Abstract
Gram-negative bacteria in the order Rickettsiales are obligate intracellular parasites that cause human diseases such typhus and spotted fever. They have evolved a dependence on essential nutrients and metabolites from the host cell as a consequence of extensive genome streamlining. However, it remains largely unknown which nutrients they require and whether their metabolic dependency can be exploited therapeutically. Here, we describe a genetic rewiring of bacterial isoprenoid biosynthetic pathways in the Rickettsiales that has resulted from reductive genome evolution. We further investigated whether the spotted fever group Rickettsia species Rickettsia parkeri scavenges isoprenoid precursors directly from the host. Using targeted mass spectrometry in uninfected and infected cells, we found decreases in host isoprenoid products and concomitant increases in bacterial isoprenoid metabolites. Additionally, we report that bacterial growth is prohibited by inhibition of the host isoprenoid pathway with the statins class of drugs. We show that growth inhibition correlates with changes in bacterial size and shape that mimic those caused by antibiotics that inhibit peptidoglycan biosynthesis, suggesting statins inhibit cell wall synthesis. Altogether, our results describe an Achilles’ heel of obligate intracellular pathogens that can be exploited with host-targeted therapeutics that interfere with metabolic pathways required for bacterial growth.
Importance Obligate intracellular parasites, which include viruses as well as certain bacteria and eukaryotes, extract essential nutrients and metabolites from their host cell. As a result, these pathogens have often lost essential biosynthetic pathways and are metabolically dependent on the host. 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 an 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 highlights a potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism.
Introduction
Gram-negative alphaproteobacteria in the family Rickettsiaceae, which includes the genera Rickettsia and Orientia, are obligate intracellular parasites that can cause human diseases including spotted fever (spotted fever group (SFG) Rickettsia), typhus (typhus group (TG) Rickettsia), and scrub typhus (Orientia species) (1). These bacteria are transmitted to mammals by arthropod vectors such as fleas, ticks, and mites. Although most pathogenic species cause moderately severe illnesses, in some cases infections can be fatal if untreated or even upon delayed treatment with first-line antibiotics (2). We study the SFG species R. parkeri, which causes an eschar at the site of the tick bite and symptoms that include fever, malaise, nausea, headaches, and an occasional rash (3, 4).
Upon invasion of host cells, the Rickettsiaceae quickly escape the primary vacuole into the host cell cytoplasm, where they grow and proliferate. One property of obligate intracellular bacteria such as Rickettsia is that adaptation to a dependency upon growth inside host cells has resulted in genome size reduction (5-9). The relatively small genomes of Rickettsia species (∼1.1-1.5 Mbp) encode for a reduced number of proteins (1273 predicted proteins in Rickettsia parkeri; NCBI reference sequence: NC_017044.1). This typically correlates with the loss of genes encoding components of metabolic biosynthetic pathways, together with the requirement to scavenge essential metabolites from the host (5, 8).
One essential class of metabolites are the isoprenoids (also known as terpenoids), which are derived from simple five carbon isoprene units that are assembled to make thousands of different molecules. The biosynthesis of the central isoprene precursor molecule, isopentenyl pyrophosphate (IPP), and its isomer, dimethylallyl diphosphate (DMAPP), occurs through two distinct pathways. The mevalonate (MEV) pathway (predominantly in archaea, Gram-positive bacteria, and animals) or the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (primarily in Gram-negative bacteria) (Figure 1). In bacteria, isoprenoid biosynthesis produces vital products such as bactoprenols (essential building blocks for peptidoglycan (PG) and other cell wall polysaccharides), ubiquinone or menaquinone (involved in the electron transport chain), or chlorophylls in cyanobacteria (10). In mammalian cells, isoprenoid biosynthesis produces a larger variety of products including cholesterol, ubiquinone, steroid hormones, prenylated proteins, heme, and vitamin K (11).
