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Previous Article | Next Article 
Applied and Environmental Microbiology, October 2007, p. 6637-6643, Vol. 73, No. 20
0099-2240/07/$08.00+0 doi:10.1128/AEM.00923-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Department of Microbiology and Immunology, School of Medicine, 660 West Redwood Street, Howard Hall, Room 324B, University of Maryland, Baltimore, Maryland 21201
Received 24 April 2007/ Accepted 16 August 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Additionally, the development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences between pathogenic and nonpathogenic species of rickettsiae. To be optimally useful, the cell line should be permissive toward fastidious endosymbiotic species, allow rapid passaging regimens, and produce large numbers of rickettsiae for genomic or experimental studies. While several rickettsial species (e.g., Rickettsia prowazekii, R. typhi, and R. montanensis) can grow in mammalian cell lines (e.g., Vero and L929), others appear to be restricted to arthropod cell lines. Vero cells are useful for growing many species but appear to be refractory to species that grow relatively slowly, such as R. peacockii (13). Tick cells are useful for studying SFG and TRG rickettsiae but not as useful for maintaining and comparing TG rickettsial growth (13, 15, 21). Tick cells also have the disadvantage of taking up to a month between passages (13, 15).
Mosquito cells have also been used in the past to culture obligate intracellular bacteria such as Wolbachia pipientis (hereafter referred to as Wolbachia) and some rickettsiae (7, 12, 17, 21, 25). If permissive to infection, mosquito cells have many desirable characteristics, including rapid passaging (
1 week) and the potential development of high bacterial titers. Recent attempts to maintain rickettsiae in mosquito cells have met with mixed results. Both R. felis and R. conorii were maintained successfully in the Aedes albopictus cell line C6/36 (12, 25). However, R. montanensis and R. peacockii have not been successfully maintained in two other Aedes albopictus cell lines (AeAl2 and C7/10, respectively) (13, 27).
In this study, we successfully established and propagated three rickettsial species (R. felis, R. montanensis, and R. peacockii) in two mosquito cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B). We chose these two cell lines because they had previously been shown to maintain diverse Wolbachia infections (17, 23). The three Rickettsia species were chosen based on the degree of pathogenicity and biological interest due to their transovarial transmission. R. felis is a putatively pathogenic TRG species horizontally transmitted by cat fleas (Ctenocephalides felis) to mammals and transmitted to flea offspring transovarially. R. peacockii serves as an interesting subject because it is presumed to be limited in cellular invasion capacity due to the lack of functional rickA and rompA genes (13, 26). Both R. montanensis and R. peacockii have been involved in transovarial exclusion, in which a primary rickettsial species invades a tick ovary and excludes a second species from being transmitted transovarially (3, 5, 14).
The use of these two mosquito cell lines may aid in answering questions regarding the mechanisms dictating rickettsial specificity for acarine or insect vectors and hosts. Among pathogenic rickettsiae, SFG species are associated exclusively with ticks, and TG species appear to be limited to fleas and lice (10). Nonpathogenic endosymbiotic rickettsiae have been found in association with both acarines and insects (20). The use of mosquito cell lines may allow us to explore the evolutionary implications of vertebrate pathogenicity with respect to the arthropod associate.
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MATERIALS AND METHODS |
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Mosquito cell lines and maintenance.
Anopheles gambiae Sua5B cells (23) and Aedes albopictus Aa23 cells (17) were passaged every week in sterile Schneider's insect medium (Invitrogen Corporation, CA) supplemented with 10% FBS in plug-capped 75-cm2 cell culture flasks. Cells were removed by scraping and pelleted at 2,500 x g for 5 min. Cells were then washed and resuspended in 20 ml of fresh medium, and 5-ml aliquots were transferred into 75-cm2 flasks containing 15 ml of fresh medium. Flasks were incubated at 34°C for cells to reach confluence (2 to 3 days). Prior to infection, cell suspensions were split and incubated at room temperature (22 to 25°C) for 2 days.
Rickettsiae.
Host cell-free rickettsiae were obtained as previously described with modifications (13, 23). Rickettsia-infected cells were grown to a sufficient quantity (one 75-cm2 flask of R. montanensis-infected L929 mouse fibroblasts or R. peacockii-infected DAE100 cells). R. felis was partially purified from 1 ml of frozen R. felis-infected Vero cells. Cells were harvested by washing with fresh media until cells detached from the flask, pelleted at 2,500 x g for 3 min, resuspended in 12 ml of fresh medium, and lysed by being subjected to a vortex with sterile 3-mm borosilicate glass beads (Sigma-Aldrich, MO) for 5 min. Cell lysates were transferred aseptically into 15-ml centrifuge tubes and centrifuged at 4°C and 2,500 x g for 10 min to pellet cellular debris. The supernatants were aseptically transferred into 10-ml syringes and serially filtered through a 5-µm-pore-size Millex syringe filter (Millipore, MA) and then through a 2.7-µm-pore-size syringe filter (Whatman Inc., NJ).
