• View in gallery

    Western immunoblot analysis of mouse anti-InvA antibodies. The membranes were probed with anti–native InvA 1:250 (A), anti–denatured InvA 1:250 (B), anti–MBP 1:75 (C), and preimmune mouse serum 1:100 (D). Lanes 1 and 2 contain 50 μg of uninfected Vero cell lysate and rickettsiae-infected Vero cell lysate. Lane 3 contains 2 ng of purified recombinant InvA. Lanes 2 and 3 contain 50 μg of rickettsiae-infected Vero cell lysate spiked with 2 ng of purified recombinant InvA. Arrow indicates size of InvA. Protein mass standards are indicated on both sides.

  • View in gallery

    Immunoadsorption of mouse anti-InvA antiserum evaluated by enzyme-linked immunosorbent assay using purified recombinant InvA (1 μg/ml) as antigen. Black bars, preadsorption anti-InvA antibodies; lightly shaded bars, postadsorption with recombinant maltose-binding protein (MBP)–InvA protein; medium shaded bars, adsorbed with purified MBP protein; heavily shaded bars, affinity purified anti-InvA antibodies.

  • View in gallery

    Immunogold labeling of InvA within R. typhi. Ultrathin sections through R. typhi–infected Vero cells stained with anti–native InvA (A), anti–denatured InvA (B), anti–maltose-binding protein (C), and adjuvant immunized mouse serum (D). All antibodies are diluted 1:50. R = R. typhi (Wilmington). Arrows indicate gold particles. (Original magnification ×20,000.)

  • 1

    Gaywee J, Xu W-L, Radulovic S, Bessman MJ, Azad AF, 2002. The Rickettsia prowazekii invasion gene homolog (invA) encodes a nudix hydrolase active on adenosine (5′)-pentaphospho-(5′)-adenosine. Mol Cell Proteomics 1 :179–185.

    • Search Google Scholar
    • Export Citation
  • 2

    Lee PC, Bochner BR, Ames BN, 1983. AppppA, heat shock stress, and cell oxidation. Proc Natl Acad Sci U S A 80 :7496–7500.

  • 3

    Bochner BR, Lee PC, Wilson SW, Cutler CW, Ames BN, 1984. AppppA and related adenylated nucleotides are synthesized as a consequence of oxidative stress. Cell. 37 :225–232.

    • Search Google Scholar
    • Export Citation
  • 4

    McLennan AG, 2000. Dinucleoside polyphosphates-friend or foe? Pharmacol Ther 87 :73–89.

  • 5

    Guranowski A, 2000. Specific and nonspecific enzymes involved in the catabolism of mono- and dinucleoside polyphosphates. Pharmacol Ther 87 :117–139.

    • Search Google Scholar
    • Export Citation
  • 6

    Mateo J, Miras-Portugal MT, Rotllan P, 1997. Ecto-enzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells. Am J Physiol 273 :C918–C927.

    • Search Google Scholar
    • Export Citation
  • 7

    Von Drygalski A, Ogilvie A, 2000. Ecto-diadenosine 5′,5′′′- P1,P4-tetraphosphate (Ap4A)-hydrolase is expressed as an ectoenzyme in a variety of mammalian cells and adds new aspects to the turnover of Ap4A. Biofactors 11 :179–187.

    • Search Google Scholar
    • Export Citation
  • 8

    Aguilar JS, Reyes R, Asensio AC, Oakin S, Rotllan P, Miledi R, 2001. Ectoenzymatic breakdown of diadenosine polyphosphates by Xenopus laevis oocytes. Eur J Biochem 268 :1289–1297.

    • Search Google Scholar
    • Export Citation
  • 9

    Feussner K, Guranowski A, Kostka S, Wasternack C, 1996. Diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) hydrolase from tomato (Lycopersicon esculentum cv. Lukullus)—purification, biochemical properties and behaviour during stress. Zeitschr Naturforsch Section C J Biosci 51 :477–486.

