Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin

Figures & data

Figure 1. Substitution of the 238th Asp with an Arg impaired the hemolytic and pore-forming activity of PLO.

(A) Structure model of PLO molecule and the location of the 238th Asp and the 376th Asn that were replaced with an Arg to construct the PLO mutants. The domains of the PLO molecule were colored red (D1), green (D2), yellow (D3), and orange (D4).(B) Determination of the hemolytic activity of rPLO and mutants. The smallest concentrations of rPLO, rPLO D238R, rPLO P499F, and rPLO N376R that cause complete hemolysis of sRBCs were 3.125, 12.5, 50, and 3.125 μg/mL, respectively. The sRBCs were incubated with PBS as hemolysis-negative controls.(C) Determination of the pore-forming activity of rPLO and the mutants on RBCMs by TEM observation. rPLO and rPLO N376R formed typical pores with diameters of approximately 30 nm. rPLO D238R formed incomplete pores (marked by black arrows). The sRBCs treated with rPLO P499F showed no typical pores on the cell membrane. Scale bar = 200 nm.

Figure 2. Determination of the cell membrane binding activity of rPLO and mutants by western blot assays.

The western blots showed that both rPLO and rPLO D238R can efficiently bind RBCM. rPLO P499F showed impaired cell membrane binding capacity (A). After 30 min of incubation, most of the rPLO P499F proteins were detected in the supernatant (B).

Figure 3. Determination of oligomers formed by rPLO and rPLO D238R by western blot assays.

Western blot assays showed that rPLO formed two types of oligomers (large and small) within 2 min when incubated with cholesterol-containing liposomes (A). When samples were heated to 100°C for 10 min before SDS-AGE analysis, the large and small oligomers split into two other types of oligomers (medium and the smallest) (B). rPLO D238R only formed one type of oligomer (C), and heating did not further disrupt the oligomers (D). Analysis of the oligomers formed by rPLO and rPLO D238R in a single Western blot showed that the molecular weight of the only type of oligomer formed by rPLO D238R was similar to that of the smallest oligomers, which can be obtained mainly by heating the large and small oligomers formed by rPLO (E). This result indicated that the smallest oligomers were possibly the intermediate product that further formed the large or small oligomers. The result also indicated that the substitution of the 238th Asp with an Arg mainly hindered the process of large oligomer formation by the smallest oligomers.

Figure 4. rPLO D238R showed decreased cytotoxicity in L929 and MDBK cells. rPLO and rPLO N376R showed evident cytoxicity in L929 cells even at a low concentration of 0.3 μg/mL (A and C). rPLO D238R did not show evident cytotoxicity in L929 cells at 2.5 μg/mL (B). rPLO P499F was more cytotoxic than rPLO D238R given that 2.5 μg/mL of rPLO P499F evidently decreased the viability of L929 cells (D). Compared with L929 cells, MDBK cells were less sensitive to the cytotoxicity of rPLO and rPLO N376R. rPLO and rPLO N376R showed evident cytotoxic effects in MDBK cells at 1.25 and 0.6125 μg/mL, respectively (E and G). rPLO D238R at 2.5 μg/mL was cytotoxic in MDBK cells (F). rPLO P499F showed cytotoxicity in MDBK cells only when concentration exceeded 5 μg/mL (H).

Figure 5. Profile of the expression of inflammation-associated cytokines in L929 cells treated by recombinant proteins.

rPLO and rPLO D238R at a concentration of 1.6 μg/mL significantly upregulated the expression of IL-1β within 8 h, but rPLO P499F did not; Meanwhile, the expression level of IL-1β in rPLO-treated cells was significantly higher than that of cells treated with rPLO D238R (A). rPLO P499F upregulated the expression level of IL-6 significantly, whereas rPLO and rPLO D238R showed no such effect (C). The expression levels of TNF-α (B), IFN-γ (D), IL-10 (E), and TGF-β1 (F) did not differ significantly between cells treated with different recombinant proteins (* p < 0.05, ** p < 0.01).

Figure 6. Images and histopathologizal analysis of tissue damage caused by different recombinant proteins in mice.

(A) Representative pictures of muscle tissues (hindleg of mice) in the periphery of injection sites of mice inoculated with different recombinant proteins. Mice inoculated with rPLO and rPLO P499F showed visible hemorrhagic damages (marked by black arrows) in the muscle tissues around the injection sites. Mice inoculated with rPLO D238R and PBS showed no visible tissue damage.(B) Histopathological analysis of muscle tissues around injection sites. Massive inflammatory cell infiltration was observed in the tissues of the mice inoculated with rPLO and rPLO P499F but not in the tissues of the other two groups. Scale bar = 100 μm.

Figure 7. Expression levels of inflammation-associated cytokines in mice inoculated with recombinant proteins.

rPLO inoculation significantly upregulated the expression levels of IL-1β, TNF-α, IL-6, and TGF-β1 in mice. rPLO P499F inoculation significantly upregulated the expression of TNF-α, IFN-γ, and IL-10 in mice. None of the six cytokines were significantly upregulated in mice inoculated with rPLO D238R according to statistical analysis. (* p < 0.05, ** p < 0.01, ns not significant)

Figure 8. Expression levels of IL-1β and IL-10 in BMDMs treated with rPLO or its mutants at sublytic concentrations.

MTT assay showed that rPLO at concentrations lower than 0.6 μg/mL did not signicantly decrease the viability of BMDMs within 16 h (A). Thus, 0.6 μg/mL was defined as the sublytic concentraiton of rPLO in such a system. Furthermore, 0.6 μg/mL rPLO treatment did not significantly affect the expression level of IL-10; by contrast, rPLO P499F treatment significantly upregulated the expression of IL-10 after 4 h, and rPLO D238R elicited signficant upregulation of IL-10 expression after 12 h. However, the expression level of IL-10 elicited by rPLO D238R was significantly lower than that elicited by rPLO P499F at any time point (B). An rPLO concentration of 0.6 μg/mL elicited upregulated IL-1β expression in BMDMs after 4 h, whereas 0.6 μg/mL rPLO P499F did not upregulate the expression of IL-1β even when the incubation period was extended to 16 h. The IL-1β expression elicited by 0.6 μg/mL rPLO D238R was higher than that elicited by rPLO P499F at 12 h but was significantly lower than that elicited by rPLO (C). (* p < 0.05, ** p < 0.01, ns not significant)

Table 1. Sequence of the PCR primers for constructing the mutants of plo gene.

Name of primer	Sequence	Mutation site
plo-P499F(F)	5ʹ-GGCCTAGCGTGGGATTTCTGGTGGACCGTT-3’	Pro (499) to Phe
plo-P499F(B)	5ʹ-GATAACGGTCCACCAGAAATCCCACGCTA-3’
plo-D238R(F)	5ʹ-TACACGGCTAGCGTACGTACACCGACATCT-3’	Asp (238) to Arg
plo-D238R(B)	5ʹ-ACGTACGCTAGCCGTGTAGTAAATCTGTTT-3’
plo-N376R(F)	5ʹ-AATTTCTTGAAGGATCGTCAGTTGGCAGCT-3’	Asn (376) to Arg
plo-N376R(B)	5ʹ-ACGATCCTTCAAGAAATTGACAGCATAGGA-3’