Identification of critical host mitochondrial-associated genes during

Identification of critical host mitochondrial-associated genes during Ehrlichia chaffeensis 1

infections 2

3

Tonia Von Ohlen1,2, Alison Luce-Fedrow1, M. Teresa Ortega1, Roman R. Ganta2, and Stephen K. 4

Chapes1* 5

6

1Kansas State University; Division of Biology; Manhattan, KS 66506 7

2Kansas State University; Department of Diagnostic Medicine and Pathobiology; Manhattan, KS 8

66506 9

10

*Corresponding author: Kansas State University, Division of Biology, Manhattan, KS 66506; 11

Telephone (785)532-6149; Fax (785)532-6653; [email protected] 12

13

Running Title: Host Factors in E. chaffeensis growth 14

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00670-12 IAI Accepts, published online ahead of print on 30 July 2012

on February 11, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

2

ABSTRACT 15

Ehrlichia chaffeensis is an obligate, intracellular bacterium that causes human monocytic 16

ehrlichiosis (HME). To determine what host components are important for bacterial replication, 17

we performed microarrays on Drosophila melanogaster S2 cells by comparing host gene 18

transcript levels between permissive and non-permissive conditions for E. chaffeensis growth. 19

Five-hundred twenty-seven genes had increased transcript levels unique to permissive growth 20

conditions 24 hours post infection. We screened adult flies which were mutants for several of 21

the “permissive” genes for their ability to support Ehrlichia replication. Three additional D. 22

melanogaster fly lines with putative mutations in pyrimidine metabolism were also tested. Ten 23

fly lines carrying mutations in genes CG6479, separation anxiety, chitinase 11, CG6364 (Uck2), 24

CG6543 (Echs1), withered (whd), CG15881 (Ccdc58), CG14806 (Apop1), CG11875 (Nup37), 25

and dumpy (dp) had increased resistance to infection with Ehrlichia. Analysis of RNA by qRT-26

PCR confirmed that the bacterial load was decreased in these mutant flies compared to wild-type 27

infected control flies. Seven of these genes (san, Cht11, Uck2, Echs1, whd, Ccdc58, and Apop1) 28

encoded for proteins that had mitochondrial function, or could be associated with proteins with 29

mitochondrial function. Treatment of THP-1 cells with double stranded RNA to silence the 30

human UCK2 gene indicates that the disruption of the uridine-cytidine kinase affects E. 31

chaffeensis replication in human macrophages. Experiments with cyclopentenyl cytosine 32

(CPEC), a CTP synthetase inhibitor and cytosine suggest that the nucleotide salvage pathway is 33

essential for E. chaffeensis replication and may be important for the provision of CTP, uridine 34

and cytidine nucleotides. 35

36

37


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

3

INTRODUCTION 38

E. chaffeensis is the causative agent of human monocytic ehrlichiosis (HME). There 39

were 1429 cases of HME in 2010 and 2011 (14). This represents a significant increase in the 40

incidence of the disease since 2003 and qualifies HME as an emerging infectious disease (25). 41

In addition to being reported in the United States, HME has also been documented in Africa, 42

Europe, China, and Brazil (9, 16, 42, 67). E. chaffeensis is an obligate intracellular bacterium. 43

However, little is known about the parasitized host requirements for bacterial replication. 44

D. melanogaster has been used to study a variety of intracellular pathogens. In 45

particular, it has been successfully manipulated for the identification of genes involved in host-46

pathogen interactions. These pathogens include Listeria monocytogenes (1, 2), Chlamydia 47

trachomatis (20), Mycobacterium marinum (1, 18, 30, 48), Francisella tularensis (51, 64), and 48

the protozoan parasite Plasmodium gallinaceum (8, 53). 49

We previously demonstrated that E. chaffeensis is capable of infecting, completing its 50

lifecycle, and maintaining its pathogenicity in both Drosophila S2 cells (39) and in adult flies 51

(40). We have also identified growth conditions that were non-permissive for the growth of E. 52

chaffeensis infection in Drosophila S2 cells (39). Therefore, we hypothesized that a 53

transcriptional microarray analysis of permissively infected and non-permissively infected S2 54

cells would reveal host genes that contribute to the replication of Ehrlichia. We used the 55

Affymetrix Drosophila 2.0 array to identify a subset of genes that were exclusively expressed 56

during E. chaffeensis infection in infected S2 cells under permissive growth conditions. We 57

screened adult flies carrying mutations in several of the candidate genes, 10 of which change the 58

fly response to E. chaffeensis compared to wild-type flies. Gene products from 7 of the 10 genes 59

identified are associated with mitochondrial function and/or location. We also describe follow- 60


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

4

up experiments to investigate how Uck2 might function and its relevance to mammalian 61

infections. 62

MATERIALS & METHODS 63

Maintenance of cell lines and E. chaffeensis infections. The canine macrophage cell line, 64

DH82 (ATCC #CRL-10389) was cultured in Eagle's minimal essential medium supplemented 65

with 7% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA))(EMEM7). THP-1 (ATCC 66

#TIB-202) cells were cultured in RPMI1640 medium supplemented with 10% FBS. Drosophila 67

S2 cells, obtained from the Drosophila RNAi Screening Center (Harvard Medical School, 68

Boston, MA) were cultivated at 25°C in Schneider’s Drosophila medium (Invitrogen, Carlsbad, 69

CA) supplemented with 10% fetal bovine serum. For experiments S2 cells were seeded at a 70

concentration of 1 x 106 cells/well in 6-well plates. 71

The E. chaffeensis Arkansas isolate was continuously cultivated in DH82 cells as has 72

been described previously at 37°C, 8% CO2 in EMEM7 medium (40). Purified bacteria were 73

used to re-infect DH82, THP-1 and S2 cells. 74

Infections in Permissive and Non-Permissive Drosophila S2 cells. To make S2 cells non-75

permissive to E. chaffeensis infection, S2 cells were allowed to adhere for at 30 min prior to 76

adding sonicated LPS (from Salmonella enterica serovar Minnesota; Sigma, St. Louis, MO) at a 77

concentration of 10 μg per ml. S2 cells plus LPS were incubated for 5 h prior to infection with E. 78

chaffeensis. Ehrlichia-infected S2 cells were monitored at 24 and 96 hours post-infection (hpi). 79

Additionally, uninfected S2 cells inactivated and activated with LPS were used as controls. S2 80

cells were centrifuged at 300 x g for 5 minutes prior to RNA isolation from cell pellets using 1 81


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

5

ml of TriReagent method (Molecular Research Center, Inc., Cincinnati, OH). The 82

TriReagent/cell mix was transferred to 2.0 ml, Heavy Phase Lock Gel tubes (5 Prime, Westbury, 83

New York). Chloroform (200 μl) was added and the mixture was gently mixed for 15 seconds. 84

After centrifugation at 12,000 x g for 10 minutes at 4°C the aqueous phase was poured to 1.5 ml 85

tubes. RNA was precipitated and washed with isopropanol and ethanol according to 86

manufacturer’s instructions. RNA pellet was suspended in 50 μl of nuclease-free water after and 87

RNA concentration was determined spectrophotometrically (NanoDrop Technologies, 88

Wilmington, DE) before and after DNA digestion using a turbo DNA-free kit (Ambion Inc. 89

(Austin, TX). 90

Determination of infection by RT-PCR and quantitative real time reverse transcription – 91

PCR (qRT-PCR). For the microarray, infections were confirmed by RT-PCR using the 92

Promega Access One-Step RT-PCR kit (Madison, WI). Total RNA (750 ng) was used for each 93

25 μl reaction which contained the following reagents: 1X buffer, 0.2 mM dNTPs, 2 μM forward 94

primer, 2 μM reverse primer, 1.5mM MgSO4, 1U per μl DNA polymerase, 1U per μl reverse 95

transcriptase, and nuclease-free water. RT-PCR reactions were performed in a thermocycler 96

(Eppendorf Mastercycler Gradient, Hauppauge, NY). Primers and probes were using the Primer 97

Quest software (Integrated DNA Technologies; http://www.idtdna.com; Coralville, IA) referring 98

data from NCBI reference sequences (Supplemental Table 1). RT PCR conditions for the 16S 99

ribosomal RNA of E. chaffeensis were: 48°C for 45 minutes, 9 4°C for 4 minutes, then 35 cycles 100

of 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute (21). Ribosomal protein 49 101

(rp49) was used as housekeeping gene in RT-PCR experiments with Drosophila S2 cells. RT-102

