LasA Protease Research Articles

Antibiotic Resistance in Pseudomonas Aeruginosa is Associated with Decreased Fitness

The number of clinical specimens containing β-lactam-resistant Pseudomonas aeruginosa isolates is increasing. However, whether resistance is associated with reduced fitness is still uncertain in clinical Pseudomonas aeruginosa isolates. In this study, we aimed to determine whether β-lactam resistance conferred a fitness cost in Pseudomonas aeruginosa. Growth rate, extracellular slime production, elastase activity, proteolytic activity, LasA protease activity, biofilm formation, and pairwise in vitro competition experiments were investigated in a collection of 11 isogenic, β-lactam-susceptible and -resistant (≥8-fold increase in minimum inhibitory concentration (MIC)) pairs of P. aeruginosa clinical isolates; each pair was recovered from a different patient treated with β-lactam antibiotics. All β-lactam-resistant Pseudomonas aeruginosa isolates showed a significant reduction in elastase activity. In addition, 90.9% (10/11) of β-lactam-resistant Pseudomonas aeruginosa isolates were associated with markedly lower growth rate and proteolytic activity, and 81.8% (9/11) of β-lactam-resistant Pseudomonas aeruginosa isolates had less extracellular slime production, compared to susceptible isolates. Meanwhile, LasA protease activity and biofilm formation ability were variable among isolates. Pairwise in vitro competition experiments showed that 72.7% (8/11) of β- lactam-susceptible strains could outgrow resistant strains. In conclusion, resistance development with β-lactam exposure confers a fitness cost, resulting in a decreased invasion potential, while the effect on viability varied. Thus, the potential for the dissemination of β-lactam-resistant Pseudomonas aeruginosa clinical isolates should not be underestimated.

Characterization of an anti‐pathogenic lactonase from Palk Bay metagenome

ObjectivesScreening of metaclones for anti‐pathogenic activity and identification of anti‐pathogenic agents.MethodsScreening for anti‐pathogenic activity using Chromobacterium violaceum (ATCC 12472) and CV026 as indicators. Mode of action by TLC (Thin Layer Chromatography), HPLC (High Performance Liquid Chromatography) and PCR (Polymerase Chain Reaction).ResultsRecombinant fosmid clones (N=10200) were propagated in E. coli DH10B. When individually 1056 clones were used to challenge C. violaceum CV026, eight clones could able to inactivate the perception of QS signal HHL (C6‐HSL) by the Chromobacterium biosensor. Additional assays revealed that the metagenomic clone M8‐70 disrupted AHL signals, including C6‐HSL, heptanoyl‐ (C7‐ ), octanoyl‐ (C8‐) and decanoyl‐homoserine lactone (C10‐HSL), as well as the hydroxy‐ or oxo‐substituted derivatives at carbon 3, such as OC6‐HSL, OC8‐HSL and OHC8‐HSL. Culture supernatants of M8‐70 was examined for its effect on P. aeruginosa PAO1 (LasA, LasB, Pyocyanin, Pyoverdin and and Total protease), S. marcescens (prodigiosin production, swarming, secreted caseinase and lipase) QS system and distinct profiles were obtained for las and rhl QS genes. Furthermore, the growth curve analysis and cell count method made it clear that the supernatant of M8‐70 had no antibacterial activity toward P. aeruginosa PAO1 and S. marcescens. The results of the ring closure assay and the sequence analysis of the PCR products from M8‐70 (GenBank accession number HQ876560) revealed that its activity as lactonase activity.ConclusionIn this study a lactonase is reported through metagenomics approach from the sediment of Palk Bay which was found responsible for the inhibition of QS mediated virulence factors of Gram negative bacterial pathogens without inhibiting their growth.

