Characterization of a profilin-like protein from Fasciola hepatica

Cloning and phylogenetic analysis of Fasciola hepatica profilin

The native F. hepatica profilin sequence (accession number D915_008168) was chemically synthesized and cloned (Bioneer, Oakland, CA, USA) via NdeI and XbaI sites into a modified pET-28 vector, resulting in an open reading frame containing an N-terminal hexahistidine tag followed by an HRV 3C protease cleavage site and the F. hepatica (FhProfilin) sequence. The Phylogeny.fr program was used to compare FhProfilin with identified profilins from other parasitic species and construct a phylogenetic tree based on multiple alignments and a neighbor-joining method as well as to estimate the confidence value of the branching patterns (Dereeper et al., 2008).

Recombinant FhProfilin expression

Plasmid DNA containing the FhProfilin sequence was transformed into BL21 (DE3) E. coli cells and plated onto a Luria–Bertani (LB) agar plate containing kanamycin (50 μg ml⁻¹). A single colony was inoculated into a starter culture and grown overnight in 10 ml LB medium containing 50 μg ml⁻¹ kanamycin with shaking at 225 rpm. The starter culture was used at a 1:100 dilution to inoculate 400 ml of fresh LB medium containing 50 μg ml⁻¹ kanamycin and grown at 37 °C until the optical density at wavelength 600 nm (OD600) reached 0.5. Expression of FhProfilin was induced with 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) and the cells were allowed to grow for a further 4 h at 37 °C. The cells were collected by centrifugation at 6,000g for 10 min at 4 °C and were stored at −20 °C.

Recombinant FhProfilin purification

The frozen cell pellet was thawed on ice and resuspended in 2 ml of lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl and 10 mM imidazole at pH 8.0) per gram of wet cell weight. After thawing, 200 µL of 25 mg/ml lysozyme and 200 µL of 2 mg/ml DNase was added and incubated on ice for 30 min. The solution was sonicated with 3 mm microprobe using a Sonics Vibracell VCX 130PB at 25–30% amplitude with 30 s bursts on ice for a total sonication time of 3 min, with 30 s of rest in between each burst. The cell debris was removed by centrifugation at 30,000g for 20 min at 4 °C. The supernatant was added to 1 ml of 50% (w/v) Ni-Sepharose resin (Clontech) pre-washed with 5 column volumes of lysis buffer and incubated for 1 h with gentle shaking at 4 °C. The lysate-nickel Sepharose mixture was loaded into a gravity flow column and the flow through collected. The column was washed with 2 column volumes of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl and 20 mM imidazole, pH 8.0) and eluted with 8 ml of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl and 250 mM Imidazole, pH 8.0) and collected as 2 ml fractions. Each step of the purification process was validated by visualization of protein fractions using SDS–PAGE.

Fractions containing FhProfilin were pooled and concentrated to 5 ml using Amicon ultracentrifugal filters (3 kDa molecular-weight cutoff; Millipore). The partially purified FhProfilin was further purified by size-exclusion chromatography with a Superdex S75 16/60 gel-filtration column (GE Healthcare Life Sciences, Chicago, IL, USA) equilibrated in TBS (10 mM Tris-HCL and 300 mM NaCl, pH 8.0) using an AKTA Basic fast protein liquid-chromatography (FPLC) system at 1 ml/min. The molecular weight, purity and identity of the FhProfilin preparation were confirmed by SDS–PAGE and Western blotting.

Actin affinity assay

The ability of FhProfilin to bind and sequester actin was investigated. Actin (5 µM) derived from bovine muscle (Sigma, St. Louis, MO, USA) was induced to polymerize with 1 mM MgCl₂ and 0.15 M KCl. FhProfilin was added in molar ratios of 1:1, 1:2 and 1:4 molar ratios to the polymerized actin in total volume of 150 μL and incubated at room temperature for 2–3 h. After incubation, the actin-profilin mixtures were centrifuged at 100,000xg for 30 min at 20 °C. Equal amounts of the supernatants and pellets fractions were analyzed by SDS-PAGE.

Phospholipid affinity assay

PIP Strips™ (Echelon Biosciences Incorporated, Salt Lake City, UT, USA) were used to assess the specificity of FhProfilin for associating with various phospholipids. Each membrane has been spotted with 15 assorted phospholipids at 100 pmol in each spot. The membrane was blocked with 5 ml of PBS (50 mM Sodium phosphate, 150 mM NaCl, pH 7.4) plus 3% (w/v) skim milk blocking solution and gently shaken for 1 h at room temperature. The blocking solution was then discarded and various concentrations of FhProfilin, starting with an initial concentration of 20 µg/ml up to 500 µg/ml, was added to 10 ml of PBS plus 3% (w/v) skim milk and gently shaken at room temperature for 1 h.