    • Search Google Scholar
    • Export Citation
  • 10

    Nielsen H, Engelbrecht J, Brunak S, Von Heijne G, 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10 :1–6.

    • Search Google Scholar
    • Export Citation
  • 11

    Radulovic S, Feng HM, Morovic M, Djelalija B, Popov V, Crocquet-Valdes P, Walker DH, 1996. Isolation of Rickettsia akari from the patient in a region where Mediterranean spotted fever is endemic. Clin Infect Dis 22 :216–220.

    • Search Google Scholar
    • Export Citation
  • 12

    Manor E, Carbonetti NH, Silverman DJ, 1994. Rickettsia rickettsii has proteins with cross-reacting epitopes to eukaryotic phospholipase A2 and phospholipase C. Microb Pathog 17 :99–109.

    • Search Google Scholar
    • Export Citation

 

 

 

 

SUBCELLULAR LOCALIZATION OF RICKETTSIAL INVASION PROTEIN, INVA

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  • 1 Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, Maryland

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.

INTRODUCTION

We identified Rickettsia prowazekii InvA to be Ap5A pyrophosphatase, a member of the Nudix hydrolase family.1 Our previous experiments have shown that invA is present and conserved among typhus group and spotted fever group rickettsiae.1 One attractive hypothesis concerning the role of InvA relates to the observation that its substrates, the diadenosine oligophosphates, are involved in cellular stress responses.2 As a result of heat shock or oxidative stress, the concentration of these compounds can increase >100-fold over their endogenous level in bacteria.3 Intracellular imbalance of these minor nucleotides results in diverse physiologic affects that are potentially lethal to the cell.4 Removal of excess amounts of these compounds would be part of the normal regulatory process in the cell, to maintain its viability.4 InvA may function by reducing the stress-induced concentration of these signaling molecules that accumulate after rickettsial host cell invasion.1

We examined the transcriptional profile of R. prowazekii invA during rickettsial host cell infection, using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and real-time fluorescent probe–based quantitative RT-PCR (J. Gaywee et al, unpublished data). A differential expression of invA was observed during rickettsial entry into eukaryotic cells, early growth phase, and exit. Although not directly related, expression of rickettsial groEL, a molecular indicator of cellular stresses, also was up-regulated during the early period of infection (J. Gaywee et al, unpublished data). These experiments have established the possible role of InvA as a dinucleoside oligophosphate pyrophosphatase, facilitating bacterial survival during entry and enhancing rickettsial survival within the cytoplasm of eukaryotic cells. The location of the enzyme in the cell would be an important determinant of its efficacy. It is assumed that the NpnN hydrolases generally are located in the cytosol.5 It has been shown, however, that a variety of the diadenosine oligophosphate hydrolases are ecto-enzymes and located on the cell surface68; in only one case, an Ap4A hydrolase from a higher plant was shown to be predominantly present in the nucleus and cytoplasm during the interphase of the cell cycle.9 Little is known regarding localization of this hydrolase subfamily in prokaryotes.

Computer analysis of the InvA amino acid sequence by the SignalP V1.1 (www.cbs.dtu.dk)10 and k-NN Prediction (http://psort.nibb.ac.jp) programs suggested that InvA was likely to be a cytoplasmic protein (52%), with a lower probability of being located to any other specific compartments. Examining the localization of InvA may help determine the biologic function of this enzyme in rickettsiae. The current study used immunoelectron microscopy to delineate the location of InvA within rickettsiae.

MATERIALS AND METHODS

Rickettsial propagation and sample preparation.

R. prowazekii (Madrid E) and R. typhi (Wilmington) were propagated in our laboratory as previously described.11 Briefly, a confluent monolayer of African green monkey kidney cells (Vero cell, ATCC C1008) in Eagle’s minimum essential medium supplemented with 5% fetal bovine serum and 2 mM of L-glutamine was inoculated with rickettsiae at multiplicity of infection (MOI) of 80 rickettsiae per cell. After absorption of rickettsiae to the host cells for 30 minutes at room temperature, freshly prepared culture medium was added, and the cultures were shifted to 37°C for 30 minutes to allow bacterial entry. Cultures subsequently were incubated at 34°C in a humidified atmosphere with 5% carbon dioxide.

