Identification of New Sphingomyelinases D in Pathogenic Fungi and Other Pathogenic Organisms

Identification of New Sphingomyelinases D in Pathogenic Fungi and Other Pathogenic Organisms Camila Dias-Lopes1☯, Izabella A. P. Neshich2☯, Goran Neshich3, José Miguel Ortega1, Claude Granier4, Carlos Chávez-Olortegui1, Franck Molina4, Liza Felicori1* 1 Departamento de Bioquímica-Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, 2 Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil, 3 Embrapa Information Technologies, Campinas, SP, Brazil, 4 SysDiag UMR 3145 CNRS/BioRad, Cap Delta, Montpellier, France Abstract Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms. Citation: Dias-Lopes C, Neshich IAP, Neshich G, Ortega JM, Granier C, et al. (2013) Identification of New Sphingomyelinases D in Pathogenic Fungi and Other Pathogenic Organisms. PLoS ONE 8(11): e79240. doi:10.1371/journal.pone.0079240 Editor: Eugene A. Permyakov, Russian Academy of Sciences, Institute for Biological Instrumentation, Russian Federation Received August 2, 2013; Accepted September 27, 2013; Published November 1, 2013 Copyright: © 2013 Dias-Lopes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by INCTTOX PROGRAM of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Centre national de la recherche scientifique (CNRS) - Projet International de Coopération Scientifique [PICS no. 2474] and Coodernação de aperfeiçoamento de pessoal de nivel superior, Brazil – CAPES (Toxinologia no. 23038000825/2011-63). Izabella Pena Neshich received a PhD fellowship from FAPESP– 12/00235-5 and Goran Neshich received support from FAPESP (research project number 2009/16376-4). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ☯ These authors contributed equally to this work. Introduction homologous enzymes in the fungi Aspergillus and Coccidioides (UniProt accession numbers Q2UAL9, Q2UKE8, Q2U8X2 and Sphingomyelinases D (SMases D; EC 3.1.4.41), also known Q1DU31) [11]. Intrigued by the presence of this toxic enzyme as sphingomyelin phosphodieasterases D and sometimes as in medically important but distantly related organisms, such as phospholipase D (PLD) [1], catalyze the hydrolytic cleavage of spiders and bacteria, Cordes and Binford [12] identified a sphingomyelin or many other lysophospholipids to produce common motif at the C-terminal end of SMase D (without choline and ceramide 1-phosphate or choline and known function), supporting their inference about the origins of lysophosphatidic acid (LPA), respectively [2,3]. These enzymes these enzymes from the broadly conserved glycerophosphoryl are found in the venom of spiders from the Sicariidae family diester phosphodiesterase (GDPD; EC 3.1.4.46) family, in and are responsible for the outcome of local dermonecrosis in which, however, this motif is absent. victims that suffer spider bites, as well as the systemic toxic In the present work, using a bioinformatics sequence effects, such as kidney failure and death [4–8]. Proteins with similarity search methodology, we identified several new SMase D activity have been identified in arachnids, in the SMases D in different pathogenic organisms, such as spiders, saliva of a tick from the genus Ixodes [9] and in a few strains of bacteria, ticks, mites and fungi. A significant number of Corynebacteria and Arcanobacterium [10]. Recently, BLAST pathogenic fungal species were found to contain SMase D-like searches, though not accompanied with corresponding sequences, presenting strictly conserved catalytically critical experimental evidence, have revealed the presence of amino acids. Thus, for the first time, an SMase D activity was PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e79240 Sphingomyelinases D in Pathogenic Organisms experimentally demonstrated in a fungi. We also infer the The phylogenetic analysis was performed using one function of the C-terminal conserved motif (SMD-tail) in representative putative SMase D sequence for each genus. stabilizing the entire internal structure of the SMase D TIM Clustal W was used for the multiple sequence alignments and barrel. This work suggests that SMases D are widely MEGA 4 [18] for the creation of Maximum Likelihood represented in several genera and may act as a common phylogenetic trees. The Jones, Taylor and Thornton (JTT) pathogenic effector for a significant diversity of organisms. model [19] and the γ-based method were used for correcting the heterogeneity rates among sites. Bootstrap replicates Methods (1000) were calculated to access the confidence of the tree. SMase D sequence similarity search and ortholog SMase D structural analysis identification To gain insights about the differences and similarities of the A bidirectional best hit (BBH) approach for automated protein enzymes from the different organisms, structural bioinformatics sequence similarity searches was performed using the SMase analyses were performed. First, the 3D structures of D protein sequences from Loxosceles laeta (GI: 60594084) Corynebacterium pseudotuberculosis and Aspergillus flavus and Corynebacterium pseudotuberculosis (GI: 300857446) as SMase D (sequence GIs: 300857446 and 220691453) were queries. The searches started with 5 iterations of PSI-BLAST predicted and comparisons of the generated models with the [13] against a downloaded NCBI nr protein database (release crystallographic structure of SMase D 1 from Loxosceles laeta number?). The sequences found as hits at the 5th iteration were performed with previously predicting secondary structure (where the e-value was set to be better than the 1×10-5 elements in the target sequences using Jpred [20]. Homology threshold) were selected for further examination. To avoid models were built using the YASARA molecular modeling misinterpretation of the PSI-BLAST results, the bidirectional package [21]. For all modeling, the “hm_build” macro of the best hit function was used to carefully select only the true YASARA package was used with the default parameters positives. The protein sequences found as hits were used as except for the oligomerization state, which was set to queries in a BLASTp search against a test set database “monomeric”. The crystal structure of L. laeta (PDB code 1XX1) containing only the true positive (SMases D from was used as a template. The models were initially refined using Corynebacterium, Loxosceles and Ixodes) and true negative YASARA and the system containing a protein immersed in a sequences (paralogs such as GDPD from diverse organisms) – water box was optimized using energy minimization to remove both recovered from the BRENDA databank [14]. The protein any steric clashes and, later, using the molecular dynamics sequences that matched the true positive sequences as best YASARA macro (“md_run”) during 1ns. The default simulation hits were classified as true orthologs and those that matched parameters were maintained at the values defined by the true negatives were not considered for further analysis. The macro. The simulation used the AMBER03 force field. The above described search was not restricted only to the protein modeled structures were then examined and the best databases. In fact, a tBLASTn against the nucleotide database, representative structure was chosen based on the calculated which does not have a direct mapping to protein databases, energy of the structure by YASARA. The quality of the final was also performed. The subject databases chosen were: model was also checked using Prosa-Web [22], space-clash NCBI WGS (Whole Genome Shotgun sequences http:// analysis and Ramachandran plot analysis using STING Java www.ncbi.nlm.nih.gov/genbank/wgs), NCBI dbEST (database Protein Dossier [23,24]. Structural alignments were calculated of Expressed Sequence Tags http://www.ncbi.nlm.nih.gov/ using the software MUSTANG [25]. SMase I (PDB code 1XX1) dbEST/) and NCBI TSA (Transcriptome Shotgun Assembly image rendering was performed using the STING Java Protein http://www.ncbi.nlm.nih.gov/genbank/tsa), all of which were Dossier, SwissPDB-Viewer

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