Mutational analysis of the zinc metalloprotease EmpA of Vibrio anguillarum

The extracellular zinc metalloprotease, EmpA, is a putative virulence factor involved in pathogenicity of the fish pathogen Vibrio anguillarum. The 611-amino acid precursor of this enzyme is encoded by the empA gene. The residues His346, His350, Glu370, Glu347, His429, Tyr361 and Asp417 are highly conserved and putatively function together at the active site of the enzyme. In this study, empA was inserted into pET24d(+) and expressed in Escherichia coli strain BL21(DE3) as a 6 x His tagged protein (r-EmpA). All the conserved residues of EmpA mentioned above were individually mutated by site-directed mutagenesis and the mutants were also expressed (m-r-EmpAs). r-EmpA and m-r-EmpAs were purified, and assayed for their proteolytic activities with azocasein as the substrate and cytotoxicities on a flounder gill cell line. m-r-EmpAs that had been mutated at His346, His350, Glu370 and Glu347 almost completely lost their proteolytic activity and cytotoxicity, pointing towards the essential roles played by these residues. In contrast, those mutated at Tyr361, His429 and Asp417 still retained a partial proteolytic activity and cytotoxicity. Our results indicate that these conserved residues play important roles in enzymatic activity and that the proteolytic activity of the enzyme is involved in the pathogenesis of V. anguillarum     

Induction of protease activity in Vibrio anguillarum by gastrointestinal mucus.

The effect of gastrointestinal mucus on protease activity in Vibrio anguillarum was investigated. Protease activity was measured by using an azocasein hydrolysis assay. Cells grown to stationary phase in mucus (200 microg of mucus protein/ml) exhibited ninefold-greater protease activity than cells grown in Luria-Bertani broth plus 2% NaCl (LB20). Protease induction was examined with cells grown in LB20 and resuspended in mucus, LB20, nine-salts solution (NSS [a carbon-, nitrogen-, and phosphorus-free salt solution]), or marine minimal medium (3M) ( approximately 10(9) CFU/ml). Induction of protease activity occurred 60 to 90 min after addition of mucus and was >/=70-fold greater than protease activity measured in cells incubated in either LB20 or 3M. Mucus was fractionated into aqueous and chloroform-methanol-soluble fractions. The aqueous fraction supported growth of V. anguillarum cells, but did not induce protease activity. The chloroform-methanol-soluble fraction did not support growth, nor did it induce protease activity. When the two fractions were mixed, protease activity was induced. The chloroform-methanol-soluble fraction did not induce protease activity in cells growing in LB20. EDTA (50 mM) inhibited the protease induced by mucus. Upon addition of divalent cations, Mg(2+) (100 mM) was more effective than equimolar amounts of either Ca(2+) or Zn(2+) in restoring activity, suggesting that the mucus-inducible protease was a magnesium-dependent metalloprotease. An empA mutant strain of V. anguillarum did not exhibit protease activity after exposure to mucus, but did grow in mucus. Southern analysis and PCR amplification confirmed that V. anguillarum M93 contained empA. These data demonstrate that the empA metalloprotease of V. anguillarum is specifically induced by gastrointestinal mucus.     

Identification of critical residues in the propeptide of LasA protease of Pseudomonas aeruginosa involved in the formation of a stable mature protease

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.     

In vivo processing of Staphylococcus aureus lipase.

The Staphylococcus aureus lipase gene encodes a 76-kDa protein. Extracellular lipase purified from culture supernatants is only 45 to 46 kDa, however. We show that the lipase is secreted in vivo as an 82-kDa protein with full enzymatic activity. It is then sequentially processed, both in culture and in cell-free supernatants, to a mature, 45- to 46-kDa protein. Protein sequencing demonstrates that the N-terminal region of the 82-kDa prolipase, comprising 295 amino acids, is cleaved from the central and C-terminal moieties, which contain the active site. A metallocysteine protease is probably responsible for initiating this processing. The extremely hydrophobic, mature lipase is resistant to further protease degradation and retains the full catalytic activity of the prolipase.     