R. typhi–infected Vero cells were used to determine sub-cellular localization of InvA. The infected cell culture was fixed in phosphate-buffered saline (PBS) containing 1% paraformaldehyde and 0.1% glutaraldehyde for 1 hour at room temperature. Fixed cells were scraped from the culture flask, transferred to microcentrifuge tubes, and pelleted by centrifugation for 2 minutes at 13,000 rpm. Thin-section grids for immunoelectron microscopy were prepared at the electron microscopy facility in the Department of Pathology, School of Medicine, University of Maryland, Baltimore. Briefly, dehydration of the cell pellet was performed with increasing concentrations (50–100%) of ethanol and LR-White resin (Polyscience, Inc, Warrington, PA). The pellet was embedded in gelatin capsules followed by heat polymerization for 24 hours at 50°C, then cut into ultra-thin sections and mounted on nickel grids.

The supernatant of heavily infected rickettsial cell cultures, containing released rickettsiae and detached host cells, was harvested and centrifuged at 14,000 rpm for 10 minutes to pellet the cells. These samples were used to prepare immunofluorescence assay (IFA) antigen slides and rickettsial lysate for Western blot analysis.

Production of InvA-specific antibodies.

Purified recombinant InvA obtained as described previously1 was used to generate InvA-specific antibodies in mice. Two forms of the proteins, native and denatured forms, were used for immunization. To prepare the denatured protein, partially purified recombinant InvA was separated on a 4–12% gradient SDS polyacrylamide gel (Invitrogen, Carlsbad, CA) and stained with Coomassie R-250 to visualize the protein. The 19-kd band of InvA was excised from the gel, and the denatured protein was electroeluted out of the gel slices using an Electroeluter, model 422 (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer’s directions. The concentration of the electroeluted InvA was quantitated using BioRad Bradford reagent (Bio-Rad Laboratories, Hercules, CA). Groups of female, 4- to 6-week-old, BALB/c mice (3–5 mice) were inoculated subcutaneously with 50 μg of protein mixed with Freund’s complete adjuvant. A booster injection, 50 μg of protein mixed with Freund’s incomplete adjuvant, was administered 2 weeks after the primary injection. The animals were boosted twice more at 2-week intervals. Preinoculation and 2-week postinoculation sera were collected and stored in aliquots at -80°C until used. Nonspecific, irrelevant antibody controls included antibodies raised against an unrelated protein, purified recombinant maltose binding protein (MBP) (J. Gaywee et al, unpublished data), and against adjuvant only. All antibodies were affinity purified using an NAB protein A spin column chromatography kit (Pierce, Rockford, IL) according to the manufacturer’s protocol.

Enzyme-linked immunosorbent assay.

The antibody titers against InvA were determined by enzyme-linked immunosorbent assay (ELISA). Briefly, 100 μl of 1 μg/ml of purified recombinant InvA in bicarbonate buffer was added to each well of a 96-well microplate and incubated overnight at 4°C. The InvA-coated wells were washed 3 times with 200 μl of PBST (PBS with 0.05% Tween-20) and blocked with 100 μl of blocking solution (PBST, 0.5% bovine serum albumin [BSA], 0.01% NaN3) at 37°C for 30 minutes. The plate was washed 3 times in PBST, then incubated at 37°C for 30 minutes with the serial dilutions of the sera to be tested. After incubation with the primary antibody, the plates were washed 3 times in PBST, and the secondary antibody, horseradish peroxidase (HRP)–conjugated goat–anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), was added and incubated at 37°C for 30 minutes. After washing 3 times, 100 μl of 3,3′,5,5′-tetramethyl benzidine (TMB) substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added, and the plate was incubated at room temperature for 10 minutes. The reaction was stopped with 100 μl of stop solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The optical density at 450 nm was recorded within 30 minutes.

