STK35L1 is highly upregulated in vitro and in vivo during P. berghei infection
To gain an insight into the role of STK35L1 in governing susceptibility to parasite infection in the liver stage of malaria, a hepatocyte cell line HepG2 were infected with P. berghei ANKA sporozoites. The expression of STK35L1 was measured by qPCR, and we found that it was significantly upregulated (~5 fold; p<0.01) after sporozoite infection in HepG2 cells (Figure 1A). The upregulation of STK35L1 was further confirmed at the protein level by Western blotting using an anti-STK35L1 antibody. We found an increased expression (~2 fold) of STK35L1 protein in infected HepG2 cells (Figure 1B; black arrow).
We asked whether infection of P. berghei sporozoites in mice liver would also enhance the expression of Stk35l1.Indeed, we observed a significant increase (~2 fold; p<0.03) of Stk35l1 gene expression in mice liver infected with P. berghei sporozoites. (Figure 1C). The parasite load in the mice livers was also determined by the copy number of the P. berghei 18S rRNA in the infected and uninfected liver. The data showed that all the mice were infected, but the infection (parasite load) was highly variable (Figure 1D). In a nutshell these data suggest that the STK35L1 upregulation during P. berghei infection might play a crucial role in facilitating the parasite’s infection and growth during the liver stage of malaria.
Inhibition of STK35L1 impairs the infection of host cells by Plasmodium sporozoites.
We asked whether STK35L1 is essential for P.berghei sporozoite infection in hepatocytes. We knocked down the expression of STK35L1 using three STK35L1 siRNAs and then measured the parasite load to address this question. We procured commercial siRNAs that were designed from different regions of STK35L1. We confirmed the knockdown efficiency of these siRNAs via qPCR and Western blotting. Two of the siRNAs (siRNA1 and siRNA2) showed the highest knockdown efficiency (~82% and ~87%, respectively) for STK35L1 (Figure 2A). The knockdown of the STK35L1 protein was also confirmed by immunoblotting (Figure 2B). These siRNAs(siRNA1 and siRNA2) were chosen for further studies.To investigate the role of STK35L1 in P.berghei infection, HepG2 cells were treated with siRNA1 and siRNA 2 for 72 hours and then infected with P.berghei sporozoites for 48 hours. The scrambled siRNA was used as a negative control. Notably, we found that the sporozoite infection (parasite load) was significantly reduced to ~20% in cells treated with siRNA1 and siRNA 2 (Figure 2C). The infection was decreased slightly but was non-significant in cells treated with scrambled siRNA (Figure 2C).
Similarly, the STK35L1 upregulation was significantly reduced after sporozoites infection in cells treated with siRNA1 and siRNA2 (Figure 2D). Scrambled siRNA could not affect the expression of stk35l1 (Figure 2D). These data showed the physiological relevance of STK35L1 as a vital host factor required for the liver infection with Plasmodium sporozoites.
STAT3 is highly upregulated during Plasmodium infection in HepG2 cells.
We hypothesized that the STK35L1 upregulation during P. berghei infection is regulated by the member(s) of the STAT transcription factor family. First, we examined the expression of STAT family transcription factor genes in HepG2 cells via qPCR. We found that STAT1 and STAT3 genes were highly abundant in HepG2 cells (Figure 3A). The STAT4 gene was relatively less expressed (~6 fold lower) in comparison to STAT1. Other family members (STAT2, STAT5A, STAT5B, and STAT6) were expressed at 50 to a 100-fold lesser extent in HepG2 cells (Figure 3A). We further studied the effect of sporozoite infection on STAT1, STAT3, and STAT4 expression in HepG2 cells. Notably, we found that only the expression of STAT3 was significantly upregulated (~7 fold; p<0.01) in infected HepG2 cells (Figure 3B). We could not find any significant changes in the expression levels of STAT1 andSTAT4 (Figure 3B). The upregulation of STAT3 in infected cells was also confirmed by Western blotting using the anti-STAT3 antibody (Figure 3C). Collectively, these data show that only STAT3 is significantly upregulated via Plasmodium sporozoite infection, and STAT3-dependent signaling might be necessary for the liver stage of malaria.
