Expression of green fluorescent protein in Rickettsia conorii

Article available online at http://www.idealibrary.com on

1

Microbial Pathogenesis 2002; 33: 17±21 doi:10.1006/mpat.2002.0508

Expression of green ¯uorescent protein in Rickettsia conorii Patricia Renestoa*, Edith Gouinb & Didier Raoulta a

Unite des Rickettsies-CNRS UMR-6020, IFR 48, Faculte de MeÂdecine, 27 Boulevard Jean Moulin, 13385 Marseille and b Unite des Interactions BacteÂries-Cellules, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France (Received February 21, 2002; accepted in revised form April 2, 2002)

Rickettsiae are obligate intracellular class III pathogens for which genetic manipulation has only recently been shown to be feasible. Such experiments were restricted to the typhus group rickettsiae, namely R. typhi and R. prowazekii. Here we report the ®rst genetic manipulation of Rickettsia conorii, the bacterial agent responsible for the Mediterranean spotted fever. A gene encoding a variant of the green ¯uorescent protein under the control of the sterically repressed promoter (srp) from E. coli was integrated into the genome of this bacteria and detected by FACS & 2002 Elsevier Science Ltd. All rights reserved. analysis. Key words: Rickettsiae, homologous recombination.

Introduction Rickettsia conorii is the causative agent of Mediterranean spotted fever [1]. The genus Rickettsia is composed of two groups, the typhus group (TG) and the spotted fever group (SFG). The TG includes only two species, namely R. prowazekii, agent of epidemic typhus and R. typhi, agent of murine typhus. The SFG comprises numerous antigenically related species, including the etiologic agents of geographically widespread human diseases among which R. rickettsii (Rocky Mountain spotted fever), R. sibirica (North Asian tick typhus), R. australis (Queensland tick typhus), and R. akari (rickettsialpox) [2]. In human, the endothelial cells are * Author for correspondence. E-mail: patricia.renesto@ medecine.univ-mrs.fr 0882±4010/02/$ ± See front matter

the targets of rickettsiae [3, 4]. Invasion of these cells induces increased vascular permeability, edema, and in severe cases, thrombocytopeniaassociated hemorrhagic manifestations [5]. Despite exciting aspects of rickettsial intracellular invasion, little is known about the molecular mechanisms of rickettsial pathogenicity. The obligate intracellular cycle of this microorganism associated with its high virulence and the lack of an ef®cient genetic manipulation system of these bacteria [6] have strongly hindered their study. The recent completion of genome sequences of both R. prowazekii [7] and R. conorii [8] has highlighted potential virulence factors. The characterization of rickettsial virulence factors will undoubtedly require genetic transformation of these bacteria. During past few years, substantial progress allowing the genetic manipulation of rickettsiae has been made [9±11] but these studies concerned either R. prowazekii & 2002 Elsevier Science Ltd. All rights reserved.

18

[9, 10] or R. typhi [11]. The ®rst successful rickettsial transformation was described by Rachek et al. [10] who demonstrated that a plasmid containing a rifampicine resistance within the R. prowazekii RNA polymerase b subunit (rpoB) and introduced into the rickettsia by electroporation recombined into the genome by homologous recombination. This method was further improved by Troyer et al. [11] who succeed to introduce non-rickettsial DNA insert ¯anked by rickettsial DNA, the GFP-rpoB fusion. The choice of rpoB was based on the fact that in E. coli the C-terminal part of this gene was shown to be not functional [12]. Because the transformation of rickettsiae was restricted to TG, we investigated the possible transformation of a SFG rickettsia, namely R. conorii.

Results and Discussion The strategy described in the Figure 1 was similar to the one used by Troyer et al. [11]. Thus, a 1094-bp fragment of the rpoB gene from R. conorii (nucleotides 3025±4119) [8, 13] was subcloned into the pGEM-T easy vector, linearized with Sma I (position 3276 of rpoB) and ligated with the srp-GFP cassette. The plasmid pEGFP2 carrying the green ¯uorescent protein (GFP) reporter gene under the control of the E. coli srp (sterically repressed) promoter [14, 15] was used as DNA template for ampli®cation of this cassette. Finally, the 1891-bp rpoB-GFP amplicon was used to electroporate Rickettsiae. Genomic DNA extracts from electroporated R. conorii were collected at different time points post-infection and analyzed by PCR using primers rpoB2800F and GFP360R. These

