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TITLE: Non-selective Persistence of a Rickettsia conorii Extrachromosomal Plasmid 1
During Mammalian Infection 2
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RUNNING TITLE: Rickettsia plasmids in mammalian infection 4
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AUTHORS: Sean P Riley1,2, Abigail I Fish1,2, Daniel A Garza1,2, Kaikhushroo H 6
Banajee1,2, Emma K Harris1,2, Fabio del Piero2, Juan J Martinez1,2,# 7
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AFFILIATIONS: 1 Vector Borne Disease Laboratories, 2 Department of Pathobiological 9
Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 10
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# CORRESPONDING AUTHOR: [email protected], (225) 578-929712
IAI Accepted Manuscript Posted Online 11 January 2016Infect. Immun. doi:10.1128/IAI.01205-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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ABSTRACT: 13
Scientific analysis of the genus Rickettsia is undergoing a rapid period of change with 14
the emergence of viable genetic tools. The development of these tools for mutagenesis 15
of pathogenic bacteria will permit forward genetic analysis of Rickettsia pathogenesis. 16
Despite these advances, uncertainty still remains regarding the use of plasmids in 17
studying these bacteria during in vivo mammalian models of infection; namely the 18
potential for virulence changes associated with presence of extrachromosomal DNA 19
and non-selective persistence of plasmids during mammalian models of infection. 20
Herein, we describe transformation of Rickettsia conorii Malish 7 with the plasmid 21
pRam18dRGA[AmTrCh]. Transformed R. conorii stably maintains this plasmid in 22
infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth 23
kinetics in cell culture similar to non-transformed R. conorii. Using a well-established 24
fatal murine model of Mediterranean spotted fever, we demonstrate that R. conorii 25
(pRam18dRGA[AmTrCh]) elicit the same fatal outcomes in animals as the 26
untransformed counterparts and ,importantly, maintain the plasmid throughout the 27
infection in the absence of selective antibiotic pressure. Interestingly, plasmid-28
transformed R. conorii were readily observed both in endothelial cells and also within 29
circulating leukocytes. Together, our data demonstrate that the presence of an 30
extrachromosomal DNA element in a pathogenic rickettsial species does not affect 31
either in vitro proliferation or in vivo infectivity in models of disease, and that plasmids 32
such as pRam18dRGA[AmTrCh] are valuable tools for further genetic manipulation of 33
pathogenic Rickettsia.34
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INTRODUCTION: 35
Rickettsia conorii is a pathogenic member of the spotted fever group (SFG) 36
Rickettsia. Multiple tick-borne SFG Rickettsia species can cause a wide range of 37
human and zoonotic diseases, with untreated cases of Rocky Mountain spotted fever 38
(R. rickettsii) disease resulting in 20-25% mortality (1-3). The similar disease in 39
Eurasia, Mediterranean spotted fever (MSF), is caused by R. conorii and results in 2-40
32% mortality in reported human cases (4). Symptoms from these diseases manifest 2-41
14 days following arthropod transmission, and are the result of widespread bacterial 42
infection of the endothelium with concurrent inflammation (5, 6). While broad-spectrum 43
antibiotic treatment can significantly reduce morbidity and mortality, untreated infections 44
can proceed to the more severe disease manifestations, including pulmonary edema, 45
interstitial pneumonia, and multi-organ failure (7). 46
Historically, analysis of the genetic determinants of SFG Rickettsia disease has 47
been hampered by a lack of viable tools for genetic manipulation of these bacteria. This 48
genetic recalcitrance was largely a consequence of lack of both anexic growth medium 49
and selectable markers. However, the development of tools for chromosomal 50
mutagenesis and selectively-maintained extrachromosomal DNA in rickettsial species 51
has been demonstrated (8). Plasmids isolated from non-pathogenic Rickettsia have 52
been adapted for replication and selection in both Rickettsia and Escherichia species 53
(9). These plasmids can potentially be useful for heterologous gene expression, 54
reporter gene expression, and in trans complementation in diverse Rickettsia species. 55
One such plasmid, pRam18dRGA[AmTrCh], is derived from the 18kbp plasmid 56
from R. amblyommii (10, 11). The nonessential portion of the naturally occurring 57
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plasmid was removed, and the remaining portion was inserted into an E. coli vector. 58
The gfpuv/rpaar2 genes, and a multiple cloning site (MCS) were introduced to produce 59
the plasmid pRam18dRGA[MCS] (9, 12, 13). The 948bp AmTrCh cassette encoding for 60
