RNAi Screen of –Associated Host Factors Reveals a Role for IRE1a in Supporting Brucella Replication

Qing-Ming Qin1*, Jianwu Pei2, Veronica Ancona1, Brian D. Shaw1, Thomas A. Ficht2,3, Paul de Figueiredo1,3,4,5* 1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, United States of America, 2 Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas, United States of America, 3 Faculty of Genetics, Texas A&M University, College Station, Texas, United States of America, 4 Professional Program in Biotechnology, Texas A&M University, College Station, Texas, United States of America, 5 Faculty of Molecular and Environmental Plant Systems, Texas A&M University, College Station, Texas, United States of America

Abstract Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident , the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 , including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded response (UPR). We employed IRE1a2/2 murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1a, and other ER- associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.

Citation: Qin Q-M, Pei J, Ancona V, Shaw BD, Ficht TA, et al. (2008) RNAi Screen of Endoplasmic Reticulum–Associated Host Factors Reveals a Role for IRE1a in Supporting Brucella Replication. PLoS Pathog 4(7): e1000110. doi:10.1371/journal.ppat.1000110 Editor: David S. Schneider, Stanford University, United States of America Received April 4, 2008; Accepted June 24, 2008; Published July 25, 2008 Copyright: ß 2008 Qin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by start-up resources from Texas A&M University (TAMU), Texas A&M Agrilife Research, and a grant from the U.S. Mexican American Latino Research Center to PdF; National Institutes of Health Grant (R01 AI048496) and 1U54AI057156-0100 through the Western Regional Center of Excellence to TAF; and start-up funds from TAMU, Texas A&M Agrilife Research, and the Texas A&M University College of Agriculture and Life Sciences to BDS. Competing Interests: Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (Q-MQ); [email protected] (PdF)

Introduction phosphate receptors (M6PRs) and cathepsin D, markers for late endosomes and lysosomes, respectively [8,11]. Instead, maturing Infectious diseases caused by intracellular bacterial pathogens are BCVs fuse with membranes that contain endoplasmic reticulum responsible for an enormous amount of worldwide pain, suffering, (ER) resident proteins, including calreticulin and calnexin [7,8,11]. and mortality. Brucella spp., for example, cause brucellosis, a global In addition, this trafficking involves BCV interactions with a zoonosis of profound importance [1,2]. Brucella melitensis, B. abortus, compartment that contains the autophagosomal marker mono- and B. suis are highly infectious and can be readily transmitted in dansylcadaverin [7,12]. Finally, Brucella spp. replicate in an ER- aerosolized form [3,4]. In addition, they have eluded systematic like compartment, and then presumably lyse the host cell to allow attempts at eradication for more than a century, even in most the infectious cycle to begin anew [8,13,14]. developed countries, and a human vaccine against brucellosis is not Bacterial lipopolysaccharides (LPS) play an important role in available [3]. Therefore, Brucella spp. have been classified as potential directing the bacterium along an intracellular trafficking pathway bioterror threat agents [5], and have generated significant interest in that enables a productive infection to be established. Brucella LPS the biosecurity and world health communities. also protects the bacterium from the harsh intracellular environ- Understanding the molecular mechanisms of Brucella pathogen- ment, suppresses pro-inflammatory and antibacterial host respons- esis and host response is critical for brucellosis control, and es, and interferes with antigen presentation in macrophages [15]. intracellular trafficking and replication of Brucella spp. play Unlike their smooth wild-type (WT) counterparts, B. melitensis or B. important roles in these processes [6–8]. First, bacteria, internal- abortus mutants harboring a deletion in the phosphomannomutase ized from the host cell plasma membrane, orchestrate the (DmanBA) lack LPS O-antigens, form rough colonies on solid biogenesis of early Brucella-containing vacuoles (BCVs) [9,10]. medium, and are rapidly internalized by macrophages via a poorly Next, BCVs acidify but nevertheless fail to accumulate mannose 6- understood pathway [16,17]. However, these mutants fail to

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Author Summary In addition, we demonstrate the power of this system by identifying novel Brucella host factors, including IRE1a, a conserved transmem- Brucella spp. are facultative intracellular pathogens that brane kinase that plays a key role in regulating the host cell unfolded cause brucellosis in a broad range of hosts, including protein response (UPR) [32–34]. Finally, we demonstrate that humans. Brucella melitensis, B. abortus, and B. suis are IRE1a is required for Brucella infection of mammalian cells, and highly infectious and can be readily transmitted in discuss a possible mechanism by which this intriguing protein may aerosolized form, and a human vaccine against brucellosis regulate bacterial infection. is unavailable. Therefore, these pathogens are recognized as potential bioterror agents. Because genetic systems for studying host–Brucella interactions have been unavailable, Results little is known about the host factors that mediate Brucella infection of Drosophila S2 and mammalian cells infection. Here, we demonstrate that a Drosophila S2 cell system and RNA interference can be exploited to study the shares striking similarities role that evolutionarily conserved Brucella host proteins If Drosophila S2 cells are to provide a model system for studying play in these processes. We also show that this system Brucella infection, then they must support bacterial entry and provides for the identification and characterization of host replication. In addition, isogenic Brucella mutants with established factors that mediate Brucella interactions with the host cell entry, intracellular trafficking and replication properties should endoplasmic reticulum. In fact, we identified 52 host behave similarly in S2 cells and mammalian macrophages. Finally, factors that, when depleted, inhibited or increased Brucella Brucella should display similar infection phenotypes in S2 and infection. Among the identified Brucella host factors, 29 mammalian cells that have been treated with compounds that have not been previously shown to support bacterial disrupt host cell functions. With these ideas in mind, we employed infection. Finally, we demonstrate that the novel host gentamicin protection assays [18] to examine the entry and factor inositol-requiring enzyme 1 (IRE1) and its mamma- replication of different B. melitensis and B. abortus WT and mutant lian ortholog (IRE1a) are required for Brucella infection of strains (listed in Table S1) in S2 cells. Because S2 cells require Drosophila S2 and mammalian cells, respectively. There- temperatures below 30uC for growth, all infection experiments fore, this work contributes to our understanding of host were performed at 29uC, unless otherwise indicated. Importantly, factors mediating Brucella infection. J774A.1 cells supported Brucella entry and intracellular replication at this temperature (Fig. S2). Brucella WT (S2308 and 16M) and mutant strains displayed establish an intracellular replicative niche and reportedly induce a strikingly similar properties when infecting S2 and mammalian cells. necrotic cytopathic effect in these cells [18,19]. The bacterial type First, B. abortus and B. melitensis strains with smooth colony IV secretion system (T4SS) is also important for bacterial morphologies (i.e., 102B2, 146D5, BA114, S2308DvirB2)(Fig.1 pathogenesis, and mutant strains lacking this system fail to traffic A1 and A2) and attenuated rough mutants (i.e., CA180, to, or replicate in, the ER [7,20–22]. S2308DmanBA and 16MDmanBA) displayed corresponding entry To date, relatively few host factors, including Rho1, Rac1, Cdc42 phenotypes in Drosophila S2 and mammalian cells (Fig. 1 A2 and data [23] and Sar1 [8], have been shown to be important for Brucella not shown). Second, B. melitensis strains harboring mutations in mucR infection. Phosphoinositide 3-kinase (PI3K) activities have also been (strain 102B2) and merR (strain 146D5) failed to replicate in both implicated in supporting Brucella infection [23]. Despite these J774A.1 [35] and S2 cells. B. abortus and B. melitensis strains lacking advances, factors that mediate Brucella infection of host cells remain the T4SS (e.g., BA114, S2308DvirB2,16MDvirB2) behaved similarly obscure. However, Brucella intracellular trafficking from the plasma (Fig. 1 A3, A4 and data not shown). Third, vaccine strains RB51 and membrane to an ER-associated replicative niche involves interac- S19 [36] displayed significantly decreased levels of replication in tions with a membrane bounded compartment that contains both host cell systems (Fig. 1 A4 and data not shown). Fourth, similar autophagosome markers [7,12]. In addition, the organism replicates cytopathic effects were observed when rough strain CA180 infected within a compartment that contains ER resident proteins [7,8,11]. S2 and J774A.1 cells [18,19] (Fig. 1B and 1C). Finally, the number of These data thereby suggest that host cell autophagic pathway bacteria that entered S2 cells was directly proportional to the proteins, and ER-associated factors, may regulate the intracellular multiplicity of infection (MOI) (Fig. S3). This feature was also trafficking and replication of the pathogen. observed in mammalian cell systems (data not shown). Recent developments in the use of evolutionarily divergent To easily visualize the intracellular trafficking and replication of Drosophila S2 cell model systems to study host-pathogen interactions, Brucella spp., we exploited a GFP-expressing 16M strain (henceforth and RNA interference (RNAi) technology for knocking down host 16M-GFP) (Fig. S4). A comparison of the intracellular trafficking of , have provided unprecedented opportunities for Brucella spp. in S2 and mammalian cells indicated that the pathogen making significant progress in elucidating Brucella host factors. follows similar pathways in both host cell systems. BCVs trafficked to Drosophila S2 cells are macrophage-like cells that recapitulate and replicated within an intracellular compartment that contained conserved aspects of innate immunity [24] and that have been ER markers (e.g., mSpitz in S2 cells) [37], and was closely associated exploited for studying mammalian host-pathogen interactions. with COPII-coatomer (Sec 23) proteins (Fig. S5A and data not RNAi-based forward genetic screens in S2 cells have, for example, shown) in both cell systems. Quantitative analysis also demonstrated identified novel host factors involved in the recognition and that the bacterium failed to accumulate late endosome, Golgi marker replication of significant human bacterial pathogens, including E. (dGRASP) [38], or lysosomal markers in S2 or mammalian cells coli [25], Listeria [26,27], Mycobacterium [28], Legionella [29], and ([7,8,12] and Fig. S5B). In addition, heat killed, formaldehyde fixed, Chlamydia [30,31]. Importantly, mammalian orthologs of hits and DvirB controls did not similarly colocalize with ER markers in identified in these screens have been shown to be important for either system (Fig. S5A and data not shown). Therefore, the bacterial infection of mammalian cells, thereby validating the utility intracellular trafficking of B. abortus and B. melitensis in S2 and of this Drosophila cell model for host-pathogen studies [28–31]. In this mammalian cells shared striking similarities. study, we show that the Drosophila S2 cell-Brucella interaction system Similar infection profiles were observed when B. abortus was recapitulates critical aspects of Brucella infection of mammalian cells. used to infect mammalian or S2 cells that were treated with several