Lagerstroemia speciosa fruit extract modulates quorum sensing-controlled virulence factor production and biofilm formation in Pseudomonas aeruginosa

Lagerstroemia speciosa (Lythraceae) is a south-east Asian tree more commonly known as 'Jarul'. Research on health benefits suggests that the L. speciosa plant contains phytomolecules that may have antioxidant, anti-diabetic and anti-obesity properties. However, antimicrobial activities have not been reported for this plant. The ability of L. speciosa fruit extract (LSFE) to antagonize cell-to-cell communication, expression of virulence genes and factors, and biofilm formation was evaluated in Pseudomonas aeruginosa strain PAO1. Our results suggested that LSFE caused downregulation of quorum sensing (QS)-related genes (las and rhl) and their respective signalling molecules, N-acylhomoserine lactones, without affecting the growth of P. aeruginosa PAO1. Significant inhibition of virulence factors: LasA protease, LasB elastase, and pyoverdin production, was also recorded. Application of LSFE to P. aeruginosa PAO1 biofilms increased bacterial susceptibility to tobramycin. These data suggest a possible role for quorum-quenching mechanisms unrelated to static or cidal effects, and also suggest that L. speciosa could serve as a cost-effective source in the development of new QS-based antibacterial drugs.

Staphylolysin is an effective therapeutic agent for Staphylococcus aureus experimental keratitis

Therapy of S. aureus keratitis is increasingly challenging due to emerging resistant strains. Staphylolysin (LasA protease) is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa. The purpose of the current study was to study the effect of treatment with staphylolysin on experimental keratitis caused by various Staphylococcus aureus strains. The therapeutic effect was studied in a keratitis model induced in rabbits by intrastromal injections of 10(3) S. aureus cells of three different methicillin-resistant S. aureus (MRSA) strains and one methicillin-susceptible S. aureus strain (MSSA). Topical treatment with either staphylolysin or bovine serum albumin (BSA; control) was applied every half hour for 5h, starting at 4h after infection. Corneas were removed for bacterial quantification. Histopathological analysis was performed on MSSA-infected rabbits, killed at either one or 84h after completion of treatment and on uninfected eyes 1h after treatment termination. The number of bacteria in the staphylolysin-treated corneas was significantly reduced in all infections with the four S. aureus strains studied as compared to controls: the staphylolysin-treated eyes infected with MRSA strains were either completely sterilized or showed a 3-4 orders of magnitude decrease in the number of cfu/cornea (p = 0.004 to 0.005); all of the staphylolysin-treated MSSA-infected eyes were sterile. Histopathological analysis of the methicillin-sensitive (MSSA) strain-infected eyes at 84h after completion of treatment showed moderate inflammation in the staphylolysin-treated eyes as compared with extensive abscess formation in the control group. The uninfected corneas showed only mild stromal edema in both the staphylolysin and BSA-treated groups. Staphylolysin provided long-lasting protection against several strains of S. aureus, evident by both its strong anti-bacterial activity and beneficial histopathological results of treatment.

Antipathogenic potential of marine Bacillus sp. SS4 on N-acyl-homoserine-lactone-mediated virulence factors production in Pseudomonas aeruginosa (PAO1)

Antipathogenic therapy is an outcome of the quorum-sensing inhibition (QSI) mechanism, which targets autoinducer-dependent virulent gene expression in bacterial pathogens. N-acyl homoserine lactone (AHL) acts as a key regulator in the production of virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1 and violacein pigment production in Chromobacterium violaceum. In the present study, the marine bacterial strain SS4 showed potential QSI activity in a concentration-dependent manner (0.5-2 mg/ml) against the AHL-mediated violacein production in C. violaceum (33-86%) and biofilm formation (33-88%), total protease (20-65%), LasA protease (59-68%), LasB elastase (36-68%), pyocyanin (17-86%) and pyoverdin productions in PAO1. The light and confocal laser scanning microscopic analyses confirmed the reduction of the biofilm-forming ability of PAO1 when treated with SS4 extract. Furthermore, the antibiofilm potential was confirmed through static biofilm ring assay, in which ethyl acetate extract of SS4 showed concentration-dependent reduction in the biofilm-forming ability of PAO1. Thus, the result of this study clearly reveals the antipathogenic and antibiofilm properties of the bacterial isolate SS4. Through 16S rDNA analysis, the strain SS4 was identified as Bacillus sp. (GenBank Accession Number: GU471751).

