly escapes from the phagosome into the host cytoplasm where it replicates and eventually causes the invaded cell to burst (3.Winkler H.H. Rickettsia species (as organisms).Annu. Rev. Microbiol. 1990; 44: 131-153Google Scholar, 4.Walker T.S. Winkler H.H. Penetration of cultured mouse fibroblasts (L cells) by Rickettsia prowazekii.Infect. Immun. 1978; 22: 200-208Google Scholar). Destruction of host cells is the basis of rickettsial pathogenesis (3.Winkler H.H. Rickettsia species (as organisms).Annu. Rev. Microbiol. 1990; 44: 131-153Google Scholar). Although systemic approaches have revealed substantial information about the biology of rickettsial host cell invasion, the molecular basis underlying the invasive mechanism remains undefined. Recently, the completion of the R. prowazekii genome project revealed the presence of the invasion gene homolog, invA (5.Andersson S.G.E. Zomorodipour A. Andersson J.O. Sicheritz-Ponten T. Alsmark U.C.M. Podowski R.M. Naslund A.K. Eriksson A.S. Winkler H.H. Kurland C.G. The genome sequence of Rickettsia prowazekii and the origin of mitochondria.Nature. 1998; 396: 133-140Google Scholar). The invA open reading frame encodes a polypeptide of 161 amino acids with a predicted molecular mass of 18,752 daltons, containing a conserved motif called the Nudix box (Nucleoside diphosphates linked to some other moiety, X) (6.Bessman M.J. Frick D.N. O’Handley S.F. The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes.J. Biol. Chem. 1996; 271: 25059-25062Google Scholar). Nudix boxes are present in the Nudix hydrolase family, a group of diverse enzymes that catalyze the hydrolysis of nucleoside diphosphate derivatives (6.Bessman M.J. Frick D.N. O’Handley S.F. The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes.J. Biol. Chem. 1996; 271: 25059-25062Google Scholar). Alignment analysis of the deduced amino acid sequence of R. prowazekii InvA demonstrated 37–44% identity to the putative invasion proteins of other invasive bacteria including Bartonella bacilliformis IalA, Neisseria meningitidis putative Ap4A 1The abbreviations used are: Ap4A, adenosine (5′)-tetraphospho-(5′)-adenosine; Ap5A, adenosine (5′)-pentaphospho-(5′)-adenosine; HPLC, high performance liquid chromatography. pyrophosphatase, Helicobactor pylori InvA, Escherichia coli YgdP, Salmonella typhimurium putative invasion protein, Haemophilus influenzae InvA, and Pseudomonas aeruginosa invasion protein homolog (Fig. 1). Among these homologous genes, only B. bacilliformis ialA and E. coli K1 ygdP have been documented to be associated with host cell invasion (7.Mitchell S.J. Minnick M.F. Characterization of a two-gene locus from Bartonella bacilliformis associated with the ability to invade human erythrocytes.Infect. Immun. 1995; 63: 1552-1562Google Scholar, 8.Badger J.L. Wass C.A. Kim K.S. Identification of Escherichia coli K1 genes contributing to human brain microvascular endothelial cell invasion by differential fluorescence induction.Mol. Microbiol. 2000; 36: 174-182Google Scholar). Furthermore, purified IalA and YgdP were shown to be members of the dinucleoside oligophosphate pyrophosphatase subfamily of the Nudix hydrolases (9.Conyers G.B. Bessman M.J. The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a Nudix hydrolase active on dinucleoside 5′polyphosphates.J. Biol. Chem. 1999; 274: 1203-1206Google Scholar, 10.Cartwright J.L. Britton P. Minnick M.F. McLennan A.G. The ialA invasion gene of Bartonella bacilliformis encodes a (di)nucleoside polyphosphate hydrolase of the MutT motif family and has homologs in other invasive bacteria.Biochem. Biophys. Res. 1999; 256: 474-479Google Scholar, 11.Bessman M.J. Walsh J.D. Dunn C.A. Swaminathan J. Weldon J.E. Shen J. The gene ygdP, associated with the invasiveness of Escherichia coli K1, designates a Nudix hydrolase, Orf176., active on adenosine(5′)-pentaphospho-(5′)-adenosine.J. Biol. Chem. 2001; 276: 37834-37838Google Scholar). Their specific substrates, dinucleoside oligophosphates, are considered to be a class of signaling molecules involved in cell stress