For the positive control, 5 µg/ml of PI(4,5)P₂ Grip™ protein in 5 ml of PBS-T (50 mM Sodium phosphate, 150 mM NaCl, 0.05% (v/v) Tween 20 pH 7.4) plus 3% (w/v) BSA was used. The protein solution was discarded, and the membrane was washed three times with 5 ml of PBS with 5 min of gentle shaking for each wash. After washing the test strip, anti-His HRP-conjugated antibody (R&D systems, Minneapolis, MN, USA) was diluted to 1:10,000 in PBS with 3% (w/v) skim milk for FhProfilin and added to the membrane and incubated for 1 h at room temperature with gentle shaking. For the positive control strip, anti-GST-HRP antibody (GenScript, Piscataway, NJ, USA) was diluted to 1:2,000 in PBS-T 3% (w/v) BSA. The antibody solution was discarded, and the membrane washed as previously stated. For both FhProfilin and positive control samples, detection was performed incubating the mebrane with 5 ml of Clarity ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA) for 5 min and imaged using the C-DiGiT blot scanner (Li-Cor).

Poly-L-proline affinity assay

The affinity of FhProfilin for proline-rich domains was investigated. pLp sepharose was prepared by coupling 50 mg of pLp (Sigma-Aldrich, St. Louis, MO, USA) to 1 g of cyanogen bromide-activated sepharose resin (Sigma-Aldrich, St. Louis, MO, USA). The pLp was dissolved in 4 ml of ice cold deionized water and the sepharose was resuspended in 8 ml of 250 mM sodium carbonate to make a 50% slurry. The pLp was added to the 50% slurry and stirred for 2 h at room temperature. The mixture was transferred to a cold room and stirred at 4 °C overnight. The reaction was quenched with 1.2 ml of 10× bead buffer (1 M NaCl, 1 M glycine and 100 mM Tris). The resin was washed in a Buchner funnel with 500 ml of deionized water, dried and stored at 4 °C in 1× storage buffer (10 mM Tris at pH 7.5, 50 mM KCl, 1 mM EDTA and 0.002% (w/v) sodium azide). A total of 50 µL aliquots of the 50% pLp sepharose slurry with PBS was incubated with 10 nM of purified FhProfilin for test samples and 10 nM of BSA for control samples. Each sample was incubated at room temperature for 15 min and analyzed by SDS PAGE. To quantify the binding effect of FhProfilin with pLp, different concentrations of FhProfilin, from 1 to 10 µg/µL, were incubated with the poly-L-proline sepharose beads for 15 min and analyzed by SDS-PAGE.

Immune profile of FhProfilin in sheep and cattle sera

Fasciola hepatica were obtained from the abattoir and whole worm extract (WE) was prepared as previously reported (Swan et al., 2019). Native GST was purified as previously described by (Wijffels et al., 1992). A total of 40 µg of WE, purified FhProfilin (3 µg) and native GST (3 µg) were loaded onto an SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) using a Trans-Blot^® Turbo™ Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% (w/v) skim milk, pooled sera from experimentally infected Merino sheep (12 animals pooled, 6 weeks post-infection, infected with 200 metacercariae), Indonesian Thin Tailed (ITT) sheep (15 animals pooled, 6 weeks post-infection, infected with 200 metacercariae) and cattle (six animals pooled from 125 days post-infection, infected with 350 metacercariae) (kindly donated by Prof. Terry Spithill) were incubated at a 1:4,000 dilution with the blots in 5% (w/v) skim milk with PBS-T for 1 h. Membranes were washed three times with PBS-T and were then incubated with either anti-sheep HRP-conjugated IgG or anti-bovine HRP-conjugated IgG (Sigma-Aldrich, St. Louis, MO, USA) diluted to 1:4,000 in 5% (w/v) skim milk in PBS-T for 1 h. Membranes were washed again as above and thens were visualized by Clarity ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA) and C-DiGiT blot scanner (Li-Cor) according to the manufacturer’s instructions.