Expression and processing of Vibrio anguillarum zinc-metalloprotease in Escherichia coli

The extracellular zinc-metalloprotease of Vibrio anguillarum is a secreted virulence factor. It is synthesized from the empA gene as a 611-residue preproprotease and processed to the active mature protease (EmpA) with concomitant secretion via the type II secretion pathway. Active EmpA has been found only in the V. anguillarum culture supernatant and the process of the activation seems to vary depending on strains analyzed. To better understand the mechanism of EmpA export and processing, the empA gene was cloned and expressed in Escherichia coli strains. Expression of empA did not have toxic effect on bacterial growth. Rupturing E. coli TOP10 cells by heating in gel-loading buffer resulted in activation of EmpA and severe proteolysis of the samples. In contrast, the same treatment of the E. coli MC4100A strain did not lead to the general proteolysis. In this strain, EmpA was exported into the periplasm via the Sec pathway. The periplasmic EmpA was detected in two active conformations. Therefore, in E. coli processing of EmpA precursor to an active enzyme did not require secretion to the media and the help of other V. anguillarum protein. Like in V. anguillarum, heterologous expression of empA in E. coli showed strain-specific activation process.     

A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion.

The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasAdelta mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.     

Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp. strain A28

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M(w)-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N'-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30 degrees C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

IL-1 beta is secreted by activated murine macrophages as biologically inactive precursor

IL-1 alpha and IL-beta are distinct cytokines, produced by activated macrophages. The temporal sequence in the processing and secretion as well as the mechanism(s) by which IL-1 is secreted from the cells remain undefined. Here we have studied the production of IL-1 from murine macrophages after stimulation with LPS or Listeria monocytogenes by two distinct methods: i) immunoprecipitation of radio-labeled IL-1 peptides from culture supernatants, and ii) determination of IL-1 activity by neutralization with monospecific antisera to either form of IL-1. We confirmed that precursor and mature forms of both IL-1 alpha and IL-1 beta can be detected in the culture supernatants after stimulation of the macrophages with 10 to 20 micrograms LPS/ml but, in addition, we report the novel finding that IL-1 beta is exclusively secreted in its unprocessed precursor form after stimulation of the cells with either 0.5 to 1 microgram LPS/ml or with L. monocytogenes. Exposure of the cells to increasing amounts of LPS led to the appearance of a 20-kDa IL-1 beta peptide in the culture supernatants concomitant with the release of a processing activity for the IL-1 beta precursor. These data therefore suggest that, in a first step, IL-1 beta is secreted as an unprocessed precursor protein that in a second, postsecretory step is cleaved by a LPS-inducible protease, thus generating the 20-kDa IL-1 beta peptide. The latter represents the biologically active IL-1 beta inasmuch as the generation of IL-1 beta activity in the culture supernatants strictly correlated with the appearance of the 20-kDa IL-1 beta peptide.     

Involvement of an Extracellular Protease in Algicidal Activity of the Marine Bacterium Pseudoalteromonas sp. Strain A28

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M_w-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N′-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.     

<Emphasis Type="Italic">Aeromonas caviae</Emphasis> CB101 Contains Four Chitinases Encoded by a Single Gene <Emphasis Type="Italic">chi1</Emphasis>

Aeromonas caviae CB101 secretes four chitinases (around 92, 82, 70, and 55 kDa) into the culture supernatant. A chitinase gene chi1 (92 kDa) was previously studied. To identify the genes encoding the remaining three chitinases, a cosmid library of CB101 was constructed to screen for putative chitinase genes. Nine cosmid clones were shown to contain a chitinase gene on chitin plates. Surprisingly, all the positive clones contained chi1. In parallel, we purified the 55-kDa chitinase (Chi55) from the CB101 culture supernatant by continuous DEAE-Sepharose and Mono-Q anion exchange chromatography. The N-terminal amino acid sequence of the purified chitinase exactly matched the N-terminal sequence of mature Chi1, indicating that the purified chitinase (Chi55) is a truncated form of Chi1. The N- and C-terminal domains of chi1 were cloned, expressed, and purified, separately. Western blots using anti-sera to the N- and C-terminal domains of chi1 on the chitinases of CB101 showed that the four chitinases in the culture supernatant are either chi1 or C-terminal truncations of Chi1. In addition, the CB101 chi1 null mutant showed no chitinolytic activity, while CB101 chi1 null mutant complemented by pUC19chi1 containing chi1 showed all four chitinases in gel activity assay. These data indicated that all four chitinases secreted by CB101 in the culture supernatant are the product of one chitinase gene chi1.     