Indirect immunofluorescense assay.

Antibodies were tested for their specificity against native antigen, and the titer was determined using immunofluorescence. To prepare the antigen slides for IFA, the rickettsial cell pellet was washed with PBS and resuspended in a small volume (100 μl). A 10-μl aliquot of the cell suspension was placed onto microscope slides and allowed to dry. Slides were fixed in acetone for 10 minutes at room temperature and stored desiccated at -20°C until used. Before use in the assay, IFA antigen slides were dried at room temperature, and blocking solution (PBS, 5 mM of magnesium chloride, 1% BSA) was applied to the cell spots for 30 minutes at room temperature. Slides were incubated with serial dilutions of primary antibodies in blocking solution for 1 hour in a humidified environment, followed by washing (3 times) with PBST. The secondary antibody, fluorescein isothiocyanate–conjugated goat–anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), was diluted 1:50 in blocking solution containing 0.01% Evans blue and incubated for 30 minutes. After washing, the slides were mounted with Vectashield mounting solution (Vector Laboratories Inc, Burtingame, CA). Photomicrographs were taken using a Nikon phase contrast, elipse E600 microscope, with SPOT RT camera and SPOT RT Software V3.2 (Diagnostic Instrument Inc, Sterling Heights, MI).

Western blot analysis.

Rickettsial lysates for Western blot analysis were prepared as follows. The cell pellet harvested from the supernatant of heavily infected rickettsial cell cultures was washed in PBS and subsequently lysed by repetitive pipetting in B-PER lysis buffer in PBS (Pierce), containing 1× Halt protease inhibitor cocktail (Pierce); lysis was facilitated further by 3 cycles of freeze-thawing. The uninfected Vero cell pellet was lysed in M-PER lysis buffer (Pierce) containing 1× protease inhibitor cocktail, according to the manufacturer’s procedures. The protein content of the lysates was measured by the bicinchoninic acid assay (Pierce, Rockford, IL). Aliquots of the lysate were stored at -20°C until used. Lysate, 50 μg, was mixed with NUPAGE LDS loading buffer (Invitrogen, Carlsbad, CA), and 1:10 of reducing agent (Invitrogen). The lysate was denatured at 70°C for 10 minutes. The samples were separated by NUPAGE 4–12% gradient SDS-PAGE (Invitrogen) under reducing conditions at 200 V for 35 minutes. Proteins from the gel were electrotransferred onto Hybond-P polyvinylidene difluoride (PVDF) membrane (Armersham Pharmacia Biotech, Buckinghamshire, UK) at 30 V for 1 hour. The blot was incubated in blocking buffer (20 mM of Tris, 500 mM of sodium chloride, pH 7.5, 5% w/v nonfat milk) for 1 hour at room temperature and washed 3 times in washing solution (20 mM of Tris, 500 mM of sodium chloride, pH 7.5, 0.05% v/v Tween-20). Antibodies were incubated with the membranes for 1 hour, followed by washing as previously. The secondary antibody, goat–anti-mouse-HRP IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), diluted 1:3,000 in blocking solution was incubated with the membranes for 30 minutes at room temperature. Membranes were washed as previously and developed with Super-signal West pico chemiluminescence detection reagent (Pierce) according to the manufacturer’s instructions.

Immunoadsorption of anti-InvA antibodies.