mCherry was subsequently added to create pRam18dRGA[AmTrCh] (9, 14). This 61
plasmid has been successfully transformed into five pathogenic and non-pathogenic 62
Rickettsia species (9, 15). 63
Herein, we utilize this plasmid for transformation of pathogenic R. conorii to 64
examine the in vivo persistence of an extrachromosomal DNA element in the absence 65
of antibiotic selection, and to determine whether the presence of the plasmid affects the 66
virulence of transformed R. conorii Malish 7 in a murine model of MSF. These analyses 67
subsequently yielded identification of new host cell types that are parasitized by R. 68
conorii in vivo.69
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MATERIALS AND METHODS: 70
Bacterial transformation. R. conorii strain Malish 7 was electroporated with 71
pRam18dRGA[AmTrCh] in a manner similar to Rackek, et.al (16). Briefly, 72
approximately 4x108 R. conorii were purified by sucrose density centrifugation (17). 73
The bacteria were made electro-competent through washing 4 times with ice-cold 74
250mM sucrose. 10µL of 1mg/mL pRam18dRGA[AmTrCh] was added to 4x107 75
electrocompetent R. conorii in a 0.1cm-gap electroporation cuvette. After 30 minutes of 76
incubation on ice, the sample was electroporated at 1700V, 150Ω, 50µF with a resulting 77
time constant of approximately 6.5ms. This mixture was immediately transferred to 1mL 78
of 8x106 Vero cells in Hank’s Balanced Salt Solution. After 1 hour of incubation at 34°C, 79
5% CO2 with rotation, the mixture was transferred to a 75cm flask containing 15mL 80
DMEM+10% BSA+ 1% Non-essential Amino Acids. After 24 hours of incubation at 81
34ºC 5% CO2 the media was changed to complete DMEM with 200ng/mL Rifampicin. 82
The media was replaced every 3 days until bacilliary growth was observed by Diff-Quik 83
staining. 84
Sorting and recovery of transformants. Single bacterium cell sorting was performed 85
as described in (18). Briefly, the PerCP/Cy5.5 fluorochrome was conjugated to the anti-86
R. conorii antibody RcPFA (19) using the Lightning-Link PerCP/Cy5.5 conjugation kit 87
(Innova Biosciences). R. conorii (pRam18dRGA[AmTrCh]) were stained with anti-R. 88
conorii antibody RcPFA-PerCP/Cy5.5 at 10 µg/mL. Labeled R. conorii were applied to a 89
FACSJazz cell sorter with forward scatter (FSC) threshold/trigger, side scatter (SSC) 90
versus FSC graph, and SSC versus log 488nm excitation 692/30nm emission graph. 91
Lysed Vero cells and non-transformed R. conorii were used as controls to set negative 92
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and positive sort gates. pRam18dRGA[AmTrCh] transformed R. conorii were applied to 93
the sorter and sorted with 1 sort event into 32 wells, 5 sort events into 32 wells, and 50 94
sort events into 32 wells of a 96 well plate containing Vero cells in complete DMEM 95
containing 200ng/mL Rifampicin. After 5 days of culture, the infected Vero cells were 96
lifted with Trypsin-EDTA and expanded into three identical 96 well plates containing 97
Vero cells in complete DMEM with 200ng/mL Rifampicin. Flow cytometric analyses and 98
cell sorting of pathogenic R. conorii were performed in a CDC-approved BSL-3 99
laboratory in the Department of Pathobiolgical Sciences at the LSU School of Veterinary 100
Medicine utilizing protocols approved by the LSU Institutional Biological and 101
Recombinant DNA Safety Committee (IBRDSC). 102
10 total days of growth after sorting, one of the three identical plates was fixed 103
with 4% paraformaldehyde. This plate was stained with the anti-R. conorii antibody 104
RcPFA (19) with anti-rabbit-AlexaFluor546 and observed under 10x magnification in an 105
Olympus IX71 inverted microscope for the presence of R. conorii (RcPFA) and GFPuv- 106
mediated fluorescence. Subsequently, DNA was extracted as described below from 107
wells containing R. conorii and demonstrating GFPuv-mediated fluorescence. Whole 108
DNA was extracted using the PureLink Genomic DNA kit (Life Technologies) according 109
to the manufacturer’s instructions. The extracted DNA was subjected to PCR using 110
gfpuv_F and gfpuv_R (Supplemental Table 1) followed by agarose gel electrophoresis 111
to ensure presence of pRam18dRGA[AmTrCh]. The clone utilized in this study came 112
from a single event sort into one well of a 96 well plate. 113
Immunofluorescence microscopy. R. conorii and R. conorii (pRam18dRGA[AmTrCh]) 114
were cultured in Vero cells. Both types of bacteria were purified by sucrose density 115
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gradient (17) and mounted onto a single glass slide using a Cytopro Dual sample 116
chamber with a Cytospin centrifuge. The bacteria were fixed with 4% paraformaldehyde 117
and stained using RcPFA with anti-rabbit-AlexaFluor350. All microscopy was captured 118
using a Leica DM4000B equipped with 100x oil objective, EvolutionOEi camera, with 119