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Figure 1. Brucella abortus and B. melitensis entry, replication and cytotoxicity in Drosophila S2 cells. A. Gentamicin protection assays were employed to assess the entry (panels 1 and 2) and replication (Panels 3 and 4) of assorted B. melitensis (Panels 1 and 3) and B. abortus (panels 2 and 4) strains. B. Rough mutant strains (CA180, S2308manBA::Tn5) induce a cytopathic effect in S2 cells. Trypan blue dye exclusion assays were used to measure the viability of S2 cells after infection with various Brucella strains. The manBA mutant induced a cytopathic effect on S2 cells. Smooth wild- type strains (16M and 2308) and strains lacking the T4SS VirB system (BA114, S2308virB10::Tn5) did not induce a similar effect. C. Viability of Brucella infected S2 cells at 48 h.p.i. Drosophila S2 cells were infected with Brucella strains, (1) uninfected control, (2) 16M, (3) S2308, (4) BA114, and (5) CA180 at an MOI of 200. Infected cells were stained with trypan blue, fixed and analyzed by Olympus IX70 inverted microscopy (magnification, 6200). Dead cells appear black. The images were taken from a representative experiment. All data represent the means 6 standard deviations from at least three independent experiments. doi:10.1371/journal.ppat.1000110.g001 compounds; these compounds disrupted host cell functions and prevents the assembly of COPI coated vesicles and disrupts did not impair the bacterial growth in culture, or the viability of vesicular transport [7,8] (Table S2 and Fig. S7A and S7B). infected S2 cells (Fig. S6). These included: cytochalasin D [23], a Treatment of S2 and J774.A1 cells with the PI3K inhibitor compound that disrupts actin polymerization; bafilomycin A1, a wortmannin (WM) significantly reduced entry of B. abortus and B. specific inhibitor of vacuolar H+-ATPase activity and endolysoso- melitensis (Fig. S7A and data not shown). However, WM treatment mal acidification [39]; brefeldin A (BFA), a fungal metabolite that of S2 and J774.A1 cells had no effect on the replication efficiency

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Table 1. Evolutionarily conserved host factors required for Brucella abortus (S2308) infection in both Drosophila S2 and mammalian cell lines

Gene name CFU (% of control) a Reference S2 cells Mammalian cells

CG # Method Internalization Replication Method Internalization

Rho1 8416 dsRNAi 25.1615.2** b 22.4611.2** Dom-neg ,2865 [23], this study Rac1 2248 dsRNAi ND d 63.7613.0**e Dom-neg ,3763 [23], this study Rac 2 8556 dsRNAi 45.9611.1** 46.0616.5** ND ND This study Cdc 42 1253 dsRNAi 51.9624.6** 56.0619.5** Dom-neg ,45617 [23], this study Sar 1 7073 dsRNAi ND 47.161.3** e Dom-neg NAf [8], this study Pi3K21B 2699 dsRNAi 59.7610.8** 65.967.5** p85abD g 9.762.7** This study p85b2/2 43.765.5** This study Pi3K92E 4141 dsRNAi 62.2614.9** 65.765.2** ND ND This study PI3Ks (2) h dsRNAi 59.5614.7** 60.4615.8** ND ND This study Pi3K68D 1162 dsRNAi ND 47.764.8** ND ND This study Pi3K59F 5373 dsRNAi 49.6610.5** 44.4616.9** ND ND This study PI3Ks (4) i dsRNAi 64.469.0** 52.865.3** ND ND This study

aColony forming units (CFUs) of the untreated control was normalized as 100%. bData represent the means6standard deviations from at least three independent experiments. **represents significant at P,0.001 compared with no-RNAi control. cDominant-negative mutant. dNot detected. eData resulted from image analysis using NIH Image J software describe in Materials and Methods. fNo accurate data, ,30% of infected cells with replicating bacteria. gMEFs are deficient in class IA PI3Ks p85a and p85b (p85a2/2p85b2/2). h Double strand RNA targeting to the two PI3Ks Pi3K21B (CG2699) and Pi3K92E (CG4141) in class IA and IB, respectively. iDouble strand RNA targeting to all the four members of PI3Ks [Pi3K21B, Pi3K92E, Pi3K68D (CG11621, class II PI3K) and Pi3K59F (CG5373, class III PI3K)] in the fly genome. doi:10.1371/journal.ppat.1000110.t001

of the internalized bacteria (Fig. S7C, and data not shown). These Rho1-depleted S2 cells appeared larger than untreated controls, findings were similar to those previously reported in mammalian contained numerous enlarged intracellular vacuoles, and also cell systems [8,23,39,40]. In addition, we performed several displayed significantly decreased levels of Brucella entry (Table 1, experiments to assess the role of sphingolipids in supporting Fig. 2B). Sar1-depleted S2 cells also displayed dramatically bacterial infection, and exploited myriocin (MR), a potent reduced levels of Brucella replication (Table 1, Table S3 and inhibitor of serine palmitoyltransferase (SPT), the first step in Fig. 2A) were observed in these cells. These findings were similar sphingosine biosynthesis [41], for these studies. B. abortus entry and to results obtained when B. abortus was used to infect mammalian survival were significantly inhibited when cells were treated with cells in which the activities of the corresponding human high MR concentrations ($1 mM). Low concentrations (#100 nM) orthologous proteins had been depleted [8,23]. Therefore, the of the compound had no effect on bacterial entry (Table S2 and activities of these evolutionarily conserved GTP-binding proteins Fig. S7A). However, the replication efficiency of the pathogen was were required to support bacterial infection of both S2 and decreased under these conditions (Fig. S7A). mammalian cells (Table 1, Table S3, Fig. 2 and Fig. S8). To assess whether PI3Ks played similar roles in supporting RNAi-mediated inactivation of host factors required for bacterial infection of mammalian and S2 cells, we performed Brucella infection several experiments. First, we treated S2 and J774A.1 cells with We employed RNAi technology to examine whether host WM and found that the levels of B. abortus and B. melitensis entry proteins that are known to support bacterial infection of decreased in a similar fashion in both host cell systems (Table S2, mammalian cells play similar roles in S2 cells. The evolutionarily Fig. S7A and data not shown). Second, we employed RNAi conserved host proteins Rho, Rac, Cdc42 and Sar1 have been technology to deplete S2 cells of individual PI3K proteins and then previously shown to be required for Brucella infection of measured bacterial entry and replication. These experiments mammalian cells [8,23], therefore, we examined whether these revealed that multiple classes of PI3Ks are required to support B. proteins were also required for Brucella entry and replication in S2 abortus and B. melitensis WT strain infection (Table 1, Table S3, cells. Fluorescence microscopy image assays were employed for Fig. 2A, Fig. 3A and 3B). However, rough and smooth strains these studies because they offered a rapid and convenient method exploit separate host molecular pathways for entry [42]. when B. for assessing bacterial infection. Importantly, similar results were abortus rough strain CA180 was used to infect PI3K-depleted S2 obtained when either fluorescence microscopy or gentamicin cells, bacterial entry was dramatically enhanced (Fig. 3C). These protection assays were performed (Table 1 and Fig. 2A). When S2 data indicated that multiple PI3Ks play differential roles in cells were depleted of Rac and Cdc42, the entry of B. abortus mediating the entry of smooth and rough Brucella strains into S2 (S2308) or B. melitensis (16M) was impaired (Table 1 and Fig. 2A). cells.

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MEFs (Fig. 3C and data not shown). These findings were similar to results obtained in experiments in which the entry of a Brucella rough mutant into class IA PI3K-depleted S2 cells was examined (Fig. 3C). Therefore, host cell PI3K isoforms differentially mediated the infection of smooth and rough organisms in both cell systems, and supported the use of the Drosophila cell system for elucidating novel Brucella host cell factors.