To determine whether the generated antibodies were specific for the rickettsial InvA, immunoadsorption studies were undertaken. The rationale was that InvA-specific antibodies would be depleted after adsorption by immobilized InvA, resulting in a decreased antibody titer that could be detected by ELISA and IFA. The purified recombinant MBP-InvA fusion protein from previous work (J. Gaywee et al, unpublished data) was used to generate the InvA-affinity, agarose-based column. The MBP-InvA first was dialyzed against an excess of phosphate buffer, pH 7.4, using the Slide-A-Lyzer dialysis unit (Pierce), overnight at 4°C. The dialyzed protein was concentrated in a Centricon Plus-20 microconcentrator (Millipore Co, Bedford, MA), and the protein concentration was measured by the bicinchoninic acid assay (Pierce). To generate the MBP-InvA coupled agarose column, 100 μg of protein was coupled with 4% activated agarose beads using the AminoLink Plus Immobilization Kit (Pierce) according to the manufacturer’s protocol. An immobilized MBP-agarose column also was produced as a control. Polyclonal antibodies to InvA were adsorbed to the specific antigen by passing them through the immobilized MBP-InvA coupled agarose column (2 μg per 1 μl of settled gel) or MBP column. The adsorbed antiserum (flow through) was collected. The antibodies that bound to the specific antigen were eluted from the immobilized antigen using ImmunoPure elution buffer (Pierce). To evaluate the adsorption yield, protein concentrations of adsorbed serum and eluted solution were determined and compared with the preadsorbed serum. Titers of the adsorbed serum and the antigen-specific, affinity-purified antibodies were determined by ELISA and IFA.

Immunoelectron microscopy.

The localization of InvA in R. typhi–infected Vero cells was examined by immunoelectron microscopy according to the modified methods of Manor et al.12 First, sections on the grids were etched by incubation with freshly prepared, saturated sodium-m-periodate for 5 minutes, followed by rinsing 3 times in deionized water. Quenching with 0.1 M of glycine in phosphate buffer for 20 minutes prevented any free aldehyde groups from binding to the primary antibody. The grids were blocked by incubating them in PBS, 1% BSA, 5% Fish gelatin (Ted Pella Inc, Redding, CA) for 30 minutes. Grids were incubated at room temperature with the primary antibody (diluted 1:50) in a humidified environment for 2 hours, followed by washing 5 times in PBS/0.1% Tween-20. The grids were incubated for 30 minutes with a goat–anti-mouse antibody conjugated to 10-nm gold particles (Ted Pella Inc, Redding, CA), then washed as described previously and stained with 2% aqueous Uranyl acetate and rinsed with deionized water. The sections were examined with a JEOL (Peabody, MA) 100 EX transmission electron microscope. Controls for this experiment included uninfected cells and the use of nonspecific, irrelevant antibodies as the primary antibody.

RESULTS AND DISCUSSION

Polyclonal antibodies were generated against recombinant R. prowazekii InvA for use in several immunoassays to define InvA localization within rickettsial cells. Native and denatured forms of recombinant proteins were used to produce antibodies. The homogeneous denatured antigen, eluted from SDS-PAGE, was inoculated in mice to produce specific antibody. One concern was, however, that the antibody against the denatured protein might not recognize the native target antigen. Native and denatured forms of recombinant InvA were found to elicit the specific antibodies. As determined by ELISA, postimmunization serum and purified antibodies produced from both types of antigens showed an end point titer of 1:10,000 against purified recombinant InvA (1 μg/ml) (Table 1). Control sera from mice before immunization and after administration with adjuvant also were tested. Neither of these antibodies reacted with InvA (Table 1). InvA-antiserum and purified antibodies against InvA showed significant reaction with the native form of recombinant R. prowazekii InvA.

To evaluate whether the generated antibodies against InvA reacted with the protein present in rickettsial cells, an indirect IFA was used. Antigen slides were incubated with serial dilutions of antibodies against native and denatured InvA used as primary antibody. The end titers of antiserum and purified antibodies against native and denatured InvA were determined to be 1:400 (Table 1) and did not react with uninfected Vero cells. Anti-MBP and adjuvant immunization mouse serum used as the controls revealed no fluorescent signal (Table 1).