ImagePro Plus software, using GFP (488/40nm ex 527/30nm em), TRITC (515-560nm 120
ex LP590nm em), and DAPI (360/40nm ex 470/40nm em) filters. Analysis of 121
fluorescent intensity was performed using the “RGB profile plot” plugin within the NIH 122
Image J freeware package (http://rsb.info.nih.gov/ij/). 123
Flow cytometric analysis. Sucrose purified bacteria were analyzed on a BD 124
FACSJazz equipped with aerosol containment and placed within a Baker class IIA 125
biosafety cabinet within the BSL-3 laboratory suite at the LSU School of Veterinary 126
Medicine. R. conorii (pRam18dRGA[AmTrCh]) were stained with anti-R. conorii 127
antibody RcPFA-PerCP/Cy5.5 at 10 µg/mL. All cytometric events were initially gated for 128
FSC and SSC to eliminate large events. PerCP/Cy5.5-mediated fluorescence was 129
measured at 488nm excitation and 692/40nm emission. GFPuv-mediated fluorescence 130
was measured at 488nm excitation and 530/40nm emission. This fluorescence 131
combination did not produce cross channel background so compensation was not 132
required. Each heat-map represents at least 50,000 FSC/SSC gated events. 133
Growth in culture. R. conorii and R. conorii (pRam18dRGA[AmTrCh]) were used to 134
infect Vero cells at a multiplicity of infection of 1 bacteria/ host cell. Bacteria were 135
allowed to grow at 34°C 5% CO2 for a total of 5 days in DMEM/10% FBS supplemented 136
with 200ng/mL Rifampicin for the transformed bacteria. At day = 1, 3, 5, 7, and 9 post-137
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infection, the infected Vero cells were scraped from the culture plate and DNA extracted 138
as above for host, R. conorii, and plasmid quantification. 139
R. conorii quantification. The chromosomal ratio of mouse or Vero (actin) to R. conorii 140
(sca1) was queried by quantitative PCR using TaqMan Master Mix at with a 95° 5 min 141
incubation followed by 55 cycles of 95°C 15sec, 53°C 15 sec, and 68°C 30 sec. Mouse 142
actin was amplified using actin_Ms_F, actin_Ms_R, and actin_Ms_Max(Vic). Vero actin 143
was amplified using actin_Vero_F, actin_Vero_R, ,and actin_V_Hex(Vic). R. conorii 144
sca1 was amplified using sca1_Rc_F, sca1_Rc_R, and sca1_Rc/Rr_Fam 145
(Supplemental Table 1). All unknowns were quantified by ∆∆Ct as compared to molar 146
standards. 147
Plasmid quantification. DNA from the above growth curve was used to quantify the 148
presence of the pRam18dRGA[AmTrCh] plasmid during growth in culture. The ratio of 149
plasmid (gfpuv) to R. conorii (sca1) was determined by quantitative PCR using SYBR 150
master mix (Roche) with a pre-incubation at 95°C for 5 min, followed by 50 cycles of 151
95°C 15sec, 55°C 15 sec, and 68°C 30 sec. Specificity of amplification was validated 152
by melting curve analysis. Gfpuv was amplified using gfpuv_F and gfpuv_R. R. conorii 153
sca1 was amplified using sca1_Rc_F and sca1_Rc_R. (Supplemental Table 1). 154
Animal infection. 5-7 week old male C3H/HeN mice were inoculated by retro-orbital 155
injection with 6x106 (1.5 LD50) R. conorii (n=5), R. conorii (pRam18dRGA[AmTrCh]) 156
(n=11) or PBS (n=5) (19). Infected mice were monitored twice daily for signs of disease 157
and daily for weight change. Animals that exhibited symptoms of severe disease that 158
were consistent with not recovering from the infection were removed from the study and 159
scored as succumbing to the infection as has been previously described (19). These 160
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symptoms included ruffled fur, hunched posture, shallow respiration, immobility when 161
touched, and weight loss of at least 15% of the initial body weight. Animal experiments 162
were conducted in accordance with protocols approved by the Institutional Biological 163
and Recombinant DNA Safety Committee (IBRDSC) and Institutional Animal Care and 164
Use Committee (IACUC) at the Louisiana State University School of Veterinary 165
Medicine. Two pre-designated mice were sacrificed at 1, 3, and 5 days post infection. 166
Spleens, kidneys, livers, hearts, lungs, brains, and blood were aseptically extracted to 167
determine the bacteria load, plasmid copy number, and assess pathological lesions. 168
Each organ was split into two sections for use in PCR analyses as described above and 169
pathological examination as described below. 170
Indirect Immunohistochemistry. Murine tissues, including lung, liver and spleen, 171
were collected immediately after euthanasia and fixed in 10% buffered formalin (1:10 172
tissue-formalin ratio). Samples were routinely processed and embedded in paraffin, 173
and 5-μm sections were cut for hematoxylin and eosin (H&E) staining. Isolated tissues 174
were additionally examined by immunohistochemistry to localize R. conorii within the 175
infected animals using anti-RcPFA, which recognizes several SFG rickettsial species 176
(19) or the macrophage-specific antigen, anti-Iba1. Primary antibody staining was 177