Although the isoprenoid biosynthetic pathway is essential, its components are missing from certain obligate pathogens including Mycoplasma species (12) and the protozoan parasite Cryptosporidium (13). Bioinformatic analysis (14) shows that genes encoding the upstream enzymes from the MEV and MEP pathways that are required to make IPP and DMAPP are also missing in Rickettsia species, with the exception of the idi gene encoding the enzyme isopentenyl diphosphate isomerase (IDI), which catalyzes the reversible conversion of IPP to DMAPP (15). Since Rickettsia have access to metabolites in the host cytoplasm, this suggests that Rickettsia may steal IPP and/or DMAPP, altering the flux of host-derived isoprenoid precursors to initiate downstream bacterial isoprenoid biogenesis. The downstream enzymes required to utilize these short-chained isoprenoid precursors for ubiquinone biosynthesis and PG biosynthesis are encoded in Rickettsia genomes.
In this study, we explore the evolutionary changes that occurred within the order Rickettsiales with respect to the presence of genes encoding components of the isoprenoid biosynthetic pathway. We also show that bacterial infection results in depletion of host isoprenoid products and the synthesis of bacterial isoprenoid products. Furthermore, we use a chemical genetic approach to reveal that inhibition of host isoprenoid biosynthesis by statin treatment prevents bacterial growth and alters bacterial shape. Our results suggest that the host MEV pathway serves as the upstream source of isoprene units for the synthesis of bacterial bactoprenols and ubiquinone, and that inhibition of the host MEV pathway by statins may represent a promising avenue for host-directed therapeutics to treat Rickettsia infection.
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 constructed a phylogenetic tree using 16S rDNA sequences across six main genera of the order as well as the closest free-living organism, Pelagibacter ubique, and assessed the presence or absence of genes encoding upstream or downstream components of the MEP pathway as well as the IDI enzyme (Figure 2A). We confirmed that the Rickettsia (14) and Orientia lineages lack the genes encoding upstream MEP pathway enzymes but have those encoding the downstream pathway components, whereas the other genera contained the full complement of MEP pathway genes. None have MEV pathway genes, with the exception of the idi gene in Rickettsia species. The absence of upstream MEP pathway genes suggests that Rickettsia must scavenge MEV/MEP pathway intermediates (IPP, DMAPP or FPP, Figure 1) from the host cell.
Additionally, the idi gene is only present in the Rickettsia lineage but not the Orientia lineage. A previous report suggested that the idi gene in Rickettsia was acquired by horizontal gene transfer (14). To further investigate this, we examined the genomic locus surrounding the idi gene in the R. parkeri str. Portsmouth genome (Figure 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 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 R. rickettsii. Interestingly, we found the two genomes were highly syntenic, with the exception of a 65kb region containing the idi gene (Figure 2C, region in lime green, idi gene in purple) that is inverted between the two species and in the R. rickettsii genome contains a 33.5 kb deletion (orange highlighted region in Figure 2C and Supplemental Table S1). These observations suggest that the locus surrounding the idi gene was acquired by horizontal gene transfer and is still under evolutionary pressure to retain the idi gene while simultaneously undergoing continuing reduction.
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-mass spectrometry (LC-MS/MS). Confluent infected and mock-infected cultures of African green monkey kidney epithelial Vero cell cultures were incubated for 4 d, collected and then extracted for lipid metabolites. For host isoprenoids, we monitored cholesterol, total cholesteryl esters, cholesteryl oleate, and ubiquinone-10 using single-reaction monitoring (SRM)-based LC-MS/MS methods (Figure 3). We observed a statistically significant approximately 2-fold decrease in total cholesteryl esters and cholesteryl oleate in the infected compared with uninfected samples. These products are generally understood to be the storage units of cholesterol packaged in intracellular lipid droplets (16). 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, which include the isoprenoid products C55-isopentyl pyrophosphate (C55-IPP) and C55-isopentyl phosphate (C55-IP). C55-IPP is initially produced by a dedicated prenyltransferase, UppS, and must be dephosphorylated to C55-IP to act as a lipid carrier (17). We also measured bacterial ubiquinone, ubiquinone-8, which contains an 8 prenyl subunit tail, in contrast with the human version containing 10 prenyl subunits, ubiquinone-10 (Figure 3) (18, 19). We observed a statistically-significant approximately 4-fold increase in both C55-IPP and C55-IP in infected compared with uninfected cells. We did not, however, find significant differences in bacterial ubiquinone (ubiquinone-8), possibly due to the presence of ubiquinone-8 intermediates from the host cell ubiquinone-10 biosynthesis pathway.
