A Metabolic Dependency for Host Isoprenoids in the Obligate Intracellular Pathogen Rickettsia parkeri Underlies a Sensitivity to the Statin Class of Host-Targeted Therapeutics

Obligate intracellular pathogens, which include viruses as well as certain bacteria and eukaryotes, are a subset of infectious microbes that are metabolically dependent on and unable to grow outside an infected host cell because they have lost or lack essential biosynthetic pathways. In this study, we describe a metabolic dependency of the bacterial pathogen Rickettsia parkeri on host isoprenoid molecules that are used in the biosynthesis of downstream products, including cholesterol, steroid hormones, and heme. Bacteria make products from isoprenoids, such as an essential lipid carrier for making the bacterial cell wall. We show that bacterial metabolic dependency can represent a potential Achilles’ heel and that inhibiting host isoprenoid biosynthesis with the FDA-approved statin class of drugs inhibits bacterial growth by interfering with the integrity of the cell wall. This work supports the potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism.


RESULTS
The isoprenoid biosynthesis pathway is under evolutionary flux in the order Rickettsiales. We first sought to determine the evolutionary conservation of the isoprenoid biosynthesis pathway in the order Rickettsiales. We analyzed six major genera of the order and assessed the presence or absence of genes encoding upstream or downstream components of the MEP pathway as well as the central isoprenoid pathway enzyme, IDI ( Fig. 2A; see also Table S1 in the supplemental material). We confirmed that the Rickettsia (14) and Orientia lineages lack the genes encoding upstream MEP pathway enzymes but retain those encoding the downstream pathway components, whereas the other genera contained the full complement of MEP pathway C. genes. None have MEV pathway genes, but the idi gene is present in Rickettsia species. The absence of upstream MEP pathway genes suggests that Rickettsia species must scavenge MEV/MEP pathway intermediates (IPP, DMAPP and/or FPP) ( Fig. 1) from the host cell. Additionally, the idi gene is present only in the Rickettsia lineage and not in the Orientia lineage. A previous report suggested that the idi gene in Rickettsia species was acquired by horizontal gene transfer (14). To further investigate this possibility, we examined the genomic locus surrounding the idi gene in the genome of the R. parkeri strain Portsmouth (Fig. 2B). We found many surrounding genes that were associated with conjugal transfer functions, such as transposases and pili. Furthermore, there are nine surrounding pseudogenes with premature stop codons, suggesting that this region is in flux and may be in the process of undergoing genomic reduction. To further test this hypothesis, we performed a whole-genome alignment between R. parkeri and the closely related yet more pathogenic species Rickettsia rickettsii. Interestingly, we found that the two genomes were highly syntenic, with the exception of a 65-kb region containing the idi gene (Fig. 2C, region in lime green and idi gene in purple) that is inverted between the two species and that in the R. rickettsii genome contains a 33.5-kb deletion ( Fig. 2C orange highlighted region; Table S2). These observations suggest that the locus surrounding the idi gene was acquired by horizontal gene transfer (in agreement with previous phylogeny estimation [14] and with reports of similar genome rearrangements in Rickettsia species [16]) and is still under evolutionary pressure to retain the idi gene while simultaneously undergoing continuing reduction.