Cell line inoculation.
To quantify rickettsiae, bacteria were stained with LIVE/DEAD BacLight stain according to the instructions of the manufacturer (Molecular Probes; Invitrogen Corporation, CA). Live rickettsiae were counted on a hemacytometer illuminated on a fluorescence microscope and were adjusted to a density of 107 rickettsiae/ml. The number of Aa23 or Sua5B cells was determined using a hemacytometer, and the density was adjusted to 106 cells/ml. A 500-µl rickettsial inoculum was applied to Sua5B or Aa23 cells grown in Falcon 24-well plates (Becton Dickinson) from which the medium had been removed. Plates were incubated for 2 h at room temperature, followed by the addition of 500 µl of medium/well. Plates were incubated at room temperature for 3 days, and then cells were transferred into 12.5-cm2 culture flasks. The cell suspension added to each flask was initiated from pooled cells from three replicate wells. Cells from an additional three replicate wells were used for PCR and staining with Diff-Quik Wright-Giménez stain (Dade Behring, IL). Two negative controls were included: (i) no inoculum and (ii) heat-killed rickettsiae. Each trial was replicated at least once. Inoculated cultures were sampled weekly and evaluated for infection using Diff-Quik staining and/or fluorescence in situ hybridization (FISH) and PCR. Infected cells were maintained in Schneider's insect medium-5% FBS at room temperature and passaged every 7 days for eight or more passages. Uninfected layers of Aa23 cells at 50% confluence were overlaid with infected Aa23 cells, while infected Sua5B cells were split into fresh medium.
To demonstrate that rickettsiae passaged through mosquito cells were still capable of infecting the cell lines from which they were derived, rickettsiae were partially purified from mosquito cells and applied to Vero, L929, or DAE100 cells as described above. Reinfection was confirmed by FISH, PCR, and reverse transcriptase PCR (RT-PCR). Because our primary objective was the confirmation of Rickettsia infection status, and not the establishment or maintenance of Rickettsia in mammalian cells, we confirmed infection only to passage 3.
DNA extraction and PCR.
DNA was extracted from cells by using the Wizard genomic DNA purification kit according to the instructions of the manufacturer (Promega Corp., WI). PCR amplification of Rickettsia rompA and of the genes encoding the 17-kDa protein and the bacterioferritin comigratory protein (bcp) was conducted on all extracted DNA by using Rickettsia genomic DNA as a positive control and a no-template negative control (Table 1). Each 25-µl reaction mixture consisted of 1 µl of template DNA, 0.4 µM concentrations of all forward and reverse primers, and Promega 2x PCR master mix. The PCR program consisted of 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 2 min followed by a final step at 72°C for 10 min. Restriction enzyme digestion of amplified rompA fragments from each species was conducted using AluI and PstI (New England Biolabs, MA). Alu digestion was conducted by adding 2.5 µl each of AluI and buffer NEB 2 (New England Biolabs, MA) to the amplified rompA product (4 µg) and incubating for 4 h at 37°C. PstI digestion was conducted by adding 2.5 µl each of PstI, 10x bovine serum albumin, and buffer NEB 3 (New England Biolabs, MA) to the amplified rompA product (4 µg) and incubating for 4 h at 37°C. All PCR products were separated on 1% Tris-acetate-EDTA agarose gel by electrophoresis and visualized by using ethidium bromide.
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Cloning and sequencing of amplified products.
PCR and RT-PCR amplicons were separated by 1% Tris-acetate-EDTA-agarose gel electrophoresis and purified using the StrataPrep DNA gel extraction kit (Stratagene, CA). Amplicons were ligated into the TOPO TA cloning vector, and Top10 chemically competent cells (Invitrogen) were transformed with the vector. Clones were selected by blue-white screening, and plasmids were extracted using the QIAprep spin miniprep kit (QIAGEN). Sequences were analyzed on an Applied Biosystems 3730XL high-throughput 96-capillary or an ABI 3100 16-capillary DNA sequencer at the University of Maryland, Baltimore, Biopolymer/Genomics Core Facility.
FISH.
