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Evidence from bioinformatics, expression and inhibition studies of phosphoinositide-3 kinase signalling in Giardia intestinalis.

Cox SS, van der Giezen M, Tarr SJ, Crompton MR, Tovar J - BMC Microbiol. (2006)

Bottom Line: The inhibitory effect of the PI3K inhibitor LY294002 on trophozoite proliferation also supports their functionality.In addition, giardial genes encoding putative homologues of phosphoinositide-metabolising enzymes such as PTEN, MTM, PIPkin and PI 5-phosphatase as well as downstream effectors with phosphoinositide-binding domains have been identified, placing GiPI3K1 and GiPI3K2 in a broader signalling context.The presence of genes encoding putative homologues of phosphoinositide-metabolising enzymes and downstream effectors in the G. intestinalis genome further suggests that the overall architecture of PI3K signalling may be comparable with pathways present in other better-studied organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, UK. s.s.e.cox@rhul.ac.uk

ABSTRACT

Background: Giardia intestinalis is a parasitic protozoan and major cause of diarrhoeal disease. Disease transmission is dependent on the ability of the parasite to differentiate back and forth between an intestine-colonising trophozoite and an environmentally-resistant infective cyst. Our current understanding of the intracellular signalling mechanisms that regulate parasite replication and differentiation is limited, yet such information could suggest new methods of disease control. Phosphoinositide-3 kinase (PI3K) signalling pathways have a central involvement in many vital eukaryotic processes, such as regulation of cell growth, intracellular membrane trafficking and cell motility. Here we present evidence for the existence of functional PI3K intracellular signalling pathways in G. intestinalis.

Results: We have identified and characterised two genes, Gipi3k1 and Gipi3k2, which encode putative PI3Ks. Both genes are expressed in trophozoites and encysting cells, suggesting a possible role of GiPI3K1 and GiPI3K2 in regulating giardial growth and differentiation. Extensive nucleotide and amino acid sequence characterisation predicts that both encoded PI3Ks are functional as indicated by the presence of highly conserved structural domains and essential catalytic residues. The inhibitory effect of the PI3K inhibitor LY294002 on trophozoite proliferation also supports their functionality. Phylogenetic analysis supports the identity of GiPI3K1 as a Class I isoform and GiPI3K2 as a Class III isoform. In addition, giardial genes encoding putative homologues of phosphoinositide-metabolising enzymes such as PTEN, MTM, PIPkin and PI 5-phosphatase as well as downstream effectors with phosphoinositide-binding domains have been identified, placing GiPI3K1 and GiPI3K2 in a broader signalling context. Compared with twenty-six PI3Ks from other organisms, GiPI3K1 and GiPI3K2 are unique in that they contain large insertions within their highly conserved kinase domains. The function of these insertions is unknown; however we show here that they are not intron-derived and would probably not hinder substrate binding. These insertions may represent a plausible drug target.

Conclusion: G. intestinalis encodes and expresses two putative PI3Ks, at least one of which appears to be required during normal parasite proliferation. The identification of Class I and Class III but not Class II isoforms suggests that both extracellularly-initiated signalling (Class I-regulated) and intracellular vesicle trafficking (Class III-regulated) might be controlled by PI3Ks in G. intestinalis. The presence of genes encoding putative homologues of phosphoinositide-metabolising enzymes and downstream effectors in the G. intestinalis genome further suggests that the overall architecture of PI3K signalling may be comparable with pathways present in other better-studied organisms.

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PI3K domain pairwise alignments for GiPI3K1 and GiPI3K2. Pairwise sequence alignment of GiPI3K1 and GiPI3K2 indicates conservation in predicted Ras Binding (a), C2 (b) and PIK (c) domains. Identical aligned residues are indicated in black shading, while conserved residues are indicated by grey shading according to the PAM250 matrix [45]. Identity/similarity percentages for the GiPI3K1 alignments are 15%/45%, 12%/31% and 20%/51% for the GiPI3K1 RBD, C2 and PIK domains respectively, for the GiPI3K2 alignments these values are 13%/31% and 16%/47% for the GiPI3K2 C2 and PIK domains respectively.
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Figure 2: PI3K domain pairwise alignments for GiPI3K1 and GiPI3K2. Pairwise sequence alignment of GiPI3K1 and GiPI3K2 indicates conservation in predicted Ras Binding (a), C2 (b) and PIK (c) domains. Identical aligned residues are indicated in black shading, while conserved residues are indicated by grey shading according to the PAM250 matrix [45]. Identity/similarity percentages for the GiPI3K1 alignments are 15%/45%, 12%/31% and 20%/51% for the GiPI3K1 RBD, C2 and PIK domains respectively, for the GiPI3K2 alignments these values are 13%/31% and 16%/47% for the GiPI3K2 C2 and PIK domains respectively.

Mentions: Full-length and domain-only sequence similarity analysis of GiPI3K1 and GiPI3K2 demonstrated clear similarity with PI3Ks from other organisms, in particular GiPI3K1 is predicted to be similar to Class I PI3Ks and GiPI3K2 to Class III PI3Ks (Table 1, see also next section). Due to low conservation of sequence similarity amongst C2 domains from various PI3Ks, similar analysis carried out for the predicted C2 domains of GiPI3K1 and GiPI3K2 did not return significant matches to the C2 domains of other PI3Ks. However, C2 pairwise sequence alignments (Figure 2) demonstrated reasonable C2 domain similarity.