Pseudogene
R. parkeri infection results in depletion of host isoprenoid products and accumulation of bacterial isoprenoid products. To test whether R. parkeri scavenges host isoprenoids to make bacterial products, we measured the presence and abundance of host and bacterial isoprenoid-derived metabolites by liquid chromatography tandem mass spectrometry (LC-MS/MS). Confluent infected and mock-infected cultures of African green monkey kidney epithelial Vero cell cultures were incubated for 4 days, collected, and then extracted for lipid metabolites. For host isoprenoids, we monitored cholesterol, total cholesteryl esters, cholesteryl oleate, and ubiquinone-10 using singlereaction monitoring (SRM)-based LC-MS/MS methods (Fig. 3). We observed a statistically significant, approximately 2-fold, decrease in total cholesteryl esters and cholesteryl oleate in the infected samples compared to levels in uninfected samples. These products are generally understood to be the storage units of cholesterol packaged in intracellular lipid droplets (17). In contrast, we saw no change in cholesterol or ubiquinone-10. Thus, infection results in the depletion of isoprenoid-derived storage forms of cholesterol. For bacterial isoprenoids, we measured bactoprenols, including the isoprenoid products C 55 pyrophosphate (C 55 -PP) and C 55 phosphate (C 55 -P) (based on targeting for the [M-H] Ϫ parent masses because there were no authentic standards available). C 55 -PP is initially produced by a dedicated prenyltransferase, UppS, and must be dephosphorylated to C 55 -P to act as a lipid carrier (18). We also measured bacterial ubiquinone, ubiquinone-8, which contains an 8-prenyl subunit tail, in contrast with the human version ubiquinone-10, which contains 10 prenyl subunits (Fig. 3) (19,20). We observed an ϳ4-fold increase in both C 55 -PP and C 55 -P in infected cells compared to levels in uninfected cells (values were normalized to internal standards [21,22]). We did not, however, find significant differences in bacterial ubiquinone (ubiquinone-8) levels, possibly due to the presence of ubiquinone-8 intermediates from the host cell ubiquinone-10 biosynthesis pathway. These results suggest that bacterial isoprenoid products, primarily bactoprenols, accumulate during R. parkeri infection. Collectively, the depletion of host isoprenoid products and accumulation of bacterial isoprenoid products, even in the absence of a bacterial isoprenoid synthesis pathway, suggest that isoprenoids are scavenged from the host by R. parkeri.
Chemical inhibition of the host mevalonate pathway inhibits bacterial growth. To determine whether host isoprenoids are necessary for bacterial growth, we sought to inhibit the host mevalonate pathway and reduce the pool of available host IPP, DMAPP, and FPP. We used statins, a class of drugs that block the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Fig. 1). A previous report showed that preincubation of Rickettsia conorii-infected mouse L929 fibroblast cells with the statin lovastatin caused a reduction in R. conorii plaque size, which was interpreted to result from a reduction in cholesterol-dependent adherence of bacteria to host cells (23). To bypass possible effects of statins on bacterial adherence and invasion, we allowed infection of Vero cells to proceed for 2 h to allow for maximal bacterial adherence/invasion (24) and then, using untreated cells and cells treated with various concentrations of the statin pitavastatin, measured bacterial numbers using an R. parkeri-specific quantitative PCR (qPCR) endpoint assay (Fig. 4A). We observed a Ͼ2-log reduction in bacterial growth with increasing pitavastatin concentrations. To ensure that statin inhibition of bacterial growth was dependent on HMG-CoA reductase inhibition by pitavastatin and not on secondary effects of the drug, we tested whether the downstream product of HMG-CoA reductase, mevalonate, could rescue this growth inhibition. Indeed, cotreatment with 400 M mevalonate and pitavastatin rescued bacterial growth (Fig. 4A).
To further assess the importance of the MEV pathway, we generated dose-response curves for the effects of lovastatin and pitavastatin, which are chemically distinct, on R. parkeri growth using a 96-well qPCR endpoint assay. We observed a dose-dependent inhibition of bacterial growth with lovastatin (50% effective concentration [EC 50 ], 2.0 M) and even more robust inhibition with pitavastatin (EC 50 , 0.5 M) (Fig. 4B). The effect of statins on bacterial growth was not due to host cell death as treatment of uninfected cells throughout the dose-response range caused no significant loss of cell viability as measured by lactate dehydrogenase release ( Fig. S1A) (although subcytotoxic concentrations of pitavastatin have been shown to alter cellular phenotypes such as the proportion of cells in different stages of the cell cycle, oxidant-induced apoptosis, or viability of different cancel cell lines [25][26][27]). Furthermore, host cells remained adherent throughout the dose-response range although there was an apparent decrease in cell-cell contact at concentrations of lovastatin and pitavastatin greater than the EC 50 for bacterial growth inhibition (Fig. S1B). Together, these data indicate that the host MEV pathway is critical for R. parkeri growth.
To further test that bacterial growth is dependent on a complex integration of the host and pathogen isoprenoid pathways (depicted in Fig. 1), we tested for dose-dependent growth inhibition using additional inhibitors of MEV and MEP pathway enzymes (Fig. 4B). Fosmidomycin, which specifically inhibits 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, a key enzyme in the bacterial MEP pathway, caused no inhibition of bacterial growth at up to 100 M. Alendronate sodium, a specific inhibitor of host farnesyl diphosphate (FPP) synthase, caused approximately 50% growth inhibition. This further suggests that R. parkeri growth is independent of bacterial isoprenoid production and partially dependent on FPP from the host.
