Down-regulation of 7SL RNA Expression and Impairment of Vesicular Protein Transport Pathways by Leishmania Infection of Macrophages*

The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display reverse transcription-PCR and cDNA microarray analyses. The genes that are down-regulated in mouse (J774G8) or human (U937) macrophages upon exposure to Leishmania include small RNA transcripts from the short interspersed element sequences. Among the short interspersed element RNAs that are down-regulated is 7SL RNA, which is the RNA component of the signal recognition particle. Because the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down-regulation of 7SL RNA may be significant in the establishment of infection by Leishmania in macrophage phagolysosomes. To evaluate whether down-regulation of 7SL RNA results in inhibition of signal recognition particle-mediated vesicular protein transport processes, we have tested and found that the targeting of proteins to the endoplasmic reticulum and plasma membrane and the secretion of proteins by macrophages are compromised in Leishmania-infected J774G8 and U937 cells. Knocking down 7SL RNA using small interfering RNA mimicked the effect of exposure of macrophages to Leishmania. The overexpression of 7SL RNA in J774G8 or U937 cells made these cells resistant to Leishmania infection, suggesting the possible biological significance of down-regulation of 7SL RNA synthesis in the establishment of infection by Leishmania. We conclude that Leishmania down-regulates 7SL RNA in macrophages to manipulate the targeting of many proteins that use the vesicular transport pathway and thus favors its successful establishment of infection in macrophages.

Pathogenic microbes often develop strategies to evade host immune responses, learn to adapt to the host environment, and/or manipulate the host system to make it more hospitable for the establishment of infection and propagation (1)(2)(3). Any microbe interacting with the mammalian immune system is subjected to ultimate phagocytosis by blood mononuclear phagocytes or tissue macrophages, and it is then cytolyzed to death inside macrophage phagolysosomes (2)(3)(4). The parasitic protozoan Leishmania has the uncanny ability to evade the immune reactions of macrophages and is able to establish infection inside macrophage phagolysosomes and to propagate inside tissue macrophages (1)(2)(3). To survive inside the macrophage and to escape immunity, Leishmania has developed mechanisms that deactivate macrophage immune functions, including inhibition of the respiratory burst and interleukin-12 and nitric oxide synthesis and down-regulation of major histocompatibility complex class II molecules as well as promotion of the synthesis of inhibitory cytokines such as transforming growth factor-␤ and interleukin-10 and induction of the suppressor of cytokine signaling (5,6). Cytokines and especially ␥-interferon are essential contributors to macrophage activation to promote the effective killing of parasites (5,6).
Many of the functions carried out by macrophages, including destruction of intracellular parasites such as Leishmania, are enhanced by their activation by extracellular factors. Proper targeting of the receptor molecules specific for those activating factors on the cell surface is thus very important for the optimal function of macrophages. We hypothesize that, in the early stages of infection, Leishmania manipulates the expression of cell-surface receptors of the macrophages, making the cells unable to respond to their external stimuli.
It has become increasingly apparent that, in addition to their phagocytic and immunomodulatory properties, macrophages have an extensive secretory capability that includes secretion of protein molecules such as lysozymes, lysosomal acid hydrolases, neutral proteases, lipases, arginase, protease inhibitors, phospholipase inhibitors, complement factors, blood coagulation factors, and several cytokines (7,8). Macrophage secretion involves synthesis on the rough endoplasmic reticulum (ER), 1 cotranslational glycosylation and translocation into the lumen of the ER, transport to the Golgi, and vesicular transport to the plasma membrane. The fusion of the secretory vesicle membrane and plasma membrane then occurs with the re-establishment of the membrane bilayer structure, thereby maintaining the integrity of the cell while expelling vesicle contents to the external milieu (9). The secretion of many cytokines by macrophages is of particular importance because those cytokines interact with macrophages and T-or B-cells and assist in the control and fine-tuning of the immune response. Our proposal is that Leishmania successfully establishes infection in macrophages by down-regulating the biosynthesis of 7SL RNA, which in turn results in inhibition of protein targeting into the ER lumen and plasma membrane and secretion of protein molecules by macrophages.
The signal recognition particle (SRP) is a ribonucleoprotein machine that delivers certain nascent polypeptides to specific recognition components on the cytoplasmic face of the ER membrane for translocation of secretory or membrane proteins (10,11). The RNA component of the SRP (7SL RNA) contains two elements related to the human and rodent Alu families of interspersed repetitive DNA sequences connected by a unique sequence, the S domain (11). 7SL RNA associates with six proteins in mammalian cells termed SRP72, SRP68, SRP54, SRP19, SRP14, and SRP9 (11).
All proteins destined for the secretory pathway must first be targeted to the ER. In mammalian cells, this targeting reaction primarily occurs cotranslationally via the SRP pathway. Proteins targeted to the cell membrane for either secretion or integration typically have an N-terminal signal peptide that directs them to their destinations. As the nascent polypeptide chain exits the ribosome, it is recognized and bound by the SRP. The SRP-ribosome complex then binds to the SRP receptor, which resides in the target membrane. Once bound, the paused translation machinery docks with a protein-translocating pore, or channel, and the SRP releases the signal peptide (10,11). Translation resumes with the growing peptide integrating into or passing through the membrane. The SRP pathway is essential in all organisms examined to date except the yeast Saccharomyces cerevisiae (12).