responses, cell growth, and cell differentiation (12.McLennan A.G. Dinucleoside polyphosphates-friend or foe?.Pharmacol. Ther. 2000; 87: 73-89Google Scholar, 13.Guranowski A. Specific and nonspecific enzymes involved in the catabolism of mono- and dinucleoside polyphosphates.Pharmacol. Ther. 2000; 87: 117-139Google Scholar). It has been proposed that these enzymes might play a role in enhancing the intracellular survival of the invading bacteria by regulating the stress-induced dinucleoside oligophosphate levels during host cell invasion (6.Bessman M.J. Frick D.N. O’Handley S.F. The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes.J. Biol. Chem. 1996; 271: 25059-25062Google Scholar, 9.Conyers G.B. Bessman M.J. The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a Nudix hydrolase active on dinucleoside 5′polyphosphates.J. Biol. Chem. 1999; 274: 1203-1206Google Scholar, 10.Cartwright J.L. Britton P. Minnick M.F. McLennan A.G. The ialA invasion gene of Bartonella bacilliformis encodes a (di)nucleoside polyphosphate hydrolase of the MutT motif family and has homologs in other invasive bacteria.Biochem. Biophys. Res. 1999; 256: 474-479Google Scholar, 11.Bessman M.J. Walsh J.D. Dunn C.A. Swaminathan J. Weldon J.E. Shen J. The gene ygdP, associated with the invasiveness of Escherichia coli K1, designates a Nudix hydrolase, Orf176., active on adenosine(5′)-pentaphospho-(5′)-adenosine.J. Biol. Chem. 2001; 276: 37834-37838Google Scholar, 12.McLennan A.G. Dinucleoside polyphosphates-friend or foe?.Pharmacol. Ther. 2000; 87: 73-89Google Scholar, 13.Guranowski A. Specific and nonspecific enzymes involved in the catabolism of mono- and dinucleoside polyphosphates.Pharmacol. Ther. 2000; 87: 117-139Google Scholar). In this paper, we have identified homologous genes in several Rickettsia species. Among these, the R. prowazekii invA was expressed, and the purified protein was characterized to be a member of the dinucleoside pentaphosphate pyrophosphatase subfamily of Nudix hydrolases. The R. prowazekii (Madrid E), Rickettsia typhi (Wilmington), Rickettsia rickettsii (Sheila Smith), Rickettsia akari (Kaplan), Rickettsia canada (CA410), and Rickettsia rhipicephali (CA871) were propagated in our laboratory (University of Maryland, Baltimore, MD). Primers utilized in this study were synthesized at the Biopolymer core facility, University of Maryland, Baltimore. The genomic DNA isolation kit was from Promega (Madison, WI). Enzymes used in standard cloning procedures were from Stratagene (La Jolla, CA) and Invitrogen. Calf intestinal alkaline phosphatase and inorganic pyrophosphatase were from Stratagene (La Jolla, CA). PCR 2.1 TA TOPO cloning vector and E. coli TOP10 competent cells were purchased from Invitrogen. The prokaryotic expression system, pET-24a(+) and E. coli DH5α and E. coli HMS174(DE3) hosts were obtained from Novagen (Madison, WI). The plasmid, pGroESL, containing the E. coli groEL and groES genes, a T7lac promoter, and a chloramphenicol resistance gene was a gift from George H. Lorimer (Dupont). All chemical reagents were purchased from Sigma. Sephadex G-100 was from Amersham Biosciences. A Centricon Plus-20 microconcentrator and a C18 reverse phase HPLC column were purchased from Millipore Co. (Bedford, MA) and YMC Inc. (Japan), respectively. Rickettsiae were propagated in African green monkey kidney (Vero) cells (ATCC C1008) as described previously (14.Radulovic S. Feng H.M. Morovic M. Djelalija B. Popov V. Crocquet-Valdes P. Walker D.H. Isolation of Rickettsia akari from the patient in a region where Mediterranean spotted fever is endemic.Clin. Infect. Dis. 1996; 22: 216-220Google Scholar). 90–95% infected cells were harvested, and rickettsiae were purified by Renografin density gradient centrifugation (15.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Google Scholar). Genomic DNAs were isolated using a WizardTM DNA isolation kit and were quantified spectrophotometrically. To investigate whether members of typhus group and spotted fever group rickettsiae, both human pathogens and non-pathogens, contain the invA gene homolog, primers based on the R. prowazekii invA sequence were used in PCR reactions. 