followed by biotinylated anti-rabbit IgG secondary antibody (1:1000, Vector BA 1000, 178
Vector Laboratories) and exposure to the detection reagent (Vectastain ABC Rabbit IgG 179
Kit, Vector PK 6102, Vector Laboratories). Slides were analyzed, micrographs 180
captured, and pathologic changes recorded by a Diplomate of the American College of 181
Veterinary Pathologists (DACVP). 182
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Identification of R. conorii in Leukocytes. Whole mouse blood was recovered by 183
cardiac puncture immediately after euthanasia. Blood was immediately anti-coagulated 184
using BD microtainer with K2EDTA, diluted 1:1 with PBS. Leukocytes were purified 185
using Ficoll-paque Premium (GE Healthcare) as directed by the manufacturer. 186
Leukocyte preparations were fixed with 4% paraformaldehyde, permeabilized with 0.1% 187
Triton X-100, and blocked with 2% Bovine Serum Albumin. For initial identification of R. 188
conorii infection, host nuclei were labeled with 1ng/mL DAPI and Phalloidin-Texas Red 189
(1:200, Life Sciences). These cells were visualized at 100x as described above. 190
Images were created by identification of fields containing GFPuv-positive bacilli, 191
followed by capture of Texas Red and DAPI images. Micrographs of the host cells 192
without the corresponding GFPuv-positive R. conorii image were scored for cell lineage 193
by a Diplomate of the American College of Veterinary Pathologists (DACVP). Only after 194
blinded identification of the host cell types, were the location and number of GFPuv-195
positive R. conorii attributed to each cell type. 196
Statistics. Bacterial growth in culture was analyzed by nonlinear fit analysis of the 197
slope and intercept. Plasmid content in culture and in mouse was analyzed by 1-way 198
ANOVA. Survival changes between R. conorii and R. conorii(pRam18dRGA[AmTrCh]) 199
were analyzed by comparison of survival curves via Log-rank (Mantel-Cox) test and 200
were analyzed from 5 mice per experimental group. All statistical analyses were 201
performed using the indicated tests within the Prism software package (GraphPad 202
Software Inc.).203
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RESULTS: 204
Transformation and isolation of pRam18dRGA[AmTrCh] transformed R. conorii in 205
cell culture. Previous reports had demonstrated the feasibility of using plasmids, 206
including pRam18dRGA[AmTrCh], to transform both recognized human pathogenic- 207
and non-pathogenic members of the genus Rickettsia (9-12, 15). Although successful, 208
these strategies have technical limitations in obtaining clonal transformed populations. 209
Use of either limiting dilution or plaque assays to isolate Rickettsia clones are time and 210
labor intensive, and repeated passage of these bacteria outside of a relevant animal 211
model can potentially contribute to attenuation (20). We sought to determine whether 212
transformation of R. conorii was achievable using this plasmid, and whether high-213
throughput fluorescence activated cell sorting (FACS) would be useful to isolate clonal 214
transformed R. conorii populations. 215
The plasmid pRam18dRGA[AmTrCh] (9) was introduced into purified R. conorii 216
Malish 7 by electroporation, and ,subsequently, transformed bacteria were subjected to 217
Rifampicin selection for a period of 10 days. After observing outgrowth of Rickettsia 218
bacilli via Diff-Quik staining and microscopic analysis, the infected host cells were lysed 219
by needle passage and unbroken cells were removed utilizing a 2µm filter. The 220
liberated bacteria were subsequently labeled with a PerCP/Cy5.5 conjugated anti-R. 221
conorii antibody (RcPFA, (19)) and subjected to flow cytometric analysis. Rifampicin-222
resistant fluorescent-labeled R. conorii were individually sorted using extremely 223
stringent single event sort using a FACS Jazz cell sorter (18). After outgrowth from the 224
single parent bacterium, clonal populations were screened for presence of GFPuv-225
positive R. conorii by PCR using primers specific for the gfpuv gene on the 226
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pRam18dRGA[AmTrCh] plasmid (data not shown). A single R. conorii 227
(pRam18dRGA[AmTrCh]) clone was propagated and purified by sucrose density 228
centrifugation for further analysis. 229
The pRam18dRGA[AmTrCh] plasmid encodes for two fluorescent proteins, 230
GFPuv and mCherry. mCherry expression is driven by the Anaplasma marginale 231
promoter tr, and the encoded protein fluoresces into the TRITC filter set (21, 22). gfpuv 232
expression is driven by the R. prowazekii ompA promoter, and GFPuv fluoresces 233
adequately into the FITC filter set (13). Accordingly, we hypothesized that transformed 234
R. conorii(pRam18dRGA[AmTrCh]) should readily fluoresce into the FITC (GFPuv) and 235
