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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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Inhibition of trophozoite proliferation by a PI3K inhibitor. a) Trophozoites treated with increasing concentrations of LY294002 were counted after 48 hours of treatment. b) To test the specificity of inhibition, the effect of LY294002 (50 μM) and DRB (100 μM final concentration) on exponentially growing trophozoites was monitored by cell counting over a 48 hour period. Error bars indicate the standard deviation of 3 counts. Asterisks denote significance at p < 0.001 for normalised data.
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Figure 8: Inhibition of trophozoite proliferation by a PI3K inhibitor. a) Trophozoites treated with increasing concentrations of LY294002 were counted after 48 hours of treatment. b) To test the specificity of inhibition, the effect of LY294002 (50 μM) and DRB (100 μM final concentration) on exponentially growing trophozoites was monitored by cell counting over a 48 hour period. Error bars indicate the standard deviation of 3 counts. Asterisks denote significance at p < 0.001 for normalised data.

Mentions: To determine the functional role of putative PI3Ks in G. intestinalis growth, we applied a commonly used PI3K inhibitor, LY294002, on G. intestinalis trophozoites. We tested a range of concentrations around those shown to selectively inhibit mammalian PI3Ks [26]. Figure 8a shows a dose-response of exponentially growing cells to the inhibitor, with concentrations of LY294002 as low as 25 μM causing a significant inhibitory effect on cell number as compared with the untreated control. Approximately 50% inhibition of cell proliferation occurred at concentrations between 25 and 75 μM. This effect is likely to be PI3K-mediated, since LY294002 concentrations within the 50 – 100 μM range have been employed for selective PI3K inhibition in mammalian cells. To understand the time course over which LY294002 exhibited its effects, we counted 50 μM LY294002 – treated cells at regular intervals over a 48 hour period. Figure 8b demonstrates that LY294002 begins to significantly effect cell number 8 hours into treatment. For the duration of the time-course, cell number remains approximately constant, whilst the untreated control continues to grow exponentially. This suggests that LY294002 may affect cell proliferation by inducing cell cycle arrest. In addition, trophozoites treated with LY294002 do not undergo any dramatic changes in their morphology or motility, thus further demonstrating the selective effect of LY294002 on Giardia's cell cycle.


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)

Inhibition of trophozoite proliferation by a PI3K inhibitor. a) Trophozoites treated with increasing concentrations of LY294002 were counted after 48 hours of treatment. b) To test the specificity of inhibition, the effect of LY294002 (50 μM) and DRB (100 μM final concentration) on exponentially growing trophozoites was monitored by cell counting over a 48 hour period. Error bars indicate the standard deviation of 3 counts. Asterisks denote significance at p < 0.001 for normalised data.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Inhibition of trophozoite proliferation by a PI3K inhibitor. a) Trophozoites treated with increasing concentrations of LY294002 were counted after 48 hours of treatment. b) To test the specificity of inhibition, the effect of LY294002 (50 μM) and DRB (100 μM final concentration) on exponentially growing trophozoites was monitored by cell counting over a 48 hour period. Error bars indicate the standard deviation of 3 counts. Asterisks denote significance at p < 0.001 for normalised data.
Mentions: To determine the functional role of putative PI3Ks in G. intestinalis growth, we applied a commonly used PI3K inhibitor, LY294002, on G. intestinalis trophozoites. We tested a range of concentrations around those shown to selectively inhibit mammalian PI3Ks [26]. Figure 8a shows a dose-response of exponentially growing cells to the inhibitor, with concentrations of LY294002 as low as 25 μM causing a significant inhibitory effect on cell number as compared with the untreated control. Approximately 50% inhibition of cell proliferation occurred at concentrations between 25 and 75 μM. This effect is likely to be PI3K-mediated, since LY294002 concentrations within the 50 – 100 μM range have been employed for selective PI3K inhibition in mammalian cells. To understand the time course over which LY294002 exhibited its effects, we counted 50 μM LY294002 – treated cells at regular intervals over a 48 hour period. Figure 8b demonstrates that LY294002 begins to significantly effect cell number 8 hours into treatment. For the duration of the time-course, cell number remains approximately constant, whilst the untreated control continues to grow exponentially. This suggests that LY294002 may affect cell proliferation by inducing cell cycle arrest. In addition, trophozoites treated with LY294002 do not undergo any dramatic changes in their morphology or motility, thus further demonstrating the selective effect of LY294002 on Giardia's cell cycle.

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