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Title: Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79

Abstract

The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn2+. When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5–9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however,more » the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases.« less

Authors:
 [1];  [2];  [3];  [2];  [4];  [1]
  1. Khon Kaen Univ. (Thailand). Faculty of Medicine. Dept. of Biochemistry and Melioidosis Research Center
  2. Khon Kaen Univ. (Thailand). Faculty of Medicine. Dept. of Microbiology and Melioidosis Research Center
  3. Khon Kaen Univ. (Thailand). Faculty of Medicine. Dept. of Microbiology
  4. Univ. of California, Davis, CA (United States). Dept. of Land, Air and Water Resources; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1627035
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
AMB Express
Additional Journal Information:
Journal Volume: 6; Journal Issue: 1; Journal ID: ISSN 2191-0855
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biotechnology & Applied Microbiology; Endolysins; Enzyme; Gene expression; Peptidoglycan

Citation Formats

Khakhum, Nittaya, Yordpratum, Umaporn, Boonmee, Atcha, Tattawasart, Unchalee, Rodrigues, Jorge L. M., and Sermswan, Rasana W. Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79. United States: N. p., 2016. Web. doi:10.1186/s13568-016-0251-7.
Khakhum, Nittaya, Yordpratum, Umaporn, Boonmee, Atcha, Tattawasart, Unchalee, Rodrigues, Jorge L. M., & Sermswan, Rasana W. Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79. United States. doi:10.1186/s13568-016-0251-7.
Khakhum, Nittaya, Yordpratum, Umaporn, Boonmee, Atcha, Tattawasart, Unchalee, Rodrigues, Jorge L. M., and Sermswan, Rasana W. Thu . "Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79". United States. doi:10.1186/s13568-016-0251-7. https://www.osti.gov/servlets/purl/1627035.
@article{osti_1627035,
title = {Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79},
author = {Khakhum, Nittaya and Yordpratum, Umaporn and Boonmee, Atcha and Tattawasart, Unchalee and Rodrigues, Jorge L. M. and Sermswan, Rasana W.},
abstractNote = {The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn2+. When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5–9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases.},
doi = {10.1186/s13568-016-0251-7},
journal = {AMB Express},
issn = {2191-0855},
number = 1,
volume = 6,
place = {United States},
year = {2016},
month = {9}
}

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Works referenced in this record:

Strigolactones Stimulate Arbuscular Mycorrhizal Fungi by Activating Mitochondria
journal, June 2006


The MEROPS batch BLAST: A tool to detect peptidases and their non-peptidase homologues in a genome
journal, February 2008


Characterization of endolysin from a Salmonella Typhimurium-infecting bacteriophage SPN1S
journal, April 2012


Phages will out: strategies of host cell lysis
journal, March 2000


Phage cocktails and the future of phage therapy
journal, June 2013

  • Chan, Benjamin K.; Abedon, Stephen T.; Loc-Carrillo, Catherine
  • Future Microbiology, Vol. 8, Issue 6
  • DOI: 10.2217/fmb.13.47

Phage lytic enzymes as therapy for antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model
journal, November 2003

  • Jado, I.
  • Journal of Antimicrobial Chemotherapy, Vol. 52, Issue 6
  • DOI: 10.1093/jac/dkg485

Correlation between biofilm production, antibiotic susceptibility and exopolysaccharide composition in Burkholderia pseudomallei bps I, ppk , and rpo S mutant strains: Biofilm components and drug resistance
journal, November 2015

  • Mongkolrob, Rungrawee; Taweechaisupapong, Suwimol; Tungpradabkul, Sumalee
  • Microbiology and Immunology, Vol. 59, Issue 11
  • DOI: 10.1111/1348-0421.12331

The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes
journal, October 2008


The Pfam protein families database: towards a more sustainable future
journal, December 2015

  • Finn, Robert D.; Coggill, Penelope; Eberhardt, Ruth Y.
  • Nucleic Acids Research, Vol. 44, Issue D1
  • DOI: 10.1093/nar/gkv1344

Consensus on the Development of Vaccines against Naturally Acquired Melioidosis
journal, June 2015

  • Limmathurotsakul, Direk; Funnell, Simon G. P.; Torres, Alfredo G.
  • Emerging Infectious Diseases, Vol. 21, Issue 6
  • DOI: 10.3201/eid2106.141480

Vancomycin Resistance in Gram-Positive Cocci
journal, January 2006

  • Courvalin, Patrice
  • Clinical Infectious Diseases, Vol. 42, Issue Supplement_1
  • DOI: 10.1086/491711

Isolation and characterization of a novel podovirus which infects burkholderia pseudomallei
journal, January 2011

  • Gatedee, Jiraporn; Kritsiriwuthinan, Kanyanan; Galyov, Edouard E.
  • Virology Journal, Vol. 8, Issue 1
  • DOI: 10.1186/1743-422X-8-366

Effects of soil pH, temperature and water content on the growth ofBurkholderia pseudomallei
journal, March 2003

  • Chen, Y. S.; Chen, S. C.; Kao, C. M.
  • Folia Microbiologica, Vol. 48, Issue 2
  • DOI: 10.1007/BF02930965

Food-grade controlled lysis of Lactococcus lactis for accelerated cheese ripening
journal, October 1997

  • Ruyter, Pascalle G. G. A. de; Kuipers, Oscar P.; Meijer, Wilco C.
  • Nature Biotechnology, Vol. 15, Issue 10
  • DOI: 10.1038/nbt1097-976

InterProScan - an integration platform for the signature-recognition methods in InterPro
journal, September 2001