TRITC (mCherry) channels as has been previously observed in other Rickettsia species 236
(9, 15). As shown in Figure 1A, pRam18dRGA[AmTrCh] transformed R. conorii labeled 237
with the RcPFA antibody (19) are observable by fluorescence microscopy under TRITC 238
and FITC filter sets while untransformed bacteria are not visible. Analysis of the 239
fluorescence signal intensity across a portion of a R. conorii (pRam18dRGA[AmTrCh]) 240
photomicrograph indicates that individual bacterium elaborate both GFPuv and mCherry 241
signals (Figure 1B). 242
Flow cytometric analysis of GFPuv fluorescence. Since GFPuv can be excited 243
under a 488nm laser and emit into common filter sets (23), we hypothesized that 244
GFPuv-mediated fluorescence should be observable by flow cytometry in R. conorii 245
(pRam18dRGA[AmTrCh]). First, we conjugated the anti-R. conorii antibody RcPFA with 246
the PerCP/Cy5.5 tandem fluorophore. We used RcPFA-PerCP/Cy5.5 to identify R. 247
conorii when excited at 488nm and detected using 692/40nm emission filter. Indeed, 248
purified R. conorii are readily detectible by flow cytometry using RcPFA-PerCP/Cy5.5 249
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(Figure 2A vs. 2B). Additionally, GFPuv-mediated fluorescence from R. conorii 250
(pRam18dRGA[AmTrCh]) can be detected using FITC filter sets (488nm excitation and 251
530/40nm emission) (Figure 2A vs. 2C). As such, this is a novel method to identify 252
Rickettsia-specific events and to identify bacteria transformed to GFPuv fluorescence 253
(Figure 2D). 254
In vitro growth of transformed R. conorii in cell culture. 255
We next sought to determine if the presence of an extrachromosomal DNA element in 256
R. conorii has a deleterious affect on fitness in cell culture. R. conorii and R. conorii 257
(pRam18dRGA[AmTrCh]) were inoculated into Vero cell cultures at a MOI of 1. 258
Samples were removed from these cultures 1, 3, 5, 7, and 9 days post inoculation and 259
genomic DNA from both host and bacteria was extracted. Q-PCR analysis of the ratio 260
of R. conorii (sca1) to Vero (actin) DNA content indicates that transformed and non-261
transformed bacteria proliferate similarly over 9 days in Vero cell culture (Figure 3). 262
We then determined whether the plasmid was stably maintained in vitro without 263
selective antibiotic pressure. The copy number of pRam18dRGA[AmTrCh] was 264
determined by Q-PCR as the ratio of plasmid (gfpuv) to R. conorii chromosomal (sca1) 265
DNA. As demonstrated in Table 2, the pRam18dRGA[AmTrCh] copy number over the 266
time course of culture indicates that the plasmid is maintained without antibiotic 267
selection. The determined plasmid copy numbers over time are similar to those 268
documented in other transformed Rickettsia species (9, 11, 15). 269
R. conorii (pRam18dRGA[AmTrCh]) experimental mammalian infection. An 270
important question pertaining to the future of rickettsial genetics is the persistence of 271
extrachromosomal DNA in animal models of infection when lacking selective pressure. 272
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Indeed, pRam18dRGA can be maintained in R. monacensis for five passages in tissue 273
culture without antibiotic selection (9), but this phenotype has not been investigated in 274
an in vivo model. Additionally, future uses of extrachromosomal DNA will be dependent 275
on establishing that the existence of the plasmid does not, in itself, modulate the 276
infectivity of the bacterium. To address these questions, we utilized a well-established 277
murine model of fatal MSF to examine the pathogenesis of R. conorii 278
(pRam18dRGA[AmTrCh]) (19, 24). Susceptible C3H/HeN mice were intravenously 279
infected with 4x106 infectious units of R. conorii or R. conorii (pRam18dRGA[AmTrCh]) 280
This dose is 1.5 LD50 for non-transformed R. conorii (19). Mice were monitored for signs 281
of disease including: ruffled fur, hunched posture, shallow respiration, immobility, and 282
weight loss. R. conorii and R. conorii (pRam18dRGA[AmTrCh]) infected mice 283
demonstrated progressive morbidity as demonstrated by persistent weight loss (Figure 284
4A). As demonstrated in Figure 4B, infected mice succumbed to infection during the 285
normal course of disease with mortality at day 4-5.5 post-infection with a slight delay to 286
time of mortality as compared to WT. Nevertheless, this finding establishes that R. 287
conorii(pRam18dRGA[AmTrCh]) retain infectivity using a mouse model of infection. 288
To confirm that R. conorii (pRam18dRGA[AmTrCh]) infected mice followed the 289
normal disease progression, we quantified the bacterial burden in major organs. 290
Bacteria DNA could be detected and quantified in the mouse spleen and liver after 1 291
day of infection (Figure 5A). Total bacterial burden increased after 3 days of infection 292
(Figure 5B), and ultimately resulted in similar rickettsial organ tropism and quantity as 293
