Host SUMOylation Pathway Negatively Regulates Protective Immune Responses and Promotes Leishmania donovani Survival

Abstract

SUMOylation is one of the post-translational modifications that have recently been described as a key regulator of various cellular, nuclear, metabolic, and immunological processes. The process of SUMOylation involves the modification of one or more lysine residues of target proteins by conjugation of a ubiquitin-like, small polypeptide known as SUMO for their degradation, stability, transcriptional regulation, cellular localization, and transport. Herein, for the first time, we report the involvement of the host SUMOylation pathway in the process of infection of Leishmania donovani, a causative agent of visceral leishmaniasis. Our data revealed that infection of L. donovani to the host macrophages leads to upregulation of SUMOylation pathway genes and downregulation of a deSUMOylating gene, SENP1. Further, to confirm the effect of the host SUMOylation on the growth of Leishmania, the genes associated with the SUMOylation pathway were silenced and parasite load was analyzed. The knockdown of the SUMOylation pathway led to a reduction in parasitic load, suggesting the role of the host SUMOylation pathway in the disease progression and parasite survival. Owing to the effect of the SUMOylation pathway in autophagy, we further investigated the status of host autophagy to gain mechanistic insights into how SUMOylation mediates the regulation of growth of L. donovani. Knockdown of genes of host SUMOylation pathway led to the reduction of the expression levels of host autophagy markers while promoting autophagosome-lysosome fusion, suggesting SUMOylation-mediated autophagy in terms of autophagy initiation and autophagy maturation during parasite survival. The levels of reactive oxygen species (ROS) generation, nitric oxide (NO) production, and pro-inflammatory cytokines were also elevated upon the knockdown of genes of the host SUMOylation pathway during L. donovani infection. This indicates the involvement of the SUMOylation pathway in the modulation of protective immune responses and thus favoring parasite survival. Taken together, the results of this study indicate the hijacking of the host SUMOylation pathway by L. donovani toward the suppression of host immune responses and facilitation of host autophagy to potentially facilitate its survival. Targeting of SUMOylation pathway can provide a starting point for the design and development of novel therapeutic interventions to combat leishmaniasis.

Keywords: Leishmania donavani; SUMOylation; SUMOylation mediated immune responses; autophagy; autophagy maturation; host–pathogen interaction.

Figure 1

Induced expression of genes involved in host SUMOylation pathway upon Leishmania donovani infection. PMA-differentiated THP-1 macrophages were infected with metacyclic stage L. donovani promastigotes at 20 MOI for 48 (h) Uninfected (Un) and L. donovani-infected (Ld) cells were processed for different evaluations. (A) RNA was isolated from uninfected and infected macrophages at 48 h post-infection, and expression analysis of genes of the host SUMOylation pathway was performed by quantitative real-time PCR (qRT-PCR). p-Value was calculated based on Student’s unpaired 2-tailed t-test comparing uninfected macrophages to infected macrophages (*p < 0.05 and **p < 0.01). (B) Cytoplasmic extracts of uninfected and infected macrophages were analyzed by Western blotting to check the expression level of SUMO-1, SUMO-2/3, AOS-1, UBA2, UBC9, and SENP1 at 48 h post-infection. GAPDH was used as the loading control. Band intensities were calculated using ImageJ software. Data from one of three experiments are shown. (C) The graph represents the relative intensity of the bands calculated by ImageJ software at 48 h post-infection; *p = 0.028 for UBC9; **p = 0.0043 and 0.0073 for SUMO-2/3 and AOS-1, respectively. p-Value was calculated based on an unpaired t-test with Welch’s correction comparing uninfected macrophages to L. donovani-infected macrophages (Un vs. LD). (D) Cytoplasmic extracts of uninfected and infected macrophages were analyzed by Western blotting to check the expression of overall SUMOylated proteins at 48 h post-infection. GAPDH was used as the loading control. The band intensities of each lane were analyzed by ImageJ software, and the values concerning band intensity are labeled on the peaks. PMA, phorbol 12-myristate 13-acetate; MOI, multiplicity of infection.

