Eimeria maxima phosphatidylinositol 4-phosphate 5-kinase: locus sequencing, characterization, and cross-phylum comparison

Abstract

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) may play an important role in host-cell invasion by the Eimeria species, protozoan parasites which can cause severe intestinal disease in livestock. Here, we report the structural organization of the PIP5K gene in Eimeria maxima (Weybridge strain). Two E. maxima BAC clones carrying the E. maxima PIP5K (EmPIP5K) coding sequences were selected for shotgun sequencing, yielding a 9.1-kb genomic segment. The EmPIP5K coding region was initially identified using in silico gene-prediction approaches and subsequently confirmed by mapping rapid amplification of cDNA ends and RT-PCR-generated cDNA sequence to its genomic segment. The putative EmPIP5K gene was located at position 710-8036 nt on the complimentary strand and comprised of 23 exons. Alignment of the 1147 amino acid sequence with previously annotated PIP5K proteins from other Apicomplexa species detected three conserved motifs encompassing the kinase core domain, which has been shown by previous protein deletion studies to be necessary for PIP5K protein function. Phylogenetic analysis provided further evidence that the putative EmPIP5K protein is orthologous to that of other Apicomplexa. Subsequent comparative gene structure characterization revealed events of intron loss/gain throughout the evolution of the apicomplexan PIP5K gene. Further scrutiny of the genomic structure revealed a possible trend towards "intron gain" between two of the motif regions. Our findings offer preliminary insights into the structural variations that have occurred during the evolution of the PIP5K locus and may aid in understanding the functional role of this gene in the cellular biology of apicomplexan parasites.

Sequence analysis of the PIP5K locus in Eimeria maxima provides further evidence for eimerian genome plasticity and segmental organization

“…A 490-bp probe derived from the E. tenella PIP5K genomic region was used to screen an EmW BAC library that was constructed in the pBACe3.6 vector, as described previously (Goh et al, 2011). Three BACs with homology to the probe, namely 1P2, 7C17, and 7K21, were identified.…”

“…By excluding the redundant clone 7C17, clones 1P2 and 7K21 were selected for DNA extraction and small insert library construction. After polymerase chain reaction (PCR) verification using the primers described by Goh et al (2011), these clones were restriction enzyme digested and mapped using BamHI, EcoRI, and HindIII, and fractionated by electrophoresis on 1% agarose gel with 1X TAE buffer at 10 V for 20 h. The restriction fragment patterns were analyzed by generating a digital fingerprint map to establish the extent of overlapping BAC clones. Common fragments observed on the agarose gel were recorded, and detailed restriction maps of BACs 1P2 and 7K21 were constructed.…”

“…Among the potential target proteins, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) has previously been reported to play important roles in diverse biological processes, including signal transduction, cell secretion, vesicular trafficking, and regulation of cytoskeleton assembly (Kunz et al, 2000). For the Eimeria genus, PIP5K coding regions have been described for Eimeria tenella (Ling et al, 2007a) and more recently for E. maxima (Goh et al, 2011), including analysis of the E. maxima (Weybridge strain) PIP5K gene structure, comparison with related apicomplexans, and structural variations that have occurred during the evolution of the locus.…”