To substantiate our hypothesis that Leishmania actively manipulates macrophages in the initial phases of its establishment of infection, we studied the differential expression of genes in cultured macrophages exposed to virulent Leishmania promastigotes (the insect form of the parasite) compared with unexposed macrophages. Exposure of cultured mouse macrophages to virulent Leishmania promastigotes for 2-6 h caused significant decreases in the levels of small RNA transcripts from short interspersed element sequences in the cells (13). We report here the data from our differential display reverse transcription (DD-RT) PCR and cDNA microarray analyses showing the differential expression of specific abundant mRNAs in Leishmania-exposed macrophages. We hypothesize that the altered expression of these genes may lead to the non-apoptotic growth arrest of host cells favoring the parasitism of Leishmania. We further characterize the down-regulation of one of the short interspersed element RNAs, 7SL RNA, in Leishmaniaexposed macrophages and present evidence that this downregulation leads to inhibition of vesicular protein transport in macrophages, perhaps favoring leishmanial parasitism in the phagolysosomes of these cells. We also show that the overexpression of 7SL RNA in macrophages confers resistance to the cells against Leishmania infection, at least in vitro.

MATERIALS AND METHODS
Leishmania, Trypanosoma, and Macrophages-The promastigotes and amastigotes of Leishmania amazonensis (LV78), Leishmania major (Friedlin), and Leishmania donovani (DD8) were used in this study. The promastigotes were grown at 25°C in M199 medium with 10% heat-inactivated fetal bovine serum (13). Amastigotes were maintained in mouse tail base lesions or in infected J774G8 cultures (14). Virulent promastigotes are those that are differentiated from amastigotes, freshly isolated from infected macrophages (14). Avirulent promastigotes are cloned laboratory stock of Leishmania that has been maintained in axenic culture medium for Ͼ10 years and that has lost its infectivity in cultured macrophages or in mice (15). Trypanosoma brucei procyclics (16) were obtained from Dr. Minu Chaudhuri (Department of Microbiology, Meharry Medical College). We used the mouse macrophage cell line J774G8 and the human monocytic leukemia cell line U937 in our study. These cells were grown in tissue culture flasks in RPMI 1640 medium with 20% heat-inactivated (56°C, 30 min) fetal bovine serum at 37°C as described (13,14). U937 cells were differen-tiated into macrophages with phorbol 12-myristate 13-acetate (10 ng/ ml) for 24 h as described (17). The macrophages were incubated with the parasite cells at a macrophage/parasite ratio of 1:10 at 37°C for 2-6 h for exposure experiments (13)(14)(15)18).
DD-RT-PCR and Microarray Analyses-Comparative gene expression analyses of unexposed and virulent L. amazonensis-exposed J774G8 cells were done by DD-RT-PCR as well as mouse cDNA microarray analyses similar to those described previously (13,19,20). Total RNA was isolated from subconfluent cultured cells using TRIzol reagent (Invitrogen). For isolation of RNA from unexposed macrophages, we added an equivalent number of parasite cells after addition of TRIzol to the macrophages so that the contribution of Leishmania RNA, if any, in the subsequent microarray hybridization was countered. The RNA quality was checked by formaldehyde-agarose gel electrophoresis. DD-RT-PCR analysis was performed as described previously (13). For cDNA microarray analysis, total RNAs (40 g) from unexposed and exposed cells were labeled in reverse transcription reactions (SuperScript II kit, Invitrogen) with Cy3-labeled dCTP and Cy5-labeled dCTP (Amersham Biosciences), respectively (19,20). Four microarrays were used for this study. In every second replicate experiment, the fluorescent deoxyribonucleotides were swapped. Purified cDNA probes labeled with Cy3 and Cy5 were mixed per pair and hybridized to mouse cDNA microarray chips (Mouse Research Genetics 5K) in the Vanderbilt Microarray Shared Resource Core Facility. The slides were scanned with a GenePix 4000A microarray scanner (Axon Instruments, Inc., Foster City, CA), and the images were analyzed using GenePix Pro 3 software. A uniform scale factor was applied to normalized signal intensities between Cy5 and Cy3. Flagged spots and spots with an average intensity below 2.5-fold above the background were not retained for further analysis. The log 2 (Cy5/Cy3) ratio of the other spots was calculated for each slide. To compare the results from the different samples, data from each slide were normalized in log space to have a mean of 0 and an S.D. of 1 by using the Cluster program (21). Genes with significant changes in mRNA levels were identified using the SAM (significant analysis of microarrays) procedure (22), a validated statistical technique for identifying differentially expressed genes across high density microarrays. This procedure provides a list of "significant" genes and an estimate of the false discovery rate, which represents the percentage of genes that could be identified by chance (22).