Finally, we tested the antibiotic tetracycline, which is used as a first-line treatment for Rickettsia species infection and targets protein synthesis, or D-cycloserine, which blocks the PG biosynthesis pathway that also requires isoprenoid biosynthesis products. Tetracycline inhibited growth, with an EC 50  parkeri genome copy numbers in the presence of various concentrations of pitavastatin, without or with mevalonate. Two independent biological replicates were performed, but four technical replicates from a single experiment are shown for simplicity. Error bars represent standard deviations. Statistical comparisons were done by an unpaired Student's t test for each concentration of pitavastatin for the wild type versus the wild type with mevalonate supplementation (ns, not significant; ***, P Ͻ 0.001; ****, P Ͻ 0.0001). R.p., R. parkeri. (B) Dose-dependent growth inhibition of R. parkeri in the presence of the indicated drugs targeting the MEV or MEP pathway enzymes or of tetracycline, normalized to that of a no-drug control. Fosmidomycin and alendronate failed to generate fit curves.
(MIC of 30 g/ml, or 290 M) (30) and Mycobacterium tuberculosis (MIC of 15 g/ml, or 150 M) (31). Taken together, the results of chemical inhibition assays support the notion that Rickettsia species are dependent on the upstream host MEV pathway but not the upstream MEP pathway. Furthermore, they are susceptible to inhibition of PG biosynthesis.
Statin treatment causes bacterial shape defects that mimic those caused by peptidoglycan-targeting antibiotics. We sought to further assess the effect of statin treatment on bacterial physiology. Limiting the availability of isoprenoids is predicted to result in reduced PG synthesis. Though Rickettsia species (28), like other Gramnegative bacteria (32), are generally resistant to PG-targeted antibiotics, one study showed that high concentrations of penicillin produced spheroplasts (33), a type of L-form bacteria that have defective PG cell walls and take on a spherical shape (34). Therefore, we sought to measure whether statin inhibition of the host MEV pathway altered bacterial shape in a similar manner to inhibition of PG synthesis. To this end, we performed immunofluorescence microscopy on 96-well plates of infected cells at 3 days postinfection (dpi) to track the alterations of bacterial shape in response to dosedependent application of statins, D-cycloserine, or tetracycline as a control. We performed automated image analysis using CellProfiler (35) and measured features of bacterial cell shape. We determined that two shape measurements, area and eccentricity (circle ϭ 0; rod/line ϭ 1), accounted for most of the shape alterations seen in the concentration ranges between the EC 50 and EC 100 . In D-cycloserine-treated cells, there was a dose-dependent increase in area and decrease in eccentricity as bacteria became more circular and less rod shaped ( Fig. 5A and B). In cells treated with either lovastatin or pitavastatin, similar changes in area and eccentricity were seen along the doseresponse curve. In contrast, upon treatment with tetracycline, we found little change in the bacterial area although there was a general decrease in eccentricity.
Upon comparison of shape at the EC 100 of each drug with that of the untreated control, we observed a significant increase in bacterial area and decrease in eccentricity in cells treated with D-cycloserine, lovastatin, and pitavastatin (Fig. 5C). For tetracycline, we found no significant differences in area or eccentricity compared to those of the untreated control group, likely because tetracycline does not directly interfere with cell wall biosynthesis. Thus, growth inhibition by statins causes bacterial shape defects that are similar to those caused by D-cycloserine, suggesting a shared mechanism of bacterial growth inhibition resulting from targeting host isoprenoid and bacterial PG synthesis pathways.

DISCUSSION
Obligate intracellular pathogens such as Rickettsia species are dependent upon nutrients and metabolites from their host cells as a consequence of reductive genome evolution (5,6,14). This suggests the possibility that this metabolic dependency represents an Achilles' heel in the host-pathogen relationship and that these pathogens could be sensitive to changes in host cell nutrient and metabolite levels. If metabolite availability is limited by the host or by extrinsic pressures, such as chemical inhibition of a host biosynthetic pathway, pathogen growth should also be reduced. Here, we report the reduction of R. parkeri replication when the host isoprenoid pathway is inhibited with statins. This growth inhibition correlates with changes in bacterial shape that are consistent with defects in cell wall biosynthesis. Our results suggest that statins interfere with Rickettsia species scavenging of host isoprenoids used for downstream biosynthetic pathways such as peptidoglycan biosynthesis, measured in this study.
