Immune recognition of human Hsp60 by Lyme disease patient sera

Girouard, Linda; Laux, David C.; Jindal, Shalini; Nelson, David R.

doi:10.1006/mpat.1993.1028

Cited by 9 publications

(6 citation statements)

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“…Gels were either stained in Bio-Safe Coomassie (Bio-Rad) or transferred to nitrocellulose membranes for immunoblotting using the mini-Protean II system (Bio-Rad). Transfers were performed as described by Towbin et al (11) at 100 V for 1.5 h. Nitrocellulose membranes were blocked, as described by Girouard et al (5), with the addition of 5% skim milk. Detection of EmpA bands was done with rabbit anti-P. aeruginosa LasB elastase antibodies at a dilution of 1:7,000.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Vibrio anguillarum Metalloprotease EmpA by Posttranslational Modification

2005

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The zinc metalloprotease EmpA is a virulence factor in the fish pathogen Vibrio anguillarum. Previous studies have shown that two strains of V. anguillarum regulate empA differently. Strain M93Sm exhibits protease activity only in the presence of fish gastrointestinal mucus, while protease activity is detected in NB10 culture supernatant under all stationary-phase conditions. In this study, we use real-time reverse transcription-PCR to show that even in conditions where no protease activity is detected, empA transcription occurs. Western blot analysis revealed that EmpA is secreted as a ϳ48-kDa proenzyme and that activation occurs extracellularly by the removal of a ϳ10-kDa peptide. The presence of stable extracellular pro-EmpA in M93Sm culture supernatants suggests that activation of EmpA is not autolytic.The marine bacterium Vibrio anguillarum is the causative agent of vibriosis, a systemic disease of fish characterized by hemorrhagic septicemia (1). Outbreaks of vibriosis result in high mortality rates of infected fish, and it is a major obstacle to the spread of commercial aquaculture (1).The zinc metalloprotease EmpA has been identified as a virulence factor for V. anguillarum (2,7,8). EmpA is a structural homologue to the secreted LasB elastase of Pseudomonas aeruginosa (7). The gastrointestinal (GI) tract has been shown to be a route of entry for V. anguillarum into fish hosts (9, 10). Incubation of V. anguillarum in gastrointestinal mucus specifically induces a number of proteins, including EmpA (3, 4). Denkin and Nelson (2) showed that the induction of protease activity by GI mucus in stationary-phase cultures varied between two strains of V. anguillarum. Strain M93Sm demonstrated protease activity only in the presence of mucus, while strain NB10 expressed protease activity under all stationaryphase conditions with and without mucus.In this report, we further describe the differences between NB10 and M93Sm in the regulation of EmpA and show that EmpA is under both transcriptional and posttranslational control. Differences in the induction of empA and the role of gastrointestinal mucus were examined using real-time reverse transcription-PCR (RT-PCR). Western blot analysis was used to describe the secretion and processing of EmpA. MATERIALS AND METHODSStrains and culture conditions. Two wild-type strains of V. anguillarum, NB10 (7) and M93Sm (3), were used in this study. Strain M03, a streptomycin-and chloramphenicol-resistant (Sm r Cm r ) rpoS mutant derived from M93Sm (2) was also used. Cultures were routinely grown in Luria-Bertani broth plus 2% NaCl (LB20) (4) with the addition of the appropriate antibiotics on a rotary shaker at 27°C. Experimental media included LB20 and nine-salt solution (NSS) plus 200 g of salmon GI mucus protein ml Ϫ1 (NSSM) (4). For experiments, 16-h cultures of V. anguillarum (grown at 27°C) were centrifuged (9,000 ϫ g, 5 min, 4°C), washed twice in NSS, and resuspended at the appropriate cell densities in either LB20 or NSSM. Cell densities were determined by serial dilution a...

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Section: Methodsmentioning

confidence: 99%

Regulation of the Vibrio anguillarum Metalloprotease EmpA by Posttranslational Modification

2005

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“…Labelled cells were then centrifuged (9000 g, 10 min, 4 mC) and washed twice in 10 mM HEPES buffer (pH 7n6, 4 mC). Cells were disrupted by sonication and protein was TCA-precipitated in order to concentrate the $&S-labelled protein as described previously (Girouard et al, 1993 ;Scorpio et al, 1994). TCA-precipitated proteins were solubilized in solubilization-reduction mixture (Laemmli, 1970).…”

Section: Methodsmentioning

confidence: 99%

“…Equal protein (20 µg) was loaded in each gel lane. The separated proteins were either stained with Coomassie blue or electroblotted to nitrocellulose by a modification of the procedure described by Towbin et al (1979) using a Bio-Rad Transblot Cell (400 mA, 60 min) (Girouard et al, 1993). The nitrocellulose membranes were briefly stained with 0n1% Ponceau S (Sigma) to mark the positions of molecular mass standards and then blocked in Tris-buffered saline (TBS : 50 mM Tris, 150 mM NaCl ; pH 7n4) plus 2 % Tween 20 (Sigma) for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Serum-starvation-induced changes in protein synthesis and morphology of Borrelia burgdorferi

2000

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“…Gels were transferred to nitrocellulose membranes for immunoblotting using the mini-Protean II system (Bio-Rad). Transfers were performed as described by Towbin et al (26) at 100 V for 1.5 h. Nitrocellulose membranes were blocked, as described by Girouard et al (11), with the addition of 5% skim milk. EmpA bands were detected with rabbit anti-P. aeruginosa LasB elastase antibodies at a dilution of 1:5,000, as described by Staroscik et al (25).…”

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

et al. 2008

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The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ϳ10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.Vibrio anguillarum, a marine bacterium, is the causative agent of vibriosis, a systemic disease of both wild and cultured marine fish characterized by hemorrhagic septicemia (2). Outbreaks of vibriosis result in high mortalities among infected fish, and this disease continues to be a major obstacle for the aquaculture industry (2). V. anguillarum enters its fish host through the gastrointestinal (GI) tract and quickly colonizes this nutrient-rich environment (21,22). Garcia et al. (9) have shown that V. anguillarum grows extremely well in salmon intestinal mucus and that mucus-grown cells specifically express a number of different proteins, including several outer membrane proteins (9) and the extracellular metalloprotease EmpA (7).The zinc metalloprotease EmpA has been identified as a virulence factor for V. anguillarum and is important for virulence during infection of the GI tract of Atlantic salmon (Salmo salar) (7,16,19). In V. anguillarum wild-type strain M93Sm, EmpA is expressed during stationary phase when cells are incubated in Atlantic salmon GI mucus (6, 7). The creation of an empA null mutant (M99) by insertional mutagenesis showed that EmpA is responsible for the protease activity observed for wild-type strain M93Sm (7). However, EmpA activity is dependent on successful secretion and processing of the nascent protein to a...

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Immune recognition of human Hsp60 by Lyme disease patient sera

Cited by 9 publications

References 0 publications

Regulation of the Vibrio anguillarum Metalloprotease EmpA by Posttranslational Modification

Regulation of the Vibrio anguillarum Metalloprotease EmpA by Posttranslational Modification

Serum-starvation-induced changes in protein synthesis and morphology of Borrelia burgdorferi

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

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