0.1 μg of genomic DNA of typhus group, R. typhi and spotted fever group rickettsiae, R. rickettsii, R. akari, R. canada, and R. rhipicephali were subjected to PCR. The amplified fragments were cloned into the TA TOPO cloning vector, PCR 2.1, and recombinant vectors were transformed into competent cells for propagation. Plasmid DNA was purified and sequenced. Alignments were compared through blastn searches from GenBankTM. Full-length R. prowazekii invA (GenBankTM accession number AJ235271) was amplified from 0.1 μg of R. prowazekii genomic DNA utilizing a forward primer incorporating an NdeI site (NdeI-invAF; nucleotide 12769–12736; 5′ GCG CGC GCC ATA TGA GGA ATT CTT CTA ACA AAT) and a reverse primer incorporating a BamHI site (BamHI-invAR; nucleotide 12284–12320; 5′ GCG CGC GGA TCC TTA CTG AAT TAA TGA TTC AAA). The amplicon was subcloned into PCR 2.1 TA TOPO cloning vector. The recombinant plasmid was transformed into E. coli TOP10 for propagation and sequenced to ensure the fidelity of the amplified gene. R. prowazekii invA was then excised from the TA cloning vector, gel-purified, and directionally cloned into the NdeI/BamHI sites of the pET-24a(+) expression vector. The resulting construct, pETinvA, was introduced into E. coli DH5α for further propagation, and the in-frame insertion of invA was confirmed by sequencing. To obtain a high quantity of native InvA, it was necessary to use an enhancing expression protocol optimizing the production of soluble protein (9.Conyers G.B. Bessman M.J. The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a Nudix hydrolase active on dinucleoside 5′polyphosphates.J. Biol. Chem. 1999; 274: 1203-1206Google Scholar). In this procedure, the expression host, E. coli HMS174(DE3), was co-transformed with pETinvA and pGroESL expressing the chaperones GroEL and GroES. The transformed clones were recovered by double antibiotic selection (kanamycin and chloramphenicol resistance acquired from pETinvA and pGroESL, respectively). To express the recombinant InvA, two liters of prewarmed LB medium containing 30 μg/ml each of kanamycin and chloramphenicol was inoculated with 20 ml of an overnight culture of E. coli HMS174(DE3) carrying pETinvA and pGroESL. The culture was incubated at 37°C to an A600 of 0.3 and then transferred to 18°C. At an A600 of 0.8, the cells were induced with 0.5 mm isopropyl-β-d-thiogalactopyranoside and incubated at 18°C overnight. The induced cells were harvested by centrifugation, washed with isotonic saline, and frozen at −80°C. To extract the protein, the frozen cells (4 g) were resuspended in 2.5 volumes of TED buffer (50 mm Tris-Cl, pH 7.5, 0.1 mm EDTA, 0.1 mm dithiothreitol) and ruptured by three repetitive freeze-thaw cycles. The cell debris was removed by centrifugation to obtain the crude extract (Fraction I). A solution of 10% streptomycin sulfate was slowly added to Fraction I to a final concentration of 1.5%. The precipitated nucleic acids were removed by centrifugation, and the supernatant, Fraction II, was to with The precipitated protein was in a of TED buffer (Fraction to a 2.5 Sephadex G-100 and with mm in TED were for enzymatic activity. The active were by in a Centricon Plus-20 and to (Fraction at −80°C. of the process was by by concentration was to A and for the of of protein utilizing the of Biol. Chem. Scholar). activity of purified InvA was as described previously (9.Conyers G.B. Bessman M.J. The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a Nudix hydrolase active on dinucleoside 5′polyphosphates.J. Biol. Chem. 1999; 274: 1203-1206Google Scholar). this the of a to a by the were incubated for at 37°C with InvA and intestinal alkaline phosphatase (4 in of a standard containing mm pH 8.5, and mm The was by of mm EDTA, and the was to the of and The role of the in the of bacteriophage Biol. Chem. Scholar). of InvA the hydrolysis of of these HPLC was used to the as described previously (9.Conyers G.B. Bessman