Western immunoblot analysis was used to determine the specificity of the antibodies generated against recombinant InvA to the rickettsial protein. Uninfected and rickettsiae-infected Vero cell lysates were separated by SDS-PAGE under reducing conditions. The resolved proteins were transferred to PVDF membrane and blotted against InvA antibodies. Purified IgG to native InvA recognized a strong single band of purified recombinant InvA at the expected size, 19 kd (Figure 1A, lane 3). Two major bands, 70 kd and 21 kd, were detected in the rickettsial lysate. The 21-kd band was suspected to be InvA, but the size was slightly greater compared with the recombinant InvA running in parallel. We believed that the difference in size resulted from the retardation of the InvA band in the rickettsiae-infected Vero cell lysate. By spiking 2 ng of recombinant InvA into the infected Vero cell lysate (Figure 1A, lanes 2 and 3), it was shown that the lower band was identical to purified InvA (Figure 1A, lane 3). Several factors, such as contamination of intact genomic DNA or sample buffer conditions, may cause retardation in protein mobility. Analysis of purified antibody against denatured InvA showed that it recognized only the expected size of InvA in the rickettsial lysate, the recombinant InvA, and a few weak bands of the Vero cells (Figure 1B). The antibody titer against denatured InvA appeared to be lower than the antibody against native InvA because the visualized band showed a weaker intensity. Antibody against MBP, used as an irrelevant, nonspecific antibody control, reacted with the 70-kd band and a 52-kd protein in the rickettsial lysate and weakly with 3 protein bands of the Vero cells (Figure 1C). The preimmune serum reacted weakly with a 31-kd band in the Vero cells (Figure 1D). Both antibodies raised against purified recombinant proteins, InvA and MBP, identified the 70-kd protein in Rickettsia. The antigens, InvA and MBP were different in size, structure, origin, and purification procedures; the only common factor that these 2 antigens shared was they both had been mixed with Freund’s complete adjuvant for the initial immunizing dose. This raised the question of whether this protein might be an immunoglobulin binding protein. Future investigation is required to address this question.

In an attempt to analyze whether the generated antibodies against InvA were specific to the protein in Rickettsia, immunoadsorption was carried out with the rationale that specific antibodies would be depleted after adsorption with the specific antigen. Antiserum to native InvA was adsorbed 3 times with immobilized recombinant MBP-InvA. The adsorbed yield was determined by protein assay, and the postadsorption antiserum was evaluated for its titer by ELISA and IFA. As shown in Figure 2, the antibody titer of the postadsorption antiserum significantly decreased compared with that of the preadsorption antiserum. Titration by IFA revealed similar results. The IFA titer of the postadsorption antiserum decreased significantly from 1:400 to 1:50 (data not shown). Adsorption with an irrelevant protein, recombinant MBP, showed no change in titer compared with the preadsorption antiserum by ELISA (Figure 2) and IFA (data not shown). Affinity-purified, InvA-specific antibodies were obtained by eluting the antibodies that bind to the InvA adsorption matrix. These antibodies reacted with InvA by ELISA (Figure 2) and IFA. All antibodies were diluted based on the protein concentration except for the affinity-purified InvA specific antibodies. We have shown that the generated antibodies to InvA were antigen specific by ELISA and IFA. These 2 techniques analyzed the interaction of antibody to native form antigen, and Western blot determined the antibody binding to the denatured antigen. Denatured antigen may exhibit different epitopes from the native protein. Antibodies against native and denatured InvA were used in immunoelectron microscopy.

