Down-regulation of 7SL RNA expression and impairment of vesicular protein transport pathways by Leishmania infection of macrophages

Abstract

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.

Fig. 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. β-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. β-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. β-Actin was used as a normalization control.

Fig. 2. RT-PCR analysis of 7SL RNA levels in 7SL RNA-knocked down or 7SL RNA-overexpressing J774G8 cells

A, siRNA-mediated knockdown of 7SL RNA compared with cells treated with mismatched control siRNA. B, overexpression of 7SL RNA in J774G8 cells stably transfected with the pSUPER-7SL plasmid. β-Actin was used as a normalization control. C, effect of exposure to virulent L. amazonensis on 7SL siRNA-treated J774G8 cells. Mismatched siRNA was used as a negative control. The results from densitometric scanning and normalization against β-actin of several RT-PCR photographs are shown. D, effect of exposure to virulent L. amazonensis promastigotes on 7SL RNA-overexpressing J774G8 cells. Cells overexpressing mutated 7SL RNA were used as a control. The results shown in C and D are means ± S.E. (n = 12). The differences in the 7SL RNA levels between cells treated with control siRNA or 7SL siRNA (as in C) or between cells with no overexpression or with either wild-type (Wt) or mutated (as in D) 7SL RNA overexpression were statistically significant (p < 0.001).

Fig. 3. Inhibition of the targeting of proteins to the ER in J774G8 cells exposed for 4 h to virulent L. amazonensis promastigotes

Recombinant J774G8 cells stably expressing ECFP-ER were tested. a and a′, control recombinant J774G8 cells without any exposure; b and b′, effect of exposure to virulent L. amazonensis promastigotes; c and c′, effect of treatment with 7SL siRNA; d and d′, effect of exposure to virulent L. amazonensis promastigotes on J774G8 cells overexpressing 7SL RNA. a–d show the phase-contrast photographs of the macrophages, and a′–d′ show the corresponding fluoromicrographs. Stably transfected, G418/hygromycin-resistant cells were used for the experiments in d and d′.

Fig. 4. Down-regulation of the levels of scavenger receptor II in J774G8 cells exposed for 4 h to virulent L. amazonensis promastigotes

A, effect of a 4-h exposure to virulent L. amazonensis promastigotes or to T. brucei procyclics; B, effect of siRNA-induced knockdown of 7SL RNA without any exposure to the Leishmania promastigotes; C, effect of the overexpression of wild-type (Wt) or mutated (Mt) 7SL RNA, followed by a 4-h exposure to virulent L. amazonensis promastigotes. The B_max for the binding of ¹²⁵I-MBSA to the scavenger receptor on the cell surface was determined by Scatchard analysis.

Fig. 5. Inhibition of the secretion of alkaline phosphatase from J774G8 cells exposed for 4 h to virulent L. amazonensis promastigotes

A, effect of exposure to different parasite cells. Stably transfected J774G8 cells were exposed to equivalent numbers of avirulent, virulent (vLam), and heat-killed (100 °C for 10 min) virulent L. amazonensis (Lam) promastigotes or T. brucei procyclics for 4 h before alkaline phosphatase assay with the cell-free supernatant. B, effect of the length of exposure of recombinant J774G8 cells to avirulent or virulent L. amazonensis promastigotes on the secretion of alkaline phosphatase. C, effect of the number of avirulent or virulent L. amazonensis promastigotes/recombinant J774G8 cell on the secretion of alkaline phosphatase. D, effect of knockdown of 7SL RNA by siRNA on the secretion of recombinant alkaline phosphatase. E, effect of exposure of recombinant J774G8 cells overexpressing either wild-type or mutated 7SL RNA to virulent L. amazonensis promastigotes on the secretion of alkaline phosphatase. Data are expressed as a percent of the data obtained with unexposed or untreated J774G8 cells. The results are the means ± S.E. (n = 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 < 0.001).

Fig. 6. Establishment of infection by avirulent L. amazonensis promastigotes in 7SL siRNA-treated macrophages

A, infection of 7SL siRNA-treated J774G8 cells by virulent or avirulent L. amazonensis promastigotes. Panel i, effect on the number of infected cells/100 macrophages checked; panel ii, effect on the total number of amastigotes/100 macrophages (infected or uninfected). B, infection of 7SL siRNA-treated, differentiated U937 cells by virulent or avirulent L. amazonensis promastigotes. Panel i, effect on the number of infected cells/100 macrophages checked; panel ii, effect on the total number of amastigotes/100 macrophages (infected or uninfected). 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 ± S.E. (n = 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 < 0.001).

Fig. 7. Development of resistance against virulent L. amazonensis promastigotes in macrophages overexpressing functional 7SL RNA

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 ± S.E. (n = 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 < 0.001).