Enolase

Placental tissues were homogenized and prepared as described in Gel-based activity-based protein profiling (ABPP) section. The chemical proteomics workflow is based on previously published protocol (29 (link)) and conducted with minor modifications. In short, cytosolic and membrane fractions of placental tissue lysates (250 µg protein, 1 mg/mL, n=5) were incubated with serine hydrolase probe cocktail (10 µM MB108, 10 µM FP-Biotin, 30 min, 37 ˚C, 300 rpm). A pool of denatured vehicle control samples (1% SDS, 5 min, 100°C) was taken along as a negative control. Following steps were preformed according to protocol, including precipitation, alkylation, avidin enrichment, on-bead digestion, and sample preparation. Dried and desalted peptide samples were stored at -20°C until LC-MS analysis. Prior to measurement, samples were reconstituted in 50 µL 97:3:0.1 solution (H2O, ACN, FA) containing 10 fmol/µL yeast enolase digest (Waters, cat# 186002325) and transferred to LC-MS vials. Additionally, a quality control sample was prepared to prevent overloading the nanoLC system and the automatic gain control (AGC) of the QExactive HF mass spectrometer. LC-MS data was analyzed by MaxQuant software 2.0 applying match between runs. For further analysis, the following cut-offs were used: unique peptides ≥ 2, identified peptides ≥ 2, ratio positive over negative control ≥ 2. Additionally, targets were filtered against a putative probe-target list including human metabolic serine hydrolases.

The animal trial comprised 20 animals, with 10 animals being vaccinated twice with the recombinant Haemonchus vaccine at the 4-week interval and 10 animals serving as the control and receiving no treatment (Figure 1B and Table S1). The recombinant Haemonchus vaccine consisted of recombinant enolase, arginine kinase, ornithine decarboxylase, malate dehydrogenase, serly tRNA synthetase, macrophage inhibition factor-2, glutamyl tRNA synthetase, aspartyl tRNA synthetase, fatty acid synthetase thioesterase domain, transcriptional co-activator (the histone acetyltransferase-HAT-domain), and vacuolar ATPase, B subunit. All antigens were expressed in a bacterial expression system (data unpublished). The eleven recombinant proteins were combined and dialysed overnight at 4 °C. As described previously [41 (link)], the antigens were formulated in the QuilA and chitin-based slow-release formulation. Each lamb was subcutaneously vaccinated with 75 µg of the vaccine on each vaccination.
Two weeks after the second vaccination, all animals were infected with 5000 L3 H. contortus. Fecal egg counts were monitored twice weekly from day 16 post-infection until the end of the trial. Animals were bled and weighed weekly throughout the course of the trial. One control animal died because of an unrelated cause and was therefore not included in the sample collection or analysis. All 19 animals were killed 8 weeks post challenge, and abomasa were collected for the adult worm counts. Each abomasum was cut open, washed, and had 1/10 worms collected for worm counts. The remaining worms were collected as previously described [9 (link)]. Briefly, the abomasal contents were mixed 2:1 with 3% agar, and the solidified agar blocks incubated at 37 °C in a saline bath. Adult male H. contortus worms were collected from the saline soon after emergence and stored in cryovials in liquid N₂ for subsequent DNA and RNA isolation.

Annotated plasmid maps for all plasmids used in this study are provided in the Supplemental Material, and GenBank-style files are available upon request. All plasmids and strains used in this study are listed in Table S1. The first set of recombinase helper plasmids were constructed by GenScript, where the gene encoding λ Int in pINT-ts (8 (link)) was replaced with the synthesized serine recombinases that were previously described (7 (link)), resulting in plasmids pGSs037, 038, 040, 041, 042, 043, 044, 045, 046, 047, 048, 050, 053, 054, and 082 (Table S1). Recombinases were codon optimized for expression in E. coli.
For the second set of helper plasmids, recombinases were cloned under the C. thermocellum DSM 1313 enolase promoter (28 (link)) with a ribosome binding site modified to AGGAGGA. First, ΦC31 was synthesized with the enolase promoter into a pUC replicating vector. The promoter and recombinase DNA were then amplified by PCR using Phusion High Fidelity master mix (Thermo Scientific) and inserted using Gibson Assembly (New England Biolabs) into pINT-ts, replacing the native promoter and lambda recombinase, resulting in pLAR047. Using pLAR047 as a backbone, the remaining serine recombinases were cloned and inserted, replacing ΦC31, using Gibson Assembly, resulting in plasmids pLAR052-62 and 074 (Table S1).
The poly-attB cassette was synthesized by GenScript with all 14 attachment sites previously described (7 (link)) (att sites are listed in Table S2) except ΦC31 (plasmid pAMGs177). In order, the attB sites are Φ370, BT1, R4, BxB1, TP901-1, RV, SpβC, TG1, ΦC1, MR11, ΦK38, A118, Wβ, and BL3. The attB cassette was PCR amplified and inserted into the multiple cloning site of pAH144 (8 (link)) using Gibson Assembly, resulting in plasmid pLAR080. The attB cassette was then integrated using the CRIM system into the HK022 phage attB site (8 (link)) of: (i) E. coli Top 10 Δdcm::frt, (ii) E. coli WM3188 Δdcm::frt, and (iii) BW25113 ΔmcrA::frt ΔmcrC-mrr::frt Δdcm::frt Δdam::frt, creating strains AG2005, AG3525, and AG4277 respectively. The HK022 core attB site (33 (link)) is located between bases 1,051,926 to 1,051,940 in BW2115 (34 (link)) at sequence 5′- CTTTAGGTGAAAAAG -3′.
The base integration plasmid, pGSs009, was synthesized and constructed by GenScript. It was constructed from pAH55 (8 (link)) and a synthesized insert containing ΦC31 attB and attP sites flanking the T5lac promoter and lacZα. The promoter in pGSs009 was replaced with P_BAD from pLA2 (8 (link)) to create pLAR067. For recombinase efficiency experiments, a synthesized cassette containing 13 attP sites, excluding SpβC, was first inserted into the BamHI and NdeI sites of pGSs009 using Gibson Assembly. Next, oligonucleotides with the Spβ attP and homology to pGSs009 were inserted into the EcoO1091 site, creating pLAR031.
C. clariflavum methyltransferases (CloclaDRAFT _2368_2369, CloclaDRAFT _1830_1831, CloclaDRAFT _1994, CloclaDRAFT _1058, CloclaDRAFT _1051, CloclaDRAFT _1868_1869, CloclaDRAFT _2133, and CloclaDRAFT _1961) were codon optimized and synthesized by GenScript, each with a different attP site. Each methyltransferase was then cloned into pLAR067, under the control of P_BAD, using NEB builder, resulting in plasmids pMTV37-44 (Table S1).