Evidence from bioinformatics, expression and inhibition studies of phosphoinositide-3 kinase signalling in Giardia intestinalis.

Cox SS, van der Giezen M, Tarr SJ, Crompton MR, Tovar J - BMC Microbiol. (2006)

PI3K domain pairwise alignments for GiPI3K1 and GiPI3K2. Pairwise sequence alignment of GiPI3K1 and GiPI3K2 indicates conservation in predicted Ras Binding (a), C2 (b) and PIK (c) domains. Identical aligned residues are indicated in black shading, while conserved residues are indicated by grey shading according to the PAM250 matrix [45]. Identity/similarity percentages for the GiPI3K1 alignments are 15%/45%, 12%/31% and 20%/51% for the GiPI3K1 RBD, C2 and PIK domains respectively, for the GiPI3K2 alignments these values are 13%/31% and 16%/47% for the GiPI3K2 C2 and PIK domains respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1483827&req=5

Figure 2: PI3K domain pairwise alignments for GiPI3K1 and GiPI3K2. Pairwise sequence alignment of GiPI3K1 and GiPI3K2 indicates conservation in predicted Ras Binding (a), C2 (b) and PIK (c) domains. Identical aligned residues are indicated in black shading, while conserved residues are indicated by grey shading according to the PAM250 matrix [45]. Identity/similarity percentages for the GiPI3K1 alignments are 15%/45%, 12%/31% and 20%/51% for the GiPI3K1 RBD, C2 and PIK domains respectively, for the GiPI3K2 alignments these values are 13%/31% and 16%/47% for the GiPI3K2 C2 and PIK domains respectively.
Mentions: Full-length and domain-only sequence similarity analysis of GiPI3K1 and GiPI3K2 demonstrated clear similarity with PI3Ks from other organisms, in particular GiPI3K1 is predicted to be similar to Class I PI3Ks and GiPI3K2 to Class III PI3Ks (Table 1, see also next section). Due to low conservation of sequence similarity amongst C2 domains from various PI3Ks, similar analysis carried out for the predicted C2 domains of GiPI3K1 and GiPI3K2 did not return significant matches to the C2 domains of other PI3Ks. However, C2 pairwise sequence alignments (Figure 2) demonstrated reasonable C2 domain similarity.

Bottom Line: The inhibitory effect of the PI3K inhibitor LY294002 on trophozoite proliferation also supports their functionality.In addition, giardial genes encoding putative homologues of phosphoinositide-metabolising enzymes such as PTEN, MTM, PIPkin and PI 5-phosphatase as well as downstream effectors with phosphoinositide-binding domains have been identified, placing GiPI3K1 and GiPI3K2 in a broader signalling context.The presence of genes encoding putative homologues of phosphoinositide-metabolising enzymes and downstream effectors in the G. intestinalis genome further suggests that the overall architecture of PI3K signalling may be comparable with pathways present in other better-studied organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, UK. s.s.e.cox@rhul.ac.uk

ABSTRACT

Background: Giardia intestinalis is a parasitic protozoan and major cause of diarrhoeal disease. Disease transmission is dependent on the ability of the parasite to differentiate back and forth between an intestine-colonising trophozoite and an environmentally-resistant infective cyst. Our current understanding of the intracellular signalling mechanisms that regulate parasite replication and differentiation is limited, yet such information could suggest new methods of disease control. Phosphoinositide-3 kinase (PI3K) signalling pathways have a central involvement in many vital eukaryotic processes, such as regulation of cell growth, intracellular membrane trafficking and cell motility. Here we present evidence for the existence of functional PI3K intracellular signalling pathways in G. intestinalis.

Results: We have identified and characterised two genes, Gipi3k1 and Gipi3k2, which encode putative PI3Ks. Both genes are expressed in trophozoites and encysting cells, suggesting a possible role of GiPI3K1 and GiPI3K2 in regulating giardial growth and differentiation. Extensive nucleotide and amino acid sequence characterisation predicts that both encoded PI3Ks are functional as indicated by the presence of highly conserved structural domains and essential catalytic residues. The inhibitory effect of the PI3K inhibitor LY294002 on trophozoite proliferation also supports their functionality. Phylogenetic analysis supports the identity of GiPI3K1 as a Class I isoform and GiPI3K2 as a Class III isoform. In addition, giardial genes encoding putative homologues of phosphoinositide-metabolising enzymes such as PTEN, MTM, PIPkin and PI 5-phosphatase as well as downstream effectors with phosphoinositide-binding domains have been identified, placing GiPI3K1 and GiPI3K2 in a broader signalling context. Compared with twenty-six PI3Ks from other organisms, GiPI3K1 and GiPI3K2 are unique in that they contain large insertions within their highly conserved kinase domains. The function of these insertions is unknown; however we show here that they are not intron-derived and would probably not hinder substrate binding. These insertions may represent a plausible drug target.

Conclusion: G. intestinalis encodes and expresses two putative PI3Ks, at least one of which appears to be required during normal parasite proliferation. The identification of Class I and Class III but not Class II isoforms suggests that both extracellularly-initiated signalling (Class I-regulated) and intracellular vesicle trafficking (Class III-regulated) might be controlled by PI3Ks in G. intestinalis. The presence of genes encoding putative homologues of phosphoinositide-metabolising enzymes and downstream effectors in the G. intestinalis genome further suggests that the overall architecture of PI3K signalling may be comparable with pathways present in other better-studied organisms.

Show MeSH
Related in: MedlinePlus