P. Renesto et al.

oligonucleotides should respectively anneal downsteam the rpoB-GFP construct and inside the inserted nonrikettsial gene. As shown in Figure 2, PCR analysis after 48 h of infection revealed a fragment with an expected size of 898-bp. This ampli®cation was not observed with DNA from uninfected Vero cells or from Vero cells infected with non-transformed R. conorii. This result which was con®rmed by the sequencing of the ampli®ed fragment (not shown), indicated that the rpoB-GFP cassette was integrated into the R. conorii genome. No ampli®cation was obtained at t ˆ 24 h postinfection, most probably because the amount of rickettsial DNA was too low at that time. The PCR ampli®cation was obtained up to 14 days post-infection. This result demonstrates the successful genetic transformation of R. conorii. Although we used the GFP gene under the control of the srp E. coli promoter [14, 15] to increase the GFP expression level, GFPtransformed rickettsia were not detectable by epi¯uorescence microscopy. Thus, we analyzed GFP expression by ¯ow cytometer cytometry. As illustrated in Figure 3, the ¯uorescence intensity was signi®cantly higher in R. conorii electroporated with the rpoB-GFP construct (black) than in bacteria electroporated without the corresponding amplicon (white). Indeed, the mean of ¯uorescence values were respectively of 98 and 305. Over the last few years, the increased awareness of the problems associated with the growth dependent analysis of bacterial populations led to the development of direct optical detection methods. In this way, ¯ow cytometry in combination with single cell sorting has allowed the interpretation and veri®cation of the properties of a number of ¯uorescent strains [16]. Such a

Figure 1. Schematic diagram of the transformation procedure. Construction of the rpoB-GFP cassette used to transform R. conorii was carried out as illustrated on this graph. The positions of the primers used for PCR ampli®cation are indicated by arrows.

Transformation of R. conorii

Figure 2. GFP insertion in R. conorii genome. Agarose gel electrophoresis of PCR products obtained using as template DNA extracted from Vero cells 24 h after their infection with electroporated rickettsiae and using primers rpoB2800F and GFP379R. These primers are respectively located on the chromosome of R. conorii and inside the exogenous inserted DNA fragment. Lane 1, molecular size markers; lane 2, R. conorii electroporated with rpoB-GFP, lane 3, R. conorii electroporated without DNA. Marker is the marker VI from Boehringer.

19

method would permit isolation of individual rickettsial transformated clones. However, and as mentioned above, these bacteria are class III pathogens which are manipulated in P3 laboratories rarely equiped with ¯ow cytometer. In this context, our perpectives are to obtain GFPtransformed rickettsiae ¯uorescent under UV light. However, in contrast with E. coli colonies transformed with the srp-GFP cassette and which were visualized under UV light, transformed R. conorii were only detectable by FACS analysis at 480 nm. An higher GFP expression level should be reached by replacing the E. coli promoter by a rickettsial one. In this respect, the recent publication of the genome of R. conorii [8] would be helpful to determine such a promotor. In conclusion, this study con®rms data published by Troyer et al. [11] and documents the ®rst genetic transformation of R. conorii, a SFG rickettsia. Future studies orientated toward the R. conorii promotor are in progress.

Materials and Methods Bacterial strains and culture conditions Bacterial strains and plasmids used in this study were listed in Table 1. The Escherichia coli JM109 were routinely grown at 37 C in Luria-Bertani (LB) medium or on LB-agar plates supplemented with ampicillin (100 mg/ml; Sigma; St. Louis, MO, U.S.A.), isopropyl-b-D-thiogalactoside (0:5 mM) and X-gal (40 mg/ml) when appropriate. Rickettsia conorii were propagated in Vero cells and puri®ed as previously reported [17]. When infected with transformed rickettsia, Vero cells in Costar 6-wells plates were incubated at 37 C in an atmosphere of 5% CO2 and 95% air.

Construction of rpoB-GFP insert

Figure 3. FACS analysis of R. conorii electroporated with rpoB-GFP DNA construct. Vero cells infected with R. conorii electroporated with rpoB-GFP amplicon were harvested at day 7 after infection and analysed by ¯ow cytometry (488 nm) in parallel with bacteria electroporated in absence of DNA as control. The data presented in the Figure 3 are representative of two distinct experiments.