Bacteriophage Endolysins as a Novel Class of Antibacterial Agents
journal, April 2006

  • Borysowski, Jan; Weber-Dąbrowska, Beata; Górski, Andrzej
  • Experimental Biology and Medicine, Vol. 231, Issue 4
  • DOI: 10.1177/153537020623100402

Biochemical Characterization and Validation of a Catalytic Site of a Highly Thermostable Ts2631 Endolysin from the Thermus scotoductus Phage vB_Tsc2631
journal, September 2015


Cloning and expression of a gene encoding the lytic functions of Bacillus amyloliquefaciens phage: Evidence of an auxiliary lysis system
journal, January 1999


Experimental Phage Therapy for Burkholderia pseudomallei Infection
journal, July 2016


Diffusion and activity of antibiotics against Burkholderia pseudomallei biofilms
journal, April 2012

  • Pibalpakdee, Phannarai; Wongratanacheewin, Surasakdi; Taweechaisupapong, Suwimol
  • International Journal of Antimicrobial Agents, Vol. 39, Issue 4
  • DOI: 10.1016/j.ijantimicag.2011.12.010

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
journal, September 1997

  • Altschul, Stephen F.; Madden, Thomas L.; Schäffer, Alejandro A.
  • Nucleic Acids Research, Vol. 25, Issue 17, p. 3389-3402
  • DOI: 10.1093/nar/25.17.3389

φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. mallei strain infectivity
journal, January 2012

  • Kvitko, Brian H.; Cox, Christopher R.; DeShazer, David
  • BMC Microbiology, Vol. 12, Issue 1
  • DOI: 10.1186/1471-2180-12-289

Glycopeptide resistance in enterococci
journal, October 1996


A standardized approach for accurate quantification of murein hydrolase activity in high-throughput assays
journal, April 2007

  • Briers, Yves; Lavigne, Rob; Volckaert, Guido
  • Journal of Biochemical and Biophysical Methods, Vol. 70, Issue 3
  • DOI: 10.1016/j.jbbm.2006.10.009

The gut microbiota shapes intestinal immune responses during health and disease
journal, May 2009

  • Round, June L.; Mazmanian, Sarkis K.
  • Nature Reviews Immunology, Vol. 9, Issue 5
  • DOI: 10.1038/nri2515

Growing Burkholderia pseudomallei in Biofilm Stimulating Conditions Significantly Induces Antimicrobial Resistance
journal, February 2010


Peptidoglycan types of bacterial cell walls and their taxonomic implications.
journal, January 1972


The gut flora as a forgotten organ
journal, July 2006


Soil physicochemical properties related to the presence of Burkholderia pseudomallei
journal, December 2008

  • Palasatien, Supapong; Lertsirivorakul, Rungruang; Royros, Phairat
  • Transactions of the Royal Society of Tropical Medicine and Hygiene, Vol. 102
  • DOI: 10.1016/S0035-9203(08)70003-8

SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information
journal, April 2014

  • Biasini, Marco; Bienert, Stefan; Waterhouse, Andrew
  • Nucleic Acids Research, Vol. 42, Issue W1
  • DOI: 10.1093/nar/gku340

Bacteriophage therapy: a potential solution for the antibiotic resistance crisis
journal, February 2014

  • Golkar, Zhabiz; Bagasra, Omar; Pace, Donald Gene
  • The Journal of Infection in Developing Countries, Vol. 8, Issue 02
  • DOI: 10.3855/jidc.3573

Identification of the Burkholderia pseudomallei bacteriophage ST79 lysis gene cassette
journal, May 2016

  • Khakhum, N.; Yordpratum, U.; Boonmee, A.
  • Journal of Applied Microbiology, Vol. 121, Issue 2
  • DOI: 10.1111/jam.13151

Identification and characterization of a highly thermostable bacteriophage lysozyme
journal, November 2004

  • Lavigne, R.; Briers, Y.; Hertveldt, K.
  • Cellular and Molecular Life Sciences, Vol. 61, Issue 21
  • DOI: 10.1007/s00018-004-4301-y

Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages ?KZ and EL
journal, September 2007


Novel lytic bacteriophages from soil that lyse Burkholderia pseudomallei: Bacteriophage of B. pseudomallei
journal, November 2010


Bacteriophage Therapy for Bacterial Infections: Rekindling a Memory from the Pre-Antibiotics Era
journal, January 2001


Melioidosis: Review and Update
journal, May 1989


Phage treatment of human infections
journal, March 2011

  • Abedon, Stephen T.; Kuhl, Sarah J.; Blasdel, Bob G.
  • Bacteriophage, Vol. 1, Issue 2
  • DOI: 10.4161/bact.1.2.15845

Characterization of LysB4, an endolysin from the Bacillus cereus-infecting bacteriophage B4
journal, January 2012


Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against Pseudomonas aeruginosa: EL188 and EDTA against Ps. aeruginosa
journal, January 2011


Modifications to the Peptidoglycan Backbone Help Bacteria To Establish Infection
journal, November 2010

  • Davis, Kimberly M.; Weiser, Jeffrey N.
  • Infection and Immunity, Vol. 79, Issue 2
  • DOI: 10.1128/IAI.00651-10

Antibacterial activity of Acinetobacter baumannii phage ϕAB2 endolysin (LysAB2) against both Gram-positive and Gram-negative bacteria
journal, January 2011

  • Lai, Meng-Jiun; Lin, Nien-Tsung; Hu, Anren
  • Applied Microbiology and Biotechnology, Vol. 90, Issue 2
  • DOI: 10.1007/s00253-011-3104-y