Fig 1.
RLip is a secreted effector harboring a conserved hydrolase motif.
(A) Comparative sequence alignment of catalytic Serine hydrolase motifs among rickettsial proteins [Pat1, Pat2, and Rickettsia Lipase (RLip, locus_tag: A1G_01170)], and other bacterial lipases VipD (Legionella pneumophila), VpdC (Legionella pneumophila), and ExoU (Pseudomonas aeruginosa). Highly conserved amino acids of the Serine hydrolase motif are highlighted in red. (B) Sequence alignment of the Serine hydrolase motif (GXSXG) and the percent sequence identity of RLip protein (R. rickettsii) with RLip molecules from other Rickettsia species are shown. (C–E) Expression kinetics of rlip (black line) and bacterial burden (red line) during R. rickettsii-infection of Vero76 (C), HMEC-1 (D), and SVEC 4-10 (E) was determined by RT-qPCR. RLip expression was normalized with respect to gltA transcription level. Bacterial burden was determined by normalized gltA transcription level with respect to host cell GAPDH transcription level as described previously [31,38]. Uninfected or R. rickettsii-infected Vero76 (F) or HMEC-1 (G) cells were lysed with 0.1% Triton X-100 treatment and separated into cytoplasmic (C) and pellet (P) fractions. Samples were immunoblotted with anti-RLip, anti-Pat1 (secreted rickettsial effector control), anti-OmpA/B (rickettsiae associated surface protein control), or anti-GAPDH Abs (host cytoplasmic protein control). Whole cell lysates (WCL) were used as expression control for all target proteins. (H) Partially purified rickettsiae were incubated with Vero76 cells for various length of time at 34°C. The harvested whole host lysates were analyzed by immunoblotting as described in the Materials and Methods section. Uninfected, R. rickettsii-infected WCL as well as partially purified rickettsiae were used as controls. Densitometry in panel H was performed using Fiji software, and RLip as well as Pat1 expression was normalized with respect to levels observed in partially purified Rickettsia (lane 9). Data shown in panel H is presented as fold change ratios between RLip/Pat1. Error bars (C–E) represent means ± standard error of the mean (SEM) from 3 independent experiments. Images in panels F-I are a representative of 3 independent experiments.
Fig 2.
RLip facilitates intracellular replication in HMEC-1 cells.
(A–C) HMEC-1 cells were infected with spotted fever group (SFG) rickettsiae, including R. rickettsii, R. parkeri, and R. montanensis (non-pathogenic) for up to 48 hrs. (A) Host cell lysates were analyzed by western blot analysis using anti-RLip, anti-Pat1, anti-OmpA/B, or anti-GAPDH Abs. Expression kinetics of rlip (B) and bacterial burden (C) during infection of R. rickettsii, R. parkeri, and R. montanensis was determined by RT-qPCR. Rlip expression was normalized with respect to gltA transcription level. Bacterial burden was determined by normalized gltA transcription level with respect to host cell GAPDH transcription level. Densitometry in panel A was performed using Fiji software, while RLip and Pat1 expression was normalized with respect to levels in uninfected HMEC-1 cell lysates (lane 1) and were presented as fold change ratios between RLip/Pat1. Error bars (B, C) represent SEM from 3 independent experiments, NS, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001. Images in panels A are a representative of 3 independent experiments.
Fig 3.
RLip is a rickettsial effector with lipase and cytotoxic activities.
(A) Lipase activity of purified codon-optimized recombinant (r) rRLip-WT-CO, rRLip-S138A-CO and heat-inactivated rRLip-WT-CO-Hi, rRLip-S138A-CO-Hi proteins in the absence or presence of HMEC-1 host cell lysate was assessed as described previously [20,21]. The rLacZ protein and a lipase derived from Chromobacterium were used as a non-specific protein and positive (+) control, respectively. Inset represents a western blot analysis of the utilized rRLip-WT-CO and rRLip-S138A-CO using an anti-His Ab. (B) Transformed yeast cells were streaked onto inducing (SC-U + Gal) or repressing (SC-U + Glu) agar and incubated at 30°C for 3 days. (C) Cytotoxicity assay in yeast strain INVSc1 transformed with plasmids expression RLip-WTGS, RLip-WT-CO or lipase mutant RLip-S138A-CO was performed as described previously [20,21]. Colonies were counted to determine the percentage of colony formation units (CFU) on inducing agar with respect to that on repressing agar. LacZ plasmid was used as control in panels B, and C. (D) Western blot analysis of V5-epitop-tagged RLip-WTGS, RLip-WT-CO or lipase mutant RLip-S138A-CO expression in yeast strain INVSc1 under inducing conditions (SC-U + Gal medium). The total proteins from yeast cells carrying the appropriate plasmid were probed with anti-V5 Ab. Error bars in panels A, and C represent means ± SEMs (standard errors of the means) from 3 independent experiments; NS, not significant; **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001. Images in A, B, and D are a representative of 3 independent experiments.
Fig 4.
RLip contributes to R. parkeri intercellular survival by facilitating the escape from phagosomes.
(A) Evaluation of the intact rlip gene and rlip transcript using R. parkeri WT or R. parkeri rlip::Tn bacterial DNA. (B) RLip protein expression was detected in HMEC-1 cells infected with R. parkeri WT or R. parkeri rlip::Tn bacteria by western blot analysis using anti-RLip, and anti-GAPDH Abs. (C, D) R. parkeri WT- or R. parkeri rlip::Tn mutant-infected HMEC-1 cells were analyzed for rlip expression (C) and bacterial burden (D) by RT-qPCR. The gene expression of RLip was normalized with respect to gltA transcription level, while bacterial burden (gltA) was normalized with respect to host cell GAPDH transcription level. (E, F) HMEC-1 cells were infected with purified R. parkeri WT and R. parkeri rlip::Tn mutant (MOI: 20) and internalization was assessed at 2 hpi by IFA. (G-H) Colocalization of R. parkeri WT or R. parkeri rlip::Tn mutant (MOI: 5) with LAMP2 was evaluated by IFA at 24 hpi. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Numbers of extracellular and intracellular rickettsiae (E, F) as well as colocalization between rickettsiae and LAMP2 (G-H) was analyzed using Coloc 2 plugin Fiji software. Bars in panels E, and G, 10 μm. Approximately 200 bacteria-infected cells were counted per condition and time point. Error bars (C, D, F and H) represent means ± standard error of the mean (SEM) from three independent experiments; NS, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001. Images in A and B are a representative of 3 independent experiments.
Fig 5.
Working model for RLip facilitating pathogenic rickettsiae intracellular survival.
Expression of RLip is low during the cell-free stage, while rapidly induced as soon as the bacteria encounters the host cell. Upon host cell invasion, RLip is predominately released into the host cytoplasm, while minimally retained by the bacteria itself. Our findings suggest that RLip plays an important role in the phagosomal escape of pathogenic Rickettsia species into the host cell cytoplasm to facilitate the establishment of a replication niche.