We observed that the isoprenoid biosynthesis pathway is under evolutionary flux in the order Rickettsiales, with the Rickettsia and Orientia lineages having lost genes encoding the upstream components of the MEP pathway and Rickettsia species having gained the idi gene, consistent with previous findings (14). Through our understanding of reductive genome evolution and gene acquisition in prokaryotes (5,6,8,36), we can surmise a most parsimonious order of loss and gain of isoprenoid biosynthetic pathway genes in the Rickettsiales. We propose that the common ancestor of the Rickettsia and Error bars represent the 95% confidence interval. (C) Graphs plotting area and eccentricity measurements at the EC 100 values calculated from the data shown in Fig. 4B and matched to measurements from panel A. Regions shaded in yellow are the calculated values of EC 50 to EC 100 from the data shown in Fig. 4B. Graphs show the mean of the sample set, and error bars represent the 95% confidence interval. Statistical comparisons were done by one-way ANOVA of results for all drug-treated samples compared to results for the no-drug (nd) sample set (ns, not significant; **, P Ͻ 0.01; ***, P Ͻ 0.001; ****, P Ͻ 0.0001). dcs, D-cycloserine; tet, tetracycline; lov, lovastatin; pit, pitavastatin.
Orientia lineages, which inhabit the host cell cytoplasm, may have gained the ability to scavenge IPP, DMAPP, and/or FPP from the cytoplasm by acquisition of a gene encoding a transporter that moves these highly charged pyrophosphate molecules into the bacterial cell. There is evidence for the presence of such transporters in plants, bacteria, and protozoan parasites although their molecular identities remain uncertain (37)(38)(39). Furthermore, previous studies in R. prowazekii have implicated metabolite transporters that allow the acquisition of triose phosphates from the host cell (40,41). Gaining an IPP transporter would have enabled the loss of the upstream MEP pathway genes to reduce the bacterial genome and reduce fitness costs of pathway redundancy. Subsequently, the idi gene was gained in the Rickettsia lineage, which may reflect a need for additional DMAPP beyond that acquired from the host MEV pathway. In contrast, Ehrlichia, Anaplasma, Wolbachia, and Neorickettsia lineages, which grow within a membrane-bound vacuole, may not have sufficient access to host IPP and/or DMAPP and therefore have retained the entire MEP pathway. We also found evidence of metabolic parasitism of host isoprenoids by R. parkeri. In particular, infected host cells became depleted of isoprenoid-derived storage forms of cholesterol, total cholesteryl esters, and choleteryl oleate, perhaps to balance free cholesterol levels. Curiously, we did not find differences in host or bacterial ubiquinone levels during infection, perhaps due to host compensation for reduced ubiquinone in response to infection. An additional complication of this metabolite analysis is the fact that ubiquinone-8 is an on-pathway intermediate in the synthesis of host ubiquinone-10, possibly masking our ability to distinguish changes in either host or bacterial ubiquinones in infected versus uninfected cells by mass spectrometry. Whether Rickettsia species also scavenge downstream host isoprenoid products such as cholesterol, as observed for other bacteria such as M. tuberculosis and Chlamydia trachomatis (42)(43)(44), remains to be determined. Furthermore, R. parkeri produces downstream isoprenoid products even though it lacks genes for the upstream components of the MEP pathway. This metabolic parasitism suggests that inhibition of host isoprenoid biosynthesis should reduce the ability of the bacteria to synthesize bacterial isoprenoid products.
In keeping with this prediction, we found that statins, which inhibit host HMG-CoA reductase, halt R. parkeri growth. Previous work had established an ability of statins to limit rickettsial plaque size (23), but the mechanism of inhibition was not explored in detail. It was suggested that statins might inhibit bacterial adherence to and/or invasion of host cells based on previous studies that had found a role for cholesterol in these processes (45,46). Our work reveals an effect of statins on intracellular bacterial growth, downstream of adherence/invasion, as well as an effect on bacterial morphology that mirrors that which is caused by the cell wall synthesis inhibitor D-cycloserine. Whether statins have a bactericidal or bacteriostatic effect remains to be determined, but we observed morphological evidence of bacterial lysis at higher concentrations of statins, similar to lysis effects seen with D-cycloserine treatment but not with tetracycline treatment. These observations led us to conclude that statins inhibit bacterial growth by reducing host metabolite availability for production of bacterial products required for PG biosynthesis, leading to lethal defects in the bacterial cell wall. Future studies are also needed to determine if there are additional effects of statins on bacterial O-antigen and ubiquinone-8.