Standard DNA cloning procedures were performed as described by Sambrook et al. [18]. All enzymes were purchased from Gibco (Gibco BRL Life Technology, Paisley, U.K.). As summarized in Figure 1, a 1094-bp fragment of the rpoB gene from R. conorii (nucleotides 3025±4119) [8, 13] was ampli®ed by PCR (Thermocycler Perkin-Elmer, Warrington, U.K.) using the Expand High Fidelity PCR system (Roche, Mannheim, Germany) and primers rpoB3025F

20

P. Renesto et al.

Table 1. Bacterial strains and plasmidsa Strains or plasmids Strains Escherichia coli JM109 Rickettsia conorii, strain Malish (seven) Plasmids pGEM-T pEGFP1 pEGFP1md pEGFP2 pPRRc1 pPRGFP5 a

Characteristics

Souce or reference

Host strain for cloning plasmid propagation

Promega ATTCaVR-613T

Ampicillin resistant Plasmid encoding an optimized mutant of the GFP pEGFP1 without EcoR I and Xba I restriction sites pGEM-T with inserted srp-GFP cassette from pEGFP1md (Oligo1-Oligo2) pGEM-T with inserted PCR fragment from R. conorii (rpoB3025F-rpoB4119R) pPRRc1 with inserted PCR fragment from pEGFP2 (GFPEcoRVF-GFPEcoRVR)

Promega [3] This study This study This study This study

American Type Culture Collection.

Table 2. Oligonucleotide primers used in this study (5 0 -3 0 ) Oligo1 Oligo2 GFPEcoRVF1 GFPEcoRVR rpoB2800F rpoB3025F rpoB4119R GFP360R GFP600F

CCC CCA ATT ATA CAT AAA ACT AAG CTG

AAG CTG AAT TAA CGC GTA TGA GGT TCC

CTT CAG ATA TAA TGC GGA AGT ATC TTT

AAT GTC GAT GAT ATG CAA TAC ACC TAC

TGA TGG ATC ATC TAC ACT TTC TTC CAG

CAT ACA AAT GGT CTT ATT AAG AAA ACA

and 4119R (Table 1). The ampli®ed fragment was subcloned into the pGEM-T easy vector, as described by the manufacturer (pGEM-T vector systems, Promega corp., Madison, WI, U.S.A.) to generate the plasmid pPRRc1. The plasmid pEGFP2 carrying the green ¯uorescent protein (GFP) reporter gene under the control of the E. coli srp (sterically repressed) promoter (Ezaaz-Nikpay) was used as DNA template for ampli®cation of the srp-GFP cassette. The PCR was performed by using primers containing engineered EcoRV (GFPEcoRVF and GFPEcoRVR). The resulting PCR fragment was then cleaved by EcoRV (797-bp), puri®ed on gel, phosphorylated and ligated to the plasmid pPRRc1 which was previously linearized with Sma I (position 2756 of rpoB) and subsequently dephosphorylated. The sequence was con®rmed by DNA sequencing (ABI 310 automated sequencer, PerkinElmer, Warrington, U.K.). Finally, the rpoB-GFP construct (1850-bp length) was ampli®ed by PCR with primers rpoB3025F and rpoB4119R.

TGT TTT TGA CTG CCG ACT C CTT ACC

GAG ATT CAT GAC G ACT

TG TGT GAG CGG ATT TAT TTG G

GAC

PCR PCR PCR PCR PCR/Sequencing PCR/Sequencing PCR/Sequencing PCR/Sequencing PCR/Sequencing

The resulting amplicon was further puri®ed (PCR puri®cation kit, Qiagen) and concentrated in H2O.

Transformation protocol Rickettsia conorii propagated in Vero cells were puri®ed [17] just prior electroporation from 5 162-cm2 cell culture ¯asks (Corning Costar, Cambridge, MA, U.S.A.). The cells were washed twice with cold H2O containing 10% glycerol, and resuspended in 500 ml. Hundred microliter of bacteria were transferred to a 0.2-cm gap electroporation cuvette (Bio-Rad Laboratories, Hercules, CA, U.S.A.) maintained at 4 C and containing 2 mg of PCR fragment. The mixture was electroporated in a Gene Pulser (Bio-Rad Laboratories, Hercule, CA, U.S.A.) using 2:5 kV, 200 O, and 25 mF, immediately supplemented with 500 ml of M4 medium and incubated on ice for 10 min before infection of Vero cells in 6well plates.