has been previously observed for R. conorii fatal infection (Figure 5C and (19)). Most 294
importantly, pRam18dRGA[AmTrCh] was readily detected at all time points and the 295
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plasmid quantity did not decrease over the course of infection (Figure 5D). Together, 296
these data demonstrate that the pRam18dRGA[AmTrCh] extrachromosomal DNA does 297
not affect R. conorii pathogenesis, and that the plasmid is stably maintained by R. 298
conorii in an animal model of infection. 299
Pathologic examination of animals after 5 days of infection demonstrated R. 300
conorii (pRam18dRGA[AmTrCh]) infection of endothelial cells in the heart and lung 301
(Supplemental Figure 1), as has been previously reported (24). The mouse liver 302
additionally demonstrated multifocal to coalescing coagulative hepatic necrosis 303
characterized by the presence of numerous neutrophils and leukocytic debris (Figure 304
6A). IHC staining of this lesion revealed the presence of numerous bacteria within the 305
cytoplasm of macrophages and neutrophils (Figure 6B). Additionally, bacterial were 306
noted to be infecting cells within vessels (Figure 6C). In the mouse liver, the 307
inflammatory cell population within vessels, sinusoids and microabscesses is mainly 308
comprised of round cells with macrophage morphology expressing the Iba1 309
macrophage marker (Figure 6D). There is no evidence of sloughed endothelial cells 310
within the lumen of blood vessels. These data demonstrate a normal distribution of 311
plasmid-transformed R. conorii in the infected animal along with a presence of these 312
bacteria within macrophages of in the liver. 313
Observation of ex vivo GFPuv-positive bacilli parasitizing leukocytes. We have 314
demonstrated that R. conorii (pRam18dRGA[AmTrCh]) is present within the 315
endothelium of the infected mammalian host (Supplemental Figure 1), but also within 316
several different cell types in infected target tissues and in circulation (Figure 6). In 317
addition, we demonstrated that the pRam18dRGA[AmTrCh] plasmid is stably 318
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maintained within infected cell cultures and in vivo (Figure 5D). We, therefore, 319
hypothesized that transformed R. conorii express genes encoded on the plasmid and 320
are readily detectable by endogenous GFPuv expression in vivo. To this end, we 321
sacrificed mice that were succumbing to the fatal R. conorii infection and isolated total 322
leukocytes from murine blood. After fixation and labeling of nucleus (DAPI) and host 323
cytoplasm (Phalloidin), GFPuv-fluorescent bacilli are readily observed within 324
granulocytic, monocytic, and lymphocytic cells (Figure 7). 325
We next sought to elucidate the different cell types present within the mammalian 326
bloodstream that are infected by R. conorii by determining the locations of the GFPuv-327
positive R. conorii within the ex vivo WBC preparation. GFPuv-positive bacilli were 328
located by fluorescence microscopy without observing location of the bacteria relative to 329
host cells. After detection of the bacteria, images of the host cells in that microscopic 330
field were acquired. A board-certified veterinary clinical pathologist identified the types 331
of leukocytes in the microscopic field without knowledge of the location of the bacteria. 332
Only after identification of host cells were the locations of the bacteria assigned. Of the 333
207 GFP-positive R. conorii that could be assigned to a host location, 110 were present 334
in lymphocytes, 67 were in monocytes, 14 were in granulocytic cells, and 16 were not 335
associated with any host cell (Table 2). Thus, these data suggest that in addition to 336
endothelial cells, cells of non-endothelial origin support R. conorii parasitism during fatal 337
mammalian infection.338
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DISCUSSION: 339
Herein, we have demonstrated the ability of R. conorii to be transformed with the 340
plasmid pRam18dRGA[AmTrCh] (9). R. conorii stably maintains this plasmid in culture 341
with no apparent affect on fitness, but with the addition of GFPuv- and mCherry-342
mediated fluorescence phenotypes (Figures 1, 2, 3, Table 1). By utilizing a well-343
characterized mouse model of fatal MSF, we were able to demonstrate that R. conorii 344
(pRam18dRGA[AmTrCh]) retains infectivity in a fatal mouse model of infection (Figure 345
4). Fatal R. conorii (pRam18dRGA[AmTrCh]) challenge results in disseminated 346
infection, and, importantly, retention of the extrachromosomal DNA (Figure 5). 347
Microscopic examination of R. conorii (pRam18dRGA[AmTrCh]) within the mouse 348
demonstrated the well-characterized endothelial-targeted infection (Supplemental 349
Figure 1), with newly-identified infection of mouse macrophages within the liver and 350
infection of non-endothelial cells within the microvascular circulation (Figure 6). Further 351
