Development of low-cost and portable detection platforms for bacterial detection

In this study, a diagnostic platform for detecting pathogens, including Listeria monocytogenes, E. coli O157:H11, Staphylococcus aureus, Legionella spp., P. gingivalis, and Pseudomonas aeruginosa, has been developed. Fluorogenic substrates were used as a potential tool for detecting the virulence of the proteases. The identified substrates were conjugated with nanomagnetic particles to develop colorimetric, flexible, and portable platforms. The lowest detection limit of the biosensing platforms was determined as Listeria 2.17 × 102 cfu/mL,7 cfu/mL for Staphylococcus aureus and 12 cfu/mL for E. coli O157:H7, P. gingivalis was 49 cfu/mL,60 cfu/mL for Legionella spp., and 102 cfu/mL. for P. aeruginosa. The sensors were tested with other bacteria to investigate the cross-reactivity and assess the specificity; most of the sensors were found to be specific. In order to use the biosensors for field diagnosis, the sensors were evaluated using different food matrices and environmental samples from various sources. The sensors showed significant stability with all the samples. In this study, the point of interest was to devise a sensitive and quick method for detecting pathogenic bacteria in complex samples. To that end, a colorimetric sensor employing nanomagnetic particles that targeted the proteolytic activity of protease enzymes secreted by bacteria, including Listeria monocytogenes, E. coli O157:H11, Staphylococcus aureus, Legionella spp., P. gingivalis, and Pseudomonas aeruginosa, was designed. The highly specific and semi-quantitative diagnostic device developed in the current study is portable and straightforward to operate by a nurse or a non-skilled clinician, making it a potential and highly suitable system for low-resource

Enzymatic Depilation of Animal Hide: Identification of Elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a Depilating Protease

Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme

The Role of the Propeptide and its Residues in Activation and Secretion of Elastase, an M4 Metalloprotease Secreted by Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes several proteases associated with pathogenesis, but the most abundant and active is elastase (M4 metalloendopeptidase). Elastase (lasB), is first synthesized as a preproenzyme, with a signal peptide, an 18-kDa N-terminal propeptide, and a 33-kDa mature domain. The propeptide functions as an intramolecular chaperone that is required for the folding and secretion of elastase, but ultimately is proteolytically removed and degraded. Previous research has identified the conserved residues in the propeptide of elastase as compared to other M4 protease precursors and showed some among them to be important for the production of active elastase. In this project, the ability of the propeptide alone to fold into a defined secondary structure was explored and a molecular model was created. Furthermore, the effects of substitutions on conserved residues in the propeptide of plasmid-encoded lasB pro alleles were assessed by expressing them in a lasB propeptide mutant. The kinetics of elastase activity in culture supernatants was quantitated using a fluorescent substrate, Abz-AGLA-p-Nitro-Benzyl-Amide, to provide an accurate assessment of the effects of mutant propeptides. In vitro refolding studies were also performed to determine the effects of specific substitutions on foldase activity of the propeptide. When wild-type propeptide and mature elastase were denatured as separate proteins in guanidine-HCl buffer and renatured together, restoration of activity of the refolded elastase was measured, which was propeptide-dependent. Several mutant propeptides have now been shown to have defects using this in vitro foldase assay. Additional mutants were near wild-type activity level suggesting their role in recognition by the secretion apparatus. Residue locations were determined on a molecular model of the complex and confirmed the role of the secretion mutants as residues on the exterior. Residues that had diminished ability to refold in the in vitro assay were found to be in the interior parts of the complex, confirming their ability to be critical residues at the interface of the proteins or important in the stability of the propeptide’s intrinsic structure. The goal was to perform a series of comprehensive analyses of the propeptide and its conserved residues in order to determine its role as an intramolecular chaperone

MOLECULAR CHARACTERISATION OF A TWO-COMPONENT REGULATORY SYSTEM FROM Burkholderia pseudomallei

Studies were undertaken to clone and characterise a two-component regulatory system from a clinical isolate (204) of the human and animal pathogen Burkholderia pseudomallei. A number of genomic libraries were constructed in E. coli host-vector systems and screened for the presence of a two-component system using oligonucleotide probes based on nucleotide sequence homology. Fragments of genomic DNA were cloned and sequenced and found to possess two open reading frames (ORFs) that overlap with a single nucleotide and are believed to encode a novel two-component regulatory system. A possible promoter region was identified upstream of the two ORFs, mrgR and mrgS, which read in the same direction and may represent an operon. The deduced translation of mrgR reveals a protein, MrgR, which possesses conserved motifs that are consistent with the phosphorylation domains and DNA-binding helix-turn-helix structure of a family of response regulatory proteins. The deduced translation of mrgS reveals that the MrgS protein possesses all the invariant amino acids that characterise other sensor regulatory proteins. Southern hybridisation studies showed that the mrgRS locus was present in 19 isolates of B. pseudomallei from a wide geographical derivation, but not in any closely related bacterial species, including Burkholderia thailandensis. The expression of the two genes was verified using antibodies developed to synthetic peptides based on sequences from the C- and N-terminal regions of MrgR and MrgS, respectively. The specificity of the antibodies was confirmed in Western blotting studies in which almost all of mrgR and the proximal quarter of mrgS were translationally fused with malE (MBP-MrgR and MBP-MrgS) and expressed in E. coli K12. The antibodies were used to probe Western blots of cellular and extracellular extracts of different isolates of B. pseudomallei and identified multiple bands in whole-cell lysates. The sizes of two of these bands were 24 kDa and 115 kDa, which may represent the unprocessed forms of MrgR and MrgS, respectively. It was proposed that the other bands represented either isoforms or degradation products of the full-length proteins. The recognition of all bands was abolished following pre-incubation of the antibodies with the immunising peptide but remained unaffected if an irrelevant peptide, was used for this purpose. Western blot analysis demonstrated that serum antibodies from a patient with acute melioidosis recognised MBP-MrgR but not MBP-MrgS suggesting a possible role for MrgR in the disease process. The expression of mrgR and mrgS was found to be constitutive in B. pseudomallei that had been cultured using different combinations of temperature, pH and NaCl suggesting that the genes perform a number of biological functions. There is some evidence that at 42°C the processing of MrgR and MrgS may be altered and the possible mechanisms for this are discussed. B. pseudomallei grew better at 42°C and pH 5 and less well at 25°C and pH 8 and this was influenced by NaCl concentration partly reflecting the environmental distribution and intracellular nature of the pathogen. Environmental and clinical isolates of B. pseudomallei differed in the pH optimum for growth at 42°C. The DNA flanking the mrgRS locus in isolate 204 was cloned, sequenced, and seven ORFs were identified including a transcriptional regulatory gene similar to bvgR of Bordetella pertussis. Southern blot analysis using three different DNA probes revealed restriction fragment length polymorphisms (RFLPs) in the region downstream of mrgRS. Two distinct RFLP patterns were identified among 16 different isolates of B. pseudomallei. The potential effects of this variation on gene expression and protein function await further investigation.Public Health Laboratory Service (PHLS), Derriford hospital, Plymout

Occupational respiratory diseases

Shipping list no.: 87-222-P."September 1986."S/N 017-033-00425-1 Item 499-F-2Also available via the World Wide Web.Includes bibliographies and index