RNAi screen for ER-associated host factors We were encouraged by our findings that previously described mammalian host proteins (i.e., Rho1, Rac, Cdc42 and Sar1) played similar roles in S2 cells. In addition, we noted that the Drosophila S2 cell system enabled the first molecular dissection of host cell PI3K isoform activity during Brucella infection. We therefore examined whether the Drosophila S2 cell system and RNAi technology could be combined to identify novel Brucella host factors. To focus our experiments, we constructed and screened 240 dsRNAs, including 110 dsRNAs that targeted the knockdown all of the genes annotated to be associated with the ER in the Drosophila RNAi Library Release 1.0 (Open Biosystems, Huntsville, AL, USA). The ER was ripe for examination because Brucella is known to replicate within a poorly characterized ER-like compartment, thereby suggesting that ER-associated host factors may be involved in regulating the intracellular replication of the pathogen. Our ER-directed RNAi screen gave several interesting results. First, our screening approach successfully identified 52 hits. A hit was defined as a sample in which the relative infection differed by more than two standard deviations from the untreated control (Table S3). Importantly, control genes (i.e., Rho1, Rac, Cdc42, Sar1 and PI3Ks) were identified as hits in the screen (Table S3). Figure 2. Evolutionarily conserved host factors mediate Therefore, our screening strategy was sufficiently robust to Brucella infection of Drosophila S2 cells. A. S2 cells that had been uncover known or suspected host factors. We were curious subjected to dsRNA-mediated gene depletion were infected with whether the hit frequency obtained in our ER-targeted screen Brucella 16M-GFP at an MOI of 50. At 72 h.p.i., the cells were then replated onto ConA-coated 96 well glass bottom plates, washed three would be the same if a set of dsRNAs that were not associated with times with 16PBS, fixed, stained and viewed with an Olympus IX70 the ER were screened. We therefore screened 130 dsRNAs that inverted microscope. For each experiment, two images containing a were randomly picked from 2 of the 76 96-well plates in the total of at least 1,000 cells were analyzed using NIH Image J software Drosophila RNAi library. Because the manufacturer randomly and the infection index was determined. Data represent the means 6 arrayed dsRNAs into the source plates, this strategy for picking standard deviations from three independent experiments. *** indicates dsRNAs to be screen introduced no bias in the functions of the significance at P,0.001. B. dsRNA-mediated Rho1 knockdown in S2 cells results in large sized cells with reduced ability to support WT targeted genes in the screen. Notably, this experiment uncovered Brucella (S2308) infection (48 h.p.i., Scale bar: 10 mm). C. Reduction of only 2 hits (,1.5% of the total) (Table S3), and therefore gave a hit Rho1 mRNA levels in dsRNA-treated S2 cells (upper panel) was assessed frequency that was comparable to that observed in the by RT-PCR and quantified by scanning densitometry (lower panel). Mycobacterium fortuitum and Listeria monocytogenes whole genome doi:10.1371/journal.ppat.1000110.g002 RNAi screens [26–28]. Interestingly, 14 out of 52 hits in our screen had been previously shown to mediate infection of S2 cells Experiments in mammalian cells confirm results obtained by Mycobacteria, Listeria, Legionella and Chlamydia infection [26–31] in Drosophila S2 cells (Table S3 and Fig. 4). On the other hand, 29 genes were identified If Drosophila S2 cells are to serve as a useful model host cell that had not been previously reported to be involved in supporting system, then results obtained using this system should mirror intracellular bacterial infection (Table S3). These novel genes were corresponding mammalian cell findings. To test this possibility, we classified according to the system of biological and examined whether a murine ortholog (p85) of a model Drosophila molecular function, cellular component, or protein domains as gene (Pi3K21B) that supports Brucella infection of insect cells reported in FlyBase (www.flybase.org). This classification revealed (Table 1, Table S3, Fig. 2A, Fig. 3A and 3B) mediates bacterial that the novel hits represented a variety of functional classes, infection of murine cells. We used immortalized mouse embryonic including kinases, chaperones, and biosynthetic/metabolic en- fibroblasts (MEFs) derived from knockout mice harboring zymes. In addition, these 29 genes were localized to either the ER deletions in class IA PI3Ks (p85a and p85b) [43] for these studies. lumen (CG9429, CG30498) or ER membrane (CG6437, CG1063) As expected, the levels of B. abortus and B. melitensis WT strains (Table S3). We re-tested some of our most interesting hits in both entry into MEF cells harboring PI3K gene deletions were fluorescence microscopy and gentamicin protection assays (Table dramatically reduced (Table 1 and data not shown). p85a2/2 S3, repeat$3 times), and also employed quantitative reverse p85b2/2 and p85b2/2 MEFs supported lower levels of B. abortus transcriptase polymerase chain reaction (Q-PCR) to verify that the and B. melitensis WT strains entry than p85+/+ controls (Table 1 expression of these genes in S2 cells was knocked down by dsRNA and data not shown). However, when these MEFs were infected treatment. We typically obtained 60–90% knockdown of target with Brucella rough mutants (CA180 and S2308DmanBA), bacterial gene expression in our screening plates (Fig. 2C and data not internalization significantly increased, especially in p85b2/2 shown).

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Figure 3. Depletion of individual host phosphoinositide 3-kinases (PI3Ks) affects Brucella abortus infection of Drosophila and mammalian host cells. A. Replication efficiency of WT Brucella (S2308) in depletion of PI3Ks of S2 cells (72 h.p.i.) and MEFs (48 h.p.i). B. Relative bacterial number (CFU) in Brucella infection of PI3K-depleted cells at 72 h.p.i. (S2 cells) or 48 h.p.i. (MEFs). C. Depletion of PI3Ks affects Brucella rough mutant CA180 entry into Drosophila and mammalian host cells. For these experiments, either Drosophila S2 cells, treated with dsRNAs to deplete individual PI3Ks, or mouse embryonic fibroblasts (MEFs) harboring deletions of the two regulatory isoforms of class IA PI3Ks (P85a and P85b), were infected with Brucella rough mutant CA180. The infected cells were lysed and subjected to gentamicin protection assays after an additional 1 hr of incubation with media supplemented with 40 mg/ml gentamicin at the indicated temperatures. The CFUs of all the treatments at the indicated time points were compared with that of the WT (S2308) control. CFUs were counted after 3 days of incubation at 37uC. Data represent the means 6 standard deviations from three independent experiments. *** indicates significance at P,0.001. doi:10.1371/journal.ppat.1000110.g003

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Figure 4. Host factors mediating intracellular bacterial infection. Shared and unique host factors were determined by analyzing published data from screens performed in S2 cells infected with Mycobacterium fortuitum [28], Listeria monocytogenes [26,27], Legionella pneumophila [29] and Chlamydia caviae [30]. Numbers in parenthesis represent the number of screened or targeted genes. doi:10.1371/journal.ppat.1000110.g004

Although each screen hit constituted a potential entry point for Brucella-infected IRE1a2/2 MEFs. Instead, they appeared to investigating the mechanism by which Brucella secures a replicative reflect a specific and important bacterial requirement for host niche, we were particularly intrigued with IRE1 (CG4583), a key cell IRE1a activity. Finally, the levels of entry and replication of signal transducer that plays an important role in regulating the Salmonella enterica serovar typhi, and the amounts of latex bead host cell UPR [32–34]. RNAi mediated knockdown of IRE1 gene internalization, were similar in control and IRE1a2/2 cells expression resulted in significant reductions in Brucella replication (Fig. 6 and data not shown). These data supported the idea that (Fig. 5A, 5B and Table S3). In addition, IRE1 had not been IRE1a2/2 cells do not possess general defects in phagocytosis, previously implicated as a bacterial host factor. These data raised and that IRE1a activity is not required to support infection by the intriguing possibility that IRE1 may play a novel role in all intracellular bacterial pathogens (Table S3 and Fig. 6). regulating Brucella infection. We therefore examined whether IRE1a (the mammalian ortholog of Drosophila IRE1) was PERK, ATF6 and BBF-2 are not required for Brucella important for Brucella infection of mammalian cells. replication Besides IRE1a, several other ER-associated transmembrane IRE1a is required for efficient Brucella replication in signaling molecules play important roles in initiating and regulating mammalian cells UPR in host cells, including PERK, ATF6, and BBF2H7 We performed several experiments to examine whether IRE1a (mammalian BBF-2 ortholog) [32–34,44–46]. We performed played a critical role in supporting Brucella infection of mammalian fluorescence microscopy and gentamicin protection assays to cells. First, we infected IRE1a-null (IRE1a2/2) and WT (IRE1a+/+) examine their role in Brucella entry and replication. B. melitensis control MEF cells with 16M-GFP (Fig. 5C), and also performed (16M) entry and replication in PERK-, ATF6-, and BBF-2-depleted gentamicin protection assays to assess bacterial entry and S2 cells were not significantly different from untreated controls replication (Fig. 5D). The level of bacterial entry in IRE1a2/2 (Fig. 5A, Fig. 7A and Table S3). Although B. melitensis replication was not statistically different from IRE1a+/+ controls (Fig. 5D). efficiency decreased in ATF- and BBF-2-depleted S2 cells, the However, bacterial replication was significantly inhibited in number of bacterial colony forming units (CFUs) in these cells at IRE1a-depleted S2 cells and in IRE1a-null MEF cells (Fig. 5). 72 hours post-infection (h.p.i.) was not significantly smaller than Trypan blue dye exclusion analysis of 16M-infected MEF cells controls (Fig. 7A and Table S3). Importantly, bacterial replication failed to reveal differences in host cell survival (data not shown). efficiencies in PERK2/2 and PERK+/+ MEF cell lines were similar Therefore, the differences in bacterial replication efficiencies in (Fig. 7B and 7C). Taken together, these results demonstrated that not these cell lines were not caused by the induction of host cell all UPR signaling molecules are required to support the replication pro-apoptotic programs or by differences in the survival of of this pathogen, and that IRE1a plays a specific role in this process.

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Figure 5. IRE1a is required for Brucella melitensis infection. A. Brucella (16M-GFP) infection of S2 cells in which the expression of the indicated host genes has been knocked down by dsRNA treatment at 72 h.p.i.. No RNAi-treated cells, and cells in which Rho1 expression was knocked down by dsRNA treatment were used as positive and negative controls, respectively. B. Depletion of IRE1 expression in S2 cells inhibits 16M-GFP replication. IRE1 has a limited role in B. melitensis entry S2 cell. C. B. melitensis 16M-GFP infection (MOI = 100) of mouse embryonic fibroblast (MEF) IRE1a-null and their wild-type counterparts at 48 h.p.i. D. IRE1a2/2 MEF cells support B. melitensis entry but not replication. *** Significant at P,0.001. Data in B and D represent the means 6 standard deviations from three independent experiments. The images in A and C were taken from a representative experiment. doi:10.1371/journal.ppat.1000110.g005

Discussion Drosophila S2 cells recapitulates important aspects of mammalian cell infection. First, isogenic mutants of Brucella spp. behaved The study of host-Brucella interactions has suffered from the similarly in S2 and mammalian cells. In addition, these divergent absence of a tractable genetic system to elucidate host factors. host cell systems displayed similar trends in infection by smooth However, data obtained in this study indicate that Drosophila S2 and rough strains with varied pathogenicity. Brucella rough cells provide a compelling model system for identifying and mutants, such as CA180, were cytopathic to both mammalian characterizing these important proteins. Brucella infection of and S2 host cells [18, 19, this study]. Therefore, these cells share

