Limits...
Comparative genomics of pneumocystis species suggests the absence of genes for myo-inositol synthesis and reliance on inositol transport and metabolism.

Porollo A, Sesterhenn TM, Collins MS, Welge JA, Cushion MT - MBio (2014)

Bottom Line: The underrepresentation of amino acid metabolism pathways compared to those in S. pombe, as well as the incomplete steroid biosynthesis pathway, were confirmed for P. carinii and P. jirovecii and extended to P. murina.The ITR and inositol metabolism genes in P. murina and P. carinii were expressed during fulminant infection as determined by reverse transcriptase real-time PCR of cDNA from infected lung tissue.These fungal infections are not responsive to standard antifungal therapy.

View Article: PubMed Central - PubMed

Affiliation: Center for Autoimmune Genomics and Etiology, Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA alexey.porollo@cchmc.org melanie.cushion@uc.edu.

Show MeSH

Related in: MedlinePlus

Representative products from reverse transcriptase real-time PCR of genes in the inositol phosphate metabolism pathway that were found to be conserved between P. murina and P. carinii but were not present in S. pombe. All products were found to be of the expected size, and all negative controls (mouse/rat cDNA or no cDNA) were negative. (A) P. murina inositol-polyphosphate multikinase, mouse cDNA negative control, and no-cDNA negative control. (B) P. murina inositol-pentakisphosphate 2-kinase, mouse cDNA negative control, and no-cDNA negative control. (C) P. murina phosphoinositide 5-phosphatase, mouse cDNA negative control, and no-cDNA negative control. (D) P. murina inositol-1,4-bisphosphate 1-phosphatase, mouse cDNA negative control, and no-cDNA negative control. (E) P. carinii inositol-polyphosphate multikinase, rat cDNA negative control, and no-cDNA negative control. (F) P. carinii inositol-pentakisphosphate 2-kinase, rat cDNA negative control, and no-cDNA negative control. (G) P. carinii phosphoinositide 5-phosphatase, rat cDNA negative control, and no-cDNA negative control. (H) P. carinii inositol-1,4-bisphosphate 1-phosphatase, rat cDNA negative control, and no-cDNA negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4222102&req=5

fig4: Representative products from reverse transcriptase real-time PCR of genes in the inositol phosphate metabolism pathway that were found to be conserved between P. murina and P. carinii but were not present in S. pombe. All products were found to be of the expected size, and all negative controls (mouse/rat cDNA or no cDNA) were negative. (A) P. murina inositol-polyphosphate multikinase, mouse cDNA negative control, and no-cDNA negative control. (B) P. murina inositol-pentakisphosphate 2-kinase, mouse cDNA negative control, and no-cDNA negative control. (C) P. murina phosphoinositide 5-phosphatase, mouse cDNA negative control, and no-cDNA negative control. (D) P. murina inositol-1,4-bisphosphate 1-phosphatase, mouse cDNA negative control, and no-cDNA negative control. (E) P. carinii inositol-polyphosphate multikinase, rat cDNA negative control, and no-cDNA negative control. (F) P. carinii inositol-pentakisphosphate 2-kinase, rat cDNA negative control, and no-cDNA negative control. (G) P. carinii phosphoinositide 5-phosphatase, rat cDNA negative control, and no-cDNA negative control. (H) P. carinii inositol-1,4-bisphosphate 1-phosphatase, rat cDNA negative control, and no-cDNA negative control.

Mentions: Reverse transcriptase real-time PCR was used to confirm the expression of the four Pneumocystis-specific genes in the inositol phosphate metabolism (IPM) pathway. Since P. jirovecii samples were not available for this study, the experimental results pertain to rodent Pneumocystis species only. When possible, the primers were designed to span an intron junction so that they would amplify cDNA exclusively. The threshold cycle (CT) value was determined by averaging the threshold cycles for the triplicate reactions. The ΔCT value between the IPM pathway gene and the thymidylate synthase (TS) gene was calculated by subtracting the average CT value of the single-copy reference gene, encoding TS, from the average CT value of the IPM gene (Table 2). This comparison was made to evaluate the expression levels of the four genes as they related to this single-copy gene/enzyme from Pneumocystis. No change in the ΔCT value would indicate that the level of expression of the gene of interest was similar to that of TS. A positive ΔCT value would indicate that the gene was expressed at a lower level than the reference gene. Conversely, a negative ΔCT value would indicate that the gene was expressed at a higher level than the reference gene. All genes except the inositol-1,4-bisphosphate 1-phosphatase gene were found to be expressed at higher levels than the TS gene in both P. murina and P. carinii. Inositol-1,4-bisphosphate 1-phosphatase, while still expressed, was expressed at a lower level than TS in both P. murina and P. carinii. Bands of the expected size were observed when representative products were run on an agarose gel, verifying that all genes were expressed (Fig. 4).