Primer Extension and Nuclear Run-on Analyses-The levels of 7SL RNA in the control and parasite-exposed macrophages were determined by primer extension analysis using avian myeloblastosis virus reverse transcriptase (Promega Corp., Madison, WI) according to the protocol supplied by the manufacturer. ␤-Actin mRNA levels were evaluated as a normalization control. The primers used were as follows: 7SL RNA, 5Ј-ATGCCGAACTTAGTGCGG-3Ј; and ␤-actin, 5Ј-TACACTGTAGCT-GTCTTCAGACA-3Ј. The primers were end-labeled with [␥-32 P]ATP and T4 polynucleotide kinase (23). Nuclear run-on analysis was performed as described (23).
Knockdown of 7SL RNA Gene Expression-The small interfering RNAs (siRNAs) for human 7SL RNA were designed based on the nucleotide sequence (GenBank TM accession number X04248) using the software available at the Invitrogen web site (rnaidesigner.invitrogen-.com/sirna) and custom-synthesized by Invitrogen. The nucleotide sequences of the siRNA pair (SR) and its respective control (SRC) are as follows (the number indicates the location of the sequence in 7SL RNA; see Ref. 24): SR110, 5Ј-UCCGCACUAAGUUCGGCAU-3Ј/5Ј-AUGC-CGAACUUAGUGCGGA-3Ј; and SRC110, 5Ј-UCCACUAAGUU-CGGGCCAU-3Ј/5Ј-AUGGCCCGAACUUAGUGGA-3Ј. We used Trans-Messenger reagent and protocols (Qiagen Inc.) for the transfection of macrophages with the siRNAs. RNAs were purified from the siRNAtreated cells using TRIzol reagent and treated with DNase I before RT-PCR analysis (19,20).
Overexpression of 7SL RNA in Cultured Macrophages-Human 7SL cDNA sequence (GenBank TM accession number X04248) (24) was amplified from cDNA (made from RNA isolated from U937 cells) using a BglII site-anchored forward primer (5Ј-GGAGATCTGCCGGGCGCG-GTGGCGCGTGC-3Ј) and a HindIII site-anchored reverse primer (5Ј-GGAAGCTTAGAGACGGGGTCTCGCTATG-3Ј). The sequence-verified, amplified cDNA (315 bp) was digested with BglII/HindIII and cloned behind the H1 RNA promoter in plasmid pSUPER (19) at the BglII/HindIII sites to obtain plasmid pSUPER-7SL. We mutated the 7SL RNA gene in pSUPER-7SL simultaneously (G193A, G194A, and G198A) to create a nonfunctional 7SL RNA. These Gly residues are highly conserved across the genera and are essential for the function of 7SL RNA (25,26). We used the QuikChange site-directed mutagenesis reagents and protocols (Stratagene, La Jolla, CA) (27) to accomplish the mutagenesis. Mutation was verified by nucleotide sequencing. We named the mutated plasmid pSUPER-7SL*. pSUPER-7SL or pSUPER-7SL* and pcDNA3.0 were cotransfected at a ratio of 10:1 into cultured macrophages using Lipofectamine 2000 (Invitrogen) as described (27). Control cells were transfected only with pcDNA3.0, which contains the G418 resistance gene. Recombinant cells were selected with G418 (200 g/ml). To overexpress 7SL RNA in cells that express either enhanced cyan fluorescent protein (ECFP) or secreted alkaline phosphatase and that are resistant to G418, we cotransfected them with pSUPER-7SL or pSUPER-7SL* and pTK-Hyg (a selection vector that confers hygromycin resistance; Clontech) and selected for cells that were resistant to both G418 (200 g/ml) and hygromycin (200 g/ml). Control cells were transfected only with pTK-Hyg. The overexpression of 7SL RNA in the transfected cells was evaluated by RT-PCR as described above.
Evaluation of Protein Targeting to the ER-To evaluate the effect of Leishmania-or siRNA-induced knockdown of 7SL RNA in macrophages on the targeting of proteins to the ER, we employed a commercially available plasmid construct (Clontech) designed to test this cellular process. The Living Color ER targeting vector pECFP-ER (Clontech) encodes a fusion protein consisting of ECFP, the ER targeting sequence of calreticulin cloned at the 5Ј-end, and the sequence encoding the ER retrieval sequence (KDEL) cloned at the 3Ј-end. ECFP-ER is a soluble protein that localizes in the lumen of the ER in transfected cells. We stably transfected macrophages with the pECFP-ER plasmid. Cultured J774G8 or undifferentiated U937 cells (in 6-well tissue culture plates) were transfected with the pECFP-ER plasmid (1 g) using Lipofectamine 2000 (27). Stable transfectants were obtained after selection with G418 (200 g/ml). U937 cells were differentiated before exposure to virulent Leishmania promastigotes as described above. Virulent Leishmania-exposed or unexposed cells were washed twice with phosphate-buffered saline and fixed with 4% paraformaldehyde. Cells were mounted on slides with ProLong mounting medium (Molecular Probes, Inc.) and visualized using an Olympus BX60 epifluorescence microscope with a ϫ100 objective lens. Images were recorded with an Optronics CCD camera (DEI-750). To evaluate whether the disappearance of the ECFP-ER protein in the Leishmania-exposed cells is due to proteasomemediated degradation, we used the potent, membrane-permeable proteasome inhibitor MG132 (Sigma) (28). MG132 was reconstituted in Me 2 SO to obtain a 10 M stock solution. Macrophages were treated with 1 M MG132 for 24 h.