PCR conditions for rp49gene amplification were: 48°C for 45 minutes, 94°C for 2 minutes, then 103

35 cycles of 94°C for 45 seconds, 50°C for 1 minute, and 72°C for 1.5 minutes. No template 104


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

6

RT-PCR controls were also included. RT-PCR product bands were identified on a ChemiImager 105

after electrophoresis in 2% agarose gels and staining with ethidium bromide. 106

Flies were anesthetized using CO2 prior to homogenization with disposable pestles 107

(Kimble Chase, Vineland, NJ), in 1 ml of TriReagent. Homogenates were transferred to 2.0 ml 108

Heavy Phase Lock Gel tubes and processed as described above for RNA isolation. 109

Ehrlichia quantification in S2 cells and fly experiments was estimated using TaqMan-110

based quantitative real-time reverse transcriptase PCR (qRT-PCR) as previously described (40, 111

55), using Drosophila ribosomal protein 15a as the housekeeping gene with primers described in 112

Supplemental Table 1. E.chaffeensis was detected as described (40, 55). The qRT-PCRs were 113

performed in the Cepheid SmartCycler System (Sunnyvale, CA). 114

Analysis of gene expression based on qRT-PCR results was performed using the method 115

described by Pfaffl (47). In short, primer efficiencies were calculated from standard dilution 116

curves plotting Ct values vs. log RNA and using the equation: 117

Efficiency = 10(-1/slope of standard curve) 118

The change in Ct values for both genes of interest and housekeeping controls was determined by 119

calculating the ΔΔCt using the following equation: 120

(Efficiency of gene interest Gene interest:ΔCt control – treated)______ 121 (Efficiency of housekeeping gene Housekeeping gene:ΔCt control – treated) 122 123

Microarray Analysis. Microarray analysis was performed at the University of Kansas Medical 124

Center Microarray Facility (Kansas City, KS) using the Affymetrix (Santa Clara, CA) 125

Drosophila 2.0 Gene chips according to manufacturer’s specifications. The analysis was 126

performed on four treatment groups (each submitted in triplicate at 24 hpi): (1) S2 cells infected 127

with E. chaffeensis; (2) S2 cells incubated with LPS and then infected with E. chaffeensis; (3) S2 128


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

7

cells incubated with LPS; and (4) untreated/uninfected S2 cells. CHP files were analyzed using 129

GeneSpring 7.3 software and normalized by “Per Gene: Normalize to median”. The “Filter on 130

volcano plot” was applied at fold change 1.5 and one-way ANOVA at significant level α = 0.05. 131

Genes up-regulated 1.5 fold or higher above basal expression levels were identified in both the 132

permissive and non-permissive conditions when compared to uninfected controls all at 24 hpi. 133

These gene sets were then compared and those exclusively up-regulated in either permissive or 134

non-permissive conditions at 24 hpi were identified. Microarray MIAMI compliant data are 135

available at the publicly accessible database by creating an account at 136

bioinformatics.kumc.edu/mdms/login.php, and accessing the experiment titled “Differential 137

Gene Expression in Ehrlichia chaffeensis-infected S2 cells”. 138

D. melanogaster. Flies were maintained on standard dextrose/molasses/yeast medium at 18-139

29°C. For all experiments, flies of the appropriate background were used as wild-type (WT) 140

controls. w;Hemese-Gal4, UASGFP flies (GFPHeme) (from Dr. Michael J. Williams; Umea 141

Centre for Molecular Pathogenesis; Umea University; Umea, Sweden), yellow-white (yw) 142

(maintained in our stock collection at Kansas State University), and/or white ocelli(wo1) (Stock 143

#634, from Bloomington Drosophila Stock Center at Indiana University; Bloomington, IN) were 144

used as WT in these experiments. Withered (FBgn0004012; stock #441, whd1), dumpy 145

(FBgn0053196; stock #276, dpov1), and tilt (FBgn0003868; stock #623, tt1wo1) mutants are all 146

the result of spontaneous mutations (35, 44, 65) and were obtained from Bloomington 147

Drosophila Stock Center at Indiana University, Bloomington, IN. The stock numbers, 148

genotypes, and associated genes of adult Drosophila screened by microinjection are described in 149

Supplemental Table 2. 150


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

8

Adult Drosophila Infections. Flies were transferred to fresh food at least 24 hours prior to 151

injection/infection. For injection/infection, adult male and female flies were anesthetized with 152

CO2 (for no longer than 15 minutes at a time). Flies were injected with approximately 50 nl of 153

sterile PBS or with >5000 bacteria, using pulled, glass capillary needles. Injections were made 154

in the abdomen of the fly, close to the junction of the thorax, and ventral to the junction between 155

the dorsal and ventral cuticles. Following injection, flies were maintained in clean bottles with 156

molasses caps that were changed every other day throughout the course of the experiments. 157

Survival of the flies was monitored daily. 158

Cyclopentenylcytosine (CPEC) treatment of S2 cells. CPEC was obtained from the National 159

Cancer Institute (Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, 160

Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD) through 161

a Materials Transfer Agreement. The CPEC was prepared to a final concentration of 15.06 mM 162

using sterile water containing 1% DMSO. Two different infection protocols were used for 163

testing the effect of CPEC on the growth of E. chaffeensis in the S2 cells. For the first protocol: 164

CPEC was added to S2 cells at a final concentration of 100, 10, 1, or 0.1 μm/well; E. chaffeensis 165

was added to the treated cells 2 days later, and RNA was extracted from the cells 2 days post 166

infection. For the second protocol: S2 cells were infected with E. chaffeensis for 2 days then 167

varying concentrations of CPEC were then added and RNA extractions were performed 2 days 168

following the CPEC treatment. S2 cells treated with diluent only and infected with Ehrlichia, 169

and uninfected S2 cells were used as controls. qRT-PCR assay was used to analyze transcript 170

levels of Ehrlichia16S rRNA and Drosophila ribosomal protein15a, as described above. 171

Addition of exogenous cytosine to Drosophila S2 cells. Cytosine was obtained from Sigma-172

Aldrich Co. The cytosine was consecutively dissolved in 500 mM hydrochloric acid and then in 173


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

9

sterile water to 100 mM, as per the manufacturer’s recommendation. For infection experiments, 174

S2 cells were incubated for 24 hours prior to cytosine addition to a final concentration of 25 mM; 175

the cells were further incubated for 24 hours. E. chaffeensis were added to the cytosine-treated 176

cells, to untreated S2 cells, and to S2 cells treated with the cytosine diluent. Untreated, 177

uninfected S2 cells were used as a negative control. RNA was extracted from cells at 24, 48, 72, 178

and 96 hpi. qRT-PCR was used to measure transcript levels of Ehrlichia16S rRNA and 179

Drosophila ribosomal protein 15a, as described earlier. 180

THP-1 cell differentiation and cell cultures. THP-1 human macrophages were cultured as 181

described above. For differentiation of THP-1 cells (6×104 THP-1/well; 24 well plates) were 182

incubated with phorbol 12-myristate 13-acetate(PMA; Sigma-Aldrich Chemical Co., St. Louis, 183

MO) at a final concentration of 50 µM. The cells were cultured for 24 h to allow final 184

differentiation into macrophages in a cell culture incubator at 37°C with 8% CO2. 185

Measurement of transfection efficiency and UCK2 knockdown. We selected the siRNA 186

transfection conditions based on the transfection efficiency in the cells of a fluorescent-labeled 187

transfection control siRNA duplex conjugated with the fluorescent marker TYE 563™. 188

Transiently transfected cells were analyzed for transfection efficiency using fluorescent 189

microscopy. 190

UCK2 transcript expression was knocked down in THP-1 cells using siRNA specific to 191

the human UCK2 transcript. We used the TriFecta RNAi kit with the pre-designed oligo sets 192

from IDT. The siRNA pre-designed oligo query was done using UCK2 RNA CDS from NCBI 193

reference sequence XM_846154.2 for Homo sapiens. We also used scrambled siRNA and 194

human HPRT siRNAs as negative and positive controls, respectively, to confirm transfection and 195

siRNA knockdown efficiency. All siRNA duplexes were dissolved in nuclease free water (pH 196


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

10

7.2) to a final concentration of 2 nM. Three UCK2-specific, an HPRT-specific and scrambled 197

siRNA duplexes were transfected into differentiated THP-1 cells using the Lipofectamine 198

transfection reagent (Invitrogen) by following the manufacturer’s protocol. 199

THP-1 cells were infected with E. chaffeensis 48 h post transfection. Transfected and 200