Transformation of R. conorii

Infected cells were collected on days 1, 2, 3, 7 and 14 after infection and examined microscopically following Gimenez staining or under ¯uorescence microscopy. Total DNA was extracted from each sample using a Qiamp blood kit (Qiagen, Courtaboeuf, France). The insertion of the rpoB-GFP construct in R. conorii genome was determined by PCR by using primers respectively designed in the GFP insert (GFP379R) and in the ¯anking sequence of the ampli®ed rpoB region (rpoB2800F). Each ampli®cation was performed in duplicate.

Flow cytometry analysis Infected Vero cells were harvested after 7 and 15 days of infection by trypsinization. Intracellular R. conorii were ®xed by overnight incubation in 0.01% formaldehyde and analyzed by ¯ow cytometry (FACScan, Becton-Dickinson Electronic Laboratories, Mountain Wiew, CA, U.S.A.) with the Cell Quest Sotfware. The data presented in the Figure 3 are representative of two distinct experiments.

Acknowledgement We thank gratefully B. Cormack for the gift of plamide pEGFP1 and C. Egile for critical reading of this manuscript.

References 1 Conor A, Brusch A. Une ®evre eÂruptive observeÂe en Tunisie. Bull Soc Pathol Exot 1910; 3: 492±6. 2 Roux V, Raoult D. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev 1997; 10: 694±719. 3 Silverman DJ. Rickettsia-rickettsii-induced cellular injury of human vascular endothelium in vitro. Infect Immun 1984; 44: 545±53.

21 4 Walker DH. Rickettsial interactions with human endothelial cells in vitro: adherence and entry. Infect Immun 1984; 44: 205±10. 5 Walker DH, Mattern WD. Rickettsial vasculitis. Am Heart J 1980; 100: 896±906. 6 Wood DA, Azad AF. Genetic manipulation of Rickettsiae: a preview. Infect Immun 2000; 68: 6091±3. 7 Andersson SGE, Zomorodipou A, Andersson JO et al. The genome sequence of Rickettsia prowazekii and the origin of the mitochondria. Nature 1998; 396: 133±40. 8 Ogata H, Audic S, Renesto-Audiffren P et al. Whole genome sequencing of Rickettsia conorii and comparative analysis with Rickettsia prowazekii reveal their mechanisms of evolution. Science 2001; 293: 2093±7. 9 Rachek LI, Hines A, Tucker AM, Winkler HH, Wood DO. Transformation of Rickettsia prowazekii to erythromycin resistance encoded by the Escherichia coli ereB gene. J Bacteriol 2000; 182: 3289±91. 10 Rachek LI, Tucker AM, Winkler HH, Wood DO. Transformation of Rickettsia prowazekii to rifampin resistance. J Bacteriol 1998; 180: 2118±24. 11 Troyer JM, Radulovic S, Azad AF. 1999. Green ¯uorescent protein as a marker in Rickettsia typhi transformation. Infect Immun 1999, 67: 3308±11. 12 Severinov K, Mustaev A, Severinova E et al. Assembly of functional Escherichia coli RNA polymerase containing b subunit fragments. Proc Natl Acad Sci USA 1995; 92: 4591±5. 13 Drancourt M, Raoult D. Characterization of mutations in the rpoB gene in naturally rifampin-resistant rickettsia species. Antimicrob Agents Chemother 1997; 43: 2400±3. 14 Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green ¯uorescent protein (GFP). Gene 1996; 173: 33±8. 15 Ezaz-Nikpay K, Uschino K, Lerner, RE, Verdine GL. Construction of an overproduction vector containing the novel srp (sterically repressed) promoter. Prot Sci 1994; 3: 132±8. 16 Nebe-von-Caron G, Stephens PJ, Hewitt CJ, Powell JR, Badley RA. Analysis of bacterial function by multicolour ¯uorescence ¯ow cytometry and single cell sorting. J Microbiol Methods 2000; 42: 97±114. 17 Roux V, Raoult D. Genotypic identi®cation and phylogenetic analysis of the spotted fever group rickettsiae by pulsed-®eld gel electrophoresis. J Bacteriol 1993; 175: 4895±904. 18 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989.