Whether statins can be effective at preventing or treating human Rickettsia species infections remains to be determined. Although statins are well tolerated in humans, we have limited understanding of the concentrations that would inhibit bacterial growth without causing toxicity. It is also unclear if statins cause the same degree of bacterial growth inhibition in different rickettsial target cell types in vivo. Additional studies in animal models will shed light on the future promise of statins as a prophylaxis or treatment for rickettsial infections. Currently, cases of rickettsial diseases are effectively treated with tetracyclines although there are reported cases of tetracycline-resistant scrub typhus (47,48), and tetracycline treatment is contraindicated for pregnant women and young children (49). Statins are also contraindicated for pregnant women although in children (50) pitavastatin has been found to be both efficacious and safe (51). Because a limited number of antibiotics are effective in treating rickettsial infections (28), host-targeted therapeutics would be useful additions to treatment regimens. Furthermore, targeting host biosynthesis of essential metabolites may limit the development of antibiotic resistance.
Beyond the scope of rickettsial infections, this study supports the hypothesis of using host-targeted therapeutics for a wide variety of infectious microbes, including viruses, bacteria, fungi, and parasites. Similar to R. parkeri, the parasite Cryptosporidium parvum lacks the canonical protozoan isoprenoid pathway and has been shown to be sensitive to statins (52). Interestingly, the facultative intracellular protozoan parasite Toxoplasma gondii, which encodes the MEP isoprenoid pathway within a membranebound organelle called the apicoplast, has been shown to be sensitive to statins when the parasite is intracellular (53,54), suggesting that host metabolite scavenging is still advantageous for pathogens with intact pathways. In the future, bioinformatic analyses may identify other intracellular bacteria that have incomplete upstream isoprenoid pathways and intact downstream isoprenoid pathways and thus may also be sensitive to statins. Furthermore, Rickettsia species and other parasites may be sensitive to chemical inhibition of other host metabolic pathways. Layering our knowledge of FDA-approved compounds that target host biosynthetic pathways onto predicted metabolic pathways hijacked by pathogens may therefore enable the systematic identification of drug classes that could be repurposed as antibiotics.
Cell culture and R. parkeri infections. Confluent low-passage-number African green monkey epithelial Vero cells were obtained from the University of California Berkeley Cell Culture Facility and grown at 37°C in 5% CO 2 and in high-glucose (4.5 g/liter) Dulbecco's modified Eagle's medium (DMEM) (11965092; Gibco/Life Technologies) supplemented with 2% fetal bovine serum (FBS; Benchmark). R. parkeri strain Portsmouth was generously provided by Chris Paddock (Centers for Disease Control and Prevention). R. parkeri was propagated by infecting monolayers of Vero cells with wild-type R. parkeri at a multiplicity of infection (MOI) of 0.1 and growing them at 33°C in 5% CO 2 in DMEM plus 2% FBS. Bacteria were purified from infected cells as described previously (56). Briefly, infected cells were lysed by Dounce homogenization in cold K-36 buffer (0.05 M KH 2 PO 4 , 0.05 M K 2 HPO 4 , pH 7, 100 mM KCl, 15 mM NaCl) to release bacteria; the lysate was overlaid onto 30% MD-76R (1317-07; Mallinckrodt, Inc. ), centrifuged at 58,300 ϫ g for 20 min at 4°C in an SW-28 swinging bucket rotor, and resuspended in cold brain heart infusion (BHI) broth (237500; BD Difco) Aliquots of bacteria were immediately frozen at -80°C after purification, and each infection was initiated from a single thawed aliquot of bacteria.