Ehrlichia-infected THP-1 were analyzed for UCK2, HPRT knockdown efficiency and level of 201

bacteria infection by qRT-PCR 48 h post infection using primers and probes described in 202

Supplemental Table 1 as explained above. 203

Transmission electron microscopy (TEM). TEM analysis of uninfected and E. chaffeensis 204

infected DH82 cultures was performed as described recently (17). 205

Statistics. Data are presented as the means ± standard errors of the means (SEM). Differences 206

in means were determined using the Mann-Whitney two-tailed, rank-sum statistical test which is 207

independent of the underlying population distribution (StatMost statistical package; Data XIOM, 208

Los Angeles, CA). Survival data were analyzed for significance using the log rank test of 209

Kaplan-Meier plots using Prism Graphpad software (La Jolla, CA). P values of <0.05 were 210

considered significant. 211

RESULTS 212

Microarray “permissive-exclusive” genes after infection with E. chaffeensis. To identify 213

host genes that are necessary for E. chaffeensis infection, we examined transcript levels of 214

Drosophila S2 cells under four different conditions at 24 hpi to assess the host factors that might 215

be needed to initiate the infection. These included: (1) uninfected S2 cells; (2) uninfected S2 216

cells activated with LPS; (3) S2 cells activated with LPS and then infected with E. chaffeensis 217

(non-permissive condition) [This experiment was performed as the previous studies 218


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

11

demonstrated the inhibition of E. chaffeensis replication in LPS activated S2 cells (43).]; and (4) 219

S2 cells infected with E. chaffeensis (permissive condition). E. chaffeensis replicated only under 220

permissive conditions, but not in non-permissive conditions for as long as 96 hpi (Figure 1). 221

Uninfected cells were used to discern the basal transcript levels in the S2 cells and comparisons 222

across the different conditions were based on this basal level of expression. In order to 223

understand which gene transcripts were specific to non-permissive S2 cells, we used the S2 cells 224

that were treated only with LPS as a comparison. Cells that received only LPS treatment 225

revealed activation-specific gene transcripts. We compared those genes to the genes that had 226

increased transcript levels in cells infected under non-permissive conditions for bacterial growth 227

to deduce transcripts that were up regulated exclusive to non-permissive conditions. We also 228

determined the gene transcripts that were exclusively up-regulated 1.5 fold or higher during 229

permissive infection conditions for the E. chaffeensis growth and 2,128 genes were 1.5 fold 230

higher than uninfected controls. Under non-permissive growth conditions, 1,742 of the genes 231

were 1.5 fold higher than uninfected S2 cells. When we compared transcripts that were up-232

regulated during permissive growth conditions to transcripts that were up-regulated under non-233

permissive growth conditions, we identified 527 genes unique to permissive conditions 234

(Supplemental Table 3). Of these, 210 had previously been ascribed some function and had 235

some characterization. Functions of the remaining 307 genes had yet to be defined and had 236

“CG” gene designations. However, a number of these genes had orthologs with some described 237

properties. These ortholog gene descriptions were used to ascribe functions for our analyses. 238

Viable and fertile adult fly mutant stocks were available for 118 of the 527 genes (37 stocks for 239

defined genes and 81 stocks for undefined genes). These flies had appropriate mutations in 240

coding exons in the genes of interest and allowed testing the importance of how the absence of a 241


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

12

functional gene would affect fly survival and/or bacterial replication in vivo. Our screen of 242

mutant fly lines was loosely based on descriptions of genes that controlled functions that we 243

guessed might be important for bacterial growth or were random choices of “CG”-designated 244

genes. 245

E. chaffeensis infection in selected mutant Drosophila fly lines. We screened 19 Drosophila 246

lines with mutations in the genes of interest (Supplemental Table 2) for bacterial replication and 247

fly survival. Flies (wild-type and mutant) were injected with cell-free E. chaffeensis or sterile 248

PBS and monitored for survival for 96-120 hpi (20 flies per treatment group per experiment; the 249

experiment was repeated at least 3 independent times). We looked for the mutants that displayed 250

increased survival of the flies after bacterial challenge compared to the wild-type flies. Our 251

hypothesis was that the gene affected in a mutant allowed increased survival because its 252

expression was needed for E. chaffeensis replication. We found that the flies having mutations 253

for Nup37, Echs1, Cht11, CG6479, Ccdc58, Apop1, san, and Uck2 displayed significantly 254

increased survival compared to wild-type flies after infection with E. chaffeensis (Figure 2). In 255

contrast, several mutants did not show increased resistance to E. chaffeensis infections. These 256

included: three G3pdh mutants, (Stock 1124 shown in Figure 2), CG14434 (Figure 2) as well as 257

CG10672, CG4743, CG9300, tsp3A, CG10992, and Gap69C (data not shown). 258

To confirm that Nup37, Echs1, Cht11, CG6479, Ccdc58, Apop1, san, and Uck2 gene 259

mutations affected E. chaffeensis infections, we also estimated bacterial replication as measured 260

by qRT-PCR (Figure 3). Experimental infection of flies with disruptions in all the 8 genes 261

resulted in the significant drop in bacteria numbers than observed in wild-type flies infected with 262

the organism (Figure 3). 263


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

13

Uridine/Cytidine Kinase mutations affect fly survival and bacterial replication. To 264

understand the physiological relevance of disruption of gene function to E. chaffeensis growth 265

in the host, we focused on Uck2, as an ortholog for this gene has been identified in mammalian 266

species (43). Stroman (56) previously reported that the Drosophila stocks dumpy, tilt, and 267

withered carried mutation(s) which affect uridine/cytidine kinase function. To confirm the 268

impact of the Uck2 on E. chaffeensis growth, the three fly lines with the putative defects in 269

uridine/cytidine kinase function were infected with E. chaffeensis and assessed for bacterial 270

replication. Two of these fly lines were significantly more resistant to E. chaffeensis challenge 271

than wild type (WT) flies (Figure 4). For example, at 96 hpi 61% of the dumpy flies were alive 272

compared to 42% of WT flies; similarly 66% of withered flies survived compared to 38% of WT 273

flies (P<0.05). Although there was a trend for the tilt flies (60±16% survival) to be more 274

resistant than the WT flies (46±7% survival), the differences were not statistically significant 275

(Figure 4) . The increased survival of the withered and dumpy mutants was accompanied by a 276

significant decrease in the replication of the Ehrlichia organisms, as measured by qRT-PCR 277

assay (Figure 3). At 96 hpi, WT flies contained significantly more bacteria compared to 278

withered and dumpy mutants (P=0.02; Figure 3, bottom panel). The tilt mutants averaged fewer 279

bacteria but there was more variation in the samples. 280

CPEC treatment increases E. chaffeensis infection. The uridine/cytidine kinase enzyme 281

functions in pyrimidine synthesis pathways, being specifically involved in the conversion of 282

uridine to uridine monophosphate and cytidine to cytidine monophosphate (52, 62, 63) (Figure 283

5). Deoxycytidine triphosphate can also be synthesized from glutamine through the de novo 284

synthesis pathway as well (52, 62)(Figure 5). To determine the importance of the de novo 285

synthesis pathway to E. chaffeensis infections, we obtained the drug cyclopentenyl cytosine, 286


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

14

which inhibits the conversion of [3H]-UTP to [3H]-CTP (28) as an inhibitor of cytidine 287

triphosphate synthetase (45, 52, 62). This drug effectively inhibits de novo synthesis of 288

pyrimidines, leaving only the salvage pathway with cytidine as the substrate for pyrimidine 289

synthesis. To explore the impact of CPEC on E. chaffeensis growth, S2 cells were treated with 290

CPEC for 48 hours at final concentrations of 0.1, 1, 10 or 100 uM. Subsequently, we infected 291

those cells with E. chaffeensis for an additional 48 hours. We found a significant increase in 292

bacterial growth in all of the treated cells compared to infected cells treated with carrier only 293

(Figure 6, solid bars; P<0.01). Similarly, when S2 cells were first infected with E. chaffeensis 294

for 48 hours and then treated with different concentrations of CPEC for 48 hours, we observed 295

significant increases in bacterial growth in cells treated with 10 and 100 μM CPEC compared to 296

infected cells treated with carrier only (Figure 6, hatched bars; P<0.01). Therefore, the de novo 297

synthesis pathway was not needed for bacterial growth in the S2 cells and usually enhanced 298

infection. 299

Cytosine treatment increases E. chaffeensis infection. Cytosine combines with ribose to form 300

cytidine in the nucleotide salvage pathway. Therefore, to determine if it is an important substrate 301

for bacterial growth, we supplemented S2 cell culture medium for 24 hours with 25 mM of 302

cytosine prior to infecting with E. chaffeensis and the bacterial replication was assessed at up to 303

