Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni

Xenia Gatsos, David Lawrence Steer, Thamradeen A Junaid, Alexander Ian Smith, Ben Adler, Manuel John Albert

Research output: Contribution to journalArticleResearchpeer-review

Abstract

ABSTRACT: BACKGROUND: Campylobater jejuni, a major foodborne diarrhoeal pathogen is reported to produce a number of cytotoxins of which only a cytolethal distending toxin (CDT) has been characterised so far. One or more additional cytotoxins other than CDT, including a Chinese hamster ovary (CHO) cell active, Vero cell inactive cytotoxin, may mediate inflammatory diarrhoea. Our objective was to develop a method to enrich and thus partially characterise this cytotoxin, as a pathway to the eventual identification and characterisation of the toxin. RESULTS: A number of biochemical methods including cation- and anion-exchange chromatography were evaluated to enrich the cytotoxin from a cell lysate of a known cytotoxin-producing C. jejuni, C31. The cytotoxin in crude lysate was initially prepared by size-exclusion desalting and then subjected to high pressure liquid chromatography (HPLC) ion-exchange fractionation. One pooled fraction (pool B) was cytotoxic for CHO cells equivalent to crude toxin (tissue culture infectivity dose 50 [TCID50] of 1-2 mug/ml). The proteins of pool B were identified by mass spectrometry (MS) after separation by SDS-PAGE and trypsin digestion. Also, pool B was directly digested with trypsin and then subjected to liquid chromatography tandem mass spectrometry (LCMS) analysis for identification of lesser abundant proteins in the fraction. A total of 41 proteins were found in the fraction, which included enzymes involved in metabolic and transport functions. Eighteen non-cytoplasmic proteins including 2 major antigenic peptide proteins (PEB2 and PEB3) and 3 proteins of unknown function were also identified in the screen. Cytotoxicity in pool B was trypsin-sensitive indicating its protein nature. The cytotoxic activity was heat-stable to 50 degrees C, and partially inactivated at 60-70 degrees C. The pool B fraction also induced fluid accumulation in the adult rabbit ileal loop assay with cytotoxicity for mucosa confirming the presence of the cytotoxin. CONCLUSIONS: We report the enrichment and partial purification of C. jejuni cytotoxin by HPLC ion-exchange chromatography. Further purification may be achieved using additional complementary chromatographic techniques. A short-list of six candidate cytotoxin proteins was identified using an LCMS screen of pool B. Successful isolation of the cytotoxin will initiate steps for the determination of the role of this cytotoxin in the pathogenesis of C. jejuni diarrhoea.
Original languageEnglish
Pages (from-to)1 - 10
Number of pages10
JournalBMC Microbiology
Volume12
Issue numberArt No.:303
DOIs
Publication statusPublished - 2012

Cite this

Gatsos, Xenia ; Steer, David Lawrence ; Junaid, Thamradeen A ; Smith, Alexander Ian ; Adler, Ben ; Albert, Manuel John. / Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni. In: BMC Microbiology. 2012 ; Vol. 12, No. Art No.:303. pp. 1 - 10.
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abstract = "ABSTRACT: BACKGROUND: Campylobater jejuni, a major foodborne diarrhoeal pathogen is reported to produce a number of cytotoxins of which only a cytolethal distending toxin (CDT) has been characterised so far. One or more additional cytotoxins other than CDT, including a Chinese hamster ovary (CHO) cell active, Vero cell inactive cytotoxin, may mediate inflammatory diarrhoea. Our objective was to develop a method to enrich and thus partially characterise this cytotoxin, as a pathway to the eventual identification and characterisation of the toxin. RESULTS: A number of biochemical methods including cation- and anion-exchange chromatography were evaluated to enrich the cytotoxin from a cell lysate of a known cytotoxin-producing C. jejuni, C31. The cytotoxin in crude lysate was initially prepared by size-exclusion desalting and then subjected to high pressure liquid chromatography (HPLC) ion-exchange fractionation. One pooled fraction (pool B) was cytotoxic for CHO cells equivalent to crude toxin (tissue culture infectivity dose 50 [TCID50] of 1-2 mug/ml). The proteins of pool B were identified by mass spectrometry (MS) after separation by SDS-PAGE and trypsin digestion. Also, pool B was directly digested with trypsin and then subjected to liquid chromatography tandem mass spectrometry (LCMS) analysis for identification of lesser abundant proteins in the fraction. A total of 41 proteins were found in the fraction, which included enzymes involved in metabolic and transport functions. Eighteen non-cytoplasmic proteins including 2 major antigenic peptide proteins (PEB2 and PEB3) and 3 proteins of unknown function were also identified in the screen. Cytotoxicity in pool B was trypsin-sensitive indicating its protein nature. The cytotoxic activity was heat-stable to 50 degrees C, and partially inactivated at 60-70 degrees C. The pool B fraction also induced fluid accumulation in the adult rabbit ileal loop assay with cytotoxicity for mucosa confirming the presence of the cytotoxin. CONCLUSIONS: We report the enrichment and partial purification of C. jejuni cytotoxin by HPLC ion-exchange chromatography. Further purification may be achieved using additional complementary chromatographic techniques. A short-list of six candidate cytotoxin proteins was identified using an LCMS screen of pool B. Successful isolation of the cytotoxin will initiate steps for the determination of the role of this cytotoxin in the pathogenesis of C. jejuni diarrhoea.",
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Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni. / Gatsos, Xenia; Steer, David Lawrence; Junaid, Thamradeen A; Smith, Alexander Ian; Adler, Ben; Albert, Manuel John.

