Isolation and characterisation of a novel Metalloprotease from Exiguobacterium oxidotolerans

Tracy White

Research output: ThesisDoctoral Thesis

Abstract

A 50 kDa thermolysin-like protease was identified from Exiguobacterium oxidotolerans, a marine derived bacteria isolated from the east coast of Northern Ireland and also a potential emerging opportunistic pathogen. The protease was shown to be sensitive to 1, 10 phenanthroline with little or no inhibition in the presence of E-64 or PMSF during the hydrolysis of azocasein and MMP2/7 fluorogenic substrate. The thermolysin-like protease was purified using hydrophobic interaction chromatography and a novel high throughput agar extraction method, before identification using LC-MS-MS, resulting in 5 peptide fragments exhibiting homology to the thermolysin of E. sibiricum. Inhibitor profiling was performed using a combinatorial 20 x 20 library of N-alpha-mercaptoamide dipeptide inhibitors, which revealed a preference for aliphatic residues and aromatic or polar residues in P1’ and P2’ respectively, with SH-CO-CH2-Met-Tyr-NH2 being the most potent inhibitor of the thermolysin-like protease, with a Ki value of 1.95 μM, while SH-CO-CH2-Pro-Arg-NH2 demonstrated the lowest Fi value of -0.0063.
The potential virulence mechanisms of E. oxidotolerans were also investigated, revealing the proteolytic disarming of PAR-1 receptors of human prostate cancer (PC-3) cells, disrupting cascade activation of the ERK signalling gene, which was prevented in the presence of SH-CO-CH2-Pro-Arg-NH2. Proteolytic degradation of IgA, IgM and IgG was observed in heat denatured immunoglobulins only, however immunoglobulins in their native state and antimicrobial peptides (cathelicidins and β-defensins) were not hydrolysed and cannot therefore be considered potential substrates of this thermolysin-like protease. Degradation of erythrocytes was also revealed by conditioned media and to a lesser extent by E. oxidotolerans directly. Therefore, this thesis describes the discovery and characterisation of a novel 50 kDa VII thermolysin-like metalloprotease from a marine isolate identified as E. oxidotolerans, which may play a role in virulence associated with this emerging opportunistic pathogen.
LanguageEnglish
QualificationPhD
Publication statusUnpublished - 13 Nov 2012
Externally publishedYes

Fingerprint

Metalloproteases
Carbon Monoxide
Thermolysin
Pathogens
Virulence
Immunoglobulins
Cathelicidins
Peptides
PAR-1 Receptor
beta-Defensins
Degradation
Northern Ireland
Peptide Fragments
Dipeptides
Conditioned Culture Medium
Chromatography
Hydrophobic and Hydrophilic Interactions
Fluorescent Dyes
Immunoglobulin A
Libraries

Keywords

  • Exiguobacterium oxidotolerans
  • Metalloprotease

Cite this

@phdthesis{bcec66a89d5c41ecac0ca96d0c5d372b,
title = "Isolation and characterisation of a novel Metalloprotease from Exiguobacterium oxidotolerans",
abstract = "A 50 kDa thermolysin-like protease was identified from Exiguobacterium oxidotolerans, a marine derived bacteria isolated from the east coast of Northern Ireland and also a potential emerging opportunistic pathogen. The protease was shown to be sensitive to 1, 10 phenanthroline with little or no inhibition in the presence of E-64 or PMSF during the hydrolysis of azocasein and MMP2/7 fluorogenic substrate. The thermolysin-like protease was purified using hydrophobic interaction chromatography and a novel high throughput agar extraction method, before identification using LC-MS-MS, resulting in 5 peptide fragments exhibiting homology to the thermolysin of E. sibiricum. Inhibitor profiling was performed using a combinatorial 20 x 20 library of N-alpha-mercaptoamide dipeptide inhibitors, which revealed a preference for aliphatic residues and aromatic or polar residues in P1’ and P2’ respectively, with SH-CO-CH2-Met-Tyr-NH2 being the most potent inhibitor of the thermolysin-like protease, with a Ki value of 1.95 μM, while SH-CO-CH2-Pro-Arg-NH2 demonstrated the lowest Fi value of -0.0063. The potential virulence mechanisms of E. oxidotolerans were also investigated, revealing the proteolytic disarming of PAR-1 receptors of human prostate cancer (PC-3) cells, disrupting cascade activation of the ERK signalling gene, which was prevented in the presence of SH-CO-CH2-Pro-Arg-NH2. Proteolytic degradation of IgA, IgM and IgG was observed in heat denatured immunoglobulins only, however immunoglobulins in their native state and antimicrobial peptides (cathelicidins and β-defensins) were not hydrolysed and cannot therefore be considered potential substrates of this thermolysin-like protease. Degradation of erythrocytes was also revealed by conditioned media and to a lesser extent by E. oxidotolerans directly. Therefore, this thesis describes the discovery and characterisation of a novel 50 kDa VII thermolysin-like metalloprotease from a marine isolate identified as E. oxidotolerans, which may play a role in virulence associated with this emerging opportunistic pathogen.",
keywords = "Exiguobacterium oxidotolerans, Metalloprotease",
author = "Tracy White",
year = "2012",
month = "11",
day = "13",
language = "English",

}

Isolation and characterisation of a novel Metalloprotease from Exiguobacterium oxidotolerans. / White, Tracy.

