Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii

<p>Abstract</p> <p>Background</p> <p><it>Sporothrix schenckii </it>is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in <it>S. schenckii </it>responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in <it>S. schenckii, sscmk1</it>, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle.</p> <p>Results</p> <p>Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of <it>sscmk1 </it>revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of <it>sscmk1 </it>revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition.</p> <p>Conclusion</p> <p>This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in <it>S. schenckii </it>and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus. The results suggest that the calcium/calmodulin kinases of yeasts are evolutionarily distinct from those in filamentous fungi.</p

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in -1

<p><b>Copyright information:</b></p><p>Taken from "Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in "</p><p>http://www.biomedcentral.com/1471-2180/7/107</p><p>BMC Microbiology 2007;7():107-107.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2242797.</p><p></p> the DASQTI/WSMGVI primer pair. Fifteen μl of each reaction were resolved in a 1.2% agarose gel electrophoresis. Lanes 3 and 6, show the results obtained from the RT-PCR using total RNA extracted from yeast and mycelium cells respectively, and show the 279 bp band. Lanes 2 and 5 represent the RT-PCR control for each time point where no M-MLV reverse transcriptase was added. Lanes 4 and 7 show a 363 bp product corresponding to a control PCR reaction using genomic DNA as template and the same primer pair. Lane 1 shows the 123 bp DNA Ladder. The position of each of the RT-PCR products is indicated by arrows

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in -7

<p><b>Copyright information:</b></p><p>Taken from "Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in "</p><p>http://www.biomedcentral.com/1471-2180/7/107</p><p>BMC Microbiology 2007;7():107-107.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2242797.</p><p></p>s, coding regions and amino acids are given in upper case letters. The derived amino acid sequence shows a protein of 407 amino acids. The invariant amino acids required by serine/threonine protein kinases are shaded in red. The potential autophosphorylation sites containing the consensus sequence {R, K}-X-X-{T, S} are shaded in light blue. The calmodulin-binding domain is shaded in gray

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in -6

<p><b>Copyright information:</b></p><p>Taken from "Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in "</p><p>http://www.biomedcentral.com/1471-2180/7/107</p><p>BMC Microbiology 2007;7():107-107.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2242797.</p><p></p> re-enter the budding cycle in a basal medium with glucose at pH 7.2 and incubated at 25°C in the presence and absence of W-7, KN-62 and lavendustin C. All values are given as the average percentage ± one SD of for at least three independent experiments. The Student's t test was used to determine the statistical significance of the data at a 95% confidence level. Values that differ significantly from those of the control at 95% confidence level are marked with an asterisk

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in -3

<p><b>Copyright information:</b></p><p>Taken from "Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in "</p><p>http://www.biomedcentral.com/1471-2180/7/107</p><p>BMC Microbiology 2007;7():107-107.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2242797.</p><p></p>hite letters indicates 75–99% identity, gray shading with black letters indicates 50–74% identity. The Ca/calmodulin binding domain is located on positions 300–319 of the alignment. The serine/threonine protein kinase domain is located between positions 22 and 280 of the alignment

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in -4

<p><b>Copyright information:</b></p><p>Taken from "Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in "</p><p>http://www.biomedcentral.com/1471-2180/7/107</p><p>BMC Microbiology 2007;7():107-107.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2242797.</p><p></p>e shown in red, the filamentous fungi CaMKs are shown in blue. The bootstrap values for each branch are shown (maximum value 1000)

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in -2

<p><b>Copyright information:</b></p><p>Taken from "Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in "</p><p>http://www.biomedcentral.com/1471-2180/7/107</p><p>BMC Microbiology 2007;7():107-107.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2242797.</p><p></p>he are shown in bold and underlined, and the type and location are shown

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in -8

<p><b>Copyright information:</b></p><p>Taken from "Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in "</p><p>http://www.biomedcentral.com/1471-2180/7/107</p><p>BMC Microbiology 2007;7():107-107.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2242797.</p><p></p> the DASQTI/WSMGVI primer pair. Fifteen μl of each reaction were resolved in a 1.2% agarose gel electrophoresis. Lanes 3 and 6, show the results obtained from the RT-PCR using total RNA extracted from yeast and mycelium cells respectively, and show the 279 bp band. Lanes 2 and 5 represent the RT-PCR control for each time point where no M-MLV reverse transcriptase was added. Lanes 4 and 7 show a 363 bp product corresponding to a control PCR reaction using genomic DNA as template and the same primer pair. Lane 1 shows the 123 bp DNA Ladder. The position of each of the RT-PCR products is indicated by arrows