These results indicate 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, suggests 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 and DMAPP. We used statins, a class of drugs that block the activity of HMG-CoA reductase (Figure 1). A previous report showed that pre-incubation of R. 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 (20). 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 (21), treated cells with (or without) various concentrations of the statin pitavastatin, and then measured bacterial numbers using a R. parkeri-specific qPCR endpoint assay (Figure 4A). We observed a >2-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 secondary effects of the drug, we tested whether the downstream product of HMG-CoA reductase, mevalonate, could rescue this growth inhibition. Indeed, co-treated with 400 µM mevalonate along with pitavastatin rescued bacterial growth (Figure 4A).
To further assess the importance of the MEV pathway, we generated dose-response curves for the effect 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 (EC50 = 2.0 µM) and even more robust inhibition with pitavastatin (EC50 = 0.5 µM) (Figure 4B). This data indicates 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 (as depicted in Figure 1), we tested for dose-dependent growth inhibition using additional inhibitors of MEV and MEP pathway enzymes (Figure 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 µM. 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 anti-bacterial antibiotics tetracycline, which is used as a first-line treatment for Rickettsia 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 EC50 = 0.7 µM, consistent with minimal inhibitory concentrations (MIC) values for SGF rickettsiae of 0.06-0.25 µg/mL (0.1-0.5 µM) (22). D-cycloserine also caused a dose-dependent inhibition of R.parkeri growth with an EC50 = 56 µM, consistent with effects on R. prowazekii (23), and with minimal inhibitory concentrations (MIC) values for Escherichia coli K12 (MIC = 30 µg/mL, 290 µM) (24) and Mycobacterium tuberculosis (15 µg/mL, 150 µM) (25). Taken together, the results of chemical inhibition assays support the notion that Rickettsia are dependent on the upstream host MEV pathway, but not the upstream MEP pathway. Furthermore, they are susceptible to inhibition of PG biosynthesis.
Statins treatment causes bacterial shape defects that mimic those caused by peptidoglycan-targeting antibiotics
We sought to further assess the effect of statin treatments on bacterial physiology. Limiting the availability of isoprenoids is predicted to result in reduced PG due to the requirement for isoprenoids in PG synthesis. Though Rickettsia (22)1, like other Gram-negative bacteria (26), are generally resistant to PG targeted antibiotics, one study showed that high concentrations of penicillin produced spheroplasts (27), a type of L-form bacteria that have a defective PG cell wall and take on a spherical shape (28). 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 dpi to track the alterations of bacterial shape in response to dose-dependent application of statins, D-cycloserine, or tetracycline as a control. We performed automated image analysis using CellProfiler (29) and measured a number of bacterial cell shape features. We determined that two shape measurements, area and eccentricity (circle = 0, rod/line = 1), accounted for most of the shape alterations seen in the concentration ranges between EC50-EC100. 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 (Figure 5A, B). In cells treated with either lovastatin or pitavastatin, similar changes in area and eccentricity were seen along the dose-response 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 shapes at the EC100 of each drug compared to the no-drug-treated control, we observed a significant increase in bacterial area and significant decrease in eccentricity in cells treated with D-cycloserine, lovastatin and pitavastatin (Figure 5C). For tetracycline, we found no significant differences in area or eccentricity compared to 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 mechanisms of bacterial growth inhibition resulting from targeting host isoprenoid and bacterial PG synthesis pathways.
Discussion
Obligate intracellular pathogens such as Rickettsia 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 scavenging of host isoprenoids used for bacterial cell wall biosynthesis.
We observe 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 having gained the idi gene. This is consistent with previous findings (14). Through our understanding of reductive genome evolution and gene acquisition in prokarykotes (5, 6, 8, 30), 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 Orientia lineages, which inhabit the host cell cytoplasm, may have gained the ability to scavenge IPP and/or DMAPP from the cytoplasm by acquisition of a gene encoding 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 (31-33). The gain of a transporter would have enabled the loss of the upstream MEP pathway genes to streamline 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 become depleted of isoprenoid-derived storage forms of cholesterol, total cholesteryl esters and choleteryl oleate, perhaps to balance free cholesterol levels. Whether Rickettsia also scavenge downstream host isoprenoid products such as cholesterol, as observed for other bacteria such as M. tuberculosis and Chlamydia trachomatis (34-36), 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 (20), but mechanism of inhibition was not explored in detail. It was suggested that statins might inhibit Rickettsia adherence to and/or invasion of host cells based on previous studies that had found a role for cholesterol these processes (37, 38). Our work, however, reveals an effect of statins on intracellular bacterial growth, downstream of adherence/invasion. Furthermore, with increasing statin concentration, we find morphological changes in the shape and size of bacteria that mirror those caused by the cell wall synthesis inhibitor D-cycloserine. These observations lead 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.