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Therefore, the differential growth of B. abortus and B. melitensis in these host cell systems likely results from differences in the growth temperature, and not from differential subversion of conserved host cell functions. Third, the most important criterion for judging the utility of a model non-mammalian host-pathogen interaction system is whether it can be exploited to shed new insights into the interaction in mammalian cells. In this regard, it should be noted that bacterial pathogens, such as Listeria monocytogenes [52], grow more slowly in Drosophila S2 cells than in mammalian cells; however, many host factors required for entry and survival of these intracellular pathogens have been identified using Drosophila S2 cells as a platform [25–31]. We expect to garner similar insights through the use of our Drosophila S2 cell-Brucella interaction system, and our demonstration that PI3Ks and IRE1a mediate Brucella infection of Drosophila S2 cells and murine embryonic fibroblasts support this view. Our RNAi screen in S2 cells for ER-associated Brucella host factors provides new insights into how Brucella secures an intracellular replicative niche. Our screen identified 52 genes that Figure 6. IRE1a is not required for Salmonella entry and +/+ 2/2 participate in this process, 29 of which had not been previously replication. MEF IRE1a and IRE1a cells were infected with suggested to support bacterial pathogen infection. In addition, we Salmonella enterica serovar typhi wild type strain SL1344 at an MOI of 50. Gentamicin protection assays were employed to assess Salmonella dissected the role of 4 PI3K isoforms. The number of identified entry [At 1.5 h.p.i, the number of CFUs for IRE1a+/+ and IRE1a2/2 was hits (50 out of 110 pre-selected ER- associated genes) was striking, (2.4960.36)6106 and (2.2360.22)6106, respectively] and replication (3, and likely reflects that sustained and multi-faceted Brucella-ER 6 and 9 h.p.i). All data represent the means 6 standard deviations from interactions are required for Brucella replication in host cells. three independent experiments. Interestingly, 14 of the genes identified in our screen were also doi:10.1371/journal.ppat.1000110.g006 required for infection of S2 cells by other intracellular bacterial pathogens, including Listeria, Mycobacteria, Legionella or Chlamydia conserved molecular mechanisms for recognizing and responding [26–31]. The fact that Brucella and Legionella share several ER- to Brucella LPS mutants. Second, Brucella entry and replication in associated host factors is perhaps not surprising, especially given S2 and mammalian cells were similarly sensitive to pharmacolog- that both organisms engage in sustained interactions with the host ical perturbation by structurally diverse compounds. Of particular ER as part of their virulence and replication programs [53,54]. interest was the observation that MR, an inhibitor of STP, the Finally, Brucella-specific ER-associated factors, such as IRE1 rate-limiting enzyme in sphingolipid biosynthesis, dramatically (CG4583), were uncovered in our screen. IRE1 may constitute a reduced the amount of Brucella infection of S2 cells (this study). species-specific host factor that plays a role in mediating the Previous studies have demonstrated an important role for unfolded protein response, thereby suggesting that the modulation sphingolipid enriched lipid rafts in pathogen infection [47–50], of this stress-response system may be critical to bacterial and our MR experiments support these observations. Third, intracellular survival and replication. Brucella infection of S2 cells required the activities of conserved In eukaryotic cells, IRE1a mediated UPR induction is associated GTP-binding proteins (Rho1, Rac, Cdc42, and Sar1), suggesting with enhanced expression of genes encoding ER chaperones and that Brucella infection of mammalian and Drosophila cells shared protein-folding catalysts, and proteins that participate in ER- similar host molecular requirements. Finally, the activities of associated degradation (ERAD) [55,56]. IRE1a activation also PI3Ks differentially regulate smooth and rough Brucella infection in induces the biosynthesis of membrane phospholipids that increase both mammalian and Drosophila S2 cells (this study). Interestingly, the surface area and volume of rough ER [57,58]. In Brucella infected the effects of PI3K knockdown in MEF cells were more dramatic cells, IRE1a mediated activity may result in the biosynthesis of ER than in S2 cells. In MEFs, PI3K genes are deleted, and thus the membrane that can be exploited by the pathogen to expand the size corresponding enzyme activities are absent. However, in Drosophila and enhance the quality of its replicative niche However, our data S2 cells, PI3K gene expression is knocked down (60–90%), and indicate that other UPR signal transducers, including PERK, are not some residual activity may remain. These differences likely required for Brucella infection in both Drosophila S2 and murine account for the differential infection of these cell types. Taken embryonic fibroblast cell systems. Therefore, not all UPR regulatory together, our data support the conclusion that S2 cells provide a proteins are important for bacterial replication (Fig. 7 and Table S3), useful model for investigating host-Brucella interactions. raising questions about the privileged status of IRE1a among these Our demonstration that Drosophila S2 cells can be used to classes of molecules. illuminate Brucella host factors is surprising because Brucella spp. do Recent reports have indicated an intriguing link between IRE1a not occupy a described environmental niche outside of the activity and autophagic vacuole biogenesis [59,60]. For example, mammalian host. In addition, the bacteria do not grow well in IRE1a is required for the autophagy observed after cells are culture at temperatures below 35–37uC. However, previous treated with the ER stress-inducing agents DTT, tunicamycin or reports have demonstrated B. suis multiplication within U937 cells thapsigargin [59,60]. However, parallel experiments using PERK- at 30uC [51]. Therefore, Brucella growth below 37uC is not deficient cells, and cells in which the expression of ATF6 had been restricted to B. melitensis and B. abortus strains. Second, Brucella knocked down, demonstrated that these UPR-associated signal replication in J774A.1 and Drosophila S2 cells at 29uC share similar transducers are not directly involved in the response to these drug kinetics (Fig. S2B and S2C). Although a difference in the treatments [60]. Therefore, IRE1a can regulate some autophagic replication efficiency of S2308DvirB2 in J774 and S2 cells at 24 events independently from input by these other ER associated and 48 h.p.i was observed, no difference was detected at 72 h.p.i. signaling molecules.

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Figure 7. Endoplasmic reticulum UPR regulators PERK, ATF6 and BBF-2 display limited roles in Brucella melitensis infection. A. Depletion of PERK, ATF6 and BBF-2 in S2 cells displays no significant difference in the number of bacteria (16M) (CFU, % of control) in entry (1.5 h.p.i) and replication (72 h.p.i), although the replication efficiency of depletion of ATF6 and BBF-2 displayed significantly reduced. B. Infection of mouse embryonic fibroblast (MEF) PERK2/2 and their WT counterparts by B. melitensis 16M-GFP (MOI = 100) displays no significant difference at 48 h.p.i. C. PERK2/2 MEF cells have no effect on B. melitensis replication although the entry of the bacterium displays significant difference. *** Significant at P,0.001. Data in A and C represent the means 6 standard deviations from three independent experiments. The images in B were taken from a representative experiment. doi:10.1371/journal.ppat.1000110.g007

The differential participation of IRE1a, ATF6 and PERK in replication (Fig. 5, Fig. 7 and Table S3). Second, the assembly of regulating the autophagy observed after cells are treated with ERAs is dependent upon early secretory pathway molecules [61–63]. stress-inducing agents is strikingly similar to their differential roles In yeast, the COPII mutants sec16, sec23,andsec24, are defective in in mediating Brucella replication. IRE1a is required for Brucella to autophagy. However, mutations in two other COPII genes, sec13 replicate efficiently; however, Brucella replication in PERK-, and sec31, do not affect ERA biogenesis and autophagy [61,62]. In ATF6-, and BBF-2-depleted S2 cells was not significantly different addition, PI3K activity is important for this process [64–66]. Our from untreated controls. This differential participation therefore data demonstrate similar host factor requirements during Brucella suggests a model in which IRE1a regulates Brucella infection by infection of Drosophila cells. Specifically, depletion of Sec23, Sec24 modulating the host cell autophagy pathway (Fig. 8). and PI3Ks in host cells dramatically reduces Brucella replication Based on findings from our dsRNA screen, we propose a multi- (Table 1, Table S3, Fig. 2 and Fig. 3). However, depletion of Sec31 step model by which IRE1a regulates Brucella replication. First, has no affect on this process (Table S3). Finally, Brucella trafficking to BCVs traffic to a compartment that contains ER resident proteins. its intracellular replicative niche involves interactions with a Concomitantly, BCVs trigger IRE1a activation, which in turn, compartment that contains the autophagosomal marker monodan- stimulates the biogenesis of ER-associated autophagosomes sylcadaverin [7,12]. These localization data thereby establish a (ERAs) [59,60]. ERAs then fuse with BCVs to form ERA-BCVs. physical interaction between internalized Brucella and the host cell This process is also regulated by the activities of PI3Ks. Finally, autophagy pathway. It should be noted, however, that although we ERA-BCVs fail to fuse with lysosomes and hence avoid cannot rule out the possibility that Brucella trafficking in MEFs differs degradation; instead, they fuse with the ER to form ER-derived from professional phagocytes, Brucella trafficking in HeLa cells and BCVs that are permissive for Brucella replication (Fig. 8). phagocytes share striking similarities [12]. Therefore, our observa- Several pieces of evidence support this view. First, IRE1a,but tions in MEFs likely shed light on Brucella infection of phagocytes. neither PERK nor ATF6, is required for the induction of autophagy Taken together, the data are consistent with the idea that IRE1a in response to treatment by ER stress-inducing agents [60]. Similar activity plays an important role in supporting Brucella interactions requirements for host proteins are observed during Brucella with the host cell ERA biogenesis machinery in mammalian cells

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Figure 8. Model describing how Brucella may exploit IRE1a to secure a replicative niche in an ER-like compartment. After PI3K- dependent Brucella internalization (1), intracellular Brucella-containing vacuoles (BCVs) traffic to the ER in a T4SS-dependent fashion (2). Accumulation of BCVs in the ER may modulate IRE1a activities (3), which then may trigger the biogenesis of ER-containing autophagosomes (ERAs) [59–60] (4). ERA biogenesis is known to require the activities of early secretory pathway components, including members of the COPII complex (Sar1- Sec23-Sec24) [61–63] (indicated in red). This process is also regulated by the activities of PI3Ks [64–66] (5). ERAs may then fuse with BCVs (6) to form ERA-BCVs (7). In addition, ER expansion may occur in response to these events. Finally, ERA-BCVs may fuse with an expanded ER membrane (8) and intercept ER proteins such as calreticulin (indicated in black) to form ER-derived BCVs that are permissive for Brucella replication. Depletion of members of COPII complex, PI3Ks and ER proteins such as calreticulin disrupted these processes and dramatically reduced Brucella replication. doi:10.1371/journal.ppat.1000110.g008