Comparative genomics of pneumocystis species suggests the absence of genes for myo-inositol synthesis and reliance on inositol transport and metabolism.

Porollo A, Sesterhenn TM, Collins MS, Welge JA, Cushion MT - MBio (2014)

Representative products from reverse transcriptase real-time PCR of genes in the inositol phosphate metabolism pathway that were found to be conserved between P. murina and P. carinii but were not present in S. pombe. All products were found to be of the expected size, and all negative controls (mouse/rat cDNA or no cDNA) were negative. (A) P. murina inositol-polyphosphate multikinase, mouse cDNA negative control, and no-cDNA negative control. (B) P. murina inositol-pentakisphosphate 2-kinase, mouse cDNA negative control, and no-cDNA negative control. (C) P. murina phosphoinositide 5-phosphatase, mouse cDNA negative control, and no-cDNA negative control. (D) P. murina inositol-1,4-bisphosphate 1-phosphatase, mouse cDNA negative control, and no-cDNA negative control. (E) P. carinii inositol-polyphosphate multikinase, rat cDNA negative control, and no-cDNA negative control. (F) P. carinii inositol-pentakisphosphate 2-kinase, rat cDNA negative control, and no-cDNA negative control. (G) P. carinii phosphoinositide 5-phosphatase, rat cDNA negative control, and no-cDNA negative control. (H) P. carinii inositol-1,4-bisphosphate 1-phosphatase, rat cDNA negative control, and no-cDNA negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222102&req=5

fig4: Representative products from reverse transcriptase real-time PCR of genes in the inositol phosphate metabolism pathway that were found to be conserved between P. murina and P. carinii but were not present in S. pombe. All products were found to be of the expected size, and all negative controls (mouse/rat cDNA or no cDNA) were negative. (A) P. murina inositol-polyphosphate multikinase, mouse cDNA negative control, and no-cDNA negative control. (B) P. murina inositol-pentakisphosphate 2-kinase, mouse cDNA negative control, and no-cDNA negative control. (C) P. murina phosphoinositide 5-phosphatase, mouse cDNA negative control, and no-cDNA negative control. (D) P. murina inositol-1,4-bisphosphate 1-phosphatase, mouse cDNA negative control, and no-cDNA negative control. (E) P. carinii inositol-polyphosphate multikinase, rat cDNA negative control, and no-cDNA negative control. (F) P. carinii inositol-pentakisphosphate 2-kinase, rat cDNA negative control, and no-cDNA negative control. (G) P. carinii phosphoinositide 5-phosphatase, rat cDNA negative control, and no-cDNA negative control. (H) P. carinii inositol-1,4-bisphosphate 1-phosphatase, rat cDNA negative control, and no-cDNA negative control.
Mentions: Reverse transcriptase real-time PCR was used to confirm the expression of the four Pneumocystis-specific genes in the inositol phosphate metabolism (IPM) pathway. Since P. jirovecii samples were not available for this study, the experimental results pertain to rodent Pneumocystis species only. When possible, the primers were designed to span an intron junction so that they would amplify cDNA exclusively. The threshold cycle (CT) value was determined by averaging the threshold cycles for the triplicate reactions. The ΔCT value between the IPM pathway gene and the thymidylate synthase (TS) gene was calculated by subtracting the average CT value of the single-copy reference gene, encoding TS, from the average CT value of the IPM gene (Table 2). This comparison was made to evaluate the expression levels of the four genes as they related to this single-copy gene/enzyme from Pneumocystis. No change in the ΔCT value would indicate that the level of expression of the gene of interest was similar to that of TS. A positive ΔCT value would indicate that the gene was expressed at a lower level than the reference gene. Conversely, a negative ΔCT value would indicate that the gene was expressed at a higher level than the reference gene. All genes except the inositol-1,4-bisphosphate 1-phosphatase gene were found to be expressed at higher levels than the TS gene in both P. murina and P. carinii. Inositol-1,4-bisphosphate 1-phosphatase, while still expressed, was expressed at a lower level than TS in both P. murina and P. carinii. Bands of the expected size were observed when representative products were run on an agarose gel, verifying that all genes were expressed (Fig. 4).

Bottom Line: The underrepresentation of amino acid metabolism pathways compared to those in S. pombe, as well as the incomplete steroid biosynthesis pathway, were confirmed for P. carinii and P. jirovecii and extended to P. murina.The ITR and inositol metabolism genes in P. murina and P. carinii were expressed during fulminant infection as determined by reverse transcriptase real-time PCR of cDNA from infected lung tissue.These fungal infections are not responsive to standard antifungal therapy.

View Article: PubMed Central - PubMed

Affiliation: Center for Autoimmune Genomics and Etiology, Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA alexey.porollo@cchmc.org melanie.cushion@uc.edu.

Show MeSH
Related in: MedlinePlus