STAT3 is activated during Plasmodium infection in HepG2 cells and autoregulates its expression
We investigated whether sporozoite infection activates STAT3 via phosphorylation in addition to enhancing its expression. We could observe that the phosphorylation of STAT3 residue tyrosine-705 was increased after sporozoite infection in HepG2 cells (Figure 4A). We found a very low STAT3 phosphorylation in non-infected cells (Figure 4A). These data indicate that sporozoite infection leads to STAT3 phosphorylation, which might regulate various host gene expressions.
Furthermore, to examine whether STAT3 activation regulates its expression, we used a specific STAT3 activator ML115 22. Notably, ML115 (4µM) could significantly increase the expression of STAT3 by ~3 fold; (p< 0.002) within 6 hours of treating HepG2 cells with ML115 (Figure 4B). To confirm that STAT3 regulates ML115-dependent its upregulation, we used highly specific STAT3 inhibitors Stattic and 5,15-DPP 23, 24. We observed that both inhibitors could not significantly affect the basal expression of STAT3 (Figure 4B). As expected, both inhibitors successfully inhibit the ML115-dependent upregulation of STAT3 by ~85% and ~80%, respectively (Figure 4B). Together, these data suggest that activation of STAT3 via sporozoite infection leads to the upregulation of its expression in a positive feedback manner.
Activation of STAT3 regulates the expression of STK35L1 in HepG2 cells.
We hypothesized that STAT3 activation might regulate the STK35L1 expression. We treated the HepG2 cells with specific STAT3 inhibitors, Stattic, and 5,15-DPP. Interestingly, the basal expression of STK35L1 was significantly down-regulated after the treatment of Stattic (~60%; p<0.013) and 5,15-DPP (~85%; p<0.0025) (Figure 5A). This result suggests that STK35L1 expression is tightly regulated via STAT3. Furthermore, we asked whether activation of STAT3 upregulates the expression of STK35L1. We incubated HepG2 cells with a specific STAT3 activator, ML115(4µM), for 6 hours and found that STK35L1 was significantly upregulated (~2 fold; p<0.005) after the treatment of cells with ML115 (Figure 5B). Next, we examined whether ML115-dependent upregulation of the STK35L1 gene can be reverted after the inhibition of STAT3 activation. Indeed, Stattic, and 5,15-DPP could completely block the upregulation of STK35L1 (Figure 5B). These data collectively confirm that STAT3 is the major transcription factor that plays a crucial role in regulating the expression of STK35L1. The STAT3/STK35L1 axis might be critical for the liver stage of malaria.
Various cell cycle genes were upregulated during Plasmodium infection, which were regulated via STK35L1
Previously, we found that STK35L1 regulates various cell cycle-related genes in endothelial cells 9. To identify the role of STK35L1 upregulation during P. berghei infection, we checked the expression of 11 genes (Table S2) during sporozoite infection. Interestingly, 10 genes (RAD51, MKI67, CDKN3, CDKN2A, CDK6, CDC20, DDX11, GADD45A, CCNB2, and CDC2) were significantly upregulated after sporozoite infection in HepG2 cells (Figure 6 and Table S2), but GTSE1 was not significantly upregulated (Figure 6; bottom right panel). Interestingly, RAD51 was upregulated ~18 fold (p<0.0001) in infected cells (Figure 6; top left panel). CDK6, DDX11, and CDC2 were ~4-5fold upregulated after sporozoite infections (Figure 6). Moreover, GADD45A, CDC20, and CDKN2A were ~3-4 fold upregulated (Figure 6). CDKN3, MKI67, and CCNB2 were ~2-2.5 fold upregulated (Figure 6). To summarize, these data indicate that these cell cycle-related genes might play a crucial role in hepatocyte growth and, consequently, on malaria’s liver stage.
We asked whether STK35L1 has a role in regulating the infection-dependent upregulation of these genes. Indeed, upregulation of the studied genes except CDC2 and GTSE1 was significantly downregulated after knockdown of the STK35L1 gene (Figure 6). The upregulation of CDC2 after sporozoites infection is independent of STK35L1. These data signify that STK35L1 is a major factor that controls the parasite infection-induced expressions of cell cycle-related genes.