(Fig. 8). Future studies will exploit the genetic power of the Drosophila Cell Culture S2 cell system to elucidate this intriguing possibility, and to define the Murine macrophage J774.A1 cells, MEFs and HeLa cells were precise molecular mechanisms by which Brucella secures an routinely cultured at 37uC in a 5% CO2 atmosphere in Dulbecco’s intracellular replicative niche. Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). S2 cells were maintained at 25uCin Materials and Methods Drosophila-SFM medium or in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% FBS. Cells were seeded in 24- Bacterial Strains well plates and cultured overnight before infection. For antibiotic Brucella melitensis strain 16M (WT) and B. abortus strain 2308 (WT), protection assays, 2.56105 cells were seeded in each well; for and their derived mutants are listed in Table S1. Bacteria were grown fluorescence microscopy assays (see below), 56104 cells were in tryptic soy broth (TSB) or on tryptic soy agar (TSA, DifcoTM) seeded on 12-mm glass coverslips (Fisherbrand) placed on the plates, supplemented with either kanamycin (Km, 50 mg/ml), or bottom of 24-well microtiter plates before infection. chloramphenicol (Cm, 25 mg/ml) when required. For infection, 4 ml of TSB was inoculated with a loop of bacteria taken from a single Brucella Infection colony grown on a freshly streaked TSA plate. Cultures were then Host cells were infected with Brucella at an MOI of 100, unless grown with shaking at 37uCovernight,oruntilOD600<3.0. otherwise indicated. Infected cells were then incubated at 29uC (S2

PLoS Pathogens | www.plospathogens.org 11 July 2008 | Volume 4 | Issue 7 | e1000110 ER Factors That Mediate Brucella Replication cells) or 37uC (mammalian cells) after centrifugation for 5 min at 16PBS, fixed with 3.7% formaldehyde (pH 7.4) at room 2006g. Thirty minutes post-infection, culture media was removed, temperature for 1 hr and processed for immunofluorescence and the cells were rinsed with 16phosphate buffered saline (PBS). microscopy. Fresh media, supplemented with 40 mg/ml gentamicin, was then added for 1 hr to kill extracellular bacteria. Infected cells were Immunofluorescence Microscopy Assay continuously incubated in this antibiotic for various lengths of time To elucidate Brucella spp. intracellular trafficking, S2 cells were at the indicated temperature. As indicated, viable bacteria in infected with the following strains: B. melitensis (strains 16M or infected cells were analyzed using the antibiotic protection assay or 16M-GFP); B. abortus (strain S2308); S2308 virB2 deletion mutants; the immunofluorence microscopy assay described below. In heat killed or 3.7% formaldehyde fixed WT strains. At various addition, Brucella replication efficiency ([# of CFUs at different post-infection time points, S2 cells were replated onto ConA- time points post infection]/[# of CFUs of Brucella entry]) in the coated 12-mm coverslips and allowed to adhere for 45 min to 1 hr. infected cells was also determined Cells were then washed, fixed as described above, and processed for immunofluorescence microscopy [18]. The primary Antibiotic Protection Assays used were as follows: goat polyclonal anti-Brucella; rabbit anti- At various times post-infection, viable bacteria present in human M6PR; rabbit anti-human cathepsin D; goat-anti rabbit infected cells were analyzed using gentamicin protection assays Sec23 (COPII marker, Affinity BioReagents, Inc., CO, USA). [18]. Briefly, infected cells were washed twice with 16PBS buffer, Samples were stained with Alexa Fluor 488-conjugated and/or lysed with 0.5% Tween 20 in sterile water, and the released Alexa Fluor 594-conjugated donkey anti-goat/rabbit (Molecular bacteria were subjected to serial dilution in peptone saline [1% Probes, 1:1000). Cover slips were then mounted in Vectashield (wt/vol) Bacto peptone and 0.85% (wt/vol) NaCl]. Next, 10 mlof mounting media (Vector Laboratories, Inc., CA, USA) and serial diluted cell lysate was plated on TSA plates. Finally, CFU visualized with an Olympus BX51 confocal microscope. For were counted after three days of incubation at 37uC. quantitative analysis, single confocal section of random fields was acquired, and colocalization of markers was scored as positive Viability Assay of Infected Host Cells when nonsaturated signals partially overlapped. Images for all S2 cells were coincubated with or without various drugs before immunofluorescence assays for Brucella spp. trafficking were 1 hr of and during Brucella infection (See below). Next, the infected acquired with a Hamamatsu ORCA-ER camera mounted on cells were centrifuged at 2006g for 5 min and then incubated the Olympus BX51 microscope and driven by Simple PCI (30 min) with assorted Brucella strains. Fresh Drosophila-SFM software (Compix Imaging Systems Inc., Cranberry Township, media, supplemented with drugs (as indicated) and 80 mg/ml PA.). Images were processed with Adobe Photoshop CS Software gentamicin, was added to kill extracellular bacteria. The infected (Adobe Systems Incorporated, San Jose, CA). and gentamicin treated cells were then incubated at 29uC for various lengths of time. To quantify the viability of S2 cells, at Drug Treatments various time points, a portion of the infected cells was removed Drosophila S2 cells or J774.A1 murine macrophages were and processed for 0.2% trypan blue vital stain analysis. At least coincubated in 24 well plates with assorted drugs including 500 cells were counted per sample. For image analysis, infected bafilomycin A1 (BAF), brefeldin A (BFA), cytochalasin D (CD), cells were replated onto ConA (Sigma)-coated 12-mm coverslips in myriocin (MR) and wortmannin (WM) at the indicated concen- 24-well plates and allowed to adhere for 1 hr. Cells were stained trations. Cells were treated with drugs 1 hr before, and during, with 0.2% trypan blue for 5 min and then fixed with 16PBS infection with the indicated Brucella strains. After infection, the containing 3.7% formaldehyde for 1 hr. Viability of infected cells treated cells were incubated at 29uC (S2 cells) or at 37uC with 5% was assessed by analyzing images obtained with an Olympus IX70 CO2 (J774.A1 macrophages). To evaluate Brucella internalization, fluorescence microscope. At least 500 infected cells per sample after 30 min of infection, fresh media, supplemented with the were used for the analysis. same concentration of the drugs and 80 mg/ml gentamicin was added to kill extracellular bacteria. After 45 min of incubation, the S2 Cell Transfection cells were lysed and the CFU per well determined by plating To visualize Brucella spp. trafficking, S2 cells were transfected dilutions on TSA plates as described above. To assess Brucella with ER marker mSpitz-GFP [37], and Golgi marker dGRASP- intracellular replication, CFU analysis was performed at 72 h.p.i. GFP [38] before infection. Specifically, S2 cells were grown to The effect that BAF-mediated inhibition of host cell endosomal ,80% confluence and then transfected using Effectene Transfec- acidification exerted on Brucella replication was also examined. tion Reagent (Qiagen) as per the manufacturer’s instructions. 0.25 Briefly, BAF was added to the culture media 2 h.p.i. and mg of each pUAS-mSpitz GFP and pAcpA-Gal4 were employed in continuously coincubated with infected cells for 72 hr. Cells were these transfection experiments. For the Golgi visualization lysed and analyzed using the gentamicin protection assay. To experiments, 0.25 mg of dGRASP-GFP was used in the investigate whether the drugs inhibit Brucella growth, the drugs transfection. Typically, 1.56106 cells were transfected and then were individually added to Brucella TSB cultures at 29uCor37uC grown in 2.2 ml of Schneider’s Drosophila medium supplemented and incubated for 1 and 72 hr. CFU plating was used to assess with 10% FBS. Three days post-transfection, cells were replated bacterial growth in the presence of drugs, and thereby to evaluate onto ConA-treated 12-mm glass coverslips placed on the bottom the potential inhibitory effects. of 24-well microtiter plates (for early time points of less than 8 hr) and immunofluorescence microscopy analysis was performed as Generation of dsRNAs previously described [18]. For later times points ($8 hr), the Primers for generating RNAi that target the knockdown of transfected cells were reseeded directly in 24-well plates and Drosophila Rac1, Rac2, Rho1, Cdc42, Sar1 and PI3Ks were allowed to adhere for 2 additional hours before infection with designed using sequence information present in flybase (http:// Brucella. At different post-infection time points, the infected cells flybase.org/). The primers were used in RT-PCR reactions to were replated onto ConA-coated 12-mm coverslips and allowed to generate cDNAs. dsRNAs targeting genes to be knocked down adhere for 1 hr. The cells were then washed three times with were generated using previously described methods [26]. Briefly,

PLoS Pathogens | www.plospathogens.org 12 July 2008 | Volume 4 | Issue 7 | e1000110 ER Factors That Mediate Brucella Replication gene-specific RNAi primers were used to amplify target sequences Mammalian Cell Infection from Drosophila cDNA mixtures. The PCR products were re- MEFs deficient of the two regulatory isoforms of class IA PI3Ks amplified using the RNAi primers with T7 RNA polymerase (p85a2/2 p85b2/2 and p85b2/2)[43],IRE1a (IRE1a2/2) [67] promoter sequences in the 59 end. The reamplified PCR products and PERK (PERK2/2) [68] and their corresponding WT control were then used as templates for the generation of dsRNAs. For p85+/+, IRE1a+/+ and PERK+/+ MEFs, were seeded in 24-well generation of dsRNAs targeting ER-associated and other genes, plates. After overnight culture, cells were infected with 16M-GFP cDNAs from commercially available Drosophila RNAi Library and/or S2308, and their derived mutant strains. Infected cells were Release 1.0-DNA templates (Open Biosystems, Huntsville, AL, centrifuged for 5 min at 2006gandthenincubatedat37uCfor USA) were directly used as templates. One or two microliters (total 60 min. Cells were washed with 16PBS buffer, and fresh media ,150 ng) of the PCR products were used to perform in vitro supplemented with 40 mg/ml gentamicin was added. Cells were transcription reactions with the T7 MEGAscript kit (Ambion, incubated for an additional 1 hr (entry) and 48 hr (replication) at Austin, TX) as per the manufacturer’s instructions. Aliquots of in 37uC. The amount of viable bacteria present in infected cells was vitro transcription products were subjected to quality control by 1% assessed using gentamicin protection assays. For fluorescence agarose gel electrophoresis analysis and dsRNA concentrations microscopy and viability assays, 56104 cells were seeded onto 12- were quantified using a NanoDropH ND-1000 UV-Vis spectro- mm coverslips in 24-well plates. At 48 h.p.i., infected cells were photometer (NanoDrop Technologies, Inc. Wilmington, DE). subjected to the appropriate assays as described above.