Rickettsiales pathway analyses, idi locus mapping, and whole-genome alignments. Cladogram schema were manually drawn based on relative distances from a previous phylogeny study of small subunit rRNA genes from the order Rickettsiales (57). The presence/absence of the MEP pathway or idi gene was determined using the KEGG pathway database (http://www.kegg.jp/) and manual annotations of the terpenoid backbone biosynthesis pathway (http://www.kegg.jp/kegg-bin/show_pathway?map ϭmap00900) for each bacterial species. Determination of intracellular niche was performed by manual literature curation. The idi locus map and annotations were examined using Geneious software, version 9.1.8, using the R. parkeri strain Portsmouth genome sequence (NCBI RefSeq NC_017044.1). Wholegenome alignments of R. parkeri strain Portsmouth and R. rickettsii strain Iowa (NCBI RefSeq NC_010263.3) were performed using the Mauve, version 2.3.1 (58), plug-in in Geneious software using the progressive Mauve algorithm with automatically calculated-minimum locally colinear block (LCBs) scores.
Mass spectrometry. Monolayers of uninfected control Vero cells or Vero cells infected with R. parkeri were plated in six-well plates as described above and were incubated for 4 days at a 33°C in 5% CO 2 . On the day of harvest, cells in each well were washed with 4 ml of 1ϫ phosphate-buffered saline (PBS). One milliliter of fresh 1ϫ PBS was added to each well and used to scrape the cells, samples were centrifuged at 1,000 ϫ g for 5 min on a microcentrifuge, and the supernatant was aspirated from the cell pellet. Cell pellets were extracted in 2:1:1 chloroform-methanol-PBS solution with addition of 10 nM dodecyl glycerol internal standard, after which the organic phase was separated, collected, and dried down under N 2 gas. The dried-down lipidome extract was then resuspended in 150 l of chloroform and stored at Ϫ80°C until analysis. For mass spectrometry analysis, an aliquot of this sample solution was injected into an Agilent 6430 LC-MS/MS instrument and was analyzed by single-reaction monitoring (SRM)-based targeted LC-MS/MS. Metabolite separation was performed using reverse phase chromatography, using a Luna reverse phase C 5 column (50 mm by 4.6 mm with 5-m diameter particles; Phenomenex), using the following mobile phases: buffer A consisting of 95:5 water-methanol and buffer B consisting of 60:35:5 2-propanol-methanol-water, both with 0.1% formic acid and 50 mM ammonium formate additives. The flow rate began at 0.1 ml/min for 5 min, followed by a gradient starting at 0% buffer B and increasing linearly to 100% buffer B over the course of 40 min with a flow rate of 0.4 ml/min, followed by an isocratic gradient of 100% buffer B for 10 min before equilibration for 5 min at 0% buffer B with a flow rate of 0.4 ml/min. MS analysis was performed using electrospray ionization (ESI) with a drying gas temperature of 350°C, drying gas flow rate of 10 liters/min, nebulizer pressure of 35 lb/in 2 , capillary voltage of 3.0 kV, and fragmentor voltage of 100 V. C 55 -P and C 55 -PP levels were measured based on targeting for the parent [M-H] Ϫ mass since there were no authentic standards available. The retention times for these metabolites correspond with the expected retention times based on those of shorter-chain pyrophosphate metabolites. For cholesterol, cholesteryl ester totals, cholesteryl oleate, and ubiquinone-10/8, we used single-reaction monitoring (SRM)-based methods monitoring the MS1-to-MS2 transitions based on fragmentation of standards. C 55 -P and C 55 -PP were measured in negative ionization mode, whereas cholesterol, total cholesteryl esters, cholesteryl oleate, and ubiquinone-10/8 were measured in positive ionization mode. Representative metabolites were quantified by SRM of the transitions from precursor to product ions at associated collision energies and retention times. Data were analyzed using Agilent Qualitative Analysis software by calculating the area under the curve (21,22), and values are in picomoles relative to the internal standard control equivalents.
