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

Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

<p>Abstract</p> <p>Background</p> <p><it>Sporothrix schenckii </it>is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in <it>S. schenckii</it>.</p> <p>Results</p> <p>The presence of RNA interference (RNAi) mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in <it>S. schenckii </it>DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in <it>S. schenckii </it>dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G) plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (<it>sscmk1</it>) gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of <it>sscmk1 </it>gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90) as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM), an inhibitor of HSP90.</p> <p>Conclusions</p> <p>Using the RNAi technology we silenced the expression of <it>sscmk1 </it>gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These results confirmed SSCMK1 as an important enzyme involved in the dimorphism of <it>S. schenckii</it>, necessary for the development of the yeast phase of this fungus. Also this study constitutes the first report of the transformation of <it>S. schenckii </it>and the use of RNAi to study gene function in this fungus.</p

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 -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 -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 -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 -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

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P &lt; 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)