In: BMC Microbiology, Vol. 12, No. Art No.:303, 2012, p. 1 - 10.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni

AU - Gatsos, Xenia

AU - Steer, David Lawrence

AU - Junaid, Thamradeen A

AU - Smith, Alexander Ian

AU - Adler, Ben

AU - Albert, Manuel John

PY - 2012

Y1 - 2012

N2 - ABSTRACT: BACKGROUND: Campylobater jejuni, a major foodborne diarrhoeal pathogen is reported to produce a number of cytotoxins of which only a cytolethal distending toxin (CDT) has been characterised so far. One or more additional cytotoxins other than CDT, including a Chinese hamster ovary (CHO) cell active, Vero cell inactive cytotoxin, may mediate inflammatory diarrhoea. Our objective was to develop a method to enrich and thus partially characterise this cytotoxin, as a pathway to the eventual identification and characterisation of the toxin. RESULTS: A number of biochemical methods including cation- and anion-exchange chromatography were evaluated to enrich the cytotoxin from a cell lysate of a known cytotoxin-producing C. jejuni, C31. The cytotoxin in crude lysate was initially prepared by size-exclusion desalting and then subjected to high pressure liquid chromatography (HPLC) ion-exchange fractionation. One pooled fraction (pool B) was cytotoxic for CHO cells equivalent to crude toxin (tissue culture infectivity dose 50 [TCID50] of 1-2 mug/ml). The proteins of pool B were identified by mass spectrometry (MS) after separation by SDS-PAGE and trypsin digestion. Also, pool B was directly digested with trypsin and then subjected to liquid chromatography tandem mass spectrometry (LCMS) analysis for identification of lesser abundant proteins in the fraction. A total of 41 proteins were found in the fraction, which included enzymes involved in metabolic and transport functions. Eighteen non-cytoplasmic proteins including 2 major antigenic peptide proteins (PEB2 and PEB3) and 3 proteins of unknown function were also identified in the screen. Cytotoxicity in pool B was trypsin-sensitive indicating its protein nature. The cytotoxic activity was heat-stable to 50 degrees C, and partially inactivated at 60-70 degrees C. The pool B fraction also induced fluid accumulation in the adult rabbit ileal loop assay with cytotoxicity for mucosa confirming the presence of the cytotoxin. CONCLUSIONS: We report the enrichment and partial purification of C. jejuni cytotoxin by HPLC ion-exchange chromatography. Further purification may be achieved using additional complementary chromatographic techniques. A short-list of six candidate cytotoxin proteins was identified using an LCMS screen of pool B. Successful isolation of the cytotoxin will initiate steps for the determination of the role of this cytotoxin in the pathogenesis of C. jejuni diarrhoea.

AB - ABSTRACT: BACKGROUND: Campylobater jejuni, a major foodborne diarrhoeal pathogen is reported to produce a number of cytotoxins of which only a cytolethal distending toxin (CDT) has been characterised so far. One or more additional cytotoxins other than CDT, including a Chinese hamster ovary (CHO) cell active, Vero cell inactive cytotoxin, may mediate inflammatory diarrhoea. Our objective was to develop a method to enrich and thus partially characterise this cytotoxin, as a pathway to the eventual identification and characterisation of the toxin. RESULTS: A number of biochemical methods including cation- and anion-exchange chromatography were evaluated to enrich the cytotoxin from a cell lysate of a known cytotoxin-producing C. jejuni, C31. The cytotoxin in crude lysate was initially prepared by size-exclusion desalting and then subjected to high pressure liquid chromatography (HPLC) ion-exchange fractionation. One pooled fraction (pool B) was cytotoxic for CHO cells equivalent to crude toxin (tissue culture infectivity dose 50 [TCID50] of 1-2 mug/ml). The proteins of pool B were identified by mass spectrometry (MS) after separation by SDS-PAGE and trypsin digestion. Also, pool B was directly digested with trypsin and then subjected to liquid chromatography tandem mass spectrometry (LCMS) analysis for identification of lesser abundant proteins in the fraction. A total of 41 proteins were found in the fraction, which included enzymes involved in metabolic and transport functions. Eighteen non-cytoplasmic proteins including 2 major antigenic peptide proteins (PEB2 and PEB3) and 3 proteins of unknown function were also identified in the screen. Cytotoxicity in pool B was trypsin-sensitive indicating its protein nature. The cytotoxic activity was heat-stable to 50 degrees C, and partially inactivated at 60-70 degrees C. The pool B fraction also induced fluid accumulation in the adult rabbit ileal loop assay with cytotoxicity for mucosa confirming the presence of the cytotoxin. CONCLUSIONS: We report the enrichment and partial purification of C. jejuni cytotoxin by HPLC ion-exchange chromatography. Further purification may be achieved using additional complementary chromatographic techniques. A short-list of six candidate cytotoxin proteins was identified using an LCMS screen of pool B. Successful isolation of the cytotoxin will initiate steps for the determination of the role of this cytotoxin in the pathogenesis of C. jejuni diarrhoea.

UR - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541203/pdf/1471-2180-12-303.pdf

U2 - 10.1186/1471-2180-12-303

DO - 10.1186/1471-2180-12-303

M3 - Article

VL - 12

SP - 1

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JO - BMC Microbiology

JF - BMC Microbiology

SN - 1471-2180

IS - Art No.:303

ER -