microscopic examination showed the presence of GFPuv-positive bacilli within 352
leukocytes (Figure 7), and pathologic examination was applied to identify the nature of 353
this host infection (Table 2). Taken together, these data indicate that use of 354
pRam18dRGA plasmids will add depth to our analysis of mammalian models of 355
Rickettsia infection. The GFPuv-mediated fluorescence phenotype will permit a more 356
complete analysis of the nature of the pathogenic process and to identify host cells 357
parasitized by pathogenic Rickettsia species. 358
In vivo maintenance of pRam18dRGA plasmids (Figures 5 and 7) and the 359
adaptable nature of the pRam18dRGA[MCS] plasmid allows additional approaches to 360
examining Rickettsia pathogenesis. The pRam18dRGA[MCS] plasmid contains a 361
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multiple cloning site for insertion of any DNA (9). This design feature makes this 362
plasmid a true shuttle vector, because the additional DNA will be maintained within the 363
bacteria during in vivo Rickettsia infection. First and foremost among potential uses of 364
shuttle vectors is proper and complete examination of gene deletion mutants, because 365
of the essential need for in trans gene complementation (25). pRam18dRGA[MCS] 366
derivatives can also be utilized to monitor bacterial gene expression through the 367
insertion of appropriate reporter genes, analysis of natural conjugation systems, or for 368
heterologous expression of many different bacterial genes (9, 26). 369
Of particular importance is our ex vivo microscopic observance of fluorescent R. 370
conorii directly from the infected animal. Analysis of in vivo or ex vivo fluorescent bacilli 371
will promote a more complete examination of Rickettsia pathogenesis, as has been 372
done in other bacteria (27). This microscopic technique can also be readily applied to: 373
examine cell types infected parasitized by pathogenic Rickettsia, observe arthropod 374
infection in real time, and follow the kinetics of rickettsemia in infected mammals. 375
SFG Rickettsia have long been known to infect the endothelium of the infected 376
animal (24, 28, 29). In this manuscript, we provide evidence of non-endothelial 377
parasitism by R. conorii (Figures 6 and 7, Table 2). We propose that infection of non-378
endothelial cells has been overlooked during prior examination of SFG Rickettsia 379
pathogenesis, and that our newly developed techniques have allowed a more complete 380
analysis of infection. This idea is supported by several in vitro and ex vivo studies 381
which have demonstrated that rickettsial species can infect and grow within 382
macrophages and hepatocytes (30-33), suggesting that the interaction with cells other 383
than the endothelium may be an important and underappreciated aspect in the 384
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pathogenesis of rickettsial diseases. Additionally, immunohistochemical and 385
immunofluorescence microscopy both display intact bacilli, and non-endothelial cells 386
frequently contain multiple plasmid-transformed R. conorii (Figures 6 and 7), suggesting 387
that these bacteria may not be readily susceptible to immune-mediated killing. A more 388
complete examination of the viability of bacilli, infection kinetics, and host tropism of R. 389
conorii parasitism is necessary. 390
In conclusion, we have utilized R. conorii (pRam18dRGA[AmTrCh]) to 391
demonstrate the value of plasmids in analysis of in vivo Rickettsia pathogenesis . The 392
techniques described herein have promoted our identification of novel host cell types 393
parasitized by pathogenic R. conorii in a fatal model of Mediterranean spotted fever. 394
Therefore, the use of pRam18dRGA[AmTrCh] and related plasmids will strongly 395
influence and enhance the course of future rickettsial research.396
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FUNDING INFORMATION: 397
These studies were funded in part by NIH-NIAID award R01AI1072606 to J.J.M. and 398
NIH-NIGMS award P30GM110760 Pilot Grant to S.P.R. The content is solely the 399
responsibility of the authors and does not necessarily represent the official views of the 400
National Institutes of Health.401
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ACKNOWLEDGEMENTS: 402
The authors would like to thank Dr. Kevin Macaluso (LSU-SVM) for helpful comments 403
on experimental design and manuscript construction, Dr. David Wood (University of 404
South Alabama) for dissemination of techniques for electrotransformation, Dr. Ulrike 405
Munderloh (University of Minnesota) for distribution of reagents, and Dr. Rebecca 406
Christofferson (LSU-SVM) for aid in the statistical analysis of data.407
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504
505
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TABLES: 506