96 hpi (Figure 7). The cytosine-treated infected cells contained an average of 456%, 1423%, 304

3352%, and 2465% more copies of Ehrlichia 16S rRNA at 24, 48, 72, and 96 hpi, respectively, 305

compared to mock infected cells. 306

Silencing of UCK2 in human THP-1 cells. The uridine-cytidine kinase (UKC2) is a conserved 307

enzyme that is present in humans as well as D. melanogaster. Therefore, to examine the 308

contribution of this enzyme for E. chaffeensis growth in human cells, we used double stranded 309


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

15

RNA to induce UCK2 gene-specific silencing to inhibit translation of UCK2. We used three 310

different RNA duplex sequences to define the specificity of the UCK2 knockdown. THP-1 cells 311

are a promyelocytic cell line that was induced to differentiate into macrophages for 24 hrs using 312

phorbol ester. After cells were silenced for 48 hrs, cells were infected with E. chaffeensis. Three 313

different siRNA duplexes targeted to the UCK2 transcript consistently inhibited E. chaffeensis 314

growth by 48 hpi in the range of 47-69% compared to cells treated with a scrambled control 315

siRNA (Table 1). We also detected improved E. chaffeensis growth in cells treated with siRNA 316

for hypoxanthine phosphoribosyltransferase (HPRT), which was used as a control for silencing 317

procedure effectiveness and to help show specificity of the UCK2-specific duplexes. 318

Mitochondrial localization around morulae in E. chaffeensis-infected cells. Mitochondria 319

surround morulae in E. chaffeensis infected cells (49). Indeed, when we examined DH82 cells 320

after infection with E. chaffeensis by TEM, one prominent feature of the infected cells was the 321

clustering of large numbers of mitochondria around the morulae (Figure 8A and B). It even 322

appears that mitochondria were closely apposed to bacteria within the parasitophorous vacuole 323

(Figure 8B). This contrasts the random distribution of mitochondria in uninfected cells (Figure 324

8C). 325

DISCUSSION 326

We used D. melanogaster to investigate host factors that are important for the replication 327

of E. chaffeensis. We combined microarray analysis and mutant fly screening to successfully 328

identify 10 genes that contribute to the replication of E. chaffeensis in vivo. Seven genes (san, 329

Cht11, Uck2, Echs1, whd, Ccdc58, and Apop1) encode for proteins with mitochondrial function, 330

serve as substrates in the mitochondria or are associated with proteins with mitochondrial 331

function. For example, the Cht11 gene encodes a glycoside hydrolase with a chitinase active site 332


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

16

(43). Cht11 is orthologous to the Chitinase 11 gene of Tribolium castaneum which has been 333

assigned to a separate classification group (group VII) from other identified chitinases (3), 334

(personal communication, Dr. Subbaratnam Muthukrishnan, Kansas State University). This 335

suggests that Cht11 has a function other than those classically described for chitinases. A two-336

hybrid screen associated this protein with the protein product encoded by Tiny tim 50 (22). 337

Ttm50 (also known as Tim50) encodes a protein that is part of a transporter located on the inner 338

mitochondrial membrane (15) (Figure 9). Chitins and their derivatives regulate cholesterol when 339

used as diet supplements (29, 74). Mice with high blood cholesterol had more severe Anaplasma 340

phagocytophilum (another tick transmitted rickettsia pathogen closely related to E. chaffeensis) 341

infections in their blood, livers, and spleens compared to mice with normal cholesterol levels 342

(72). E. chaffeensis and A. phagocytophilum cannot synthesize cholesterol and scavenge it from 343

host cells during infections (34). Therefore, Cht11 may function to regulate cholesterol levels to 344

facilitate infections. Cholesterol can also affect both the inner and outer mitochondrial 345

membranes and affect ATPase activity (10, 19). A human homolog of Cht11 has not been 346

identified, but homologs have been identified in ticks and body lice (43). Both of these species 347

serve as vectors for several different Rickettsia bacteria (4), and it will be interesting to explore 348

the function of this gene for the survival of E. chaffeensis and other rickettsial agents in their 349

respective arthropod hosts. 350

Several of the genes that impact E. chaffeensis infections encode for key mitochondrial 351

enzymes needed for fatty acid metabolism and acetyl-CoA production. The whd gene was 352

originally associated with the uridine kinase (56). However, recent annotation (43) and 353

experimentation (58) have identified whd as the palmitoyltransferase 1 (CPT 1) gene. 354

Palmitoyltransferase 1 is the rate-limiting enzyme for palmitoyl-CoA uptake into mitochondria 355


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

17

(24) (Figure 9). Echs1 in Drosophila is an ortholog to the ECHS1 gene in humans and other 356

mammals (43) which encodes a enoyl-CoA hydratase in the second step of the β-oxidation 357

pathway in the mitochondrion (27) (Figure 9). Therefore, when we experimentally challenged 358

flies mutant for the Echs1 gene, the immediate downstream effect might be on the supply of 359

acetyl CoA into the citric acid cycle and implies that the supply of acetyl CoA may be critical to 360

E. chaffeensis replication. san is the ortholog to the Human NAA50 gene which encodes for 361

N(alpha)-acetyltransferase 50 (43). These acetyltransferases are involved in the conversion of 362

glycerol-3-phosphate to phosphatidic acid (13). This biochemical step is upstream of the CDP-363

DAG intermediate in the synthesis of cardiolipin on the outer mitochondrial membrane. It is also 364

possible that the acetyltransferase could also be active in the β-oxidation pathway. However, this 365

has yet to be proven experimentally. 366

CG15881 or Ccdc58 is an ortholog to the human gene known as coiled-coil domain-367

containing protein 58 (CCDC58). Although the exact function for Ccdc58 is not known, its 368

structure likely allows it to interact with other coiled-coil molecules (41). Indeed, the “STRING” 369

association tool (60) shows that CCDC58 has a direct relationship with MRPS28, mitochondrial 370

ribosomal protein S28 and translocase of inner mitochondrial membrane 9 homolog (TIMM9), 371

which encode for an inner mitochondrial membrane chaperone protein, SSBP1, a protein 372

putatively thought to be involved in mitochondrial DNA replication (60). The BioGraph gene 373

association tool (33) assigned a rank of 23 out of 18,180 gene concepts (top 0.13%) when it was 374

assigned to “mitochondria”. Other coiled-coil domain containing proteins have been associated 375

with the inner mitochondrial membrane (70) and CCDC58 has also been associated with the 376

mitochondrial single nucleotide polymorphisms that impact the progression of acquired immune 377


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

18

deficiency syndrome (26). Therefore, it is highly likely that the impact of gene disruption 378

Ccdc58 on E. chaffeensis growth is due to its impact on mitochondrial function. 379

CG14806 or Apopt1 has no known function in Drosophila. It is an ortholog of the 380

Apoptogenic 1 gene (APOPT1 or Apop1) in humans and rodents (66). It is involved in the 381

release of cytochrome C through permeability transition pores in the outer membrane of the 382

mitochondrion and the activation of apoptosis (59, 73) (Figure 9). It seems plausible that 383

inhibiting apoptosis would be to the bacterium’s advantage. There is increased transcription of 384

some apoptotic genes during Ehrlichia infections (75) and there is delayed apoptosis in 385

neutrophils after infection with E. ewingii (71). It is possible that Apop1 is needed to help in the 386

release of bacteria at the end of the replicative phase. E. chaffeensis organisms disseminate by 387

lysing host cells or by exocytosis in order to spread to uninfected cells (17). More empirical 388

analyses will be needed to support this hypothesis. 389

Uck2 (CG6364) is orthologous to the mammalian UCK2 gene with the molecular 390

function of a uridine kinase (43). Functionally, it has been associated with the phagocytosis of 391

Candida albicans (57). Using the “STRING” association tool (60), we found that its predicted 392

functional partners are all involved in nucleotide/nucleoside modification. These functions 393

include nucleoside di- and tri-phosphate activity (CG5276), hydrolase activity (CG8891), uracil 394

phosphoribiosyl transferase activity (CG5537), uridine phosphorylase (CG3788, CG8349), 395

cytidylate/uridylate kinase activity (Dak1), uridine phosphorylase (CG6330) and cytidine 396

deaminase activity (CG8349). The BioGraph gene association tool (33) assigned a rank of 397