Co-regulation of β-lactam resistance, alginate production and quorum sensing in Pseudomonas aeruginosa

Development of β-lactam resistance, production of alginate and modulation of virulence factor expression that alters host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. In this study, we propose that a co-regulatory network exists between these mechanisms. We compared the promoter activities of ampR, algT/U, lasR, lasI, rhlR, rhlI and lasA genes, representing the β-lactam antibiotic resistance master regulatory gene, the alginate switch operon, the las and rhl quorum-sensing (QS) genes, and the LasA staphylolytic protease, respectively. Four isogenic P. aeruginosa strains, the prototypic Alg− PAO1, Alg− PAOampR, the mucoid Alg+ PAOmucA22 (Alg+ PDO300) and Alg+ PAOmucA22ampR (Alg+ PDOampR) were used. We found that in the presence of AmpR regulator and β-lactam antibiotic, the extracytoplasmic function sigma factor AlgT/U positively regulated PampR, whereas AmpR negatively regulated PalgT/U. On the basis of this finding we suggest the presence of a negative feedback loop to limit algT/U expression. In addition, the functional AlgT/U caused a significant decrease in the expression of QS genes, whereas loss of ampR only resulted in increased PlasI and PlasR transcription. The upregulation of the las QS system is likely to be responsible for the increased lasA promoter and the LasA protease activities in Alg− PAOampR and Alg+ PDOampR. The enhanced expression of virulence factors in the ampR strains correlated with a higher rate of Caenorhabditis elegans paralysis. Hence, this study shows that the loss of ampR results in increased virulence, and is indicative of the existence of a co-regulatory network between β-lactam resistance, alginate production, QS and virulence factor production, with AmpR playing a central role.

Identification of mutants with altered phenazine production in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that causes serious and chronic infections. Many secondary metabolites are secreted throughout its growth, among which phenazine is a known virulence factor and signalling molecule. Phenazine is coordinately controlled by the global regulatory quorum-sensing (QS) systems. Despite the detailed understanding of phenazine biosynthesis pathways in P. aeruginosa, the regulatory networks are still not fully clear. In the present study, the regulation of the phzA1B1C1D1E1F1G1 operon (phzA1) has been investigated. Screening of 5000 transposon mutants revealed 14 interrupted genes with altered phzA1 expression, including PA2593 (QteE), which has been identified as a novel regulator of the QS system. Overexpression of qteE in P. aeruginosa significantly reduced the accumulation of homoserine lactone signals and affected the QS-controlled phenotypes such as the production of pyocyanin, rhamnolipids and LasA protease and swarming motility. Indeed, overexpression of qteE in P. aeruginosa attenuated its pathogenicity in the potato and fruit fly infection models. These findings suggest that qteE plays an important role in P. aeruginosa pathogenicity and is part of the regulatory networks controlling phenazine production.

Bacillus pumilus of Palk Bay origin inhibits quorum-sensing-mediated virulence factors in Gram-negative bacteria

The aim of the current study was to inhibit quoring-sensing(QS)-mediated virulence factors of representative Gram-negative bacteria by marine bacterial isolates. Bacteria isolated from Palk Bay sediments were screened for anti-QS activity. Eleven strains inhibited QS signals in Chromobacterium violaceum (ATCC 12472) and C. violaceum CV026. The marine bacterial strain S8-07 reduced the accumulation of N-acyl homoserine lactone (AHLs) and showed significant inhibition of LasA protease(76%), LasB elastase(84%), caseinase(70%), pyocyanin (84%), pyoverdin and biofilm formation(87%) in Pseudomonas aeruginosa PAO1. Strain S8-07 also showed highly significant reduction (90%) in prodigiosin, secreted casienase (92%), hemolytic activity (73%) and biofilm formation (61%) in Serratia marcescens. Strain S8-07, identified as Bacillus pumilus (accession number FJ584416), showed distinct profiles of inhibition against the virulence factors of both P. aeruginosa PAO1 ( las, rhl) and S. marcescens ( shl). Polar extraction and proteinase K treatment of the culture supernatant confirmed that the anti-QS activity of S8-07 was indeed due to a protein molecule. Acidification assay and HPLC analysis revealed that the degradation of AHL was not due to lactonase activity, but rather, was due to acylase activity of S8-07. Thus, novel anti-QS acylase activity is reported for the first time from a B. pumilus strain of marine origin.

