Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

Abstract

Cholera toxin gene-negativeVibrio cholerae non-O1, non-O139 strains are an intriguing group that can cause typical cholera-like disease but do not produce cholera toxin (CT) (23). The virulence factor in V. cholerae non-O1, non-O139 strains that do not produce cholera toxin and yet cause symptoms similar to cholera has not yet been identified (6). It was found that none of the previously described virulence-related genes like the RTX (repeats in toxin) toxin gene cluster, the hlyA gene coding for hemolysin, the mshA gene for mannose-sensitive hemagglutinin pilus, and the stn gene encoding a heat-stable enterotoxin of non-O1 vibrios (NAG-ST) was specifically associated with the pathogenesis of TCP Ϫ CTX Ϫ non-O1, non-O139 strains that colonized mice and caused fluid accumulation in rabbits (6). In the above-mentioned study, the role of the hapA gene (coding for hemagglutinin protease [HAP]) in the disease process was not investigated. Studies by Benítez et al. with vaccine strain 638, which bears a core deletion of the ctx gene and an insertional mutation of the hapA gene, showed fewer symptoms and a decrease in diarrhea in volunteers compared to those in previous studies using only the core deletion strain (2). In addition, strain 638 induced lower levels of interleukin-8 (IL-8) than its parent wild-type strain did in HT29 cells (2, 30). Cholera vaccine strain CVD 110, a mutant of El Tor Ogawa strain E7946, which is deleted of all known virulence genes except hap, gives rise to a highly inflammatory reaction (36). Concentrated proteins derived from the culture supernatant of CVD 110 caused morphological changes in cultured MDCK-1 epithelial cells and altered their arrangement of filamentous actin and zonula occludens-associated protein (ZO-1) (38). The drastic morphological changes were inhibited by Zincov, a specific bacterial metalloprotease inhibitor. After fast-performance liquid chromatography, the cytotoxic fractions of the sample showed two visible bands with molecular masses of approximately 34 and 32 kDa. Microsequencing of these proteins revealed that they were in fact HAP. Culture supernatants prepared from the reactogenic strains of V. cholerae cause a decrease in the transcellular epithelial resistance of T84 intestinal cells (21). This decrease correlates with the presence of HAP but not with the presence of other potential accessory toxins or proteases (21). Microarray studies of the global transcription pattern of V. cholerae grown in vivo in the rabbit ileal loop (RIL) model have shown that the hemagglutinin protease (hap) gene is one of the 12 genes that belong to the pathogenesis functional group and are highly expressed compared to their levels of expression under laboratory conditions (29). V. cholerae O1 classical and El Tor biotypes as well as non-O1 serotypes produce HAP (3). The secreted HAP has been purified, cloned, and sequenced (9, 14). The hap gene consists of 1,827 nucleotides with a predicted molecular mass of 69.3 kDa. This is larger than the molecular mass of the purified HAP (3...

Conservation of the Host-Interacting Proteins Tp0750 and Pallilysin among Treponemes and Restriction of Proteolytic Capacity to Treponema pallidum

“…The expression of host-interacting adhesins/proteases by pathogenic bacteria is an important virulence mechanism that promotes host attachment, host protein degradation, tissue destruction, pathogen invasion, and dissemination of infection (56)(57)(58). To date, the only host component binding/degrading adhesins that have been identified in T. pallidum are Tp0750 and pallilysin (23)(24)(25).…”

Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

“…This raises the possibility that pro-EmpA has a role during infection by V. anguillarum that differs from that for mature EmpA metalloprotease. Perhaps EmpA, similar to Hap protease of V. cholerae, has bifunctional properties (10). How pro-EmpA may contribute to virulence of V. anguillarum is currently under investigation.…”

“…In in vivo systems, the 45-kDa form can be processed to the 35-kDa form autolytically as well as by other general proteases found in the intestine. Additional studies have shown that HA/protease has bifunctional properties; the 45-kDa form is responsible for the enterotoxic response and the fully processed 35-kDa protease causes the HA activity responsible for the cytotoxic effects caused by this organism (10). We note that the processing of the pro-LasA to mature LasA protease in P. aeruginosa has been shown to involve the action of other secreted proteases, including elas-tase, lysine-specific protease (protease IV or PrpL), and alkaline proteinase (14).…”

Bifunctional Role of theTreponema pallidumExtracellular Matrix Binding Adhesin Tp0751

“…This process has been linked to the virulence of many pathogenic bacteria, including Vibrio cholerae (27), Pseudomonas aeruginosa (48), and Legionella pneumophila (13,51), by observations that it promotes bacterial invasion and dissemination of infection via the degradation of structural tissue components and plasma proteins. Hostinteracting proteases have yet to be identified within T. pallidum at either an experimental or a bioinformatic level, although the highly invasive nature of this bacterium suggests their existence.…”