Purification, characterization, and primary structure of Clostridium perfringens lambda-toxin, a thermolysin-like metalloprotease

Abstract

The lambda-toxin of Clostridium perfringens type B NCIB10691 was purified by ammonium sulfate precipitation, followed by size exclusion, anion-exchange, and hydrophobic interaction chromatography. The purified toxin had an apparent molecular mass of 36 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The toxin possessed casein-hydrolyzing activity, which was inhibited specifically by metal chelators, indicating that the toxin is a metalloprotease. The gene encoding the lambda-toxin (lam), which was shown by Southern analysis to be located on a 70-kb plasmid, was cloned into Escherichia coli cells. Nucleotide and N-terminal amino acid sequencing revealed that the lam gene encodes a 553-amino-acid protein, which is processed into a mature form, the molecular mass of which was calculated to be 35,722 Da. The deduced amino acid sequence of the mature enzyme contains an HEXXH motif characteristic of zinc metalloproteases and is homologous to other known enzymes belonging to the thermolysin family. The purified toxin degraded various biologically important substances, such as collagen, fibronectin, fibrinogen, immunoglobulin A, and the complement C3 component. It caused an increase in vascular permeability and hemorrhagic edema on injection into the dorsal skin of mice. These results suggest that the toxin contributes to the pathogenesis of histolytic infection by lambda-toxin-producing C. perfringens.

Rapid autocatalytic activation of the M4 metalloprotease aureolysin is controlled by a conserved N‐terminal fungalysin‐thermolysin‐propeptide domain

“…Aur is structurally similar to the zinc metalloprotease thermolysin (Banbula et al ., 1998), and both belong to the M4 family of metallopeptidases (Rawlings and Barrett, 1995; Rawlings et al ., 2006), which typically undergo autocatalytic activation (McIver et al ., 1993; Marie‐Claire et al ., 1998; Kooi et al ., 2005; 2006). As with the role of Aur in activating proSspA, other M4 metalloproteases in diverse genera of pathogenic bacteria also function like proprotein convertases, including activation of cholera toxin by the Vibrio haemagglutinin metalloprotease (Booth et al ., 1984), activation of Clostridium perfringens epsilon‐ and iota‐toxins by the lambda‐toxin metalloprotease (Jin et al ., 1996; Minami et al ., 1997; Gibert et al ., 2000), activation of phospholipase C by the Mpl metalloprotease of Listeria (Poyart et al ., 1993; Marquis et al ., 1997) and activation of the Enterococcus faecalis SprE serine protease by GelE, which is coexpressed in an operon with SprE (Qin et al ., 2000; Kawalec et al ., 2005). With structurally related metalloproteases contributing to virulence of diverse genera of bacteria, it may be necessary to tailor the zymogen activation mechanism to suit the physiology and virulence strategy of each pathogen.…”

Lambda‐Toxin of Clostridium perfringens Activates the Precursor of Epsilon‐Toxin by Releasing Its N‐ and C‐Terminal Peptides

“…Samples after incubation with proteases were heated in a sample buffer consisting of 62.5 mm Tris-HC1 (pH 6.8), 1% 2-mercaptoethanol, 1% sodium dodecylsulfate, 10% glycerol and 0.001% bromophenol blue at 100 C for 3 min. They were then applied to a slab gel, comprising a stacking 3% acrylamide gel over a 12.5% resolving gel, and then electrophoresed as described previously (15).…”

“…These samples were subjected to SDS-PAGE in the same manner as described above. The proteins were each electrophoretically transferred to a polyvinylidene difluoride membrane, and then N-terminal sequencing was performed as described previously (15). However, the purified E-prototoxin was subjected to N-terminal sequencing without any treatment.…”

Identificação de proteases de Leptospira envolvidas com mecanismos de escape do sistema complemento humano.

“…Enterococcus faecalis exerce atividade proteolítica sobre o componente C3 e os fragmentos C3b, C3a e iC3b, resultando em uma redução substancial na fagocitose do patógeno por leucócitos polimorfonucleares humanos (Park et al, , 2008. Além de clivar a molécula C3, a toxina lambda de Clostridium perfringens também atua em outros importantes substratos biológicos como o colágeno e a fibronectina, favorecendo o processo de invasão e disseminação da bactéria pelo organismo (Jin et al, 1996).…”

“…A cisteíno protease SpeB (streptococcal pyrogenic exotoxin B) de Streptococcus pyogenes destaca-se pela sua capacidade de degradação de diversas proteínas do complemento, resultando em uma inibição eficaz das três vias de ativação (Kuo et al, 2004;Lukomski et al, 1998;Honda-Ogawa et al, 2013;Terao et al, 2008). Já a aspártico protease PgtE (outer membrane protein E) de Salmonella enterica cliva os componentes C3b, C4b e C5 contribuindo para a disseminação da bactéria durante a infecção sistêmica (Ramu et al, 2007 Jin et al, 1996;Jusko et al, 2012;Laarman et al, , 2012Park et al, , 2008. .…”