Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase - PubMed (original) (raw)

Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase

Barbara C Figueiredo et al. PLoS Pathog. 2015.

Abstract

Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease of global public health importance. These relatively large parasites are able to survive prolonged periods in the human vasculature without inducing stable blood clots around them. We show here that the intravascular life stages (schistosomula and adult males and females) can all promote significant plasminogen (PLMG) activation in the presence of tissue plasminogen activator (tPA). This results in the generation of the potent fibrinolytic agent plasmin which could degrade blood clots forming around the worms in vivo. We demonstrate that S. mansoni enolase (SmEno) is a host-interactive tegumental enzyme that, in recombinant form, can bind PLMG and promote its activation. Like classical members of the enolase protein family, SmEno can catalyze the interconversion of 2-phospho-D-glycerate (2-PGA) and phosphoenolpyruvate (PEP). The enzyme has maximal activity at pH 7.5, requires Mg2+ for optimal activity and can be inhibited by NaF but not mefloquin. Suppressing expression of the SmEno gene significantly diminishes enolase mRNA levels, protein levels and surface enzyme activity but, surprisingly, does not affect the ability of the worms to promote PLMG activation. Thus, while SmEno can enhance PLMG activation, our analysis suggests that it is not the only contributor to the parasite's ability to perform this function. We show that the worms possess several other PLMG-binding proteins in addition to SmEno and these may have a greater importance in schistosome-driven PLMG activation.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Plasminogen (PLMG) activation by schistosomes in the presence of tissue plasminogen activator (tPA).

(A) Plasmin activity (mean OD405 value +/- SD; 60 min) detected in the presence (+) or absence (-) of live schistosomula (1000 parasites per well, n≥5). (B) Plasmin activity (mean OD405 value +/- SD; 60 min) detected in the absence (white bar) or presence of male (black bar) or female (gray bar) adult schistosomes. In each case, ≥5 adult worms were evaluated individually. (C) Plasmin activity (mean OD405 value +/- SD; 30 min) detected in the presence of different numbers of male parasites as indicated, n≥5. (D) Plasmin activity (mean OD405 value +/- SD; 60 min) detected in the absence (white bar) or presence of individual male S. japonicum (Sj), S. haematobium (Sh) or S. mansoni (Sm), n ≥10. Significant differences from control conditions (reagents themselves without parasites) are denoted by *, p<0.05, and ***, p <0.001.

Fig 2. Expression profile of SmEno at different stages in the S. mansoni life cycle.

Quantitative RT-PCR data showing relative expression level (mean +/- SD) of SmEno at different stages in the S. mansoni life cycle: eggs, cercariae, schistosomula (7-day cultured larvae), adult female worms, and adult males (set at 100%). Results are representative of two independent experiments. Significant differences between male adult worms and other life stages is denoted by ***, p <0.001.

Fig 3. Immunolocalization of _Sm_Eno in S. mansoni adult worms and schistosomula.

Indirect immunofluorescent labeling of native _Sm_Eno protein in sections of (A) an adult male and (B) a whole fixed schistosomulum using polyclonal anti-ENO1 antibody (and secondary anti-rabbit IgG antibody conjugated to Alexa 488 (green)). Enlargements of the areas shown in white boxes in the top row are presented in the middle row. Arrows indicate clear tegumental staining. As a control, secondary antibody alone was used on sections of (C) adult males and (D), whole fixed schistosomula. Scale bars = 100 μm or 50 μm in insets (middle row).

Fig 4. Schistosome enolase activity (mean PEP generated (μg/ml) +/- SD).

(A) Enolase activity exhibited by live adult male or adult female worms (individuals) or schistosomula (~1000 parasites/sample) (n ≥ 10 replicates/sample). (B) Enolase activity exhibited by live S. japonicum (Sj), S. haematobium (Sh) and S. mansoni (Sm) adult males after 4 hours, n ≥ 12 replicates/sample. (C) Enolase activity exhibited by live schistosomula (~1,000/sample, black squares) versus total schistosomula lysate (open squares) over 2 hours, n ≥ 5/condition. (D) Enolase activity exhibited by live schistosomula (~1,000/sample, black circles) versus 2 h (open diamond) or 48 h (closed diamond), conditioned medium, n ≥ 5/condition. (E) Effect of varying divalent ion (Mg2+ or Ca2+) concentration (1, 10 or 50 mM as indicated) on mean relative enolase activity. The highest activity value (in 50 mM Mg2+) was set at 100% and activities relative to this are presented (n ≥ 5/condition). Significant differences relative to equivalent measurements containing Ca2+ are denoted by *** for p <0.001. EDTA is Ethylenediaminetetraacetic acid.

Fig 5. Heterologous expression, purification and kinetics of r_Sm_Eno.