12.63% (2297th out of 18180 gene objects) when Uck2 was associated with mitochondria; 398

indicating a highly probable association. Cytosine triphosphate, a down-stream product of the 399

uridine kinase (38, 62) is a substrate used for the conversion of phosphatidic acid to cytidine 400


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

19

diphosphate diacylglycerol (CDP-DAG) (31, 46) (Figure 9). CDP-DAG is synthesized in the 401

inner mitochondrial membrane and is necessary for the synthesis of cardiolipin, a major 402

component of the inner mitochondrial membrane (46). Therefore, we reason that the uridine 403

kinase must be essential to providing substrates for E. chaffeensis, because it can’t be provided 404

by the de novo synthesis pathway. When we grew E. chaffeensis in the presence of 405

cyclopentenyl cytosine (CPEC), a CTP synthetase inhibitor (52, 62), which inhibits the synthesis 406

of CTP from glutamate (Figure 5), the bacterial replication was significantly enhanced (Figure 407

6). These data suggest that the bacterium does not require the host to use glutamine as a 408

substrate during replication. Supplementation of cytosine to cells enhanced Ehrlichia replication 409

which further implicates the use of the salvage pathway through cytidine. The enhanced growth 410

of another rickettsia, Rickettsia felis, from pyrimidines in tryptose phosphate broth is also 411

consistent with the involvement of mitochondrial enzymes in obligate intracellular bacterial 412

growth (50). Moreover, cytidine is the least abundant nucleoside in cells (32). The depletion of 413

the cytidine pools can disrupt the balance of ribonucleotides in cells, leading to alterations in 414

cellular homeostasis and apoptosis (52). These observations suggest that E. chaffeensis needs to 415

regulate cytidine in order to control apoptosis until its replication is completed within a 416

phagosome. 417

The enhanced growth of E. chaffeensis after treatment with CPEC instead of an inhibitory 418

effect suggests that the bacterium requires the uridine/cytidine salvage pathway to phosphorylate 419

cytidine and uridine (61) for its replication. More importantly, the disruption of E. chaffeensis 420

growth in UCK2-silenced THP-1 macrophages reinforces that the UCK2 target has human 421

relevance. Therefore, we focused our attention on Uck2 because it has the potential to be a target 422

for chemotherapy. UCK2 has been found to be more active in certain types of cancers (54), and 423


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

20

acts to phosphorylate nucleoside analog drugs used for treatment of cancers and hepatitis C virus 424

infections (23, 61). Uninfected host cells may be unaffected by UCK2 inhibitors since 425

nucleotides could be synthesized by the de novo synthesis pathway. 426

The clustering of mitochondria adjacent to morulae in E. chaffeensis infected 427

macrophages supports the hypothesis that it has physiological relevance and is consistent with 428

previous observations (37, 49). Mitochondrial membrane permeability and membrane potential 429

was not disrupted by either E. chaffeensis (37) or E. ewengii infections (71). However, Liu et al. 430

found that mitochondrial biochemical activity was reduced as evidenced from the measurement 431

of mitochondrial DNA synthesis or transcription of several mitochondrial genes (37). There is 432

also evidence that the E. chaffeensis type IV secretion system is used to insert bacterial effector 433

proteins into host mitochondria (36). These observations suggest that the organism inhibits 434

mitochondrial activity; perhaps to inhibit the generation of mitochondrial produced oxidative 435

products that would be harmful to the bacteria (5, 68). In contrast, we have disrupted several 436

genes which are associated with mitochondrial function that negatively affect E. chaffeensis 437

infections. The gene products of san and Uck2 affect cardiolipin synthesis on the inner 438

mitochondrial membrane (Figure 9). Cht11 encodes protein for part of a transport molecule on 439

that same inner mitochondrial membrane. The gene products of whd and Echs1 affect 440

steroidogenesis, ketogenesis and the Krebs cycle (Figure 9); the latter of which has clear 441

mitochondrial dependence. Although Ccdc58 and Apopot1 are associated with mitochondria 442

(Figure 9), we have yet to fully understand their significance. It is also interesting that genes like 443

Nup37 and dumpy which don’t have direct mitochondrial connections also affect E. chaffeensis 444

infections. The NUP37 gene product is part of nucleopores. One of the molecules it transports, 445

glucose, is also important to mitochondrial functions. The dumpy gene has been characterized 446


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

21

genetically and appears to impact wing and other body part development (11, 12). It has no 447

known mammalian orthologs (43) and dumpy encodes a large extracellular molecule needed for 448

structural integrity in wing epithelial tissue (12, 69). Interestingly, one of the properties listed for 449

dumpy is that it binds iron and sulfur (43); properties of mitochondrial proteins involved in 450

oxidation-reduction reactions (6). Clearly, more work is necessary to understand these 451

molecules and their host-bacteria interactions. Nevertheless, our findings provide clues about the 452

mitochondrial processes that are needed by the bacteria. Interestly, at least one α-453

proteobacterium closely related to Ehrlichia, designated as IricES1, infects the mitochondria of 454

ovarian cells of Ixodes ricinus (7). Perhaps E. chaffeensis is one step away from a similar 455

symbiotic relationship. 456

457

ACKNOWLEDGEMENTS 458

We would like to thank Mr. Rishi Drolia, Mr. Taylor Kinney and Ms. Rachel Nichols for 459

their help in the lab with the flies, screening and bacteria. We thank Mr. Lloyd Willard for his 460

help with the electron microscopy. We thank Ms. Nanyan Lu and the K-INBRE bioinformatics 461

center for help in analyzing the microarray data, Ms. Mal Rooks Hoover for her design of Figure 462

9 and Dr. David Rintoul for helpful comments concerning the manuscript. We thank the Kansas 463

University Medical Center-Microarray Facility (KUMC-MF) for generating array data sets. The 464

Microarray Facility is supported by the Kansas University-School of Medicine, KUMC 465

Biotechnology Support Facility, the Smith Intellectual and Developmental Disabilities Research 466

Center (HD02528), and the Kansas IDeA Network of Biomedical Research Excellence 467

(RR016475). This project has been supported by NIH grants AI088070, AI55052, AI052206, 468

AI070908, RR16475 and RR17686, the Kansas Agriculture Experiment Station Animal Health 469


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

22

Project grant 481848, the Kansas Space Grant Consortium, NASA grants NAG2-1274 and 470

NNX08BA91G, American Heart Association grant 0950036G, the Terry C. Johnson Center for 471

Basic Cancer Research and the Kansas Agriculture Experiment Station. This is Kansas 472

Agriculture Experiment Station publication 12-447-J. 473

474

475

476


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

23

FIGURE LEGENDS 477

Figure 1. Infection of microarray samples by E. chaffeensis. S2 cells ± LPS treatment were 478

assayed for E. chaffeensis infection after 24 or 96 hours; uninfected S2 cells were also assessed. 479

E. chaffeensis infection was confirmed by assessing the ribosomal 16S RNA as described in the 480

Materials and Methods. D. melanogaster rp49 transcript was used as a loading control. 481

482

Figure 2. Infection of mutant flies with E.chaffeensis. D. melanogaster were mutant for the 483

indicated gene and observed for sensitivity to infection. Wild-type flies injected with PBS (Δ) or 484

bacteria (▲), mutant flies injected with PBS (○) or bacteria (●). Numbers represent mean±SEM 485

of three or more independent experiments, 20 flies per treatment group per experiment. † 486

indicates survival is significantly different from E. chaffeensis-infected wild-type flies, P<0.05 487

using the log rank test of the Kaplan-Meier plots. 488

489

Figure 3. Bacteria growth in Drosophila mutants. Bacteria numbers were quantified 96 hrs 490

after infection using qRT-PCR as described in the Materials and Methods. Numbers represent 491

mean±SEM of three assessments for Cht11, Uck2, Echs1, san, CG6479, tilt, withered and dumpy 492

and one assessment for Ccdc58, Nup37 and Apop1. 493

494

Figure 4. Effect of withered, tilt and dumpy gene mutations on E. chaffeensis infections. 495

