We report the comparative proteomic characterization of the venoms of adult and newborn specimens of the lancehead pitviper Bothrops asper from two geographically isolated populations from the Caribbean and the Pacific versants of Costa Rica. The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The two B. asper populations, separated since the late Miocene or early Pliocene (8-5 mya) by the Guanacaste Mountain Range, Central Mountain Range, and Talamanca Mountain Range, contain both identical and different (iso)enzymes from the PLA 2 , serine proteinase, and SVMP families. Using a similarity coefficient, we estimate that the similarity of venom proteins between the two B. asper populations may be around 52%. Compositional differences between venoms among different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. To investigate venom variability among specimens from the two B. asper populations, the reverse-phase HPLC protein profiles of 15 venoms from Caribbean specimens and 11 venoms from snakes from Pacific regions were compared. Within each B. asper geographic populations, all major venom protein families appeared to be subjected to individual variations. The occurrence of intraspecific individual allopatric variability highlights the concept that a species, B. asper in our case, should be considered as a group of metapopulations. Analysis of pooled venoms of neonate specimens from Caribbean and Pacific regions with those of adult snakes from the same geographical habitat revealed prominent ontogenetic changes in both geographical populations. Major ontogenetic changes appear to be a shift from a PIII-SVMP-rich to a PI-SVMP-rich venom and the secretion in adults of a distinct set of PLA 2 molecules than in the neonates. In addition, the ontogenetic venom composition shift results in increasing venom complexity, indicating that the requirement for the venom to immobilize prey and initiate digestion may change with the size (age) of the snake. Besides ecological and taxonomical implications, the geographical venom variability reported here may have an impact in the treatment of bite victims and in the selection of specimens for antivenom production. The occurrence of intraspecies variability in the biochemical composition and symptomatology after envenomation by snakes from different gegraphical location and age has long been apreciated by herpetologist and toxinologists, though detailed comparative proteomic analysis are scarce. Our study represents the first detailed characterization of individual and ontogenetic venom protein profile variations in two geographical isolated B. asper populations, and highlights the necessity of using pooled venoms as a statistically representative venom for antivenom production.

We report a comparative venomic and antivenomic characterization of the venoms of newborn and adult specimens of the Central American rattlesnake, Crotalus simus, and of the subspecies cumanensis, durissus, ruruima, and terrificus of South American Crotalus durissus. Neonate and adult C. simus share about 50% of their venom proteome. The venom proteome of 6-week-old C. simus is predominantly made of the neurotoxic heterodimeric phospholipase A(2) (PLA(2) crotoxin) (55.9%) and serine proteinases (36%), whereas snake venom Zn(2+)-metalloproteinases (SVMPs), exclusively of class PIII, represent only 2% of the total venom proteins. In marked contrast, venom from adult C. simus comprises toxins from 7 protein families. A large proportion (71.7%) of these toxins are SVMPs, two-thirds of which belong to the PIII class. These toxin profiles correlate well with the overall biochemical and pharmacological features of venoms from adult (hemorrhagic) and newborn (neurotoxic) C. simus specimens. The venoms of the South American Crotalus subspecies belong to one of two distinct phenotypes. C. d. cumanensis exhibits high levels of SVMPs and low lethal potency (LD(50)), whereas C. d. subspecies terrificus, ruruima, and durissus have low SVMP activity and high neurotoxicity to mice. Their overall toxin compositions explain the outcome of envenomation by these species. Further, in all C. simus and C. durissus venoms, the concentration of neurotoxins (crotoxin and crotamine) is directly related with lethal activity, whereas lethality and metalloproteinase activity show an inverse relationship. The similar venom toxin profiles of newborn C. simus and adult C. durissus terrificus, ruruima, and durissus subspecies strongly suggests that the South American taxa have retained juvenile venom characteristics in the adult form (paedomorphism) along their North-South stepping-stone dispersal. The driving force behind paedomorphism is often competition or predation pressure. The increased concentration of the neurotoxins crotoxin and crotamine in South American rattlesnake venoms strongly argues that the gain of neurotoxicity and lethal venom activities to mammals may have represented the key axis along which overall venom toxicity has evolved during Crotalus durissus invasion of South America. The paedomorphic trend is supported by a decreasing LNC (lethal neurotoxicity coefficient, defined as the ratio between the average LD(50) of the venom and the crotoxin + crotamine concentration) along the North-South axis, coincident with the evolutionary dispersal pattern of the Neotropical rattlesnakes. The indistinguisable immunoreactivity patterns of Costa Rican and Venezuelan polyvalent antivenoms toward C. simus and C. durissus venoms strongly suggest the possibility of using these antivenoms indistinctly for the management of snakebites by adult C. simus and by certain C. d. cumanensis populations exhibiting a hemorrhagic venom phenotype. The antivenomic results also explain why the antivenoms effectively neutralize the hemorrhagic activity of...