Evaluation of Protein Targeting to the Plasma Membrane-To evaluate the effect of Leishmania-or siRNA-induced knockdown of 7SL RNA in macrophages on the concentration of a membrane receptor, we assayed the level of macrophage scavenger receptor AII molecules on the surface of these cells. Scavenger receptor AII is highly abundant on the surface of macrophages (29). We used maleylated bovine serum albumin (MBSA), an artificial ligand for this receptor (29), to probe this molecule. BSA was maleylated (ϳ55 maleyl groups/molecule) using maleic anhydride (29). MBSA was radiolabeled with 125 I using the ICl-catalyzed reaction (29). Binding of 125 I-MBSA to macrophages was measured as follows. After an overnight culture, the medium was removed from the 24-well dishes, and cells were washed twice with 1 ml of phosphate-buffered saline and incubated for 3 h at 4°C with 0 -50 g of protein/ml of 125 I-MBSA in 125 l of RPMI 1640 medium containing 4% fatty acid-free BSA and 25 mM Hepes (pH 7.4), in a total volume of 250 l (total binding). Nonspecific binding was determined by the addition of 1 mg of unlabeled MBSA/ml of incubation mixture. At the end of the incubation, the cell monolayer was washed twice with 1 ml of phosphate-buffered saline containing 0.2% BSA and then twice with 1 ml of phosphate-buffered saline. The cells were solubilized in 1.5 ml of 1 M NaOH and assayed for protein content, and the radioactivity was counted (29). The specific binding was calculated by subtracting the nonspecific binding of 125 I-MBSA from the total binding. The curves generated by the specific binding data were transformed into plots of the ratio of cell-bound to free 125 I-MBSA versus cell-bound 125 I-MBSA according to the method of Scatchard (see Ref. 30). The ordinate of the Scatchard plots (bound/free) represents the amount of specifically bound ligand (fmol of MBSA/mg of cellular protein) divided by the concentration of unbound ligand in the reaction mixture (fmol of MBSA/ ml). The dissociation constant (K d ) was calculated from the slopes, and the maximal binding capacity (B max ) was obtained from the x axis intercept (30).
Evaluation of the Rate of Protein Secretion by Macrophages-To evaluate the effect of 7SL RNA knockdown on the secretion of proteins by macrophages, we stably transfected cells with the pSEAP2-Control plasmid (Clontech) and evaluated the level of alkaline phosphatase in the culture medium. pSEAP2-Control is a positive control vector expressing secreted alkaline phosphatase under the control of the SV40 early promoter and the SV40 enhancer. pSEAP2-Control and pcDNA3.0 were cotransfected at a ratio of 10:1 into cultured macrophages using Lipofectamine 2000 as recommended by the manufacturer. Control cells were transfected only with pcDNA3.0, which contains the neomycin resistance gene. Transfected cells were selected with G418 (200 g/ml). Secreted alkaline phosphatase activity in the growth medium and cell extracts was quantitated by the Great Escape chemiluminescent assay in a Turner Design 20/20 luminometer.
Evaluation of Leishmania Infectivity of Macrophages-We tested both J774G8 and U937 cells for this purpose. U937 cells were differentiated with phorbol 12-myristate 13-acetate in antibiotic-free medium before infection. A monolayer of macrophages was incubated with or without parasite cells in antibiotic-free growth medium at a macrophage/parasite ratio of 1:10 continuously for 5 days. 100 macrophages were examined microscopically for the number of infected macrophages and the total number of amastigotes (14,18).
Statistical Analysis-All experimental data are expressed as the means Ϯ S.E. A one-way analysis of variance, a two-way repeated measure analysis of variance, and Student's t test were used to determine the significance of the difference (31).

Specific Alterations in the Transcriptome Are Induced in Macrophages by Their Interactions with Virulent Leishmania
Promastigotes-Parasites often manipulate the biology of their host cells to make the host environment hospitable for their growth and development. Many researchers have documented this over the past years for the interactions between Leishmania and its host macrophage. We employed DD-RT-PCR and cDNA microarray techniques to evaluate whether, at the early stages of exposure to host cells, Leishmania induces differential gene expression in the mouse macrophage cell line J774G8. Our data suggest that indeed the exposure of macrophages to Leishmania induces the up-and down-regulation of many genes (Table I). These inductions are reproducible and specific, as these changes were not revealed when J774G8 cells were exposed to avirulent promastigotes of L. amazonensis or to T. brucei procyclics (data not shown). We positively identified and validated 22 differentially regulated genes in virulent L. amazonensis-exposed J774G8 cells by independent RNA evaluation techniques such as RNase protection assays and realtime and end-point RT-PCRs (Table I). The majority of these genes were detected by both DD-RT-PCR and cDNA microarray analyses ( Table I). Some of them were detected either by cDNA microarray analysis or by DD-RT-PCR-analysis (Table  I). Details of the evaluations of all these differentially expressed genes and the significance of their differential expression individually or as a group in leishmanial parasitism of macrophages are under study. We report here the significance of the down-regulation of 7SL RNA in macrophages by Leishmania exposure.