Reduced expression of virulence factors in multidrug-resistant Pseudomonas aeruginosa strains

MDR Pseudomonas aeruginosa strains are isolated from clinical specimens with increasing frequency. It seems that acquiring genes which determine antibiotic resistance usually comes at a biological cost of impaired bacterial physiology. There is no information on investigations comparing phenotypic differences in MDR and MDS P. aeruginosa strains in literature. The study included 150 clinical P. aeruginosa isolates (75 classified as MDS and 75 as MDR). PFGE analysis revealed five pairs of identical isolates in the group of MDR strains and the results obtained for these strains were not included in the statistical analyses. MDR strains adhered to polystyrene to a lesser extent than MDS strains. The growth rate in the liquid medium was significantly lower for MDR strains. Detectable amounts of alginate were present in the culture supernatants of seven MDS and six MDR strains. The MDR P. aeruginosa strains which were investigated produced significantly lower amounts of extracellular material binding Congo Red, lower lipolytic, elastase, LasA protease, phospholipase C activity and pyocyanin quantity in culture supernatants when compared with MDS strains. No significant differences were observed between MDR and MDS strains in proteolytic activity. In conclusion, the MDR P. aeruginosa strains have impaired virulence when compared to MDS strains.

GidA Posttranscriptionally Regulates rhl Quorum Sensing in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa utilizes two interconnected acyl-homoserine lactone quorum-sensing (acyl-HSL QS) systems, LasRI and RhlRI, to regulate the expression of hundreds of genes. The QS circuitry itself is integrated into a complex network of regulation by other factors. However, our understanding of this network is still unlikely to be complete, as a comprehensive, saturating approach to identifying regulatory components has never been attempted. Here, we utilized a nonredundant P. aeruginosa PA14 transposon library to identify additional genes that regulate QS at the level of LasRI/RhlRI. We initially screened all 5,459 mutants for loss of function in one QS-controlled trait (skim milk proteolysis) and then rescreened attenuated candidates for defects in other QS phenotypes (LasA protease, rhamnolipid, and pyocyanin production) to exclude mutants defective in functions other than QS. We identified several known and novel genes, but only two novel genes, gidA and pcnB, affected all of the traits assayed. We characterized gidA, which exhibited the most striking QS phenotypes, further. This gene is predicted to encode a conserved flavin adenine dinucleotide-binding protein involved in tRNA modification. Inactivation of the gene primarily affected rhlR-dependent QS phenotypes such as LasA, pyocyanin, and rhamnolipid production. GidA affected RhlR protein but not transcript levels and also had no impact on LasR and acyl-HSL production. Overexpression of rhlR in a gidA mutant partially restored QS-dependent phenotypes. Taken together, these results indicate that GidA selectively controls QS gene expression posttranscriptionally via RhlR-dependent and -independent pathways.

Evaluation of Pseudomonas aeruginosa staphylolysin (LasA protease) in the treatment of methicillin-resistant Staphylococcus aureus endophthalmitis in a rat model

Therapy of S. aureus ocular infections is increasingly challenging due to emerging resistant strains. Staphylolysin (also called LasA protease) is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa. The purpose of this study was to evaluate the efficacy of staphylolysin as a therapy for experimental methicillin-resistant Staphylococcus aureus (MRSA) endophthalmitis, focusing on its bactericidal activity. Endophthalmitis was induced in the right eyes of 46 rats by an intravitreal injection of 50-160 MRSA cells. Two therapeutic regimens were evaluated: (i) an intravitreal injection of staphylolysin at 6 hours post-infection; (ii) two successive intravitreal injections of staphylolysin given at 6 and 30 hours post-infection. Control eyes were injected with vehicle alone at the same times. The rats were sacrificed 48 hours after infection, and the vitreous was withdrawn for determination of colony forming units (CFU). Potential adverse effects of intravitreal staphylolysin injection were assessed histopathologically in four uninfected eyes, enucleated from rats sacrificed 1 month after intravitreal staphylolysin injection. In eyes treated by the single-injection regimen, staphylolysin reduced the mean CFU value per vitreous threefold as compared to control (2,055 +/- 3,144 and 6,432 +/- 6,389 CFU/vitreous, respectively; P = 0.02). The repeated injection protocol was more effective, reducing the mean CFU value per vitreous by two orders of magnitude as compared to control (1,148 +/- 3,096 and 143,519 +/- 151,358 CFU/vitreous, respectively; P = 0.0005). Histopathological analysis showed no structural damage in eyes injected intravitreally with staphylolysin. Staphylolysin is effective in the treatment of experimental MRSA-induced endophthalmitis in rats, and causes no morphological adverse effects to ocular tissues. Staphylolysin may be beneficial in the treatment of S. aureus endophthalmitis in humans.