Whether statins can be effective at preventing or treating human Rickettsia infections remains to be determined. Although statins are well tolerated in humans, and both statins tested in this study are FDA-approved, we have limited understanding of the dose response curves for inhibition of bacterial growth versus host toxicities that can include myopathy and increased risk of diabetes and stroke (39). Furthermore, it is unclear if statins have the same degree of bacterial growth inhibition in different rickettsial target cell types in vivo. Additional studies in animal models will shed light on 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 (40, 41), and tetracycline treatment is contraindicated for pregnant women and young children (42). Because a limited number of antibiotics are effective in treating rickettsial infections (22), 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 points to a potential to use 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 (43). 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 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 re-purposed as antibiotics.
Materials and Methods
Reagents
Hoechst 33342 (B2261), D-cycloserine (C6880), tetracycline (T7660), fosmidomycin (F8682), mevalonate (M4667), and alendronate sodium hydrate (A4978) were obtained from Sigma-Aldrich. ProLong Gold Antifade (P36930), Pitavastatin (494210), Lovastatin InSolution (4381875), goat anti-mouse Alexa-488 secondary antibody (R37120), 0.25% Trypsin-EDTA phenol red (25200056) and Dynamo HS SYBR Green qPCR kit (F-410L) were obtained from Thermo-Fisher Scientific. Anti-Rickettsia 14-13 antibody was generously provided by Ted Hackstadt, NIH/NIAID Rocky Mountain Laboratories (44).
Cell culture and R. parkeri infections
Confluent low-passage African green monkey epithelial Vero cells were obtained from the UC Berkeley Cell Culture Facility and grown at 37°C, 5% CO2, in high glucose (4.5 g/l) DMEM (Gibco, Life Technologies, 11965092) 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 an MOI = 0.1 of wild-type R. parkeri and growing them at 33°C, 5% CO2 in DMEM plus 2% FBS. Bacteria were purified from infected cells as described previously (45). Briefly, infected cells were lysed by dounce homogenization in cold K-36 buffer (0.05 M KH2PO4, 0.05 M K2HPO4, pH 7, 100 mM KCl, 15 mM NaCl) to release bacteria, the lysate was overlaid onto 30% MD-76R (Mallinckrodt Inc., 1317-07), centrifuged at 58,300 x g for 20 min at 4°C in a SW-28 swinging bucket rotor, and resuspended in cold brain heart infusion (BHI) media (BD Difco, 237500) broth. Aliquots of bacteria were immediately frozen at −80°C after purification and each infection was initiated from a single thawed aliquot of bacteria.
Phylogenetic analyses, idi locus mapping, and whole genome alignments
Phylogenetic relationships between bacterial species were analyzed using 16S rDNA sequences downloaded from the SILVA database (https://www.arb-silva.de/). Phylogenetic trees were built with the Tree Builder program on Geneious software version 9.1.8 using the neighbor-joining method with Pelagibacter ubique as the outgroup. 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=map00900) for each bacterial species. Determination of intracellular niche was performed by manual literature curation. Idi locus map and annotations were examined using Geneious software version 9.1.8 using the R. parkeri str. Portsmouth genome sequence (NCBI RefSeq: NC_017044.1). Whole genome alignments of R. parkeri str. Portsmouth and R. rickettsii str. Iowa (NCBI RefSeq: NC_010263.3) were performed using the Mauve 2.3.1 (46) plugin in Geneious software using the progressive Mauve algorithm with automatically-calculated-minimum locally collinear block (LCBs) scores.