RNAi-mediated Gene Knock Down and Assays Statistical Analysis 1.06106 S2 cells were seeded in 12-well plates. dsRNAs (i.e., All quantitative data were derived from results obtained in Rho1, Rac, Cdc42, Sar1 and PI3Ks) were added to each well at a triplicate wells for at least three independent experiments. The final concentration of 15 mg/ml. After 4 days of incubation with significance of the data was assessed using Student’s t-test, and all the dsRNA, an aliquot of the S2 cells was removed to check the analyzed data were normalized with internal controls before analysis. efficiency of dsRNA mediated gene knock down by quantitative RT-PCR (Q-PCR). dsRNA-treated S2 cells in the same well were Supporting Information also re-plated in 24-well plates and allowed to adhere for at least 2 Table S1 Brucella strains used in this study hr before infection. At the selected time points, the dsRNA-treated Found at: doi:10.1371/journal.ppat.1000110.s001 (0.14 MB DOC) and Brucella infected cells were lysed and antibiotic protection assays or fluorescence microscopy image assays were performed as Table S2 Comparison of Drosophila S2 and mammalian cells described. treated with drugs that inhibit Brucella abortus (S2308) entry and To evaluate the utility of the combination of S2 cells and replication dsRNA technology, and the consistency of the results from Found at: doi:10.1371/journal.ppat.1000110.s002 (0.07 MB DOC) antibiotic protection assays, we analyzed Brucella infection using Table S3 Effect of ER-associated cellular factors on Brucella fluorescence microscopy image assays. dsRNAs that target ER- melitensis replication associated genes or other known or unknown genes were added to Found at: doi:10.1371/journal.ppat.1000110.s003 (0.10 MB XLS) 96-well microplates at a final concentration of 15 mg/ml (dsRNAs were added in duplicate in two different plates). S2 cells were then Figure S1 Schematic representation of image analysis using seeded in the plates with 5.06104 cells/well in 200 ml Drosophila- Image J to calculate the relative infection (RIF). 1. Threshold of bacterial replication in infected cells in an image using the same SFM medium. dsRNA-treated cells were incubated at 25uC for 4 days to allow for knockdown of target gene expression. The setting. 2. Analysis of particles in a thresholed image (i.e., the dsRNA-treated cells (100 ml) were replated into 96 well plates, number of infected cells with bacterial replication). 3. Histogram of the image (cell numbers were adjusted via color density). 4. infected with B. melitensis 16M-GFP at an MOI of 50. After 30 min Calculation of the infection index and RIF (% of control) of the of infection, the same amount of fresh media supplemented with samples. For example, infection index of sample B31C07 = 39/ 80 mg/ml gentamicin was added to each well and the infected cells 68.69; RIF of B31C07 = 1006[39/68.69]/[106/68.41] = 36.12. were incubated at 29uC. At 72 h.p.i., infected cells were replated Found at: doi:10.1371/journal.ppat.1000110.s004 (8.15 MB TIF) onto 96 well glass bottom plates (Greiner), that had been coated with ConA, and allowed to adhere for 1 hr. The infected S2 cells Figure S2 Brucella abortus and B. melitensis growth, and infection were washed 3 times with 16PBS, fixed with 3.7% formaldehyde of host cells, at 29uC and 37uC. A. B. abortus growth in liquid in 16PBS at 4uC overnight, and stained with phalloidin-Texas red culture (TSB) at 29uC and 37uC. B. Replication of Brucella wild- (1:1000) for 1 hr to visualize the host cell actin cytoskeleton. type strains S2308 and 16M in Drosophila S2 and J774.A1 murine Brucella infected S2 cells were viewed with an Olympus IX70 macrophages at 29uC. The number of CFUs of 16M and S2308 5 5 inverted microscope and two 4006 images from each well were for S2 [(4.1860.54) 610 /well and (3.5160.95) 610 /well, 5 acquired for image analysis. Images were analyzed using NIH respectively] and for J774.A1 [(5.1760.25) 610 /well and 5 Image J software (http://rsb.info.nih.gov/ij/), and the relative (7.7760.47) 610 /well, respectively] cells at 1 h.p.i was normal- infection (RIF) [1006(% of infected dsRNA-treated cells)/(% of ized as 100%. Brucella replication efficiency was defined as the infected cells in the untreated control)] was determined. More than number of CFUs at different time points post infection/the 1,000 S2 cells were counted to obtain the percentage of infection number of CFUs of bacterial entry (1 h.p.i). C. Entry and or infection index [(number of infected cells (at least 10 brucellae replication of S2308 derived mutants in S2 and J774.A1 murine within the cell))/(number of total cells)] in a sample. The detailed macrophages at 29uC. Data represent the means 6 standard process by which image analysis was performed is shown in Fig. deviations from three independent experiments. S1. dsRNA screen was repeated once, and some of hits identified Found at: doi:10.1371/journal.ppat.1000110.s005 (0.50 MB PDF) in both two round of screens were picked out to re-test in triplicate Figure S3 Infection of Drosophila S2 cells increases with rising in fluorescence microcopy and gentamicin protection assay as multiplicity of infection (MOI). A. With increasing MOI, the described above. number of S2 cells containing replicating Brucella melitensis (16M-

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GFP) increases at 72 h.p.i. The images were taken from a control. B. Viability of Brucella (S2308) infected and drug-treated representative experiment. B. Infection index (i.e., Number of S2 cells. S2 cells were pretreated with drugs for 1 hr, and then infected S2 cells with replicating Brucella/total cell number based infected with bacteria. At 72 h.p.i., cells were stained with trypan on image analysis) and MOI display a linear relationship in the blue, fixed, and the percentage of viable cells was determined range of tested MOI. Data represent the means 6 standard (Panel C). Two images, containing a total of at least 500 cells in deviations from three independent experiments. each sample, were analyzed in each experiment. C. Images of Found at: doi:10.1371/journal.ppat.1000110.s006 (10.28 MB TIF) infected S2 cells coincubated with the indicated drugs at 72 h.p.i., Figure S4 GFP expression has no effect on bacterial entry and (1) BAF (100 nM), (2) BFA, (3) CD, (4) WM, (5) MR, and (6) No replication. The entry (1.5 h.p.i) and replication (72 h.p.i.) of drug control. Data represent the means 6 standard deviations Brucella melitensis strains 16M and 16M-GFP in Drosophila S2 and from three independent experiments. The images were taken from J774.A1 murine macrophages at 29uC and 37uC, respectively, a representative experiment. were compared using gentamicin protection assays. The number Found at: doi:10.1371/journal.ppat.1000110.s009 (0.49 MB PDF) of 16M CFUs of entry and replication in Drosophila S2 cells Figure S7 Effect of selected drugs on Brucella entry and 5 6 [(4.8560.46) 610 /well and (8.8961.23) 610 /well, respectively] replication. A. Drosophila S2 cells were conincubated with assorted 5 and in J774.A1 cells [(3.6860.29) 610 /well and (1.7760.21) drugs at the indicated concentrations 1 hr before and during 7 610 /well, respectively] were normalized as 100%. No significant infection with B. abortus S2308 at an MOI of 100. The infected differences in entry or replication in S2 or J774.A1 cells were cells were lysed after 1.5 hr (entry) or 72 hr (replication) of observed in the two strains. Data represent the means 6 standard incubation at 29uCinDrosophila-SFM supplemented with 40 mg/ deviations from three independent experiments. ml gentamicin and the indicated concentrations of drugs. ** and Found at: doi:10.1371/journal.ppat.1000110.s007 (0.30 MB TIF) *** indicates significance at P,0.01 and at P,0.001, respectively. Figure S5 Brucella melitensis intracellular trafficking in Drosophila BAF, BFA, MR, and WM indicate treatment of S2 cells with S2 cells. A. 1. S2 cells expressing a GFP-tagged variant of the baliformycin A1, brefeldin A, myriocin and wortmannin at the Drosophila ER maker mSpitz (mSpitz-GFP). 2. B. melitensis (16M) indicated concentrations, respectively. B. Relative CFUs (% of infection of S2 cells (24 h.p.i). 3. A tight association between B. control) for Brucella (S2308) infection of BAF treated S2 cells at melitensis cells and host cell ER membranes (arrow) in the main and 72 h.p.i. Brucella infected cells were treated with BAF at 2 h.p.i.. C. inset panels is observed in the merged image. 4. Immunofluores- Pretreatment of S2 cells with the indicated concentration of WM cence localization of COPII in S2 cells using Sec23 polyclonal has no effect on internalized Brucella replication efficiency. Data antibodies. 5. 16M-GFP localization in S2 cells at 12 h.p.i. (Green, represent the means 6 standard deviations from at least three Panel A). 6. A merged image showing COPII proteins and B. independent experiments. melitensis (16M-GFP) localization. 7. mSpitz-GFP localization in S2 Found at: doi:10.1371/journal.ppat.1000110.s010 (0.75 MB PDF) cells. 8. Fixed and killed B. melitensis (16M) in S2 cells at 24 h.p.i. 9. Figure S8 RNAi-mediated knockdown of Drosophila S2 cell gene Merged panels 7 and 8. Markers used in the panels indicated in expression alters Brucella abortus entry and replication. Depletion of parenthesis are shown on the left. B. Brucella trafficking in S2 cells. known Brucella host factors disrupts BA114 (S2308virB10::Tn5, A) Double label immunofluorescence microscopy of: 1. Brucella (16M, and CA180 (S2308manBA::Tn5, C) entry but not replication (B red) (at 6 h.p.i.) and the late endosome marker mannose 6- and D). Data represent the means 6 standard deviations from phosphate receptor (M6PR, green); 2. Brucella-GFP (at 24 h.p.i) three independent experiments. *** represents significant at and the lysosomal marker cathepsin D (red); 3. Brucella (red, at P,0.001 compared with no RNAi control. 24 h.p.i) and the GFP-tagged Golgi marker D-GRASP (green). Found at: doi:10.1371/journal.ppat.1000110.s011 (1.35 MB TIF) Scale bar: 5 mm. Found at: doi:10.1371/journal.ppat.1000110.s008 (8.23 MB TIF) Acknowledgments Figure S6 Effects of selected drugs on Brucella or Drosophila S2 host cell growth. No: No drug control, 1% ddH2O (V/V); The authors gratefully thank Drs. L.C. Cantley (Harvard Medical School, Boston, MA), G. Marque´s (the University of Alabama, Birmingham), W.J. DMSO: dimethyl sulfoxide, 1% (V/V); MT: Methanol, 1% (V/ Brown (Cornell University, Ithaca, NY), V. Kondylis (Universitair Medisch V); BAF: balifomycin A1, 200 nM; BFA: brefeldin A, 2.5 mg/ml; Centrum Utrecht, The Netherlands), and J. Weeks (Texas A&M CD: cytochalasin D, 2.5 mg/ml; WM: wortmannin, 100 nM; MR: University, College Station, TX), J.C. Bell (Ottawa Health Research myriocin, 10 mM. A. The indicated drugs were added into fresh Institute, Ottawa, Ontario, Canada), and R. J. Kaufman (Howard Hughes TSB and Brucella (S2308) was incubated in this medium for the medical Institute at University of Michigan Medical School, Ann Arbor, indicated periods of time. The effects of the drugs on Brucella MI) for reagents. growth were determined using gentamicin protection assays. 1 hr (white bars) and 72 hr (gray bars) represents the relative amount of Author Contributions Brucella in the drug-treated media (CFU/ml) compared with the Conceived and designed the experiments: QMQ TAF PdF. Performed the untreated control at 1 and 72 hrs post coincubation, respectively. experiments: QMQ JP VA. Analyzed the data: QMQ PdF. Contributed Replication efficiency (black bars) indicates relative Brucella reagents/materials/analysis tools: QMQ BDS TAF PdF. Wrote the paper: replication efficiency in drug-treated medium and no drug treated QMQ PdF.