Figure 2

Host SUMOylation pathway favors the growth of Leishmania donovani in macrophages. PMA-differentiated THP-1 macrophages were transiently transfected with siRNA against specific genes for 36 h MOCK here represents the transfection with a control siRNA. (A) Total RNA was enriched using TRIzol (Qiagen). mRNA levels of transfected macrophages with target genes compared to MOCK were quantified by qRT-PCR. *p = 0.02, 0.03, and 0.04 for MOCK vs. SUMO-1, AOS-1, and UBA2, respectively; **p = 0.0012 and 0.01 for MOCK vs. UBC9 and SENP1, respectively. (B) Cell lysates of transfected macrophages were used to check the transfection efficiency by Western blotting. GAPDH was used as the loading control for each set separately. (C) Effect of transfection on cell viability measured by MTT assay. THP-1 macrophages were transfected with specific siRNAs and control siRNA (MOCK) for 36 (h) No significant changes in cell viability were observed upon the transfection of siRNAs against target genes compared to MOCK. (D) PMA-differentiated macrophages were transfected with specific siRNAs followed by L. donovani infection for 6 (h) Macrophages were stained with propidium iodide after 24 h post-infection, and parasitic load in the infected macrophages was calculated under confocal microscopy. Scale bar, 5 µm. Data from one of three experiments are shown. Parasite load was counted in 100 macrophages. Statistical significance was quantified using the unpaired t-test with Welch’s correction, *p = 0.0286, 0.0222, and 0.0266 for MOCK vs. SUMO-1, SUMO-2/3, and UBC9, respectively; **p = 0.0087 and 0.0034 for MOCK vs. AOS-1 and UBA2, respectively. PMA, phorbol 12-myristate 13-acetate.

Figure 3

Host SUMOylation regulates the growth of Leishmania donovani by modulating autophagy initiation. (A) 1 × 10⁶ THP-1 cells were differentiated into macrophages and were transfected with the specific siRNAs (B) followed by L. donovani infection. MOCK here represents the transfection with a control siRNA. Cell lysates were prepared at 48 h post-infection and were processed to check the expression level of different autophagy markers Beclin-1, Atg5, and LC3A/B. Data from one of three experiments are shown. Band intensities were quantified by ImageJ software and were plotted in GraphPad Prism 8. Statistical significance was quantified using the unpaired t-test with Welch’s correction, (A) *p = 0.03 and 0.016 for MOCK vs. UBC9 and SENP1, respectively, for the expression of LC3A/B-II. (B) *p = 0.038 and 0.018 for MOCK vs. UBA2 and UBC9, respectively, and **p = 0.01 for MOCK vs. AOS-1 for the expression of Beclin-1; *p = 0.035 and 0.043 for MOCK vs. UBA2 and UBC9, respectively, and **p = 0.01 for MOCK vs. AOS-1 for the expression of Atg5; *p = 0.027 and 0.011 for MOCK vs. AOS-1 and UBA2, respectively, and **p = 0.006 for MOCK vs. UBC9 for the expression of LC3A/B-II.

Figure 4

Host SUMOylation favors the growth of Leishmania donovani by modulating autophagy maturation. Colocalization studies of autophagosome marker, LC3A/B, and lysosome marker, LAMP-1, in L. donovani-infected macrophages were performed. (A) 0.5 × 10⁶ THP-1 macrophages were transfected with specific siRNAs followed by the infection of 20 MOI of L. donovani for 24 h MOCK here represents the transfection with a control siRNA. Macrophages were incubated with the antibodies to LAMP-1 and LC3A/B with their respective secondary antibodies (see the Materials and Methods section). Images were acquired under confocal microscopy. Here, the green color represents the expression level of LAMP-1, the red color represents the expression level of LC3A/B, and the yellow color represents the colocalization of LAMP-1 and LC3A/B. Scale bar, 10 µm. A ×5 zoom image represents colocalization in a single cell. (B) The graph represents the fluorescence intensity measurement of LAMP-1. Statistical significance was quantified using the unpaired t-test with Welch’s correction, *p = 0.04, 0.017, 0.044, 0.043, and 0.011 for MOCK vs. SUMO-1, SUMO-2/3, AOS-1, UBC9, and SENP1, respectively; **p = 0.004 for MOCK vs. UBA2. (C) Graphical representation of colocalization measured by Pearson’s correlation coefficient; values range between −1 and +1. A value of +1 indicates a positive and strong correlation, while a value of −1 indicates a negative and weak correlation. Statistical significance was quantified using the unpaired t-test with Welch’s correction, *p = 0.011 for MOCK vs. SENP1; **p = 0.0014, 0.0017, and 0.0024 for MOCK vs. AOS-1, UBA2, and UBC9, respectively; ***p = 0.0002 for MOCK vs. SUMO-2/3. MOI, multiplicity of infection.