M.J. The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a Nudix hydrolase active on dinucleoside 5′polyphosphates.J. Biol. Chem. 1999; 274: 1203-1206Google Scholar). containing mm pH 8.5, mm Ap5A, and of InvA protein was incubated to 20 at at were on a reverse phase and the were identified by to the R. prowazekii invA was and its nucleotide sequence was to the To whether invA homologs are present in other rickettsiae, PCR utilizing R. prowazekii invA primers were on the genomic DNA of human R. R. rickettsii, R. akari, and the Rickettsia R. canada and R. of PCR were amplified (Fig. The fragments were cloned and sequenced. Alignment analysis demonstrated that the nucleic acid of the invA homologs in other rickettsiae were to the R. prowazekii cloning of the R. prowazekii invA into pET-24a(+) at the NdeI and BamHI sites for the expression of protein the of the cells were and induced at a of InvA was only a was present in the soluble of the cell extract (Fig. Similar was in InvA expression at or 37°C in the presence of host chaperones The the expression and using a host in a substantial in the native of R. prowazekii invA (Fig. analysis of the soluble of the crude cell extract revealed protein at and InvA, and (Fig. of InvA was by the that of the enzyme was from the cell by a freeze-thaw the protocol as described and InvA was purified to near homogeneity as shown in analysis of the sequence demonstrated high to the Nudix hydrolases, we tested a number of shown previously to be by members of the family. or activity was with or I). as with IalA and YgdP, the InvA protein the hydrolysis of members of the dinucleoside oligophosphate family. in InvA degrades adenosine It has activity on dinucleoside oligophosphates Bartonella IalA The activity of InvA on and with is also with IalA and of the dinucleoside oligophosphate adenosine dinucleoside oligophosphates are prowazekii bacilliformis data of B. bacilliformis IalA and E. coli YgdP for are from and Bessman and Bessman coli data of B. bacilliformis IalA and E. coli YgdP for are from and Bessman and Bessman Ap5A, adenosine dinucleoside oligophosphates are The data of B. bacilliformis IalA and E. coli YgdP for are from and Bessman (9.Conyers G.B. Bessman M.J. The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a Nudix hydrolase active on dinucleoside 5′polyphosphates.J. Biol. Chem. 1999; 274: 1203-1206Google and Bessman M.J. Walsh J.D. Dunn C.A. Swaminathan J. Weldon J.E. Shen J. The gene ygdP, associated with the invasiveness of Escherichia coli K1, designates a Nudix hydrolase, Orf176., active on adenosine(5′)-pentaphospho-(5′)-adenosine.J. Biol. Chem. 2001; 276: 37834-37838Google Scholar). in a To the from the hydrolysis of the Ap5A, a standard was alkaline were for and 20 and subsequently by was a in the during the and of ATP and ADP (Fig. of adenosine was the of the The of the hydrolytic be described by of hydrolysis by InvA, with of YgdP, for are in The of InvA is to the of 1.9 compared with for analysis of the pentaphosphate prowazekii coli data are from Bessman of activity is of These data are from Bessman M.J. Walsh J.D. Dunn C.A. Swaminathan J. Weldon J.E. Shen J. The gene ygdP, associated with the invasiveness of Escherichia coli K1, designates a Nudix hydrolase, Orf176., active on adenosine(5′)-pentaphospho-(5′)-adenosine.J. Biol. Chem. 2001; 276: 37834-37838Google A of activity is of in a InvA from the column as a protein, as a in of the other characterized Nudix hydrolases, it an alkaline at 8.5, and a divalent cation, Mg2+ or Zn2+, at mm to be the hydrolytic activity of InvA it is for IalA and A of the of InvA and the other two characterized invasion proteins is in of the dinucleoside oligophosphate prowazekii bacilliformis data are from and Bessman and Bessman coli data are from and Bessman and Bessman mass Zn2+, Zn2+, These data are from and Bessman (9.Conyers G.B. Bessman M.J. The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a Nudix hydrolase active on dinucleoside 5′polyphosphates.J. Biol. Chem. 1999; 274: 1203-1206Google and