Withered, tilt and dumpy flies were screened for their sensitivity to infection. Numbers represent 496

mean±SEM of 4 or 5 independent experiments, 20 flies per treatment group per experiment. # 497

indicates survival is significantly different from E. chaffeensis-infected wild-type flies, P<0.05 498

using the log rank test of the Kaplan-Meier plots. 499


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

24

500

Figure 5. Nucleotide de novo and salvage pathways for production of dCTP (deoxycytidine 501

triphosphate). De novo synthesis through glutamine and UMP (uridine monophosphate) is 502

represented by bold arrows. The salvage pathways through uridine or cytidine are represented 503

by thin/hatched arrows. The star represents the enzyme CTP synthetase; and the black triangles 504

represent uridine/cytidine kinase. Inhibition by CPEC occurs at the conversion of UTP to CTP. 505

506

Figure 6. Effect of cyclopentenyl cytosine on E. chaffeensis infections. Percent change in E. 507

chaffeensis 16S rRNA copies in S2 cells treated with CPEC compared to S2 cells treated with 508

carrier only. Two treatment schemes: 1) “Day 1: CPEC”, S2 cells were first treated with CPEC 509

(at indicated concentration) for 48 hours, and then infected with E. chaffeensis for an additional 510

48 hours. 2) “Day 1: Ec”, S2 cells were first infected with E. chaffeensis for 48 hours, and then 511

treated with CPEC (at indicated concentration) for 48 hours. RNA extraction was performed 512

following both treatment schemes. Data presented represent the mean ± SEM of 3 independent 513

experiments. 514

515

Figure 7. Effect of cytosine on E. chaffeensis infections Percent change in Ehrlichia 16S 516

rRNA copies in S2 cells treated with cytosine and infected with E. chaffeensis organisms 517

compared to S2 cells treated with carrier only and infected with bacteria. Data presented 518

represent the mean of 3 independent experiments. † indicates a significant difference P<0.03. 519

The “Ehrlichia only” bar represents the baseline control which was the same at each time so only 520

one control was presented. 521

522


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

25

Figure 8. Localization of mitochondria around morulae of E. chaffeensis-infected DH82 523

cells. Transmission electron microscopy of DH82 cells infected with E. chaffeensis 96 (A; 524

5,000X magnification) or 168 (B; 15,000X magnification) hours after infection. Uninfected 525

DH82 cells (C; 5,000X magnification). Morulae (M) are surrounded by mitochondria indicated 526

by arrows. 527

528

Figure 9. Identification of mitochondria-associated genes that impact E. chaffeensis 529

infection. san (N-(alpha) acetyl transferase 50) and Uck2 (uridine/cytidine kinase) are needed 530

for the synthesis of cardiolipin, Phophatidic acid (PA) a product of san activity and CTP which is 531

produced from CMP, a product of Uck2, are both necessary substrates for cardiolipin 532

biosynthesis, the major inner mitochondrial membrane lipid. Cht11 (chitinase 11) encodes part 533

of a protein transporter in the inner mitochondrial membrane. whd (withered) encodes the 534

carnitine palmitoyl transferase 1 and Echs1 encodes the enoyl-CoA hydratase; both enzymes are 535

needed for the production of acetyl CoA. Ccdc58 encodes coiled-coil domain-containing 536

protein 58, which is found in complex with inner mitochondrial membrane molecules MRPS28 537

and SSBP1. Apopot1 (Apoptogenic 1) induces the release of cytochrome C through the 538

permeability transition pore and triggers apoptosis. Nup37 (Nucleoporin 37) is not found in the 539

mitochondrion but is a part of a nucleopore complex that transports hexose molecules including 540

glucose, an important molecule in energy metabolism. 541

REFERENCES 542

1. Agaisse, H., L. S. Burrack, J. A. Philips, E. J. Rubin, N. Perrimon, and D. E. 543 Higgins. 2005. Genome-wide RNAi screen for host factors required for intracellular 544 bacterial infection. Science 309:1248-1251. 545


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

26

2. Alvarez-Dominguez, C., A. M. Barbieri, W. Beron, A. Wandinger-Ness, and P. D. 546 Stahl. 1996. Phagocytosed live Listeria monocytogenes influences Rab5-regulated in 547 vitro phagosome-endosome fusion. J Biol Chem 271:13834-13843. 548

3. Arakane, Y., and S. Muthukrishnan. 2010. Insect chitinase and chitinase-like proteins. 549 Cellular and Molecular Life Sciences 67:201-216. 550

4. Azad, A., and C. Beard. 1998. Rickettsial pathogens and their arthropod vectors. 551 A. F. Azad and C. B. Beard. Emerging Infectious Diseases 4:179–186. 552 5. Basu Ball, W., S. Kar, M. Mukherjee, A. G. Chande, R. Mukhopadhyaya, and P. K. 553

Das. 2011. Uncoupling protein 2 negatively regulates mitochondrial reactive oxygen 554 species generation and induces phosphatase-mediated anti-inflammatory response in 555 experimental visceral leishmaniasis. The Journal of Immunology 187:1322-1332. 556

6. Beinert, H., R. H. Holm, and E. Münck. 1997. Iron-Sulfur clusters: Nature's modular, 557 multipurpose structures. Science 277:653-659. 558

7. Beninati, T., N. Lo, L. Sacchi, C. Genchi, H. Noda, and C. Bandi. 2004. A novel 559 alpha-proteobacterium resides in the mitochondria of ovarian cells of the tick Ixodes 560 ricinus. Applied and Environmental Microbiology 70:2596-2602. 561

8. Brandt, S. M., G. Jaramillo-Gutierrez, S. Kumar, C. Barillas-Mury, and D. S. 562 Schneider. 2008. Use of a Drosophila model to identify genes regulating Plasmodium 563 growth in the mosquito. Genetics 180:1671-1678. 564

9. Brouqui, P., C. Lecam, J. Olson, and D. Raoult. 1994. Serologic diagnosis of human 565 monocytic ehrlichiosis by immunoblot analysis. Clin Diagn Lab Immunol 1:645-649. 566

10. Calanni Rindina, F., A. Baracca, G. Solaini, A. Rabbi, and G. Parenti Castelli. 1986. 567 Effects of cholesterol on the kinetics of mitochondrial ATPase. FEBS Lett 198:353-356. 568

11. Carmon, A., M. J. Guertin, O. Grushko, B. Marshall, and R. MacIntyre. 2010. A 569 molecular analysis of mutations at the complex dumpy Locus in Drosophila 570 melanogaster. PloS One 5:e12319. 571

12. Carmon, A., F. Topbas, M. Baron, and R. J. MacIntyre. 2010. dumpy interacts with a 572 large number of genes in the developing wing of Drosophila melanogaster. Fly 4:117-573 127. 574

13. Castorena, K., K. Stapleford, and D. Miller. 2010. Complementary transcriptomic, 575 lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight 576 the role of glycerophospholipid metabolism in Flock House virus RNA replication. BMC 577 Genomics 11:183. 578

14. Centers for Disease Control and Prevention, C. 2012. Notifiable Diseases and 579 Mortality Tables. MMWR Morb. Mortal Wkly Rep 60:1729-1776. 580

15. Chacinska, A., C. M. Koehler, D. Milenkovic, T. Lithgow, and N. Pfanner. 2009. 581 Importing mitochondrial proteins: machineries and mechanisms. Cell 138:628-644. 582

16. Cinco, M., F. Barbone, M. Grazia Ciufolini, M. Mascioli, M. Anguero Rosenfeld, P. 583 Stefanel, and R. Luzzati. 2004. Seroprevalence of tick-borne infections in forestry 584 rangers from northeastern Italy. Clin Microbiol Infect 10:1056-1061. 585

17. Dedonder, S. E., C. Cheng, L. H. Willard, D. L. Boyle, and R. R. Ganta. 2012. 586 Transmission electron microscopy reveals distinct macrophage- and tick cell-specific 587 morphological stages of Ehrlichia chaffeensis. PloS One 7:e36749. 588

18. Dionne, M. S., N. Ghori, and D. S. Schneider. 2003. Drosophila melanogaster is a 589 genetically tractable model host for Mycobacterium marinum. Infect and Immun 590 71:3540-3550. 591


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

27

19. Echegoyen, S., E. B. Oliva, J. Sepulveda, J. C. Diaz-Zagoya, M. T. Espinosa-Garcia, 592 J. P. Pardo, and F. Martinez. 1993. Cholesterol increase in mitochondria: its effect on 593 inner-membrane functions, submitochondrial localization and ultrastructural morphology. 594 The Biochemical Journal 289 ( Pt 3):703-708. 595