Microscopy. To measure bacterial shape, Vero cells were plated at 50% confluence on 96-well glass-bottom plates in DMEM plus 2% FBS and allowed to settle overnight at 37°C in 5% CO 2 . The following day, cells were infected at an MOI of 0.1 of wild-type R. parkeri, centrifuged at 300 ϫ g for 5 min, and incubated for 2 h at 33°C in 5% CO 2 . Medium containing bacteria was aspirated and replaced with medium with or without the appropriate concentration of drug (see above), and cells were further incubated for 72 h at 33°C in 5% CO 2 . Cells were fixed with 4% paraformaldehyde in 1ϫ PBS for 20 min at room temperature, washed with 1ϫ PBS, and permeabilized with 0.05% Triton X-100 in 1ϫ PBS for 5 min. Cells were then stained for immunofluorescence with mouse anti-Rickettsia primary antibody 14-13 and goat anti-mouse Alexa-488 secondary antibody to stain bacteria and Hoechst 33342 to stain DNA. Infected cells were imaged on a Nikon Ti Eclipse microscope with a Yokogawa CSU-XI spinning disc confocal, 100ϫ (1.4 numerical aperture [NA]) plan apo objective, a Clara Interline charge-coupled-device (CCD) camera, and MetaMorph software taking 0.15-m z-slices across 5 m in the z-plane. Maximum z-projections for each channel were made using ImageJ, and a custom pipeline was created in CellProfiler software (35) to identify individual bacteria in each image. The CellProfiler module MeasureObjectSize-Shape was used to calculate size and shape parameters of each bacterium. Bright-field monolayer images were acquired using an Olympus IX71 microscope equipped with a 20ϫ LUCPlanFLN objective and a Photometrics CoolSnap HQ camera and MicroManager software.
R. parkeri qPCR growth and drug dose-response curves. Confluent Vero cells grown in 96-well tissue culture plates were infected with R. parkeri at an MOI of 0.1 and incubated in DMEM plus 2% FBS with or without the appropriate concentration of drug (see above) for 72 h at 33°C in 5% CO 2 . To harvest cells at the appropriate time point, medium was aspirated, and cells were lifted with 50 l of 0.25% trypsin-EDTA followed by incubation at 37°C for 5 min. Lifted cells were resuspended with an additional 50 l of DMEM before being added to 50 l of Nuclei Lysis Solution (Wizard Genomic DNA Purification kit; Promega) and frozen at Ϫ20°C overnight. Cells were then thawed and boiled for 10 min to release genomic DNA. To remove RNA, 20 g/ml RNase A was added to each sample, and samples were incubated for 15 min at 37°C and then cooled to room temperature. Protein was removed by addition of 50 l of protein precipitation solution, mixed by pipetting, and then centrifuged in a microcentrifuge for 15 min at 1,500 ϫ g at 4°C. DNA was precipitated by addition of 100 l of the resulting supernatant to 100 l of isopropanol, mixed by pipetting, and centrifuged for 15 min at 1,500 ϫ g at 4°C. Isopropanol was removed, and the DNA pellet was washed with 70% ethanol and centrifuged for 15 min at 1,500 ϫ g. Resulting DNA pellets were dried, resuspended in 50 l of H 2 O, and allowed to rehydrate overnight at 4°C. For quantitative real-time PCR, 5 l of genomic DNA was used with primers to the R. parkeri gene encoding the 17-kDa antigen (59), and runs were carried out on a Bio-Rad CFX96 Touch real-time PCR detection system. Rickettsia genome copy number was quantified against a standard curve of a plasmid containing a single copy of the R. parkeri 17-kDa gene. Regression analysis and EC 50 calculations were performed using GraphPad Prism, version 7, software.
LDH release assays. For lactate dehydrogenase (LDH) release assays, Vero cells were plated and grown to confluence in a 96-well plate in 100 l of DMEM (Gibco) containing 2% FBS (GemCell). The following day, medium was aspirated and replaced with 100 l of fresh medium containing pitavastatin or lovastatin and incubated at 33°C. After 72 h, 60 l of supernatant from wells containing Vero cells was collected into 96-well plates and 60 l of LDH buffer ( Sigma ]) was added to each well. Supernatants from untreated cells and from cells lysed with 1% Triton X-100 were used as controls. Reaction mixtures were incubated at room temperature for 20 min prior to reading the absorbance at 490 nm using an Infinite F200 Pro plate reader (Tecan). Values for untreated Vero cells were subtracted from the experimental values, divided by the difference of Triton-lysed and untreated cells, and multiplied by 100 to obtain percent lysis. Each experiment was performed, and data were averaged between biological triplicates.
Statistical analyses. Statistical analysis was performed in GraphPad Prism, version 7, and statistical parameters and significance are reported in the figures and figure legends. Statistical significance was determined either by an unpaired Student's t test or a one-way analysis of variance (ANOVA) where indicated in the figure legends.