RESUMO
The Estuary of Bahía Blanca (EBB), Argentina, is an important wetland under intense sewage pollution. We investigated the occurrence of Clostridium perfringens (CP) in populations of two benthic crabs (Neohelice granulata and Cyrtograpsus angulatus) and in sediment from the EBB. CP was found in 49.1% of the crabs and all of the isolates were identified as type A. The alpha (cpa) and enterotoxin (cpe) encoding genes were identified. Genetic analyses identified 13 novel sequence types, and found no clustering among isolates, suggesting that CP is not part of the crabs' commensal flora. CP carriage was 51 times more likely in crabs from the area nearest sewage outfalls compared with crabs from a reference site. Our in vitro experiments suggest that the carriage of CP in crabs is transient. The use of these benthic crabs as monitoring organisms of sewage pollution in coastal habitats is proposed.
Assuntos
Braquiúros/microbiologia , Clostridium perfringens , Esgotos/microbiologia , Animais , Argentina , Toxinas Bacterianas/genética , Proteínas de Ligação ao Cálcio/genética , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Ecossistema , Estuários , Sedimentos Geológicos/microbiologia , Filogenia , Fosfolipases Tipo C/genética , Poluição da ÁguaRESUMO
Clostridium perfringens alpha-toxin is a 370-residue, zinc-dependent, phospholipase C that is the key virulence determinant in gas gangrene. It is also implicated in the pathogenesis of sudden death syndrome in young animals and necrotic enteritis in chickens. Previously characterized alpha-toxins from different strains of C. perfringens are almost identical in sequence and biochemical properties. We describe the cloning, nucleotide sequencing, expression, characterization, and crystal structure of alpha-toxin from an avian strain, SWan C. perfringens (SWCP), which has a large degree of sequence variation and altered substrate specificity compared to these strains. The structure of alpha-toxin from strain CER89L43 has been previously reported in open (active site accessible to substrate) and closed (active site obscured by loop movements) conformations. The SWCP structure is in an open-form conformation, with three zinc ions in the active site. This is the first example of an open form of alpha-toxin crystallizing without the addition of divalent cations to the crystallization buffer, indicating that the protein can retain three zinc ions bound in the active site. The topology of the calcium binding site formed by residues 269, 271, 336, and 337, which is essential for membrane binding, is significantly altered in comparison with both the open and closed alpha-toxin structures. We are able to relate these structural changes to the different substrate specificity and membrane binding properties of this divergent alpha-toxin. This will provide essential information when developing an effective vaccine that will protect against C. perfringens infection in a wide range of domestic livestock.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Aves/microbiologia , Proteínas de Ligação ao Cálcio , Clostridium perfringens/química , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Doenças das Aves/microbiologia , Cádmio/metabolismo , Bovinos , Clonagem Molecular , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Cristalização , Cristalografia por Raios X , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/veterinária , Genes Bacterianos , Hemólise/efeitos dos fármacos , Cinética , Masculino , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/toxicidade , Zinco/metabolismoRESUMO
The oprF gene, expressing Pseudomonas aeruginosa major outer membrane protein OprF, was subjected to semi-random linker mutagenesis by insertion of a 1.3 kb Hincll kanamycin-resistance fragment from plasmid pUC4KAPA into multiple blunt-ended restriction sites in the oprF gene. The kanamycin-resistance gene was then removed by Pstl digestion, which left a 12 nucleotide pair linker residue. Nine unique clones were identified that contained such linkers at different locations within the oprF gene and were permissive for the production of full-length OprF variants. In addition, one permissive site-directed insertion, one non-permissive insertion and one carboxy-terminal insertion leading to proteolytic truncation were also identified. These mutants were characterized by DNA sequencing and reactivity of the OprF variants with a bank of 10 OprF-specific monoclonal antibodies. Permissive clones produced OprF variants that were shown to be reactive with the majority of these monoclonal antibodies, except where the insertion was suspected of interrupting the epitope for the specific monoclonal antibody. In addition, these variants were shown to be 2-mercaptoethanol modifiable, to be resistant to trypsin cleavage in intact cells and partly cleaved to a high-molecular-weight core fragment in outer membranes and, where studied, to be accessible to indirect immunofluorescenee labelling in intact cells by monoclonal antibodies specific for surface epitopes. Based on these data, a revised structural model for OprF is proposed.