2012. 283 p.

Research output: ThesisDoctoral Thesis

TY - THES

T1 - Isolation and characterisation of a novel Metalloprotease from Exiguobacterium oxidotolerans

AU - White, Tracy

PY - 2012/11/13

Y1 - 2012/11/13

N2 - A 50 kDa thermolysin-like protease was identified from Exiguobacterium oxidotolerans, a marine derived bacteria isolated from the east coast of Northern Ireland and also a potential emerging opportunistic pathogen. The protease was shown to be sensitive to 1, 10 phenanthroline with little or no inhibition in the presence of E-64 or PMSF during the hydrolysis of azocasein and MMP2/7 fluorogenic substrate. The thermolysin-like protease was purified using hydrophobic interaction chromatography and a novel high throughput agar extraction method, before identification using LC-MS-MS, resulting in 5 peptide fragments exhibiting homology to the thermolysin of E. sibiricum. Inhibitor profiling was performed using a combinatorial 20 x 20 library of N-alpha-mercaptoamide dipeptide inhibitors, which revealed a preference for aliphatic residues and aromatic or polar residues in P1’ and P2’ respectively, with SH-CO-CH2-Met-Tyr-NH2 being the most potent inhibitor of the thermolysin-like protease, with a Ki value of 1.95 μM, while SH-CO-CH2-Pro-Arg-NH2 demonstrated the lowest Fi value of -0.0063. The potential virulence mechanisms of E. oxidotolerans were also investigated, revealing the proteolytic disarming of PAR-1 receptors of human prostate cancer (PC-3) cells, disrupting cascade activation of the ERK signalling gene, which was prevented in the presence of SH-CO-CH2-Pro-Arg-NH2. Proteolytic degradation of IgA, IgM and IgG was observed in heat denatured immunoglobulins only, however immunoglobulins in their native state and antimicrobial peptides (cathelicidins and β-defensins) were not hydrolysed and cannot therefore be considered potential substrates of this thermolysin-like protease. Degradation of erythrocytes was also revealed by conditioned media and to a lesser extent by E. oxidotolerans directly. Therefore, this thesis describes the discovery and characterisation of a novel 50 kDa VII thermolysin-like metalloprotease from a marine isolate identified as E. oxidotolerans, which may play a role in virulence associated with this emerging opportunistic pathogen.

AB - A 50 kDa thermolysin-like protease was identified from Exiguobacterium oxidotolerans, a marine derived bacteria isolated from the east coast of Northern Ireland and also a potential emerging opportunistic pathogen. The protease was shown to be sensitive to 1, 10 phenanthroline with little or no inhibition in the presence of E-64 or PMSF during the hydrolysis of azocasein and MMP2/7 fluorogenic substrate. The thermolysin-like protease was purified using hydrophobic interaction chromatography and a novel high throughput agar extraction method, before identification using LC-MS-MS, resulting in 5 peptide fragments exhibiting homology to the thermolysin of E. sibiricum. Inhibitor profiling was performed using a combinatorial 20 x 20 library of N-alpha-mercaptoamide dipeptide inhibitors, which revealed a preference for aliphatic residues and aromatic or polar residues in P1’ and P2’ respectively, with SH-CO-CH2-Met-Tyr-NH2 being the most potent inhibitor of the thermolysin-like protease, with a Ki value of 1.95 μM, while SH-CO-CH2-Pro-Arg-NH2 demonstrated the lowest Fi value of -0.0063. The potential virulence mechanisms of E. oxidotolerans were also investigated, revealing the proteolytic disarming of PAR-1 receptors of human prostate cancer (PC-3) cells, disrupting cascade activation of the ERK signalling gene, which was prevented in the presence of SH-CO-CH2-Pro-Arg-NH2. Proteolytic degradation of IgA, IgM and IgG was observed in heat denatured immunoglobulins only, however immunoglobulins in their native state and antimicrobial peptides (cathelicidins and β-defensins) were not hydrolysed and cannot therefore be considered potential substrates of this thermolysin-like protease. Degradation of erythrocytes was also revealed by conditioned media and to a lesser extent by E. oxidotolerans directly. Therefore, this thesis describes the discovery and characterisation of a novel 50 kDa VII thermolysin-like metalloprotease from a marine isolate identified as E. oxidotolerans, which may play a role in virulence associated with this emerging opportunistic pathogen.

KW - Exiguobacterium oxidotolerans

KW - Metalloprotease

M3 - Doctoral Thesis

ER -