Purification, characterization, and comparison of the immunoglobulin A1 proteases of Neisseria gonorrhoeae.

Each isolate of Neisseria gonorrhoeae produces one of two distinct immunoglobulin A1 (IgA1) proteases, type 1 or type 2, which are known to possess different cleavage specificities for peptide bonds in the hinge region of human IgA1. Both proteases were secreted into the culture medium throughout exponential growth; however, the activity level of the type 2 protease was 10-fold that observed for the type 1 enzyme. The type 2 protease was quite stable and resistant to a variety of inhibitors. In contrast, the type 1 enzyme was highly unstable and inhibited by low concentrations of metal chelators, salts, and thiol- or serine-specific chemical reagents. Both types of gonococcal IgA1 protease were purified from broth culture supernatants by a combination of anion-exchange, chromatofocusing, and molecular sieve chromatography techniques. The stable type 2 enzyme comprised a 114-kilodalton (kDa) peptide which converted to a still active 109-kDa peptide during isolation. In contrast, the type 1 protease possessed a 112-kDa peptide which did not convert to a smaller form and which could not be dissociated from peptides of 34 and 31 kDa without complete loss of enzyme activity.     

Introduction of a cysteine protease active site into trypsin

Active site serine 195 of rat anionic trypsin was replaced with a cysteine by site-specific mutagenesis in order to determine if a thiol group could function as the catalytic nucleophile in serine protease active site environment. Two genetically modified rat thiol trypsins were generated; the first variant contained a single substitution of Ser195 with Cys (trypsin S195C) while the second variant contained the Ser195 to Cys as well as an Asp102 to Asn substitution (trypsin D102N,S195C) that more fully mimics the putative catalytic triad of papain. Both variants were expressed as his J signal peptide-trypsin fusion proteins to high levels under the control of the tac promoter. The mature forms of both variants were secreted into the periplasmic space of Escherichia coli. Trypsin S195C shows a low level of activity toward the activated ester substrate Z-Lys-pNP, while both trypsin S195C and trypsin D102N,S195C were active toward the fluorogenic tripeptide substrate Z-GPR-AMC. Esterase and peptidase activities of both thiol trypsin variants were inhibited by known Cys protease inhibitors as well as by specific trypsin inhibitors. The kcat of trypsin S195C was reduced by a factor of 6.4 x 10(5) relative to that of trypsin while the kcat of trypsin D102N,S195C was lowered by a factor of 3.4 x 10(7) with Z-GPR-AMC as substrate. Km values were unaffected. The loss of activity of trypsin D102N,S195C was partially attributed to an inappropriate Asn102-His57 interaction that precludes the formation of the catalytically competent His57-Cys195 ion pair although loss of the negative charge of D102 at the active site probably contributes to diminished activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Initial characterization of two extracellular autolysins from Pseudomonas aeruginosa PAO1.

Two extracellular autolysins have been detected in the spent culture supernatants of Pseudomonas aeruginosa PAO1 by using renaturing polyacrylamide gel electrophoresis. The two autolysins were isolated from the culture supernatant by trichloroacetic acid precipitation and were shown to have apparent molecular masses of 26 and 29 kDa. The 26-kDa autolysin first appears during the early exponential phase of growth and then declines sharply, while the 29-kDa autolysin first appears in the late exponential phase of growth and continues well into the stationary phase. Fractionation of whole cells indicated that the 26-kDa enzyme was also localized within the periplasm, with a lesser amount of activity associated with the cytoplasmic membrane. The 29-kDa autolytic activity was distributed within the cell equally between the periplasm and the cytoplasmic membrane. The pH optima of the isolated 26- and 29-kDa autolysins are 6.0 and 5.0, respectively. Further evidence from both protease susceptibility and inhibition studies confirms that these two extracellular autolysins isolated from P. aeruginosa PAO1 are separate and distinct.     

Purification of cloned proaerolysin released by a low protease mutant of Aeromonas salmonicida

The precursor to the hole-forming toxin aerolysin has been purified in high yield from culture supernatants of a mutant of Aeromonas salmonicida containing the cloned structural gene. The mutant strain was generated by Tn5 mutagenesis. It released little or no protease or other extracellular proteins, including phospholipase, suggesting that it is a regulatory mutant. The absence of protease allowed the isolation of protoxin free from contaminating aerolysin. Typically, more than 50 mg of pure proaerolysin was obtained from 2 L of culture supernatant. The purified protein was completely unable to lyse human erythrocytes without prior activation with trypsin.     