Bioinformatic analysis of the Fasciola hepatica profilin gene

The profilin gene identified (accession number D915_008168) in F. hepatica (FhProfilin) has an open reading frame (ORF) of 393 bp, encoding a 130 amino acid protein with a predicted molecular mass of 14 kDa and a predicted isoelectric point of 5.35, which was predicted using the “ProtParam” tool from ExPASy Bioinformatics Resource Portal (https://web.expasy.org/protparam/). A domain search using InterPro (http://www.ebi.ac.uk/interpro/), revealed the presence of a conserved profilin domain, containing putative actin binding sites (Fig. 1A). Structural homology modeling revealed that FhProfilin is structural indentical to Saccharomyces cerevisiae profilin with conservation of residues involved in proline binding however residues in a putative phosphatidylinositol 4,5-bisphosphate (PIP2)-interaction site are not conserved (Fig. 1B; Fig. S1).

In a phylogenetic analysis of profilin proteins from other parasite species, it was revealed that FhProfilin clustered in a clade alongside other trematode species with identities ranging from 31.1% (Schistosoma japonicum) to 41.5% (Clonorchis sinensis), while profilins from apicomplexan parasites clustered in a separate clade (Fig. 2).

Protein expression and purification of FhProfilin

F. hepatica was recombinantly expressed in E. coli and purified via immobilized metal-ion affinity chromatography, and when visualized using SDS-PAGE resulted in a single protein with an expected molecular mass of 14 kDa showing that FhProfilin was successfully expressed and purified as a soluble protein (Fig. 2A). To further purify and characterize FhProfilin it was subjected to size-exclusion chromatography. A typical trace (Fig. 2B) revealed that FhProfilin elutes at the volume of approximately 75 ml which corresponds to a molecular weight of approximately 30 kDa, suggesting that FhProfilin is a dimer in solution. Several previous studies have reported that profilin from different species such as yeast, birch pollen and humans can form dimers and tetramers in solution (Babich et al., 1996; Mittermann et al., 1998; Wopfner et al., 2002). The oligomeric state of profilin is important for the function of certain profilins, for example, the allergenic potential of birch pollen is higher if profilin is in a dimeric state (Mares-Mejia et al., 2016). In addition, interactions with phosphoinositides and pLp is regulated by an oligomeric form of profilin, with dimers having a weaker affinity to pLp than tetrameric profilin (Korupolu et al., 2009), suggesting the oligomeric state of FhProfilin may regulate its function.

Biochemical characterization of FhProfilin

Profilins are characterized by having three major biochemical functions (Krishnan & Moens, 2009; Moreau et al., 2017, 2020); firstly, profilins bind to and sequester actin monomers, therefore affecting how actin filaments polymerize. Secondly, profilins have an affinity for proline-rich domains contained within their interacting ligands and lastly, they have an affinity for phosphatidylinositol lipids that dissociate the actin:profilin complexes.

To determine if FhProfilin is able to perform the classic functional profile of other profilins, several in vitro assays were performed. Recombinant FhProfilin was added to polymerizing bovine actin at different ratios and monomeric actin was separated from polymerized actin by centrifugation. At all ratios of actin:profilin, the majority of actin appeared in the soluble supernatant fraction with very little polymerized actin being observed in the pellet fractions (Fig. 3). This indicates that FhProfilin has a strong ability to sequester actin similar to other profilins from yeast and humans (Eads et al., 1998; Pinto-Costa & Sousa, 2020). A major defense mechanism of F. hepatica is the ability to shed its tegument proteins after binding by antibodies, which is highly dependent on cytoskeletal rearrangement, thus making FhProfilin an ideal vaccine candidate or drug target (Hanna, 1980). Human and Plasmodium profilin have been explored as possible drug targets and many of the major drugs against Fasciola target tubulin, thus cytoskeletal components present ideal drug targets (Fairweather et al., 2020; Kumpula & Kursula, 2015; Moens & Coumans, 2015). In the future, the actin-profilin interaction could be explored as possible drug target in Fasciola.

Profilin interacts with its many ligands via proline-rich sequences (Bjorkegren et al., 1993; Kursula et al., 2008b; Mahoney, Janmey & Almo, 1997). The majority of residues involved in polyproline binding are conserved in FhProfilin suggesting it interacts with a variety of partner proteins (Fig. 1A). To assess the ability of FhProfilin to interact with polyproline, it was subjected to pulldown assays using polyproline-sepharose beads (Fig. 4). FhProfilin was successfully detected in the bound fraction of the assay, whereas BSA was not (Fig. 4), showing that the interaction with polyproline-sepharose is specific to FhProfilin. Not all FhProfilin was bound to the resin, suggesting either the interaction was weak, or capacity of the resin was exceeded (Fig. 4). It appears that FhProfilin may bind to similar physiological substrates as human profilin and therefore is likely to be involved in other cellular functions such as ribonucleoparticle processing (Giesemann et al., 1999), mRNA splicing (Skare et al., 2003) and nuclear export (Stuven, Hartmann & Gorlich, 2003).