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PLoS Pathogens | www.plospathogens.org 16 July 2008 | Volume 4 | Issue 7 | e1000110 Table S1. Brucella strains used in this study

Species/strain Characteristics of the strains Reference Brucella abortus S2308 Wild type strain [1, 2] S19 Vaccine strain [2] RB51 Vaccine strain [2] BA114 S2308virB10::Tn5 [3] CA180 S2308manB::Tn5 [1, 4] S2308∆manBA manBA::Km, derived from S2308 [2] S2308∆virB2 ΔvirB2, derived from S2308 [2]

B. melitensis 16M Wild type strain [2] 16M-GFP GFP expressing strain, 16M-pBBR1MCS-6Y Weeks et al. a 16MΔmanBA ∆manBA, derived from 16M [2] 16MΔvirB2 ∆virB2, derived from 16M [2] 102B2 (BMEI1364) 16MmucR::Himar1, derived from 16M [5] 146D5 (BMEI1178) 16MmerR::Himar1, derived from 16M [5] a Unpublished data

1. Pei J, and Ficht TA (2004) Brucella abortus rough mutants are cytopathic for

macrophages in culture. Infect Immun 72: 440-450.

2. Kahl-McDonagh MM, Ficht TA (2006) Evaluation of protection afforded by Brucella

abortus and Brucella melitensis unmarked deletion mutants exhibiting different rates

of clearance in BALB/c mice. Infect Immun 74:4048-4057.

3. Hong PC, Tsolis RM, and Ficht TA (2000) Identification of Genes Required for

Chronic Persistence of Brucella abortus in Mice. Infect Immun 68: 4102–4107.

4. Hong CA, Adams LG, and Ficht TA (1998) Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival. Infect Immun 66:1008-1016. 5. Wu Q, Pei J, Turse C, Ficht TA (2006) Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival. BMC Microbiol 6: 102. Table S2. Comparison of Drosophila S2 and mammalian cells treated with drugs that inhibit Brucella abortus (S2308) entry and replication

CFU (% of control) a Concentration S2 cells Mammalian cells Drug Name Target/Process (μM) Entry Replication Entry Reference Balifomycin A1 Vacuolar H+-ATPase 0.1 29.1±5.5***b 30.6±2.5*** 45.7 c [1, this study] Brefeldin A Secretion endocytosis 8.9 86.3±28.6 87.0±21.6 NA d [2, this study] Cytochalasin D Actin polymerization 5.0 1.8±0.7*** ND e 1.2±0.4 [3, this study] Myriocin Sphingolipid biosynthesis 10.0 51.2±9.2*** 24.0±2.8*** ND This study Wortmannin PI3-kinases 0.1 31.6±5.9*** 34.4±6.4*** 10.0 f [3, this study] a Colony forming units (CFU) of the untreated control [(3.1±0.4)×105/well, (1.6±0.1)×107/well for entry and replication, respectively] was normalized as 100%. b Data represent the means ± standard deviations from three independent experiments. *** represents significant at P<0.001 compared with the untreated control. c Result from murine macrophage J774.A1 cells infected with B. suis at 4 h.p.i. d Mice bone marrow derived macrophages (BMDMs) infected with wild type B. abortus and the treatment of brefeldin A has no effect on Brucella entry, replication and intracellular trafficking, and no accurate statistic data shown in the reference. e Not detected. f HeLa cells pretreated with 50 nM Wortmannin and infection with B. abortus.

1. Porte F, Liautard JP, Köhler S. (1999) Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages. Infect Immun 67: 4041-4047. 2. Celli J, Salcedo SP, and Gorvel JP (2005) Brucella coopts the small GTPase Sar1 for intracellular replication. Proc Natl Acad Sci U S A 102: 1673-1678. 3. Guzmán-Verri C, Chaves-Olarte E, von Eichel-Streiber C, Lopez-Goni I, Thelestam M, et al. (2001) GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes. J Biol Chem 276: 44435-44443. Table S3 Effect of ER-associated Cellular Factors on Brucella melitensis Replication Pathogens B. melitensis Mfc Lme Lpf Ccg Cth Hom Repeat Symbol CG # Description/Functiona RIF±SDb [1, 2]d [2, 3] [4] [5] [6] Mouse Host factors identified as hits in RNAi Screen Lipid Biosynthesis and Methabolism Fad2 CG7923 Fatty acid metabolism, stearoyl-coa 9-desaturase activity 8.72±1.54 3 down down Ni NN Scd3 HLH106 CG8522 Fatty acid biosynthesis 15.67±6.78 2 down down N down N Srebf1 Hmgcr CG10367 HMG Coenzyme A reductase 12.83±0.98 2 down down N NNHmgcr CG8630 CG8630 Fatty acid biosynthesis, metabolism 60.27±1.30 2NNNNN ? Vesicle Trafficking boca CG30498 Vesicle-mediated transport, to membrane 5.70±1.32 5NNN N down Mesdc2 Ero1L CG1333 Vesicle-mediated transport 48.13±17.95 2NNN N down Ero1l KdelR CG5183 Retrograde vesicle-mediated transport, Golgi to ER 62.75±3.24 2NNN NN Kdelr1 2 N down Rab1 CG3320 GTPase activity, endocytosis, vesicle-mediated transport 2.00±0.60 down down N mKIAA3012 sec23 CG1250 COPII vesicle coat 1.75±0.05 2 down down N NNSec23b sec24 CG1472 COPII vesicle coat 26.42±9.38 4 NNNNNSec24b Syx5 CG4214 T-SNARE 1.98±0.15 3 down down N NN Stx5a TER94 CG2331 ATPase, microtubule organization and biogenesis, exocytosis 16.19±6.58 4 down down down N down Vcp CHOp24 CG3564 COPI vesicle coatomer protein 48.56±11.33 2 NNNNNTmed2 CG1418 CG1418 Rab GTPase binding, vesicle-mediated transport 63.03±24.28 4 NNNNNRabac1 CG7011 CG7011 ER to Golgi transport vesicle 54.13±2.74 2 NNNNN Ergic3 CG8860 CG8860 Vesicle-mediated transport, SRP-dependent protein translation 20.19±6.05 3 NNNNNSec61g CG1924 CG1924 Exocytosis, intracellular protein transport 10.40±2.90 2 NNNNN ? TRAM CG11642 SRP-dependent cotranslational protein targeting to membrane 138.49±34.11 2 NNNNNTram1l1 Protein Biosynthesis and Methabolism Asph CG8421 Peptide-aspartate beta-dioxygenase activity 39.84±6.66 2 NNNNN Asph p115 CG1422 Protein transporter activity 31.35±6.45 3 NNNNN Vdp SCAP CG8356 Protein processing, 28.90±6.03 2 NNNNN Scap torp4a CG3024 Protein folding 63.16±11.90 2 NNNNN Tor1b Trp1 CG4758 Protein transporter activity 42.69±12.43 2 NNNNN Tloc1 ttv CG10117 Glycosaminoglycan biosynthesis 31.97±3.75 2 NNNNdown Ext1 Sec61γ CG14214 Protein translocase activity 141.57±39.83 2 NNNNNSec61g CG6370 CG6370 Protein modification 162.06±31.63 2 NNNNN Rpn2 CG8412 CG8412 Protein amino acid glycosylation 211.77±13.63 2 NNNNN Alg12 CG4542 CG4542 Protein amino acid N-linked glycosylation, oligosaccharyl transfer 43.33±7.21 2 NNNNN Alg8 Miscellaneous Ire-1 CG4583 mRNA processing, protein amino acid phosphorylation 55.44±10.37 5 NNNNN Ern1 Lam CG6944 Cytoskeleton organization and biogenesis 21.93±5.89 2 N down N NNLamnb1 ninaA CG3966 Cytoplasmic membrane-bound vesicle 142.76±34.64 2 NNNNN ? Ca-P60A CG3725 Calcium-transporting ATPase activity 34.27±4.83 2 NNNNNAtp2a1 Cnx99A CG11958 Calcium ion binding, unfolded protein binding, exocytosis 137.76±5.19 2 NNNNN Canx Cpr CG11567 Cyt-P450 reductase, NADPH-hemoprotein reductase activity 20.02±2.71 2 NNNNN Por Crc CG9429 Calcium ion binding, unfolded protein binding, exocytosis 39.26±10.78 5 NNNNN Calr ecd CG5714 Ecdysone biosynthesis 41.34±12.63 2 NNNNN Ecd GlcT-1 CG6437 Ceramide glucosyltransferase activity, regulation of apoptosis 15.28±1.99 2 NNNNN Ugcg Gp93 CG5520 DNA metabolism, defense response 28.89±7.89 2 NNNNNHsp90b1 Itp-r83A CG1063 Inositol-1,4,5-triphosphate receptor activity 45.61±1.26 3 NNNNN Itpr2 Khc CG7765 Cytoplasmic transport, 62.21±6.77 2 NNNNN Kif5c l(2)gl CG2671 Myosin binding 159.46±17.65 2 NNNNdown Llgl1 O-fut1 CG12366 Fucosyltransferase activity 52.62±0.37 2 NNNNN Pofut1 por CG6205 Acyltransferase activity, protein amino acid lipidation 145.78±10.92 2 NNNNN Porcn rdgA CG10966 Phosphoinositide biosynthesis, actin filament organization 55.74±18.21 2 NNNNN Dgki Rya-r44F CG10844 Calcium ion homeostasis 144.72±10.19 2 NNNNN Ryr2 CG6414 CG6414 Carboxylesterase activity 25.10±14.82 2 NNNNN Ces7 CG1193 CG1193 ATPase activity, microtubule associated complex 49.11±0.52 2 NNNNN ? CG9376 CG9376 GPI anchor biosynthesis 34.14±14.05 2 NNNNN Pigf CG11190 CG11190 GPI-anchor transamidase complex 149.60±10.25 2 N N N down N Pigt CG12010 CG12010 ATPase activity 162.24±23.78 2 NNNNN ? TrfPj CG18780 Srb-mediator complex 43.53±0.11 4 NNNNN Trfp CG10960j CG10960 Carbohydrate metabolism 41.54±3.45 5 N down N N N Slc2a8 Control Host Factors Cdc 42 CG12530 Ras small gtpase, Rho type 60.78±4.28 3 down down N down N Cdc42 Pi3K21B CG2699 Phosphoinositide 3-kinase complex, class IA 54.66±11.54 3 NNNNN Pik3r3 Pi3K92B CG4141 Phosphoinositide 3-kinase complex, class IA 57.68±3.51 3 NNNNNPik3cb Pi3K59F CG5373 Phosphoinositide 3-kinase complex, class III 48.4±15.5 3 NNNNNPik3c3 Pi3k68D CG11621 Phosphoinositide 3-kinase complex, class II 43.09±8.43 3 NNNNNPik3c2b Rho1 CG8416 Ras small GTPase, Rho type 25.38±6.82 3 NupN NN ArhA Rac 1 CG2248 Ras small GTPase, Rho type 53.7±13.0 3 NNNN down Rac2 Rac 2 CG8556 Ras small GTPase, Rho type 49.52±8.98 3 down N N NN ? Sar 1 CG7073 COPII vesicle coat 47.09±1.36 3 down down down NN Sara2