Autoproteolytic processing of aspartic proteinase from sunflower seeds

The autoproteolytic processing of mature aspartic proteinase from sunflower seeds was investigated. The mature aspartic proteinase (48 kDa) was processed at N65s-D66s in the plant-specific region of the enzyme to form 34-kDa and 14-kDa subunits. The next step was the hydrolysis of the A25s-Q26s and N97s-E98s bonds to form a 39-kDa enzyme that consisted of 29-kDa and 9-kDa disulfide-bonded subunits. Finally, bonds including V1s-M2s, M2s-S3s, C100s-D101s, and D101s-R102s were cleaved to form non-covalently bound subunits (29 kDa and 9 kDa) by eliminating the disulfide bonds in the plant-specific region of the protein.     

Cloning and characterization of the gene (empV) encoding extracellular metalloprotease from Vibrio vulnificus

A gene (empV) encoding the extracellular metalloprotease of Vibrio vulnificus CKM-1 has been cloned and sequenced. When the empV gene was expressed in minicells, a unique peptide of approx. 46 kDa was identified. Protease activity staining experiments also indicated a similar M_r for the protease. The empV gene product (EmpV) is secreted into the periplasm of Escherichia coli, but not out of it. The crude enzyme prepared from the periplasmic fraction of recombinant E. coli was inhibited by a metalloprotease inhibitor and Zn²⁺ is essential for its protease activity. Nucleotide sequence analysis predicted a single open reading frame (ORF) of 1818 bp encoding a 606 amino acid (aa) polypeptide, with a potential 24 aa signal peptide followed by a long `pro' sequence consisting of 172 aa. The N-terminal 20 aa sequence for the elastolytic protease (EepV), purified from the culture supernatant of V. vulnificus ATCC 29307, completely identified the beginning of the predicted mature protein within the deduced aa sequence except for 1 aa residue difference. The estimated pI and molecular weight of the predicted mature protein were 5.86 and 44.3 kDa, respectively, which are nearly identical to those of V. vulnificus L-180 extracellular neutral metalloprotease (EnmV) and of strain ATCC 29307 EepV. The estimated molecular weight also closely matches that determined by SDS-PAGE analysis of the minicells and by protease activity staining. The deduced aa sequence of EmpV showed high homology to V. anguillarum metalloprotease (EmpA), V. cholerae HA/protease (HprC), and V. proteolyticus neutral protease (NprP), particularly with respect to active-site residues, zinc-binding residues, and cysteine residues.     

A metalloprotease secreted by the insect pathogen Photorhabdus luminescens induces melanization

Photorhabdus luminescens is a gram-negative insect pathogen that enters the hemocoel of infected hosts and produces a number of secreted proteins that promote colonization and subsequent death of the insect. In initial studies to determine the exact role of individual secreted proteins in insect pathogenesis, concentrated culture supernatants from various P. luminescens strains were injected into the tobacco hornworm Manduca sexta. Culture supernatants from P. luminescens TT01, the genome-sequenced strain, stimulated a rapid melanization reaction in M. sexta. Comparison of the profiles of secreted proteins from the various Photorhabdus strains revealed a single protein of approximately 37 kDa that was significantly overrepresented in the TT01 culture supernatant. This protein was purified by DEAE ion-exchange and Superdex 75 gel filtration chromatography and identified by matrix-assisted laser desorption ionization-time of flight analysis as the product of the TT01 gene plu1382 (NCBI accession number NC_005126); we refer to it here as PrtS. PrtS is a member of the M4 metalloprotease family. Injection of PrtS into larvae of M. sexta and Galleria mellonella and into adult Drosophila melanogaster and D. melanogaster melanization mutants (Bc) confirmed that the purified protein induced the melanization reaction. The prtS gene was transcribed by P. luminescens injected into M. sexta before death of the insect, suggesting that the protein was produced during infection. The exact function of this protease during infection is not clear. The bacteria might survive inside the insect despite the melanization process, or it might be that the bacterium is specifically activating melanization in an attempt to circumvent this innate immune response.