Table 1. Plasmid copy number during Vero cell growth. 507
Days of culture Plasmid copy number (Average ± Standard Deviation)*
3 3.09 ± 0.52
5 2.82 ± 2.03
7 0.52 ± 0.30
9 0.55 ± 0.14
*Changes in copy number are not significant. 508
509
Table 2. Analysis of the cellular location of GFPuv-positive bacilli in an ex vivo 510
leukocyte preparation. 511
Total number of bacteria counted 207
Bacteria present in lymphocytes 110
Bacteria in monocytes 67
Bacteria in granulocytes 14
Bacteria not associated with a host cell 16
512
513
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FIGURE LEGENDS: 514
Figure 1. in vitro fluorescent properties of R. conorii (pRam18dRGA[AmTrCh]). 515
A. R. conorii and R. conorii (pRam18dRGA[AmTrCh]) were purified and stained with the 516
anti-Rickettsia antibody RcPFA with corresponding anti-rabbit AlexaFluor350 (blue). 517
Surface staining of the bacteria is apparent in both anti-RcPFA panels, but only R. 518
conorii (pRam18dRGA[AmTrCh]) demonstrate GFPuv- (green) and mCherry- (red) 519
mediated fluorescence. B. Unstained R. conorii (pRam18dRGA[AmTrCh]) were 520
analyzed for GFPuv- and mCherry- mediated fluorescence. Analysis of fluorescence 521
intensity along the magenta line indicates that individual bacteria elaborate both GFPuv- 522
and mCherry- mediated fluorescence as demonstrated by overlapping peaks. However, 523
the mCherry fluorescence is not always above the limit of detection. 524
525
Figure 2. Flow cytometric analysis of GFPuv- mediated fluorescence. A. Non-526
labeled R. conorii, B. RcPFA-PerCP/Cy5.5- labeled R. conorii, C. Non-labeled R. conorii 527
(pRam18dRGA[AmTrCh]) and, D. RcPFA-PerCP/Cy5.-labeled R. conorii 528
(pRam18dRGA[AmTrCh]) were analyzed by flow cytometry for identification of R. 529
conorii specific events (RcPFA) and GFPuv fluorescence. RcPFA-PerCP/Cy5.5 labels 530
both R. conorii (A vs. B) and R. conorii (pRam18dRGA[AmTrCh]) (C vs. D). R. conorii 531
(pRam18dRGA[AmTrCh]) demonstrates significant intrinsic GFPuv-mediated 532
fluorescence (A vs. C and B vs. D). 533
534
Figure 3. Presence of pRam18dRGA[AmTrCh] does not alter R. conorii fitness in 535
culture. R. conorii (pRam18dRGA[AmTrCh]) (dashed line) and plasmid-free R. conorii 536
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(solid line) proliferate similarly in Vero cell culture. Quantitative PCR data are 537
expressed as the ratio of R. conorii sca1 versus Vero actin DNA after 3, 5, 7, and 9 538
days of culture in Vero cells. Differences in slope or intercept are not significant. 539
540
Figure 4. R. conorii (pRam18dRGA[AmTrCh]) retains virulence in a fatal mouse 541
model of infection. A. Mice were infected with R. conorii (dashed line), R. conorii 542
(pRam18dRGA[AmTrCh]) (dotted line) or PBS (solid line) and were monitored for 543
morbidity as determined by weight change over the course of infection. B. The same 544
infected mice were monitored for survival. R. conorii harboring pRam18dRGA[AmTrCh] 545
are capable of producing fatal disease. Time to euthanasia p<0.05. 546
547
Figure 5. R. conorii(pRam18dRGA[AmTrCh]) proliferates within infected organs 548
and maintains extrachromosomal plasmid. R. conorii (pRam18dRGA[AmTrCh]) 549
dissemination into mouse organs after 1 (A.), 3 (B.), and 5 (C.) days of infection. 550
Chromosomal equivalents of R. conorii sca1 and mouse actin were determined by 551
quantitative PCR. D. Non-selective persistence of pRam18dRGA[AmTrCh] throughout 552
the mammalian infection as determined by quantitative PCR ratio of chromosomal R. 553
conorii sca1 and the plasmid gene gfpuv. Change in plasmid copy number is not 554
significant. 555
556
Figure 6. Pathological examination of mouse liver. A. Hematoxylin and eosin stain 557
of mouse liver demonstrates microabscess development when animal is succumbing to 558
infection. B. Immunohistochemical staining using anti-R. conorii antibody with 559
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hematoxylin counter stain demonstrates presence of Rickettsia antigen-positive intact 560
bacilli (brown) within liver microabscess. C. Rickettsia antigen- positive intact bacilli 561
(brown) within circulating cells of liver capillary. D. Iba1- positive macrophages (brown) 562
within both a liver venule (lower right) and microabscesses (arrow heads). All images 563
were captured at 60x. 564
565
Figure 7. ex vivo fluorescence of R. conorii (pRam18dRGA[AmTrCh]). Microscopic 566
identification of R. conorii (pRam18dRGA[AmTrCh]) infected cells from a white blood 567
cell preparation. Cells are stained with DAPI (blue) to identify host nuclei and Phalloidin 568
(red) to illuminate the cytoplasm. Green fluorescence results from intrinsic GFPuv-569
mediated fluorescence as encoded by pRam18dRGA[AmTrCh]. Infected host cells 570
demonstrate intact GFPuv-positive bacilli. White bars = 10μm. All images were 571
captured at 100x. 572
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