Figure 5

Host SUMOylation favors Leishmania donovani survival by modulating the generation levels of ROS and nitric oxide. THP-1-differentiated human macrophages were transfected with siRNAs for 36 h (A) in a 96-well plate followed by the infection of L. donovani promastigotes at 20 MOI for 30- and 60-min time points. Thirty minutes before completing the incubation period, cells were loaded with 10 µM of DCFH-DA. Samples were immediately analyzed for ROS levels by fluorometry with excitation/emission at 485/535 nm. The graph was plotted in GraphPad Prism 8. Statistical significance was quantified using the unpaired t-test with Welch’s correction, *p = 0.02, 0.05, and 0.04 for MOCK vs. SUMO-1, SUMO-2/3, and UBC9, respectively; **p = 0.005 for MOCK vs. UBA2 at 30 min post-infection and *p = 0.02 for MOCK vs. SUMO-2/3; **p = 0.003 and 0.004 for MOCK vs. SUMO-1 and UBC9, respectively; ***p = 0.001 and 0.0007 for MOCK vs. AOS-1 and UBA2, respectively, at 60 min post-infection. (B) Transfected macrophages were infected with L. donovani promastigotes at 20 MOI along with the stimulation of LPS (100 ng/ml) and hIFN-γ (20 ng/ml) for 24 h Griess reagent was used to estimate the NO level in the supernatant. The graph was plotted in GraphPad Prism 8. Statistical significance was quantified using the unpaired t-test with Welch’s correction (*p < 0.05 and **p < 0.01). ROS, reactive oxygen species; MOI, multiplicity of infection; LPS, lipopolysaccharide.

Figure 6

Host SUMOylation promotes the growth of Leishmania donovani by suppressing pro-inflammatory cytokines. THP-1 macrophages were transfected with specific siRNAs followed by the infection of L. donovani along with or without the stimulation of LPS (100 ng/ml) for 24 h MOCK here represents the transfection with a control siRNA. Culture supernatants were collected to measure different cytokines, while cells were used to check the expression of inflammatory markers at the transcript level. (A) Graph represents cytokine levels measured by sandwich ELISA. Statistical significance was quantified using the unpaired t-test with Welch’s correction, *p = 0.012 for MOCK vs. AOS-1 and ***p = 0.0003 for MOCK vs. SENP1 for IL-10; **p = 0.0029, 0.0032, 0.0028, and 0.0074 for MOCK vs. SUMO-1, AOS-1, UBA2, and SENP1, respectively, and ***p = 0.0002 for MOCK vs. SUMO-2/3 for IL-12p40; *p = 0.025 for MOCK vs. SUMO-2/3, **p = 0.002 for MOCK vs. SENP1, and ***p = 0.0002 and 0.0008 for MOCK vs. UBA2 and UBC9, respectively, for IFN-γ; *p = 0.049 for MOCK vs. UBC9, **p = 0.0012 for MOCK vs. AOS-1, ****p < 0.0001 for SUMO-2/3 and UBA2 for TNF-α in LPS-stimulated and Ld-infected macrophages. (B) RNA was isolated for gene expression analysis of inflammatory cytokines by quantitative real-time PCR (qRT-PCR). *p = 0.04 for MOCK vs. SUMO-2/3 or UBC9 for IL-10; *p = 0.04 and 0.05 for MOCK vs. UBA2 and SENP1, respectively; **p = 0.008 for MOCK vs. UBC9 for IL-12; *p = 0.02 for MOCK vs. SUMO-2/3 and 0.04 for MOCK vs. SUMO-1 or UBA2 or UBC9 for IL-32γ; *p = 0.05 and 0.02 for MOCK vs. UBA2 and SENP1, respectively; **p = 0.01 and 0.008 for MOCK vs. SUMO-1 and SUMO-2/3 for TNF-α in LPS-stimulated and Ld-infected macrophages. p-Value was calculated based on Student’s unpaired 2-tailed t-test. LPS, lipopolysaccharide.

Figure 7

Schematic representation of Leishmania donovani survival and growth in the macrophages regulated by the host SUMOylation pathway. Host SUMOylation promotes autophagy initiation while suppressing autophagy maturation, ROS generation, and nitric oxide production as well the secretion of pro-inflammatory cytokines, thus favoring the parasite growth and survival. Targeting this pathway could be a potential target for developing novel drugs to restrict the parasite’s survival and growth. ROS, reactive oxygen species.