General FISH to detect the presence of rickettsiae in 4% formalin-fixed cells was conducted as described by Gottlieb et al. (11). Infected cells were cytocentrifuged onto a slide (Wescor, Inc., UT) or grown overnight in Labtek chamber slides (8-well Permanox slides; Nalge Nunc International). In the latter case, the chamber walls and the gasket were removed after fixing cells for 20 min in 4% formalin-phosphate-buffered saline at room temperature. The Rickettsia-specific FISH probe was conjugated with a 5'-end 6-carboxyfluorescein label and is specific to all known rickettsial 16S rRNA genes (5'-6-carboxyfluorescein-TCCACGTCGCCGTCTTGC) (11). The hybridization probe was prepared to a final concentration of 10 pmol/ml as described by Rasgon et al. (23). Probed slides were incubated in a humid chamber at 37°C overnight. Slides were washed once in 1x SSC (0.15 M NaCl plus 0.015 M sodium citrate)-10 mM dithiothreitol, counterstained with Evans blue (0.1% in 1x phosphate-buffered saline), and incubated at 37°C for 15 min. Slides were then subjected to a series of washes as described by Rasgon et al. (23). Slides were mounted in 50 µl of ProLong (Invitrogen) containing DAPI (4',6'-diamidino-2-phenylindole; 1 ng/ml) and observed under oil immersion (100x objective) by using an epifluorescence microscope fitted with a green (fluorescein isothiocyanate) filter, a blue (UV DAPI) filter, and a red (tetramethyl rhodamine isocyanate) filter. Pictures taken were exposed for 0.5 to 0.75 s in a 2- by 2-pixel bin at gamma 1 by using a SPOT RT digital camera (Diagnostic Instruments, Inc., MI), and images were merged with SPOT advanced imaging software (Universal Imaging, PA). Uninfected mosquito cells, Aa23 and Sua5B cells mixed with Escherichia coli that had been grown in Luria-Bertani broth overnight, and Wolbachia-infected Aa23 cells were used as negative controls to demonstrate the specificity of the probe for Rickettsia.
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RESULTS |
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Rickettsial morphology varied with growth stage, an observation previously made by Wisseman et al. (28) and Wisseman and Waddell (29). Log-phase rickettsiae were dividing, often appearing bacillary (Fig. 1a and e) or forming short chains (Fig. 1c). Stationary-phase rickettsiae were smaller coccobacilli (Fig. 1b and f). We observed rickettsiae surrounding the cell nuclei (Fig. 1b and d), dispersed throughout the cytoplasm (Fig. 1), located within filopodia (Fig. 1e), and positioned in cell-to-cell bridges between adjacent cells (Fig. 1a and d) that were grown on Labtek chamber slides. Because we did not perform confocal microscopy, we could not discern whether rickettsiae had in fact entered the nuclei. However, we observed rickettsiae surrounding cell nuclei (stained with DAPI) and localizing in the nuclear region (Fig. 2). In later stages of infection, extracellular rickettsiae were also observed (Fig. 2).
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R. peacockii.
R. peacockii was maintained most efficiently in mosquito cells at room temperature in 5% FBS with semipersistent passaging for Aa23 cells or persistent passaging for Sua5B cells. While initial exposure to R. peacockii caused many cells in both lines to detach and round up, subsequent passages resulted in good R. peacockii growth without significant cell death. Counts of rickettsiae ranged from 0 to more than 50 per cell, with at least 80% of cells being infected. Molecular confirmation of R. peacockii infection was achieved by restriction digestion of PCR-amplified rompA and by RT-PCR (Table 2). We sequenced PCR products of the 17-kDa-protein gene, rompA, and bcp from both mosquito cell lines infected with R. peacockii and RT-PCR products of bcp from R. peacockii-infected cells (Table 2). R. peacockii was able to reinfect DAE100 cells when partially purified from mosquito cells at passage 9 (Table 2). R. peacockii was detected in L929 cells in the third passage by using RT-PCR, although it was lost after the second passage in Vero cells (both grown at 34°C with 5% CO2).
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DISCUSSION |
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To our knowledge, neither R. montanensis nor R. peacockii has been maintained previously in mosquito cell systems. A prior attempt to culture R. montanensis in Aedes albopictus AeAl2 cells was unsuccessful, although attachment did occur (27). Similarly, R. peacockii was not maintained in Aedes albopictus C7/10 cells, although it grew well in several tick species and one lepidopteran line (Trichoplusia ni) (13). Differences in Aedes albopictus cell line characteristics may explain why both R. peacockii and R. montanensis were able to grow in the particular Aedes albopictus cell line we used but did not grow in either of the cell lines used by Kurtti et al. and Uchiyama (13, 27).
The culturing of R. peacockii in mosquito cells is significant because R. peacockii is considered to be deficient in the abilities to invade and grow in mammalian cells. Unlike other SFG species, R. peacockii does not polymerize actin owing to the deletion of a rickA gene (26). Nor does R. peacockii express the outer membrane protein encoded by rompA, although it does possess the rompA gene and transcripts have been detected previously (4, 26). R. peacockii is not known to adhere to tick cells, and tick cells exposed to R. peacockii have not been observed to exhibit phagocytic behavior, suggesting that R. peacockii may have a disadvantage in entering host cells (15). Both Sua5B and Aa23 cell lines have hemocyte-like characteristics and are capable of phagocytosis (8, 17). Since many rickettsiae enter cells by inducing endocytosis, it is conceivable that R. peacockii could infect mosquito cells that exhibit phagocytic behavior provided that R. peacockii could avoid digestion. Based on RT-PCR and FISH visualization of R. peacockii after nine passages, it would appear that R. peacockii somehow avoids digestion and grows in the cytoplasm of mosquito cells. Somewhat paradoxically, R. peacockii was transcriptionally active in L929 cells after the third passage. It remains to be seen if R. peacockii infections can be sustained for several more passages in L929 or other mammalian cell lines.