The ability of profilins to bind phosphatidylinositol lipids is important as it can regulate phosphoinositide metabolism and its ability to move from the membrane to the cytosol where it can interact with actin or other ligands (Chaudhary et al., 1998; Lambrechts et al., 2002). The phosphatidylinositol lipid specificity of FhProfilin was assessed using a mini phosphatidylinositol lipid array (Fig. 5). There was no detectable phosphatidylinositol lipid binding by FhProfilin even at the highest concentration of 500 μg/ml (Fig. 5B). However, the positive control protein Grip supplied with the array was positively identified binding to the appropriate lipid phosphatidylinositol 4, 5-bisphosphate (Fig. 5C) confirming the validity of the assay and suggesting that FhProfilin has very weak or no association with phosphatidylinositol lipids. It is not surprising that FhProfilin does not bind phosphatidylinositol lipids as only two out of five PIP-2 binding sites are conserved (Fig. S1), which are normally seen in other members of the profilin family (Munkhjargal et al., 2016). Human profilin has been shown to bind to PI(3,4)P₂, PI(3,4)P₂ and PI(3,4,5)P₃ which is silimar to bovine profilin that binds PI(3,4,5)P₃ and PI(4,5)P₂ with some binding to PI(3)P, PI(4)P, and PI(5)P (Kursula et al., 2008a; Lu et al., 1996). This different which has been observed between the more closely related profilins from the apicomplexan parasites Plasmodium and T. gondii that Plasmodium profilin can bind phosphatidylinositol lipids (PI(4)P, and PI(5)P) whereas T. gondii profilin cannot (Kucera et al., 2010; Kursula et al., 2008a). The lack of binding of FhProfilin to phosphatidylinositol lipids may suggest the diffenence in phosphatidylinositol lipid metabolism in F. hepatica.

Immune profile in cattle and sheep

Apicomplexan parasitic profilins have the ability to stimulate the immune system via toll-like receptor 11 (TLR11) due to the presence of a parasite-specific surface motif consisting of an acidic loop followed by a long β-hairpin insert (Fig. S1) (Kucera et al., 2010). Due to this immune modulation activity, profilins have been used as potential vaccine candidates and adjuvants, in particular against T. gondii and Eimeria spp. (Jang et al., 2011a, 2011b, 2011c; Tanaka et al., 2014). To ascertain if FhProfilin was exposed to the host immune system, pooled sera from experimentally Fasciola infected animals was tested via Western Blot (Fig. 6). Immune sera from infected animals consisting of the susceptible sheep breed (Merino), the F. gigantica-resistant Indonesian Thin Tail (ITT) sheep and cattle did not recognize the recombinant FhProfilin while native F. hepatica glutathione S-transferase (nFhGST), a major parasite excretory-secretory antigen, was recognized (LaCourse et al., 2012). This suggests that Fasciola profilin is not exposed to immune system during infection (Fig. 6). The lack of FhProfilin-specific antibodies from exposed animals is unexpected as profilin from other parasites such Babesia spp. (Munkhjargal et al., 2016) and Schistosoma japonicum do elicit an immune response post infection (Zhang et al., 2008). Further the use of electron microscopy to perform ultrastructural studies to determine where within the tegument FhProfilin is located and whether this protein is exposed to the host immune system would help to assess the validity of FhProfilin as a vaccine candidate. A lack of direct exposure on the tegument should not exclude FhProfilin as a vaccine candidate, as the “hidden” antigen vaccines are against the nematode Haemonchus contortus (LeJambre, Windon & Smith, 2008; Munn, 1997) and the tick Rhiphicephalus microplus (Willadsen & Kemp, 1988) demonstrate the commercial viability of targeting antigens of this nature, as long they are essential in function to the parasite. Profilin is essential for the survival of P. falciparum (Kursula et al., 2008a) and necessary for virulence of T. gondii (Plattner et al., 2008), suggesting that profilin could also be essential for the survival and pathogenesis of F. hepatica.