The genes that were significantly down-regulated in Leishmania-exposed J774G8 cells include DNA primase p49 (catalytic subunit), replication protein A p14, proliferating cell nuclear antigen, p160 c-Myb-binding protein, RAD50, cyclin-dependent kinase regulatory protein-1, p70 S6 kinase, cyclin-dependent kinase-2, and a protein of the replication origin recognition complex (Table I). Upregulated genes include histone H1.1 and protein phosphatase 1A. Apoptotic marker genes that were down-regulated include Pdcd8 (programmed cell 9eath 9; apoptosis-inducing factor) and mNapor (mouse neuroblastoma apoptosis-related RNA-binding protein; an apoptosis-inducing ELAV-type RNA-binding protein) ( Table I). The profiling may indicate non-apoptotic growth arrest of the Leishmania-exposed J774G8 cells at the non-dividing stage.
Significant Inhibition of the Expression of 7SL RNA in Leishmania-exposed Macrophages-An important observation we have made during our previous differential gene expression analysis is the down-regulation of the short interspersed element RNAs induced in J774G8 cells by short exposure (2-6 h) to Leishmania (13). One of the short interspersed element RNAs in mammals is 7SL RNA, which is the RNA component of the SRP (10,11). The nucleotide sequence of 7SL RNA is highly conserved between mouse and human. Thus, we were able to use the same primers for primer extension and RT-PCR analyses of 7SL RNA in both J774G8 and U937 cells. We found that 7SL RNA was down-regulated in mouse as well as human macrophages during exposure to virulent Leishmania promastigotes (Fig. 1A). U937 cells were differentiated to macrophages before exposure to virulent L. amazonensis promastigotes. The down-regulation of 7SL RNA in J774G8 cells by virulent L. amazonensis was at the transcriptional level, as revealed by nuclear run-on analysis (Fig.  1B). We have made similar observations with differentiated U937 cells and other virulent Leishmania promastigotes (data not shown). RT-PCR analysis with the RNAs isolated from exposed and unexposed J774G8 cells showed knockdown of the 7SL transcript in virulent L. amazonensis-, L. major-, and L. donovani-exposed cells, but not in avirulent L. amazonensis-exposed cells (Fig. 1C). Procyclics of T. brucei and avirulent stock of L. donovani promastigotes, maintained in the laboratory for Ͼ10 years in axenic culture medium, failed to knockdown the 7SL RNA levels in J774G8 cells (data not shown). Similar observations were made with differentiated U937 cells and virulent or avirulent Leishmania promastigotes (data not shown). We evaluated ␤-actin mRNA levels to use as a normalization control in all these experiments.
7SL RNA Is Knocked Down or Overexpressed in Macrophages in Attempts to Mimic or Alleviate, Respectively, the Action of Leishmania-To mimic the action of Leishmania in knocking down the 7SL RNA levels, we treated macrophages (J774G8 and differentiated U937 cells) with siRNA specific to 7SL RNA. Compared with the cells treated with control siRNA containing several mismatches, the siRNA specific to 7SL RNA was able to ablate the expression of this RNA in J774G8 cells ( Fig. 2A). Similar results were obtained with differentiated U937 cells (data not shown). To evaluate whether the consequences of leishmanial knockdown of 7SL RNA may be alleviated by the overexpression of 7SL RNA in macrophages, we developed stable J774G8 and undifferentiated U937 cell lines in which 7SL RNA was overexpressed from the H1 RNA promoter. The RT-PCR data indicated that J774G8 cells that were stably cotransfected with pSUPER-7SL and pcDNA3.0 had 2-3-fold higher 7SL RNA levels than the cells that were not transfected or those that were transfected with pcDNA3.0 alone (Fig. 2B). We obtained similar results with undifferentiated U937 cells (data not shown). To overexpress a 7SL RNA that is not functional, we made three point mutations in the 7SL RNA gene in pSU-PER-7SL and stably transfected the macrophage cell lines with this mutated plasmid along with pcDNA3.0. RT-PCR analysis suggested that mutated 7SL RNA was expressed in the stably transfected cells in an amount comparable with that in the cells transfected with the wild-type construct (Fig. 2B). Exposure of the 7SL siRNA-treated J774G8 cells to virulent L. amazonensis promastigotes for 2 h further reduced the levels of 7SL RNA (Fig. 2C). On the other hand, exposure of macrophages overexpressing 7SL RNA from the H1 RNA promoter to virulent L. amazonensis promastigotes for 2 h decreased only endogenous 7SL RNA synthesis, and the 7SL RNA levels remained at least similar to those in the control untransfected cells (Fig. 2D).