Previous studies have demonstrated the successful maintenance of R. felis and R. conorii in the Aedes albopictus cell line C6/36 (12, 25). We therefore anticipated that R. felis would grow in Aa23 cells. Similarly, R. felis infection of Sua5B cells was also expected as Sua5B cells have previously supported the growth of an intracellular endosymbiont Wolbachia species (21). The R. felis LSU strain we used was originally derived from colonies of R. felis-infected cat fleas and has been transovarially maintained in these fleas for several years. The strain may therefore have characteristics of an adapted flea colony rather than those of a wild-type strain. We have recently acquired the Pedreira strain of R. felis and are currently maintaining it in Aa23 and Sua5B cells (seven passages to date). Again, this outcome was not surprising since the Pedreira strain was originally isolated from wild-caught R. felis-infected cat fleas and maintained in Aedes albopictus C6/36 cells (12).
R. felis falls phylogenetically into a transitional group between the spotted fever and typhus groups and is transmitted by fleas (10). The use of an insect cell line to study this TRG rickettsial pathogen may provide insight into the evolution of Rickettsia-vector interactions and perhaps host jumps from acarine to insect vectors or visa versa. Additionally, we may be able to explain why mosquitoes are not infected with Rickettsia spp. but many are infected with Wolbachia. For example, if infection-dependent gene expression in mosquitoes elicited by Wolbachia differs from that elicited by exposure to Rickettsia spp., this finding may explain why Rickettsia spp. have not become established in mosquitoes. Furthermore, the use of the R. felis-insect interactions may shed light into what genes and/or environmental cues cause rickettsial species to exist as endosymbionts, reproductive parasites, or pathogens.
Because these two cell lines were supportive of R. peacockii growth, it is conceivable that they can be used to isolate and maintain the growing number of newly described nonpathogenic rickettsiae from a diverse range of invertebrate and protozoan hosts. The significance of nonpathogenic rickettsiae extends beyond ecological or evolutionary interests and may have implications relevant to public health. First, nonpathogenic rickettsiae may serve as sources of emerging vertebrate pathogens. Perlman et al. suggested that since the majority of rickettsial diversity lies in species associated with invertebrates, ancestral rickettsiae are associated with invertebrates and only secondarily develop into vertebrate pathogens by rare horizontal transmission events in evolutionary time (20). Second, rickettsiae commonly referred to as nonpathogenic may in fact be pathogenic rickettsiae whose pathogenicity was not immediately apparent upon discovery. R. parkeri is one such example, having been considered nonpathogenic until 60 years after its discovery (18). Likewise, R. canadensis and R. montanensis have been referred to as being of unknown pathogenicity (19, 22). Third, some nonpathogenic rickettsiae appear to affect the epidemiology of pathogenic rickettsiae through competition for the same tick niche. The classic example of this relationship is the geographic limitation of R. rickettsii to D. andersoni populations on the west side of the Bitterroot Valley of Montana, presumably through transovarial exclusion by the closely related nonpathogenic R. peacockii (13, 15, 16). Last, nonpathogenic rickettsiae may serve as useful model systems and provide vital clues into the genetic components of rickettsial pathogenicity. Thus, the availability of these cell lines that will potentially support diverse Rickettsia species of various degrees of pathogenicity for rickettsial maintenance is highly advantageous for the study of nonpathogenic species.
The three Rickettsia species grown in these two cell lines vary in genetic characteristics and the degree of infectivity in eukaryotic cells. It is often difficult to make conclusions about their behavior without considering the cell line in which they were grown as a variable of their pathogenicity (or lack thereof). The use of a standard cell line such as Aa23 or Sua5B to grow all three species would remove the variation due to cell line differences as a factor. Further, it would be ideal to have a cell line that can support the growth of both insect-associated and tick-associated rickettsiae for studying broader questions of Rickettsia-host interactions. We propose that since both Aa23 and Sua5B cells support diverse rickettsiae of various degrees of pathogenicity derived from insect and tick sources, they are excellent candidates for future studies into the evolution of rickettsial pathogenicity and the coevolution of Rickettsia species with their arthropod vectors and/or hosts.
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ACKNOWLEDGMENTS |
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The work presented here is supported by NIH/NIAID grants AI017828 and AI063534.
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FOOTNOTES |
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Published ahead of print on 31 August 2007.
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