RNAi-mediatated depletion of the ER-associated host factors did not affect Brucella melitensis infection of S Lipid Biosynthesis and Methabolism CG5265 CG5265 Carnitine O-acetyltransferase activity, fatty acid metabolism 103.56±6.64 2NN N NN ?i CG9743 CG9743 Stearoyl-coa 9-desaturase activity, fatty acid metabolism 128.99±1.76 2NNN NN ? CG9747 CG9747 Stearoyl-coa 9-desaturase activity, fatty acid metabolism 109.05±8.45 2NNN NN ? CG31915 CG31915 Lipopolysaccharide biosynthesis 88.09±1.85 2NNN NNGlt25d1 Vesicle trafficking eyc CG4556 Vesicle-mediated transport, 105.67±21.60 2NN N NNProm2 sec31 CG8266 Exocytosis, ER to Golgi transport vesicle 119.83±26.06 2 NNupNNSec31a Snap CG6625 Cytokinesis, ER to Golgi vesicle-mediated transport 126.19±17.54 2 down down N down N Napa SsRβ CG5474 Receptor activity, vesicle-mediated transport 96.07±14.69 2 NNNNN Ssr2 αCOP CG7961 COPI vesicle coatomer protein less cellsk 2 down down N down N Copa ®COP CG6223 COPI vesicle coatomer protein less cells 2 down down N down N Copb1 ©COP CG1528 COPI vesicle coatomer protein less cells 2 down down N down down Copg δCOP CG14813 COPI vesicle coatomer protein less cells 2 down down N NN Arcn1 ⎛COP CG3948 COPI vesicle coatomer protein less cells 2 down down N down N Coppz1 ∑COP CG9543 COPI vesicle coatomer protein 120.61±3.21 2 NNNNN Cope Protein Biosynthesis and Methabolism Alg10 CG32076 Protein amino acid N-linked glycosylation 95.79±2.23 2 NNNNNAlg10b ari-1 CG5659 Ubiquitin-protein ligase activity 83.31±3.40 2 NNNNN Arih1 ERp60 CG8983 Protein modification, protein disulfide oxidoreductase activity 72.23±7.91 2 NNNNN Pdia3 Ost48 CG9022 Protein amino acid N-linked glycosylation 114.31±14.74 2 NNNNN Ddost Pdi CG6988 Protein folding, protein disulfide oxidoreductase activity 104.61±13.77 2 NNNNN P4hb Sras CG4852 Proteolysis 78.16±22.29 2 NNNNN Rce1 Ugt CG6850 Protein amino acid glycosylation 70.53±0.52 2 NNNNNUgcgl1 p47 CG1837 Protein modification 96.01±8.83 2 NNNNNTxndc5 wbl CG7225 Protein processing, Exocytosis, Toll signaling pathway 86.99±9.65 2 NNNNN Erp29 CG10908 CG10908 ER-associated protein catabolism 115.23±5.71 2 NNNNN Derl1 CG5091 CG5091 Protein amino acid N-linked glycosylation 88.95±5.11 2 NNNNN Alg6 CG9911 CG9911 Protein disulfide isomerase, oxidoreductase activity 67.78±10.51 2 NNNNNTxndc4 CG10029 CG10029 Protein disulfide isomerase, oxidoreductase activity 78.10±21.46 2 NNNNN ? CG10166 CG10166 Protein amino acid glycosylation 116.51±26.95 2 NNNNN ? Signal Transduction ATF6 CG3136 Transcription factor activity 94.69±6.77 2 NNNNN Atf6 BBF-2 CG7450 Cyclic-AMP response element binding protein A 115.19±3.93 2 NNNNNCrebl1 Dl CG3619 Signal transducer activity 66.61±15.20 2 NNNNN Dll1 PERK CG2087 Elongation factor-2 kinase activity, response to unfolded protein 110.84±11.87 2 NNNNNEif2ak3 vav CG7893 Rho protein signal transduction, Rho GTPase activator activity 106.48±23.99 2 NNNNdown Vav2 wg CG4889 Signal transducer activity, 88.29±5.00 2 NNNNN Wnt4 Miscellaneous ninaE CG4550 G-protein coupled receptor activity, membrane-bound vesicle 122.05±12.60 2 NNNNN Opn4 Adh CG3481 Alcohol dehydrogenase activity 118.09±30.55 2 NNNNN ? botv CG15110 Acetylglucosaminyltransferase activity 116.17±12.85 2 NNNNdown Extl3 Eip74EF CG6285 RNA polymerase II transcription factor activity 133.88±4.04 2 NNNNN Elf3 Ext2 CG33038 N-acetylglucosamine metabolism 128.29±0.20 2 NNNNN Ext2 fng CG10580 UDP-glycosyltransferase activity, Notch signaling pathway 110.41±4.99 2 NNNNN Rfng g CG10986 Golgi to endosome transport, eye pigment biosynthesis 91.27±21.82 2 NNNNN Ap3d1 GM130 CG11061 Golgi organization and biogenesis 101.41±30.57 2 NNNNNGolga2 Grasp65 CG7809 Protein binding 98.90±0.66 2 NNNNNGorasp2 Gtp-bp CG2522 GTPase activity 110.70±8.55 2 NNNNN Srpr Hsc70-3 CG4147 Heat shock protein cognate 3 95.20±55.13 2 down up N NN Hspa5 hts CG9325 Actin binding 71.54±8.37 2 NNNNN Add1 nmd CG5395 Intracellular protein transport, ATPase activity 91.02±12.49 2 NNNNdown Atad1 phm CG6578 Electron carrier activity 125.62±8.37 2 NNNNN 2J5 rho CG1004 Cytokinesis, receptor signaling protein activity, proteolysis 67.04±20.48 2 NNNNNRhbdl1 Spp CG11840 Signal peptide peptidase activity 122.62±11.28 2 NNNNN H13 Sulf1 CG6725 Polysaccharide metabolism 96.27±0.63 2 NNNNup Sulf2 Surf4 CG6202 Receptor signaling protein activity 105.51±22.05 2 NNNNN Surf4 CG2135 CG2135 Beta-glucuronidase activity 64.55±13.34 2 NNNNN ? CG3033 CG3033 GPI-anchor transamidase complex 130.43±32.58 2 NNNNNGpaa1 CG5776 CG5776 ATPase activity 99.85±14.39 2 NNNNNSpata5 CG6401 CG6401 GPI anchor biosynthesis 113.53±1.50 2 NNNNN Piga CG6453 CG6453 Alpha-glucosidase activity 106.79±7.61 2 NNNNNPrkcsh CG6512 CG6512 ATPase activity 104.65±0.77 2 NNNNN Afg3l2 CG13887 CG13887 Receptor binding, defense response 123.29±9.23 2 NNNNdown Bcap31 CG31152 CG31152 Unknown function 127.37±13.16 2 NNNNN Ktelc1

Qin et al., Figure S1 Qin et al., Figure S2 Qin et al., Figure S3 Qin et al., Figure S4 Qin et al., Figure S5 Qin et al., Figure S6 Qin et al., Figure S7 Qin et al., Figure S8