Leishmania-induced Down-regulation of 7SL RNA Impairs the Targeting of Proteins to the ER in Macrophages-To under-  1. Evaluation of the levels of 7SL RNA in unexposed and Leishmania-exposed macrophages. A, primer extension analysis of 7SL RNA in J774G8 and differentiated U937 cells that were either unexposed or exposed to virulent L. amazonensis promastigotes for 2 h before RNA isolation. ␤-Actin was used as a loading control. B, nuclear run-on analysis of 7SL RNA in J774G8 cells that were either unexposed or exposed to virulent L. amazonensis promastigotes, showing apparent transcriptional regulation of the 7SL RNA gene in Leishmania-exposed macrophages. ␤-Actin was used as a control. C, RT-PCR analysis of the levels of 7SL RNA in J774G8 cells that were unexposed or exposed for 2 h to virulent L. amazonensis, L. major, or L. donovani promastigotes or to avirulent L. amazonensis (La) promastigotes. ␤-Actin was used as a normalization control. stand the consequences of the down-regulation of 7SL RNA in J774G8 cells by Leishmania, we tested the targeting of proteins to the ER. The pECFP-ER plasmid encodes a fusion protein consisting of ECFP containing the ER targeting sequence of calreticulin. J774G8 cells permanently expressing this protein were developed. These cells were exposed to virulent L. amazonensis promastigotes for 4 h. The expression of ECFP in the ER was monitored by fluorescence microscopy. Leishmania exposure appeared to inhibit (by 50 -80%) the ER targeting of ECFP to the ER (Fig. 3, aЈ and bЈ). Knockdown of 7SL RNA with a specific siRNA in the ECFP-ER-expressing cells similarly inhibited the expression of ECFP fluorescence (Fig. 3cЈ). On the other hand, the overexpression of 7SL RNA in these cells alleviated the effect of Leishmania exposure (Fig. 3dЈ). The alleviating effect of overexpressed 7SL RNA depends upon the functionality of this RNA, as the overexpression of mutated 7SL RNA instead failed to alleviate the effect of Leishmania exposure on the targeting of ECFP to the ER (data not shown). The disappearance of ECFP in the Leishmania-exposed cells is probably due to the proteasomal degradation of the untargeted protein, as pretreatment of the ECFP-ER-expressing J774G8 cells with the proteasome inhibitor MG132 (1 M) for 24 h before Leishmania exposure prevented the disappearance of the fluorescence (data not shown).

Leishmania-induced Down-regulation of 7SL RNA Decreases the Levels of Receptor Protein Molecules on the Macrophage Cell
Surface-One of the major roles of the SRP is to help in the targeting of receptor proteins to the plasma membrane. To understand the effect on the level of a receptor on the cell surface, Leishmania-exposed (4 h) and unexposed J774G8 cells were assayed for 125 I-MBSA binding to the scavenger receptors at 4°C for 5 h with different concentrations of the ligand. The levels of scavenger receptor AII were reduced by 2-3-fold on the surface of Leishmania-exposed J774G8 cells (Fig. 4A). A similar down-regulation of the scavenger receptor concentration was observed when J774G8 cells were treated with 7SL siRNA (Fig. 4B). The effect of Leishmania exposure on the lowering of scavenger receptor levels was alleviated when wild-type (but not mutated) 7SL RNA was overexpressed in J774G8 cells (Fig.  4C). T. brucei procyclics could not lower the scavenger receptor levels in J774G8 cells (Fig. 4A). Similarly, avirulent promastigotes of L. amazonensis or L. donovani could not lower the scavenger receptor levels in J774G8 cells or differentiated U937 cells (data not shown).
Leishmania-induced Knockdown of 7SL RNA Inhibits the Secretion of Proteins by Macrophages-Another consequence of the down-regulation of 7SL RNA expression should be the impairment of the secretion of protein molecules by macrophages. To evaluate the effect of Leishmania-induced reduction of 7SL RNA levels on the secretion of proteins by J774G8 cells, we stably transfected J774G8 cells with the pSEAP2-Control plasmid. These cells were exposed for 4 h to virulent L. amazonensis promastigotes, and the levels of thermostable alkaline phosphatase (coded by the plasmid) secreted in the growth medium were assayed and compared with those in the uninfected cells using a fluorescent substrate (Clontech). Alkaline phosphatase secretion was inhibited by 60 -85% upon Leishmania exposure (Fig. 5A). On the other hand, exposure of the transfected cells to T. brucei did result in any significant change in alkaline phosphatase secretion (Fig. 5A). Inhibition of alkaline phosphatase secretion occurred only when the macrophages were exposed to virulent promastigotes of Leishmania, but not when the corresponding avirulent stocks were used (Fig. 5A). The degree of inhibition of alkaline phosphate secretion was dependent upon the time of exposure to Leishmania promastigotes (Fig. 5B) and on the macrophage/parasite ratio (Fig. 5C). Knockdown of 7SL RNA by siRNA also decreased alkaline phosphatase secretion, whereas mismatched control siRNA had no effect (Fig. 5D). Leishmania-induced inhibition of alkaline phosphatase secretion by J774G8 cells was alleviated in cells overexpressing wild-type 7SL RNA, but not in those expressing mutated 7SL RNA (Fig. 5E). These experiments were repeated with U937 cells, and we obtained similar results (data not shown).
siRNA-mediated Knockdown of 7SL RNA in Macrophages Transiently Helps Avirulent Leishmania Promastigotes to Establish Infection-Avirulent Leishmania promastigotes could not down-regulate the expression of 7SL RNA in macrophages.