20. Elwell, C., and J. N. Engel. 2005. Drosophila melanogaster S2 cells: a model system to 596 study Chlamydia interaction with host cells. Cellular Microbiology 7:725-739. 597

21. Ganta, R. R., C. Cheng, M. J. Wilkerson, and S. K. Chapes. 2004. Delayed clearance 598 of Ehrlichia chaffeensis infection in CD4+ T-cell knockout mice. Infect Immun 72:159-599 167. 600

22. Giot, L., J. S. Bader, C. Brouwer, A. Chaudhuri, B. Kuang, Y. Li, Y. L. Hao, C. E. 601 Ooi, B. Godwin, E. Vitols, G. Vijayadamodar, P. Pochart, H. Machineni, M. Welsh, 602 Y. Kong, B. Zerhusen, R. Malcolm, Z. Varrone, A. Collis, M. Minto, S. Burgess, L. 603 McDaniel, E. Stimpson, F. Spriggs, J. Williams, K. Neurath, N. Ioime, M. Agee, E. 604 Voss, K. Furtak, R. Renzulli, N. Aanensen, S. Carrolla, E. Bickelhaupt, Y. 605 Lazovatsky, A. DaSilva, J. Zhong, C. A. Stanyon, R. L. Finley, Jr., K. P. White, M. 606 Braverman, T. Jarvie, S. Gold, M. Leach, J. Knight, R. A. Shimkets, M. P. 607 McKenna, J. Chant, and J. M. Rothberg. 2003. A protein interaction map of 608 Drosophila melanogaster. Science 302:1727-1736. 609

23. Golitsina, N. L., F. T. Danehy, Jr., R. Fellows, E. Cretton-Scott, and D. N. 610 Standring. Evaluation of the role of three candidate human kinases in the conversion of 611 the hepatitis C virus inhibitor 2'-C-methyl-cytidine to its 5'-monophosphate metabolite. 612 Antiviral Res 85:470-481. 613

24. Gomez, L. A., S. H. Heath, and T. M. Hagen. 2012. Acetyl-L-carnitine 614 supplementation reverses the age-related decline in carnitine palmitoyltransferase 1 615 (CPT1) activity in interfibrillar mitochondria without changing the L-carnitine content in 616 the rat heart. Mechanisms of Ageing and Development 133:99-106. 617

25. Hall-Baker, P. A., J. Enrique Nieves, D. R. Jajosky, D. A. Adams, P. Shar, W. J. 618 Anderson, J. J. Aponte, G. F. Jones, M. A. E. Aranas, M. A. Rey, M. B. Lane, and 619 M. S. Wodajo. 2009. Summary of Notifiable Diseases -United States, 2007. MMWR 620 56:1-94 621

26. Hendrickson, S. L., J. A. Lautenberger, L. W. Chinn, M. Malasky, E. Sezgin, L. A. 622 Kingsley, J. J. Goedert, G. D. Kirk, E. D. Gomperts, S. P. Buchbinder, J. L. Troyer, 623 and S. J. O'Brien. 2010. Genetic variants in nuclear-encoded mitochondrial genes 624 influence AIDS progression. PloS One 5:e12862. 625

27. Janssen, U., E. M. Davis, M. M. Le Beau, and W. Stoffel. 1997. Human mitochondrial 626 enoyl-CoA hydratase gene (ECHS1): structural organization and assignment to 627 chromosome 10q26.2-q26.3. Genomics 40:470-475. 628

28. Kang, G. J., D. A. Cooney, J. D. Moyer, J. A. Kelley, H. Y. Kim, V. E. Marquez, and 629 D. G. Johns. 1989. Cyclopentenylcytosine triphosphate. Formation and inhibition of 630 CTP synthetase. J Biol Chem 264:713-718. 631

29. Koide, S. 1998. Chitin-chitosan: Properties, benefits and risks. Nutrition Research 632 18:1091-1101. 633

30. Koo, I. C., Y. M. Ohol, P. Wu, J. H. Morisaki, J. S. Cox, and E. J. Brown. 2008. Role 634 for lysosomal enzyme beta-hexosaminidase in the control of mycobacteria infection. Proc 635 Natl Acad Sci U S A 105:710-715. 636


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

28

31. Kopka, J., M. Ludewig, and B. Muller-Rober. 1997. Complementary DNAs encoding 637 eukaryotic-type cytidine-5'-diphosphate-diacylglycerol synthases of two plant species. 638 Plant Physiology 113:997-1002. 639

32. Korte, D., W. Haverkort, A. van Gennip, and D. Roos. 1985. Nucleotide profiles of 640 normal human blood cells determined by high-performance liquid chromatography. Anal. 641 Biochem. 147:197-209. 642

33. Liekens, A., J. De Knijf, W. Daelemans, B. Goethals, P. De Rijk, and J. Del-Favero. 643 2011. BioGraph: unsupervised biomedical knowledge discovery via automated 644 hypothesis generation. Genome Biology 12:R57. 645

34. Lin, M., and Y. Rikihisa. 2003. Ehrlichia chaffeensis and Anaplasma phagocytophilum 646 lack genes for lipid A biosynthesis and incorporate cholesterol for their survival. Infect 647 Immun 71:5324-5331. 648

35. Lindsley, D., and G. Zimm. 1992. The Genome of Drosophila melanogaster. Academic 649 Press, Inc., San Diego, CA. 650

36. Liu, H., W. Bao, M. Lin, H. Niu, and Y. Rikihisa. 2012. Ehrlichia type IV secretion 651 effector ECH0825 is translocated to mitochondria and curbs ROS and apoptosis by 652 upregulating host MnSOD. Cellular Microbiology 14:1037-1050. 653

37. Liu, Y., Z. Zhang, Y. Jiang, L. Zhang, V. L. Popov, J. Zhang, D. H. Walker, and X.-654 j. Yu. 2011. Obligate intracellular bacterium Ehrlichia inhibiting mitochondrial activity. 655 Microbes and Infection 13:232-238. 656

38. Loffler, M., L. D. Fairbanks, E. Zameitat, A. M. Marinaki, and H. A. Simmonds. 657 2005. Pyrimidine pathways in health and disease. Trends in Molecular Medicine 11:430-658 437. 659

39. Luce-Fedrow, A., T. Von Ohlen, D. Boyle, R. R. Ganta, and S. K. Chapes. 2008. 660 Drosophila S2 cells as a model for studying Ehrlichia chaffeensis infections. Appl. 661 Environ. Microbiol. 74:1886-1891. 662

40. Luce-Fedrow, A., T. Von Ohlen, and S. K. Chapes. 2009. Ehrlichia chaffeensis 663 infections in Drosophila melanogaster. Infection and immunity 77:4815-4826. 664

41. Lupas, A. N., and M. Gruber. 2005. The Structure of α-Helical Coiled Coils, p. 37-38. 665 In A. D. P. David and M. S. John (ed.), Advances in Protein Chemistry, vol. Volume 70. 666 Academic Press. 667

42. Machado, R. Z., J. M. Duartes, A. S. Dagnone, and M. P. Szabo. 2006. Detection of 668 Ehrlichia chaffeensis in Brazilain marsh deer (Blastocerus dichotomus. Vet Parasitol 669 139:262-266. 670

43. McQuilton, P., S. E. St Pierre, J. Thurmond, and C. FlyBase. 2012. FlyBase 101--the 671 basics of navigating FlyBase. Nucleic Acids Research 40:D706-714. 672

44. Morgan, T. 1929. Contributions to the genetics of Drosophila simulans and Drosophila 673 melanogaster. Publs Carnegie Instn 399:169-199. 674

45. Moyer, J., N. Malinowski, S. Treanor, and V. Marquez. 1986. Antitumor activity and 675 biochemical effects of cyclopentenyl cytosine in mice. Cancer Res 46:3325-3329. 676

46. Osman, C., M. Haag, F. T. Wieland, B. Brugger, and T. Langer. 2010. A 677 mitochondrial phosphatase required for cardiolipin biosynthesis: the PGP phosphatase 678 Gep4. The EMBO Journal 29:1976-1987. 679

47. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time 680 RT-PCR. Nucleic Acids Research 29:e45. 681


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

29

48. Philips, J. A., E. J. Rubin, and N. Perrimon. 2005. Drosophila RNAi screen reveals 682 CD36 family member required for mycobacterial infection. Science 309:1251-1253. 683