Inhibition of Quorum Sensing-Controlled Virulence Factor Production in Pseudomonas aeruginosa by South Florida Plant Extracts

Quorum sensing (QS) is a key regulator of virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Aqueous extracts of six plants, Conocarpus erectus, Chamaesyce hypericifolia, Callistemon viminalis, Bucida buceras, Tetrazygia bicolor, and Quercus virginiana, were examined in this study for their effects on P. aeruginosa virulence factors and the QS system. C. erectus, B. buceras, and C. viminalis caused a significant inhibition of LasA protease, LasB elastase, pyoverdin production, and biofilm formation. Additionally, each plant presented a distinct effect profile on the las and rhl QS genes and their respective signaling molecules, suggesting that different mechanisms are responsible for efficacy. Extracts of all plants caused the inhibition of QS genes and QS-controlled factors, with marginal effects on bacterial growth, suggesting that the quorum-quenching mechanisms are unrelated to static or cidal effects.

Widespread pyocyanin over-production among isolates of a cystic fibrosis epidemic strain

BackgroundSome isolates of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa exhibit an unusual virulence-related phenotype, characterized by over-production of quorum sensing-regulated exoproducts such as pyocyanin and LasA protease. Our aim was to determine the prevalence of this unusual phenotype amongst isolates of the epidemic strain, and to study other intraclonal phenotypic and genotypic variations.ResultsThe unusual phenotype was detected in at least one epidemic strain isolate from the majority of cystic fibrosis patients tested, and can be retained for up to seven years during chronic infection. Multiple sequential isolates of the epidemic strain taken from six patients over a period of up to nine years exhibited a wide range of phenotypes, including different antimicrobial susceptibilities. Our data suggest that each sputum sample contains a mixture of phenotypes and genotypes within the epidemic strain population, including within colony morphotypes. Many isolates exhibit premature (during early rather than late exponential growth) and over-production of pyocyanin, which has a number of toxic effects directly relevant to cystic fibrosis.ConclusionThe widespread occurrence of this unusual phenotype suggests that it may play an important role in the success of the epidemic strain.

New continuous and specific fluorometric assays for Pseudomonas aeruginosa elastase and LasA protease

A highly sensitive assay based on new internally quenched fluorogenic peptide substrates has been developed for monitoring protease activities. These novel substrates comprise an Edans (5-(2-aminoethylamino)-1-naphthalenesulfonic acid) group at the C terminus and a Dabsyl (4-(dimethylamino)azobenzene-4′-sulfonyl chloride) fluorophore at the N terminus of the peptide chains. The Edans fluorescence increases upon peptide hydrolysis by Pseudomonas aeruginosa proteases, and this increase is directly proportional to the amount of substrate cleaved, i.e., protease activity. The substrates Dabsyl-Ala-Ala-Phe-Ala-Edans and Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans were used for testing the peptidasic activities of P. aeruginosa elastase and LasA protease, respectively. Elastase and LasA kinetic parameters were calculated and a sensitive assay was designed for the detection of P. aeruginosa proteases in bacterial supernatants. The sensitivity and the small sample requirements make the assay suitable for high-throughput screening of biological samples. Furthermore, this P. aeruginosa protease assay improves upon existing assays because it is simple, it requires only one step, and even more significantly it is enzyme specific.

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.

Pseudomonas aeruginosaAmpR Is a Global Transcriptional Factor That Regulates Expression of AmpC and PoxB β-Lactamases, Proteases, Quorum Sensing, and Other Virulence Factors

In members of the family Enterobacteriaceae, ampC, which encodes a beta-lactamase, is regulated by an upstream, divergently transcribed gene, ampR. However, in Pseudomonas aeruginosa, the regulation of ampC is not understood. In this study, we compared the characteristics of a P. aeruginosa ampR mutant, PAOampR, with that of an isogenic ampR+ parent. The ampR mutation greatly altered AmpC production. In the absence of antibiotic, PAOampR expressed increased basal beta-lactamase levels. However, this increase was not followed by a concomitant increase in the P(ampC) promoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated beta-lactamase, PoxB. We found that the expression of P. aeruginosa ampR greatly altered the beta-lactamase production from ampC and poxB in Escherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive P(ampR) promoter activity in PAOampR indicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying the ampR mutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the P(lasA), P(lasI), and P(lasR) expression. The reduction in the LasB activity was positively correlated with the P(rhlR) expression. Thus, AmpR plays a dual role, positively regulating the ampC, lasB, and rhlR expression levels and negatively regulating the poxB, lasA, lasI, and lasR expression levels.