SUMMARYBacterial sphingomyelinases and phospholipases are a heterogeneous group of esterases which are usually surface associated or secreted by a wide variety of Gram-positive and Gram-negative bacteria. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, generating products identical to the ones produced by eukaryotic enzymes which play crucial roles in distinct physiological processes, including membrane dynamics, cellular signaling, migration, growth, and death. Several bacterial sphingomyelinases and phospholipases are essential for virulence of extracellular, facultative, or obligate intracellular pathogens, as these enzymes contribute to phagosomal escape or phagosomal maturation avoidance, favoring tissue colonization, infection establishment and progression, or immune response evasion. This work presents a classification proposal for bacterial sphingomyelinases and phospholipases that considers not only their enzymatic activities but also their structural aspects. An overview of the main physiopathological activities is provided for each enzyme type, as are examples in which inactivation of a sphingomyelinase- or a phospholipase-encoding gene impairs the virulence of a pathogen. The identification of sphingomyelinases and phospholipases important for bacterial pathogenesis and the development of inhibitors for these enzymes could generate candidate vaccines and therapeutic agents, which will diminish the impacts of the associated human and animal diseases.

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We previously isolated a mutant cell that is the only mammalian cell reported to have a persistently low level of UDP-glucose. In this work we obtained a spontaneous revertant whose UDP-glucose level lies between those found in the wild type and the mutant cell. The activity of UDP-glucose pyrophosphorylase (UDPG:PP), the enzyme that catalyzes the formation of UDP-glucose, was in the mutant 4% and in the revertant 56% of the activity found in the wild type cell. Sequence analysis of UDPG: PP cDNAs from the mutant cell showed one missense mutation, which changes amino acid residue 115 from glycine to aspartic acid. The substituted glycine is located within the largest stretch of strictly conserved residues among eukaryotic UDPG:PPs. The analysis of the cDNAs from the revertant cell indicated the presence of an equimolar mixture of the wild type and the mutated mRNAs, suggesting that the mutation has reverted in only one of the alleles. In summary, we demonstrate that the G115D substitution in the Chinese hamster UDPG:PP dramatically impairs its enzymatic activity, thereby causing cellular UDP-glucose deficiency.

The essential toxin in Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis is alphatoxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ, encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI, nanJ, and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence.Clostridium perfringens type A is the causative agent of human gas gangrene, or clostridial myonecrosis, and human food poisoning (25,27). It produces many secreted hydrolytic enzymes and toxins, including alpha-toxin and perfringolysin O. C. perfringens strains can also encode up to three sialidases, but the three sialidase genes, nanH, nanI, and nanJ, are not present in all of the strains that have been completely sequenced. Strain ATCC 13124 encodes all three sialidases (18), while strain 13 encodes both of the large sialidases, NanI and NanJ, but not the smaller NanH enzyme (32). The food poisoning isolate SM101 encodes NanH but not NanI or NanJ (18).Sialidases have been implicated in the virulence of several bacterial pathogens. They have been shown to enhance the pathogenesis of disease through synergistic effects with other bacterial factors. For example, Vibrio cholerae sialidase enhances the activity of cholera toxin (10), Pseudomonas aeruginosa sialidase increases the binding of this organism to the cells of susceptible patients (6), and the two sialidases of Streptococcus pneumoniae contribute to the progression of infection in several animal models (16,23,37). More recently, a surfaceexposed sialidase was shown to be required for persistence of the canine pathogen Capnophagia canimorsus (15). Alpha-toxin is an essential virulence factor in gas gangrene (2), and perfringolysin O, although not essential, has been found to have a synergistic role with alpha-toxin, enhancing the disease process (3). Synergy between alpha-toxin and the NanI sialidase was also observed in experiment...

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BackgroundA long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects.ResultsThe venom gland transcriptomes of 8 Costa Rican taxa from 5 genera (Crotalus, Bothrops, Atropoides, Cerrophidion, and Bothriechis) of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27%) were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements) and class II (DNA transposons) mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes, of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large divergence between A. mexicanus and A. picadoi, and a closer kinship between A. mexicanus and C. godmani.ConclusionsOur comparative next-generation sequencing (NGS) analysis reveals taxon-specific trends governing the formulation of the venom arsenal. Knowledge of the venom proteome provides hints on the translation efficiency of toxin-coding transcripts, contributing thereby to a more accurate interpretation of the transcriptome. The application of NGS to the analysis of snake venom transcriptomes, may represent the tool for opening the door to systems venomics.