49. Popov, V. L., S.-M. Chen, H.-M. Feng, and O. H. Walker. 1995. Ultrastructural 684 variation of cultured Ehrlichia chaffeensis. Journal of Medical Microbiology 43:411-421. 685

50. Saisongkorh, W., K. E. Karkouri, J.-Y. Patrice, A. Bernard, J.-M. Rolain, and D. 686 Raoult. 2012. Tryptose phosphate broth improves Rickettsia felis replication in 687 mammalian cells. FEMS Immunology & Medical Microbiology 64:111-114. 688

51. Santic, M., C. Akimana, R. Asare, J. C. Kouokam, S. Atay, and Y. A. Kwaik. 2009. 689 Intracellular fate of Francisella tularensis within arthropod-derived cells. Environ 690 Microbiol 11:1473-1481. 691

52. Schimmel, K. J., H. Gelderblom, and H. J. Guchelaar. 2007. Cyclopentenyl cytosine 692 (CPEC): an overview of its in vitro and in vivo activity. Current Cancer Drug Targets 693 7:504-509. 694

53. Schneider, D., and M. Shahabuddin. 2000. Malaria parasite development in a 695 Drosophila model. Science 288:2376-2379. 696

54. Shimamoto, Y., K. Koizumi, H. Okabe, H. Kazuno, Y. Murakami, F. Nakagawa, A. 697 Matsuda, T. Sasaki, and M. Fukushima. 2005. Sensitivity of human cancer cells to the 698 new anticancer ribo-nucleoside TAS–106 is correlated with expression of uridine-699 cytidine Kinase 2. Cancer Science 93:825-833. 700

55. Sirigireddy, K. R., and R. R. Ganta. 2005. Multiplex detection of Ehrlichia and 701 Anaplasma species pathogens in peripheral blood by real-time reverse transcriptase-702 polymerase chain reaction. J Mol Diagn 7:308-316. 703

56. Stroman, P. 1974. Pyrimidine-sensitive drosophila wing mutants: withered (whd), tilt (tt) 704 and dumpy (dp). Hereditas 78:157-168. 705

57. Stroschein-Stevenson, S. L., E. Foley, P. H. O'Farrell, and A. D. Johnson. 2006. 706 Identification of Drosophila gene products required for phagocytosis of Candida albicans. 707 PLoS Biology 4:e4. 708

58. Strub, B. R., T. L. Parkes, S. T. Mukai, S. Bahadorani, A. B. Coulthard, N. Hall, J. 709 P. Phillips, and A. J. Hilliker. 2008. Mutations of the withered (whd) gene in 710 Drosophila melanogaster confer hypersensitivity to oxidative stress and are lesions of the 711 carnitine palmitoyltransferase I (CPT I) gene. Genome / National Research Council 712 Canada = Genome / Conseil national de recherches Canada 51:409-420. 713

59. Sun, X., O. Yasuda, Y. Takemura, H. Kawamoto, M. Higuchi, Y. Baba, T. Katsuya, 714 K. Fukuo, T. Ogihara, and H. Rakugi. 2008. Akt activation prevents Apop-1-induced 715 death of cells. Biochemical and Biophysical Research Communications 377:1097-1101. 716

60. Szklarczyk, D., A. Franceschini, M. Kuhn, M. Simonovic, A. Roth, P. Minguez, T. 717 Doerks, M. Stark, J. Muller, P. Bork, L. J. Jensen, and C. v. Mering. 2011. The 718 STRING database in 2011: functional interaction networks of proteins, globally 719 integrated and scored. Nucleic Acids Research 39:D561-D568. 720

61. Van Rompay, A. R., A. Norda, K. Linden, M. Johansson, and A. Karlsson. 2001. 721 Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-722 cytidine kinases. Mol Pharmacol 59:1181-1186. 723

62. Verschurr, A. C. 2007. Cytidine triphosphage synthetase (CTP synthetase) as a 724 druggable target in cancer. Drugs of the Future 32:1071-1080. 725

63. Verschuur, A. C., A. H. Van Gennip, R. Leen, R. Meinsma, P. A. Voute, and A. B. 726 van Kuilenburg. 2000. In vitro inhibition of cytidine triphosphate synthetase activity by 727


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

30

cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia. Br J Haematol 728 110:161-169. 729

64. Vonkavaara, M., M. V. Telepnev, P. Ryden, A. Sjostedt, and S. Stoven. 2008. 730 Drosophila melanogaster as a model for elucidating the pathogenicity of Francisella 731 tularensis. Cell Microbiol 10:1327-1338. 732

65. Waddington, C. 1940. The genetic control of wing development in Drosophila. J Genet 733 41:75-139. 734

66. Waterhouse, R. M., E. M. Zdobnov, F. Tegenfeldt, J. Li, and E. V. Kriventseva. 735 2011. OrthoDB: the hierarchical catalog of eukaryotic orthologs in 2011. Nucleic Acids 736 Research 39:D283-288. 737

67. Wen, B., W. Cao, and H. Pan. 2003. Ehrlichiae and ehrlichial diseases in china. Ann N 738 Y Acad Sci 990:45-53. 739

68. West, A. P., I. E. Brodsky, C. Rahner, D. K. Woo, H. Erdjument-Bromage, P. 740 Tempst, M. C. Walsh, Y. Choi, G. S. Shadel, and S. Ghosh. 2011. TLR signalling 741 augments macrophage bactericidal activity through mitochondrial ROS. Nature 472:476-742 480. 743

69. Wilkin, M. B., M. N. Becker, D. Mulvey, I. Phan, A. Chao, K. Cooper, H. J. Chung, 744 I. D. Campbell, M. Baron, and R. MacIntyre. 2000. Drosophila dumpy is a gigantic 745 extracellular protein required to maintain tension at epidermal-cuticle attachment sites. 746 Current biology : CB 10:559-567. 747

70. Xie, J., M. F. Marusich, P. Souda, J. Whitelegge, and R. A. Capaldi. 2007. The 748 mitochondrial inner membrane protein Mitofilin exists as a complex with SAM50, 749 metaxins 1 and 2, coiled-coil-helix coiled-coil-helix domain-containing protein 3 and 6 750 and DnaJC11. FEBS Letters 581:3545-3549. 751

71. Xiong, Q., W. Bao, Y. Ge, and Y. Rikihisa. 2008. Ehrlichia ewingii infection delays 752 spontaneous neutrophil apoptosis through stabilization of mitochondria. Journal of 753 Infectious Diseases 197:1110-1118. 754

72. Xiong, Q., X. Wang, and Y. Rikihisa. 2007. High-cholesterol diet facilitates Anaplasma 755 phagocytophilum infection and up-regulates macrophage inflammatory protein-2 and 756 CXCR2 expression in apolipoprotein E-deficient mice. J Infect Dis 195:1497-1503. 757

73. Yasuda, O., K. Fukuo, X. Sun, M. Nishitani, T. Yotsui, M. Higuchi, T. Suzuki, H. 758 Rakugi, O. Smithies, N. Maeda, and T. Ogihara. 2006. Apop-1, a novel protein 759 inducing cyclophilin D-dependent but Bax/Bak-related channel-independent apoptosis. 760 Journal of Biological Chemistry 281:23899-23907. 761

74. Zacour, A., M. Silva, P. Cecon, E. Bambirra, and E. Vieira. 1992. Effect of dietary 762 chitin on cholesterol absorption and metabolism in rats. J Nutr Sci Vitaminol (Tokyo). 763 32:609-613. 764

75. Zhang, J. Z., M. Sinha, B. A. Luxon, and X. J. Yu. 2004. Survival strategy of 765 obligately intracellular Ehrlichia chaffeensis: novel modulation of immune response and 766 host cell cycles. Infect Immun 72:498-507. 767 768 769


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

Table 1. Impact of UCK2 dsRNA silencing on E. chaffeensis growth in THP-1 Cells

1Silencing efficiency compared to scrambled dsRNA. 2Numbers represent mean "SEM (median) of 5 independent experiments. Control E. chaffeensis numbers: 26,348±4,147 3Silencing efficiency of HPRT dsRNA. HPRT used as a control to assure transfection and silencing protocol effectiveness.

Treatment % Inhibition % E. chaffeensis of scrambled dsRNA Control

Scrambled dsRNA

Duplex 1 99"11,2 31"21 (9)2

Duplex 2 97"51 46"32 (24)

Duplex 3 96"31 53"30 (29)

HPRT 100"03 225"86 (108)


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

Documents

Identification of critical host mitochondrial-associated genes during