Previous experiments had shown the reactivity of anti–R. prowazekii InvA antibodies against R. typhi–infected Vero cells (data not shown). Additionally, because of the high homology between invA of R. prowazekii and R. typhi (98% identity),1 localization of InvA was established with R. typhi–infected Vero cells. Immunoelectron microscopy was conducted using polyclonal antibodies generated against native and denatured InvA as the primary antibody and goat–anti-mouse conjugated with gold particles as the secondary antibody. Gold particle labeling was found only in the cytoplasm of the Rickettsia when antibodies against InvA were used as the primary antibody (Figure 3A and B). Nonspecific, irrelevant antibody controls, including anti-MBP antiserum and adjuvant immunization mouse serum, used in place of anti-InvA antibodies showed low background (Figure 3C and D). Although a few gold particles were found within the rickettsial cytoplasm of the InvA-stained samples, it was significantly different from the comparable controls. The thin-section grids used here were prepared from a 4-day postinfection time point. Based on the results of gene expression studies, transcriptional levels of invA were low during this period (J. Gaywee et al, unpublished data). It is likely that low levels of protein would be found in the rickettsiae, explaining why only a small quantity of gold particles was found. This result corresponds with the computer prediction that InvA is likely to be a cytoplasmic protein. Location of the protein may reflect its possible function. The presence of InvA in the rickettsial cytoplasm may allow for a free interaction with its substrates, which are located in the cytosol.4 These results suggest that InvA may function in response to stress during rickettsial host cell internalization to facilitate bacterial survival during entry and may act as a buffer, enhancing rickettsial survival within the cytoplasm of the eukaryotic cells.

Authors’ addresses: Jariyanart Gaywee, Department of Microbiology, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand. John B. Sacci, Jr, Suzana Radulovic, Magda S. Beier, and Abdu F. Azad, Department of Microbiology and Immunology, School of Medicine, University of Maryland, 655 West Baltimore Street, Baltimore, MD 21201.

Table 1

Titers of generated mouse anti-InvA antibodies*

ImmunogenELISA against recombinant InvAIFA against rickettsiae-infected Vero cells
ELISA = enzyme-linked immunosorbent assay; IFA = immunofluorescence assay; MBP = maltose-binding protein; NR = nonreactive.
* End point titer.
Recombinant R. prowazekii InvA (native form)1:10,0001:400
Recombinant R. prowazekii InvA (denatured form)1:10,0001:400
Recombinant MBPNRNR
AdjuvantNRNR
Figure 1.
Figure 1.

Western immunoblot analysis of mouse anti-InvA antibodies. The membranes were probed with anti–native InvA 1:250 (A), anti–denatured InvA 1:250 (B), anti–MBP 1:75 (C), and preimmune mouse serum 1:100 (D). Lanes 1 and 2 contain 50 μg of uninfected Vero cell lysate and rickettsiae-infected Vero cell lysate. Lane 3 contains 2 ng of purified recombinant InvA. Lanes 2 and 3 contain 50 μg of rickettsiae-infected Vero cell lysate spiked with 2 ng of purified recombinant InvA. Arrow indicates size of InvA. Protein mass standards are indicated on both sides.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 68, 1; 10.4269/ajtmh.2003.68.92

Figure 2.
Figure 2.

Immunoadsorption of mouse anti-InvA antiserum evaluated by enzyme-linked immunosorbent assay using purified recombinant InvA (1 μg/ml) as antigen. Black bars, preadsorption anti-InvA antibodies; lightly shaded bars, postadsorption with recombinant maltose-binding protein (MBP)–InvA protein; medium shaded bars, adsorbed with purified MBP protein; heavily shaded bars, affinity purified anti-InvA antibodies.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 68, 1; 10.4269/ajtmh.2003.68.92

Figure 3.
Figure 3.

Immunogold labeling of InvA within R. typhi. Ultrathin sections through R. typhi–infected Vero cells stained with anti–native InvA (A), anti–denatured InvA (B), anti–maltose-binding protein (C), and adjuvant immunized mouse serum (D). All antibodies are diluted 1:50. R = R. typhi (Wilmington). Arrows indicate gold particles. (Original magnification ×20,000.)

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 68, 1; 10.4269/ajtmh.2003.68.92

Financial support: This study was supported in part by National Institutes of Health grant AI 17828. Jariyanart Gaywee received pre-doctoral support from the Royal Thai Army, Ministry of Defense, Thailand.