To determine whether the macrophages will be more hospitable to infection by avirulent promastigotes if 7SL RNA expression is artificially knocked down, we exposed control siRNA-or 7SL siRNA-treated J774G8 or differentiated U937 cells to virulent or avirulent L. amazonensis promastigotes. We counted the amastigotes inside the macrophages to evaluate the number of infected cells as well as the number of amastigotes/100 macrophages. When the macrophages were treated with control siRNA, only the virulent promastigotes were able to establish infection (Fig. 6, A and B). The avirulent promastigotes apparently could establish infection in the 7SL siRNA-treated macrophages, but could not multiply compared with the virulent  (n ϭ 12). The differences in the levels of alkaline phosphatase activities between virulent and avirulent L. amazonensis-exposed cells (A), between 7SL siRNA-treated and control siRNA-treated cells (D), and between wild-type and mutated 7SL RNA-overexpressing cells (E) were statistically significant (p Ͻ 0.001). promastigotes (Fig. 6, A and B). The establishment of infection by the avirulent promastigotes was indicted by the increased number of infected macrophages in the population (Fig. 6, A  and B). On the other hand, their inability to grow in the macrophages was indicated by the lower number of amastigotes/100 macrophages (Fig. 6, A and B). These data thus indicate that transient ablation of 7SL RNA expression may be beneficial for the establishment of infection by Leishmania promastigotes, but the ability of the transformed amastigotes to multiply inside the phagolysosomes of the infected macrophages may be determined by other phenotypes of the parasite.
Macrophages Overexpressing 7SL RNA Are Resistant to Leishmania Infection-The ability to down-regulate the expression of 7SL RNA in macrophages seems to be associated with the virulent phenotype of Leishmania. If the parasite cells could not down-regulate the 7SL RNA levels, they could not establish infection in the macrophages. We tested whether macrophage cells overexpressing 7SL RNA are resistant to infection by virulent Leishmania promastigotes. Our data show that the virulent L. amazonensis or L. major promastigotes could not establish infection in either J774G8 (Fig. 7A) or differentiated U937 (Fig. 7B) cells overexpressing functional 7SL RNA, but mutated nonfunctional 7SL RNA. DISCUSSION The molecular events involved in the parasite-host interactions between Leishmania and macrophages have captivated scientists for the last half-century. The mechanism of the successful establishment of infection by Leishmania in phagolysosomes of human or rodent macrophages is far from clear. It is apparent from the studies of several scientists that Leishmania takes an active part in manipulating the cell biology of the host . The results are expressed as a percent of the macrophages that were not treated with any siRNA and that were infected by virulent L. amazonensis promastigotes. In these control cells, typically 80 -85% of the macrophages were infected, and there were 485-515 amastigotes/100 macrophages. The results are the means Ϯ S.E. (n ϭ 12). The differences in the number of macrophages infected by avirulent L. amazonensis promastigotes between those that were treated with control siRNA and those that were treated with 7SL siRNA were statistically significant (p Ͻ 0.001). macrophages to make them more hospitable to infection (32). The results from this study show that virulent Leishmania promastigotes indeed specifically altered the transcriptome profile of the macrophages at the early stages of their encounter with the host cells. The ability of the promastigotes to manipulate macrophage gene expression in such a way may determine whether they will be able to establish infection in the host cells. The reproducible changes in the transcriptome profile induced by short (2 h) exposure to virulent Leishmania promastigotes indicated non-apoptotic growth arrest of the cells at the non-dividing stage. Detailed analysis of the individual regulated genes is needed before we will be able to precisely determine their roles in the Leishmania-macrophage interactions. We do not know at present the molecule(s) in virulent Leishmania promastigotes responsible for induction of the changes in the transcriptome of the macrophages. Abundant parasite surface molecules, such as the major surface proteinase gp63 and lipophosphoglycan (33), did not individually bring about these changes. 2 It is possible that a combinatorial display of several molecules on the parasite surface and their simultaneous and/or sequential/progressive interac-tions with the macrophage surface molecules may bring about these changes.
We are intrigued by the significant decrease in the level of 7SL RNA in macrophages exposed to the virulent (but not avirulent) Leishmania promastigotes. The mechanism of this regulation remains elusive at present. We know from our nuclear run-on experiment that the regulation is at the transcriptional level. The 7SL RNA gene is transcribed by RNA polymerase III (34 -36). Our preliminary experiments suggested that the gene internal promoters of RNA polymerase III, such as that of the tRNAs, and the gene external promoters of RNA polymerase III, such as that of the H1 RNA, are not affected by Leishmania exposure. 2 The mammalian 7SL RNA gene has outside as well as inside transcriptional elements (34 -36). Because Leishmania specifically inhibits transcription of the 7SL RNA gene, it may not inhibit RNA polymerase III transcription in general. The specific molecular target in this RNA polymerase III transcription pathway, which is manipulated by exposure of macrophages to Leishmania to down-regulate the 7SL RNA gene promoter, has yet to be identified.