Mass spectrometry
Monolayers of Vero cells plated in 6-well plates were infected (or an uninfected control) with R. parkeri as described above and were incubated for 4 d at a 33°C 5% CO2. On the day of harvest, cells in each well were washed with 4 ml of 1 x phosphate buffered saline (PBS). 1 ml of fresh 1 x PBS was added to each well and used to scrape the cells, centrifuged at 1000 x g for 5 min on a microfuge 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 dodecylglycerol internal standard, after which the organic phase was separated, collected, and dried down under N2 gas. The dried-down lipidome extract was then resuspended in 150 µl chloroform and stored at −80°C until analysis. For mass spectrometry analysis, an aliquot of this sample solution was injected into an Agilent 6430 LC-MS/MS and metabolites were chromatographically separated and analyzed by SRM-based targeted LC-MS/MS as previously described (47, 48).
Immunofluorescence microscopy
To measure bacterial shape, Vero cells were plated at 50% confluency on 96-well glass bottom plates in DMEM plus 2% FBS and allowed to settle overnight at 37°C, 5% CO2. The following day, cells were infected with an MOI = 0.1 of wild-type R. parkeri, centrifuged at 300 x g for 5 min and incubated for 2 h at 33°C, 5% CO2. Media containing bacteria was aspirated and replaced with media with or without the appropriate concentration of drug (see above), and cells were further incubated for 72 h at 33°C, 5% CO2. Cells were fixed with 4% paraformaldehyde in 1 x PBS for 20 min at room temperature, washed with 1 x PBS, and permeabilized with 0.05% Triton X-100 in 1 x 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, 100X (1.4 NA) Plan Apo objective, a Clara Interline CCD Camera and MetaMorph software taking 0.15 µm z-slices across 5 µm in the z-plane. Maximum Z-projections for each channel were made using ImageJ and a custom pipeline was created in CellProfiler software (29) to identify individual bacteria in each image. The CellProfiler module MeasureObjectSizeShape was used to calculate size and shape parameters of each bacterium.
R. parkeri qPCR growth and drug dose response curves
Confluent Vero cells grown in 96-well tissue culture plates were infected with an MOI = 0.1 of R. parkeri and incubated in DMEM plus 2% FBS with or without the appropriate concentration of drug (see above) for 72 h at 33°C, 5% CO2. To harvest cells at the appropriate time point, media was aspirated and cells lifted with 50 µl of 0.25% trypsin-EDTA followed by incubation at 37°C for 5 min. Lifted cells were resuspended with an additional 50 µl of DMEM before adding to 50 µl Nuclei Lysis Buffer (Wizard Genomic DNA Purification kit; Promega) and frozen at −20°C overnight. Cells were then thawed and boiled for 10 min to release genomic DNA. To remove RNA, 20 µg/mL RNAse A was added to each sample, incubated for 15 min at 37°C, and then samples were cooled to room temperature. Protein was removed by adding 50 µl of protein precipitation solution, mixing by pipetting, then centrifuging in a microfuge for 15 min at 1500 x g at 4°C. DNA was precipitated by adding 100 µl of the resulting supernatant to 100 µl of isopropanol, mixing by pipetting, and centrifuging for 15 min at 1500 x g at 4°C. Isopropanol was removed and the DNA pellet was washed with 70% ethanol and centrifuged for 15 min at 1500 x g. Resulting DNA pellets were dried, then resuspended in 50 µl of H2O and allowed to rehydrate overnight at 4°C. For quantitative real-time PCR, 5 µl genomic DNA was used with primers to the R. parkeri gene encoding the 17kDa antigen (49) 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 17kDa gene. Regression analysis and EC50 calculations were performed using GraphPad Prism 7 software.
Statistical analyses
Statistical analysis was performed in GraphPad PRISM 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 ANOVA where indicated. Statistical significance was denoted by asterisks as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, compared to controls.
Acknowledgments
We are grateful to Rebecca Lamason for critical reading of the manuscript. We thank Eric Lee and Dan Portnoy for reagents. We thank Matthew Knight, Eric Lee, Ellen Yeh, David Booth, Thibaut Brunet, and members of the Welch lab for helpful technical advice and discussions. This work was performed in part at the CRL MIC, which is supported by NIH grant S10RR027696-01. V.A. was supported by the NIH/NIAID grant F32 AI133912A. M.D.W. was supported by NIH/NIAID grants R01 AI109044 and R21 AI109270. C.A.B. and D.K.N. were supported by NIH/NCI grant R01 CA172667.