Bessman M.J. Walsh J.D. Dunn C.A. Swaminathan J. Weldon J.E. Shen J. The gene ygdP, associated with the invasiveness of Escherichia coli K1, designates a Nudix hydrolase, Orf176., active on adenosine(5′)-pentaphospho-(5′)-adenosine.J. Biol. Chem. 2001; 276: 37834-37838Google Scholar). in a of our is to the molecular basis underlying rickettsial host cell invasion. have on the of the R. prowazekii invA and have shown that this gene is present and conserved the and members of the typhus and spotted fever that this gene might be for host cell invasion and the obligate intracellular by To characterize its R. prowazekii InvA was expressed, and for its enzymatic to express R. prowazekii InvA as a protein for protein was of an expression of InvA as a protein with the protein in a high quantity of soluble the purified protein a specific enzyme activity that of the native M. and A. at the for Similar have been previously for in that the native protein a specific activity that of the protein and of the expressed coli. J. Biol. Chem. 1996; 271: Scholar). the system, in with the protocol described for the expression of high native InvA, was for data that R. prowazekii invA encodes a dinucleoside pentaphosphate pyrophosphatase, it as a member of the subfamily of the Nudix hydrolases. is with in the genes, Bartonella ialA and E. coli K1 ygdP, were also shown to members of this subfamily (9.Conyers G.B. Bessman M.J. The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a Nudix hydrolase active on dinucleoside 5′polyphosphates.J. Biol. Chem. 1999; 274: 1203-1206Google Scholar, 10.Cartwright J.L. Britton P. Minnick M.F. McLennan A.G. The ialA invasion gene of Bartonella bacilliformis encodes a (di)nucleoside polyphosphate hydrolase of the MutT motif family and has homologs in other invasive bacteria.Biochem. Biophys. Res. 1999; 256: 474-479Google Scholar, 11.Bessman M.J. Walsh J.D. Dunn C.A. Swaminathan J. Weldon J.E. Shen J. The gene ygdP, associated with the invasiveness of Escherichia coli K1, designates a Nudix hydrolase, Orf176., active on adenosine(5′)-pentaphospho-(5′)-adenosine.J. Biol. Chem. 2001; 276: 37834-37838Google Scholar). its enzymatic might InvA be involved in host cell invasion or its The dinucleoside oligophosphates are of the found in prokaryotic and eukaryotic cells in (12.McLennan A.G. Dinucleoside polyphosphates-friend or foe?.Pharmacol. Ther. 2000; 87: 73-89Google Scholar, of and with a purified Biophys. Res. Scholar). during cellular as or the concentration of dinucleoside oligophosphates to several and cell S. A. Scholar, and are synthesized as a of Scholar). of these in diverse as of cell and cell differentiation A. H. of cell differentiation and on in human cell of cell A. The of cell Ap4A as a 1998; of A. A. a family of of the and of activity a of Biol. Chem. Scholar). To its the cell has to this It has been proposed that the Nudix hydrolases have an function in the cell of and in the of in These enzymes might as the cell from resulting from the presence of (6.Bessman M.J. Frick D.N. O’Handley S.F. The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes.J. Biol. Chem. 1996; 271: 25059-25062Google Scholar). In this the rickettsial invasion protein play a role in the levels of intracellular dinucleoside oligophosphates that during the stress following bacterial invasion. of dinucleoside oligophosphates might be for the intracellular survival of hydrolysis of oligophosphates by Rickettsia and as as E. coli K1 invasion in ATP as of the high as a for the Furthermore, the other of also be for ATP through the of rickettsiae H.H. Rickettsial an Biol. Chem. Scholar). be an of bacterial in rickettsiae have to intracellular life by a to a from the enzymatic activity described other of the function of InvA to be the of InvA, as as its expression during the invasion as to a dinucleoside oligophosphate pyrophosphatase and dinucleoside oligophosphates are involved in rickettsial these including utilizing against purified InvA and reverse PCR are

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.