(A) Heterologous expression of r_Sm_Eno in E. coli BL21 Star (DE3). Coomassie-stained gel showing SDS-PAGE resolution of lysates of E. coli bacteria harboring pTrcHisB::_Sm_Eno before (Gel, left lane) or 4 hours after (Gel, right lane) protein expression induction. The arrow indicates r_Sm_Eno in the induced lane. In western blot analysis of these lysates probed with monoclonal anti-His tag antibody, a prominent ~50 kDa protein (_Sm_Eno) is detected (Western Blot, anti-His, arrow). Recombinant _Sm_Eno protein was purified from bacterial lysate by immobilized metal affinity chromatography. A single ~50 kDa pure protein (_Sm_Eno) is resolved following Coomassie brilliant blue staining of an SDS-PAGE gel and this protein binds anti-ENO antibody, as determined by western blot analysis (right lane, arrow). Positions of migration of molecular mass markers are indicated on the left (kDa). Michaelis-Menten kinetic curve generated using PGA as substrate (B, catalyzing the forward reaction) or using PEP as substrate (C, catalyzing the reverse reaction). The apparent Km and Vmax values shown represent the mean +/- SD of three independent experiments. (D) Recombinant _Sm_Eno activity in a buffer system covering the pH range 5.5–9.5. Enzymatic activity is maximal at pH 7.5. (E) Impact of divalent ion (Mg2+ or Ca2+, as indicated) concentration on mean r_Sm_Eno activity (± SD). The highest activity value (at 100 mM Mg2+) was set at 100% and relative activities were calculated and are presented. Significant differences relative to equivalent measurements containing Ca2+ are denoted by *** for p <0.001. EDTA is Ethylenediaminetetraacetic acid. (F) r_Sm_Eno activity in the presence of increasing concentrations of NaF (white bars) compared to its activity in the absence of inhibitors (set at 100%, black bar). Significant differences relative to the untreated control are denoted by *** for p <0.001. (G) Influence of increasing concentrations of mefloquine (MFQ) on r_Sm_Eno activity (left bars) or on schistosomula lysate enolase activity (right bars). Activity measured in the absence of MFQ was set at 100% and relative activities were calculated. Significant differences relative to the untreated control are denoted by * for p<0.05 and ** for p<0.01. In E-F, bars represent mean relative activity ± SD, n = 3.

Fig 6. Recombinant _Sm_Eno enhances plasminogen (PLMG) activation.

(A) Plasmin activity (mean OD405 value +/- SD, n = 3) detected in the presence (“+”, right gray bars) or absence (“-”, left gray bars) of r_Sm_Eno. BSA served as negative control, (white bar). tPA is tissue plasminogen activator (B) Plasmin activity (mean OD405 value +/- SD, n = 3) detected in the presence of increasing concentrations of r_Sm_Eno (black bars) or control protein (BSA) (gray bars) or no protein (white bar). Significant differences from control conditions (reagents themselves without protein) are denoted by ***, p <0.001.

Fig 7. SmEno gene suppression using RNA interference.

(A) Mean level of SmEno gene expression (+/-SD, n = 3) in cultured adult schistosome males (left), females (center) or schistosomula (right) at 72 hours after treatment with control, irrelevant siRNA (“Cont” black bars, set at 100%) or siRNA targeting _Sm_Eno (“Eno”, white bars), as determined by qRT-PCR. (B) Detection by western blot of _Sm_Eno protein (top row), in extracts prepared from parasites 72 h after treatment with _Sm_Eno (Eno) or control (Cont) siRNAs. Diminished levels of _Sm_Eno protein is seen in the first lane of each group of samples. Western blot analysis detecting a control schistosome protein (lower row) shows roughly equivalent protein amounts per lane. (C) Mean (+/-SD, n = 3) surface _Sm_Eno enzyme activity in live adult male (left) or female (center) parasites or schistosomula (right) after treatment with control siRNA (“Cont”, black bars) or siRNA targeting _Sm_Eno (“Eno”, white bars). Significant differences between suppressed compared to control parasites are denoted by ***, p <0.001. (D) Plasmin activity (mean OD405 value +/- SD, n = 3) detected in live adult male (left) or female (center) parasites or schistosomula (right) after treatment with control siRNA (“Cont”, black bars) or siRNA targeting _Sm_Eno (“Eno”, white bars). All conditions contain plasminogen (PLMG), and tissue plasminogen activator (tPA).

Fig 8. Plasminogen (PLMG) interaction with rSmEno and with schistosome lysates (A) by ELISA or (B) by western blotting.

(A) ELISA plates were coated with r_Sm_Eno (0.5 μg/well) or the indicated parasite extracts (1.0 μg/well each) and wells were incubated with increasing concentrations of PLMG (0–1 μg) in triplicate. As a negative control, some wells were coated with BSA (0.5 μg/well). Anti-PLMG antibody was used to detect PLMG following standard ELISA conditions. The lines represent the mean absorbance values at OD 450 nm (± SD). (B) Detection by western blot of schistosome PLMG-binding proteins (left panel). Lanes contain extracts from males (M), females (F) and schistosomula (S), as well as pure r_Sm_Eno (E), BSA (“-”, negative control) and commercially-obtained PLMG (“+”, a control for anti-PLMG antibody binding). Multiple bands in the schistosome extracts bind PLMG. The arrow indicates the position of migration of _Sm_Eno, here revealed to be a PLMG-binder. No binding to the negative control protein (BSA) is seen. The membrane was stripped and re-probed with anti-ENO1 antibody (right panel). A single, prominent ~47-kDa SmEno band is detected (arrow) in the extracts of males (M), females (F) and schistosomula (S) and in the case of purified rSmEno (E). Images are representative of three replicate experiments.

References

1. Lustigman S, Prichard RK, Gazzinelli A, Grant WN, Boatin BA, et al. (2012) A research agenda for helminth diseases of humans: the problem of helminthiases. PLoS Negl Trop Dis 6: e1582 10.1371/journal.pntd.0001582 - DOI - PMC - PubMed
1. van der Werf MJ, de Vlas SJ, Brooker S, Looman CW, Nagelkerke NJ, et al. (2003) Quantification of clinical morbidity associated with schistosome infection in sub-Saharan Africa. Acta Trop 86: 125–139. - PubMed
1. Hotez PJ, Ferris MT (2006) The antipoverty vaccines. Vaccine 24: 5787–5799. - PubMed
1. Steinmann P, Keiser J, Bos R, Tanner M, Utzinger J (2006) Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect Dis 6: 411–425. - PubMed
1. Gryseels B, Polman K, Clerinx J, Kestens L (2006) Human schistosomiasis. Lancet 368: 1106–1118. - PubMed

Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase - PubMed (original) (raw)