Pseudomonas aeruginosa LasA protease in treatment of experimental staphylococcal keratitis.

LasA protease is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa. We have examined the effectiveness of LasA protease in the treatment of staphylococcal keratitis caused by methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates in a rabbit model. Keratitis was induced by intrastromal injection of the bacteria. The eyes were treated topically, and the efficacy of LasA protease was compared to those of lysostaphin (a staphylolytic protease secreted by Staphylococcus simulans) and vancomycin. When treatment was initiated early (4 h) after infection, practically all of the MSSA- and MRSA-infected corneas were sterilized by LasA protease, and its efficacy in eradicating the bacteria was comparable to those of lysostaphin and vancomycin. By contrast, most of the control corneas were heavily infected, with median values of 4.5 x 10(6) (MSSA) and 5 x 10(5) (MRSA) CFU/cornea (P < 0.001). When treatment was initiated late (10 h) after infection, LasA protease reduced the numbers of CFU in both MSSA- and MRSA-infected corneas by 3 to 4 orders of magnitude compared to the numbers of CFU for the controls (median values, 1,380 and 30 CFU/cornea, respectively, for the treated animals compared to 1.2 x 10(6) and 5 x 10(5) CFU/cornea for the respective controls [P = 0.001]), and it was more effective than vancomycin in eradicating MRSA cells (P = 0.02). In both the early- and the late-treatment protocols, the clinical scores for eyes treated with LasA protease were significantly lower than those for the eyes of the corresponding controls and comparable to those for the lysostaphin- and vancomycin-treated eyes. We conclude that LasA protease is effective in the treatment of experimental S. aureus keratitis in rabbits and may have potential for the treatment of disease in humans.

A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction

Pseudomonas aeruginosa LasA protease is a secreted metalloendopeptidase that can lyse Staphylococcus aureus cells by cleaving the pentaglycine bridges of their peptidoglycan. It can also degrade elastin and stimulate shedding of cell-surface proteoglycans, activities implicated in pathogenesis of P. aeruginosa infections. The activity of LasA protease can be assayed spectrophotometrically by following the reduction in turbidity of S. aureus cell suspensions. This assay, however, does not permit kinetic studies and its reproducibility is poor. Here we describe a two-stage enzymatic reaction for the continuous measurement of LasA protease activity using a defined substrate, succinyl-Gly-Gly-Phe-4-nitroanilide, supplemented with Streptomyces griseus aminopeptidase. Cleavage of the Gly-Phe bond by LasA protease is followed by hydrolysis of the product Phe-4-nitroanilide by the aminopeptidase and the rate of release of the chromophore (4-nitroaniline) is measured spectrophotometrically using a 96-well microplate reader. Activity of nanogram amounts of LasA protease could be determined within a few minutes. Furthermore, this assay permitted the determination of K m and k cat values for LasA protease, which were 0.46 mM and 11.8 s −1, respectively. Pseudomonas elastase was also active in the assay. However, it was less effective than LasA protease and its activity was inhibited by phosphoramidon. The assay is highly sensitive and reproducible, providing a convenient tool for further studies of LasA protease function(s) and mechanism of action.

Role of bacterial proteases in pseudomonal and serratial keratitis

Pseudomonas aeruginosa and Serratia marcescens can cause refractory keratitis resulting in corneal perforation and blindness. These bacteria produce various kinds of proteases. In addition to pseudomonal elastase (LasB) and alkaline protease, LasA protease and protease IV have recently been found to be more important virulence factors of P. aeruginosa . S. marcescens produces a cysteine protease in addition to metalloproteases. These bacterial proteases have a number of biological activities, such as degradation of tissue constituents and host defense-oriented proteins, as well as activation of zymogens (Hageman factor, prekallikrein and pro-matrix metalloproteinases) through limited proteolysis. In this article, the properties of these bacterial proteases are reviewed and the pathogenic roles of these proteases in pseudomonal keratitis are discussed.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1.

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhlRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown QS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Deltalas mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-I) could not be detected when any of the lasRI or rhlRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of QS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a QS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.