The SRP is a soluble ribonucleoprotein complex that was originally identified as an important intermediary in the transport of proteins into the secretory pathway in mammalian cells (37). 2 S. Misra and G. Chaudhuri, unpublished data. Shown is the effect of the overexpression of wild-type (wt) or mutated 7SL RNA in J774G8 (A) and differentiated U937 (B) cells on their infection by the virulent L. amazonensis or L. major promastigotes, respectively. J774G8 and undifferentiated U937 cells were stably transfected with the pSUPER-7SL or pSUPER-7SL* plasmid construct and maintained in culture medium. Recombinant U937 cells were differentiated with phorbol 12-myristate 13-acetate before infection with the parasite cells. The cells were not treated with G418 during their incubation with the parasite cells. We randomly counted 100 macrophages and determined the number of macrophages that were infected by the parasite (panels i) as well as the total number of amastigotes/100 cells (panels ii) 5 days post-infection. In the control non-recombinant cells, typically 80 -85% of the macrophages were infected, and there were 485-515 amastigotes/100 macrophages. The results are the means Ϯ S.E. (n ϭ 12). The differences in the number of infected macrophages and the number of amastigotes/100 macrophages between the cells overexpressing mutated and wild-type 7SL RNAs were statistically significant (p Ͻ 0.001).
During translation, the 54-kDa polypeptide subunit of SRP (SRP54) binds to hydrophobic targeting signals that are found in both the presecretory and integral membrane proteins (38). The targeting signals are generally either N-terminal signal sequences (39) or, in the case of many membrane proteins that lack discrete signal peptides, the first transmembrane segment (40). Subsequently, the SRP targets the ribosome-nascent chain complexes to the ER, where an interaction between SRP54 and a heterodimeric SRP receptor catalyzes the release of the nascent polypeptides and their insertion into a translocation channel or "translocon" (37)(38)(39)(40)(41). In the final step of the targeting cycle, the SRP dissociates from the ER membrane. In mammalian cells, the entry of the vast majority of proteins into the secretory pathway is completely dependent on the SRP targeting pathway. Partial and gradual inhibition of 7SL RNA biosynthesis will reduce the steady supply of the SRPs from the nucleus, and thus, the vesicular transport of the proteins, which is extremely important for macrophage immune functions, will be paralyzed. Conceivably, this should make the macrophages more hospitable to Leishmania than before.
We have evaluated the possible consequences of the downregulation of 7SL RNA by Leishmania exposure to macrophages. We have measured the targeting of a microsomal protein to the ER, the targeting of a receptor protein on the surface of macrophages, and the secretion of a protein from the cells. We have determined that the decrease in the efficiency of the protein secretory pathway in macrophages is indeed a consequence of the ablation of 7SL RNA expression. Knockdown of this RNA by RNA interference produced results very comparable with those obtained upon virulent Leishmania exposure. We have also shown that the overexpression of wild-type (but not mutated) 7SL RNA abrogated the negative effects of Leishmania exposure on the protein secretory pathway in macrophages. Earlier studies by others have shown that Leishmania down-regulates the expression of both major histocompatibility complex class I and II molecules on the macrophage cell surface and also leads to less production of interleukin-1 (32). The ability of the parasite to inhibit both major histocompatibility complex expression and interleukin-1 production should be beneficial to the parasite because the macrophages would thus be less capable of activating parasite-specific T-cell immunity. Our findings offer an explanation of these decreases in protein expression and/or secretion.
Our study has also revealed that the ability of Leishmania to down-regulate the expression of 7SL RNA is critical for the ability of the parasite to establish infection in macrophage phagolysosomes. Knockdown of 7SL RNA expression by siRNA in macrophages made them amenable to infection by even otherwise attenuated Leishmania promastigotes. Although the parasites were able to establish infection in 7SL RNA-ablated macrophages, they were unable to propagate therein. It is thus possible that down-regulation of 7SL RNA alone is not sufficient for Leishmania to parasitize the macrophages. On the other hand, if virulent Leishmania promastigotes were prevented from down-regulating the level of 7SL RNA in macrophages by artificial overexpression of this RNA in these cells, they could not establish infection. The macrophages that had elevated levels of 7SL RNA were apparently resistant to Leishmania infection. Overexpressed 7SL RNA must be biologically active to confer such resistance, as macrophages expressing mutated 7SL RNA were not resistant to Leishmania infection. Chemical manipulation of macrophages to overproduce 7SL RNA may thus well be a means to combat Leishmania infection of mammalian macrophages. Understanding the mechanism of the regulation of 7SL RNA biosynthesis in detail may help us achieve this goal.
The down-regulation of 7SL RNA synthesis in macrophages may not be the primary event of Leishmania-macrophage interactions, and it is not the only molecular event that happens as a result of the parasite-host encounter. However, is could be a pivotal event that translates the hostile behavior of the macrophages toward Leishmania into moderate hospitable interactions. It is also possible that several secondary and tertiary biochemical changes happen inside and outside of the infected macrophages as a direct or indirect result of the down-regulation of 7SL RNA biosynthesis, which may in turn make the macrophages more vulnerable to leishmanial parasitism.