Midi kit

Ehcp112 complete gene was PCR amplified with specific primers containing BamHI and KpnI restriction sites (forward: 5′-GGGGTACCATGACAGCGATTGTTGTCGCTTTTTT-3′, reverse: 5′-CGGGATCCTTACTTATCGTTCGTCATCCTTGTAATCGATTGTATGATTGTAGAATTGG-3′) under the following conditions: 95°C 15 s, 60°C 30 s, 72°C 90 s during 30 cycles. Then, the PCR product was cloned into pJET1.2 plasmid (Fermentas) according to the manufacturer recommendations. The Ehcp112/pJET construction was digested and the Ehcp112 fragment was subcloned in the pExEhNeo plasmid (Hamann et al., 1995 (link)). This plasmid was amplified in E. coli DH5α, purified by Qiagen Midi Kit (Qiagen) and automatically sequenced. Transfection was performed as described (Avalos-Padilla et al., 2015 (link)). Transfected trophozoites were selected with increasing concentrations of G-418 (a neomycin analog) until stable growth was achieved (20 μg/ml). Overexpression of the Ehcp112 gene was verified by RT-PCR by specific primers and using s2 ribosomal gene as internal control. WB assays were performed using α-EhCP112 (1:5,000) and α-actin (1:3,000) antibodies. Laser confocal microscopy assays were performed using α-EhCP112 (1:100) antibodies in paraformaldehyde fixed and triton X-100 permeabilized trophozoites and nuclei were stained by propidium iodide.

Plasmid (pBluescript II SK (−) containing the T7 promoter; Stratagene, La Jolla, CA, USA: pBSII) DNA templates were purified using a QIAGEN Midi Kit (QIAGEN, Hilden, Germany). For the purpose of acetaldehyde treatment, DNA templates were incubated with 1 M acetaldehyde at 37 °C for 1 h. Although the boiling point of acetaldehyde is 20.2 °C, we used 37 °C that is an optimal temperature for commonly used enzymes. For UV treatment, UV-light (254 nm, 450 J/m²) was used. Next, the templates were purified using Sephadex G-25 columns according to the manufacturer’s instructions (GE Healthcare, Amersham, Buckinghamshire, UK).

The WGS data of the QBB study participants was provided by the Qatar Genome Project (QGP) [46 (link)]. Details of WGS and quality control measures have been described previously [47 (link)]. Briefly, genomic DNA from peripheral blood samples was extracted using Qiagen MIDI kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol and using the automated QIASymphony SP instrument (Qiagen). DNA integrity was evaluated using Caliper Labchip GXII (Perkin Elmer, Waltham, MA, USA) Genomic DNA assay and was quantified using the Quant-iT dsDNA Assay (Invitrogen, Waltham, MA, USA). Whole-genome libraries were prepared using Illumina TruSeq DNA Nano kit (Illumina, San Diego, CA, USA). HiSeq X Ten (Illumina) was used to sequence genomic libraries to achieve a minimum average coverage of 30×. Library construction and sequencing was performed at Sidra Clinical Genomics Laboratory Sequencing Facility (Sidra Medicine, Doha, Qatar). The generated files were subjected to quality control using FastQC (v0.11.2) and reads were aligned to the CRCh38 reference genome. Quality control on mapped reads was performed using Picard (v1.117). A combined variant call file (gVCF) was generated for all study subjects, which contained all genetic variations detected in the QBB study participants.

Genomic DNA was extracted using the QIAGEN Midi Kit (Qiagen, Hilden, Germany). The DNA of Sag27 was sequenced using the PacBio RS II (Pacific Biosciences, Menlo Park, CA, USA). The reads were de novo assembled using HGAP 3.0 SMRT™ Pipe. Sag3 and Sag4104 were sequenced using the Illumina HiSeq sequencing approach. SOAP de novo (http://soap.genomics.org.cn/, v2.01) was used for genome assembly. Sequence similarity was evaluated by BLAST searches (http://www.ncbi.nlm.nih.gov). Open reading frames (ORFs) were predicted using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Protein-coding genes were initially identified and annotated using RAST. Antimicrobial resistance genes were identified using ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/). Multi-locus sequence typing was identified using MLST 2.0 (https://cge.cbs.dtu.dk/services/MLST/).

PCR-amplified Ehvps32 full gene with the hemagglutinin (HA) tag in the 3’ end were cloned into pJET1.2/blunt plasmid (Fermentas), accordingly to manufacturer’s instructions. Then, Ehvps32-HA gene was subcloned into BamHI and KpnI sites of pExEhNeo (pNeo) plasmid [59 (link)], producing the pNeoEhvps32-HA construct. E. coli DH5α bacteria were transformed with pNeoEhvps32-HA or pNeo plasmids. Plasmids were purified using Qiagen Midi kit (Qiagen) and automatically sequenced. To perform transfection, 3 x 10⁵ trophozoites were cultivated ON with 5% CO₂, then, washed with M199 medium (Sigma) and incubated with M199 medium supplemented with 15% FBS. Subsequently, transfection mix (20 μg of plasmid, 20 μl of Superfect [Qiagen] in 100 μl of M199 medium) was added and incubated 10 min at RT. Trophozoites were cultivated for 3 h at 37°C and 5% CO₂. Finally, cells were cooled and transferred to a tube with 10 ml of TYI pre-warmed medium and cultivated for 12 h at 37°C.

Plasmid (pBluescript II SK (−) containing the T7 promoter; Stratagene, La Jolla, CA, USA) DNA templates were purified using the QIAGEN Midi Kit (QIAGEN, Hilden, Germany). For UV irradiation of DNA templates, UV-light (254 nm, 450 J/m²) was used. For cisplatin treatment, DNA templates were incubated with cisplatin (drug/nucleotide ratio = 0.005) at 37 °C overnight.

pcDNA3.1-v5-hYSK1 and deletion fragments of hYSK1 were generated by PCR using the human cDNA clone-hYSK1 (Origene Technologies, MD, USA) as a template. The PCR product was purified, digested with EcoRI/XhoI, and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen, MA, USA). For TOPO-GFP-hYSK1, the PCR product was inserted into the pcDNA3.1-NT-GFP-TOPO vector (Invitrogen, MA, USA). The GST-p16^INK4a and GST-deletion fragments of p16^INK4a were inserted in-frame into the BamHI/XhoI site of the pGEX-5X-1 vector (Amersham Biosciences, PA, USA). For pDsRed-p16^INK4a, the PCR product was cloned into the ApeI site of the pDsRed vector (Clontech Laboratories, CA, USA). HA-SP-1 was cloned into the EcoRI/XhoI site of the pCMV-HA and pCMV-Myc vectors (Clontech Laboratories, CA, USA). The shRNA-hYSK1 plasmid was constructed into the pSilencer 4.1-CMV-hyg vector (Ambion, NY, USA). The pGL2-p16-luc vector was a gift from Dr. Gordon Peters (Cancer Research UK, London) and the pGL2-MMP-2-luc vector was a gift from Dr. Etty N. Benveniste (The University of Alabama at Birmingham, Birmingham, AL). Various expression vectors were amplified in E. coli XL1-blue or BL21 cells and plasmids were purified using a Qiagen midi kit (Qiagen, Hilden, Germany). The DNA sequences of all plasmids were confirmed by sequencing (Dye Terminator ABI Type Seq., Bionex, NJ, USA)