REFERENCES

  • 1

    Gaywee J, Xu W-L, Radulovic S, Bessman MJ, Azad AF, 2002. The Rickettsia prowazekii invasion gene homolog (invA) encodes a nudix hydrolase active on adenosine (5′)-pentaphospho-(5′)-adenosine. Mol Cell Proteomics 1 :179–185.

    • Search Google Scholar
    • Export Citation
  • 2

    Lee PC, Bochner BR, Ames BN, 1983. AppppA, heat shock stress, and cell oxidation. Proc Natl Acad Sci U S A 80 :7496–7500.

  • 3

    Bochner BR, Lee PC, Wilson SW, Cutler CW, Ames BN, 1984. AppppA and related adenylated nucleotides are synthesized as a consequence of oxidative stress. Cell. 37 :225–232.

    • Search Google Scholar
    • Export Citation
  • 4

    McLennan AG, 2000. Dinucleoside polyphosphates-friend or foe? Pharmacol Ther 87 :73–89.

  • 5

    Guranowski A, 2000. Specific and nonspecific enzymes involved in the catabolism of mono- and dinucleoside polyphosphates. Pharmacol Ther 87 :117–139.

    • Search Google Scholar
    • Export Citation
  • 6

    Mateo J, Miras-Portugal MT, Rotllan P, 1997. Ecto-enzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells. Am J Physiol 273 :C918–C927.

    • Search Google Scholar
    • Export Citation
  • 7

    Von Drygalski A, Ogilvie A, 2000. Ecto-diadenosine 5′,5′′′- P1,P4-tetraphosphate (Ap4A)-hydrolase is expressed as an ectoenzyme in a variety of mammalian cells and adds new aspects to the turnover of Ap4A. Biofactors 11 :179–187.

    • Search Google Scholar
    • Export Citation
  • 8

    Aguilar JS, Reyes R, Asensio AC, Oakin S, Rotllan P, Miledi R, 2001. Ectoenzymatic breakdown of diadenosine polyphosphates by Xenopus laevis oocytes. Eur J Biochem 268 :1289–1297.

    • Search Google Scholar
    • Export Citation
  • 9

    Feussner K, Guranowski A, Kostka S, Wasternack C, 1996. Diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) hydrolase from tomato (Lycopersicon esculentum cv. Lukullus)—purification, biochemical properties and behaviour during stress. Zeitschr Naturforsch Section C J Biosci 51 :477–486.

    • Search Google Scholar
    • Export Citation
  • 10

    Nielsen H, Engelbrecht J, Brunak S, Von Heijne G, 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10 :1–6.

    • Search Google Scholar
    • Export Citation
  • 11

    Radulovic S, Feng HM, Morovic M, Djelalija B, Popov V, Crocquet-Valdes P, Walker DH, 1996. Isolation of Rickettsia akari from the patient in a region where Mediterranean spotted fever is endemic. Clin Infect Dis 22 :216–220.

    • Search Google Scholar
    • Export Citation
  • 12

    Manor E, Carbonetti NH, Silverman DJ, 1994. Rickettsia rickettsii has proteins with cross-reacting epitopes to eukaryotic phospholipase A2 and phospholipase C. Microb Pathog 17 :99–109.

    • Search Google Scholar
    • Export Citation

Footnotes

Authors’ addresses: Jariyanart Gaywee, Department of Microbiology, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand. John B. Sacci, Jr, Suzana Radulovic, Magda S. Beier, and Abdu F. Azad, Department of Microbiology and Immunology, School of Medicine, University of Maryland, 655 West Baltimore Street, Baltimore, MD 21201.

Author Notes

Reprint requests: Abdu F. Azad, Department of Microbiology and Immunology, School of Medicine, University of Maryland, 655 West Baltimore Street, Baltimore, MD 21201, Telephone: 410-706-3335, Fax: 410-706-0282, E-mail: aazad@umaryland.edu
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