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Quantitative proteomics of food pathogenic Bacilli - a quest for biomarkers

Stelder, S.K.

Publication date2016Document VersionFinal published version

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Citation for published version (APA):Stelder, S. K. (2016). Quantitative proteomics of food pathogenic Bacilli - a quest forbiomarkers.

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Published work

Stelder, S. K, .; Mahmud, S. A.; Dekker, H. L.; de Koning, L. J.; Brul, S.; de Koster, C.

Pathogenic Food Spoiler Bacillus weihenstephanensis Type Strain WSBC 10204. Journal of Proteome Research 2015, 14 (5), 2169–2176.

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SummaryQuantitative proteomics of food pathogenic Bacilli– a quest for biomarkers

Food manufacturers have a variety of methods at their disposal to ensure foodsare microbially safe. However, due to increasing demands from consumersfor minimally processed foods of high sensory or textural quality the severityof these treatments is frequently mild. Frequently being able to survive suchtreatments, bacteria of the genus Bacillus present a pressing issue. Comprisingspore-forming, Gram-positive, aerobic or facultative anaerobic bacteria thegenus contains a group of pathogenic bacteria known as Bacillus cereus sensu lato.Within this group Bacillus cereus and weihenstephanensis are both notorious foodpathogens indicated to be of particular concerns to the dairy and processedready-to-eat food industry. Both potentially produce cereulide toxin as well asseveral enterotoxins which are harmful to humans. Upon ingestion of the toxinsor the spores of these bacteria, they can cause either vomiting or diarrhoearespectively. Spores are a metabolically dormant, extremely resistant cell typecapable of surviving in extreme environments The mild treatments used in thefood industry used to eliminate bacteria are then unable to kill or inactivatespores present.

To detect Bacillus cereus and ensure food safety, current ISO guidelines (ISO 7932, ISO 21871) are based around incubation on agar plates for 24 hours or evenlonger. Developing a platform for direct detection of spores would allow for better, faster and ultimately cheaper detection methods. In order to develop such

reliable interaction with the sensor, and be accessible to the receptor moleculeof the sensor it is supposed to bind to.

Spores acquire their unique resistance properties from the characteristicintricate structure of protective layers. The outermost layers, the inner coat, outer coat and exosporium, primarily consist of proteins. Therefore we set outto identify and quantify the proteome of spores of food pathogenic Bacilli, as well obtain information on protein localisation within in the spore.

In Chapter 2, in order to make B. weihenstephanensis tractable for studies thetype strain Bacillus weihenstephanensis WSBC10204 (DSM11821) has been sequenced. Annotation of the newly created genome sequence allowed for acomplete proteome prediction and subsequent analysis by bottom-up mass

6°C or 30°C have been compared which revealed considerable differences andindicated several hundred (uncharacterized) proteins as being subproteome

processes have been newly

routes and a different role for the urea cycle. Furthermore, a possible post-translational regulatory function for acetylation is suggested. Toxin productionwas determined to be largely independent of growth temperature.

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In Chapter 3, spore proteome analysis of B. weihenstephanensis gives animpression of the reproducibility of the spore proteome under varyingsporulation conditions, in this case temperature, and the robustness of

which are sporulation temperature dependent. Several proteome variations(N-acetylmuramoyl-L-alanine amidase, N-acetylmuramoyl-L-alanine amidasefamily protein, GerD, SpoVAD) have been correlated to spore characteristics

B. weihenstephanensis

In Chapter 4, the abundance of 21 spore proteins of B. cereus has been

QconCAT in a heterogeneous system. In this manner 5.66% ±0.51 of the total

to classify proteins by function, i.e. structural or otherwise. Furthermore, determining protein stoichiometry provides insight into the way either knownor potential interactors may relate to each other. Finally, the potential of themost abundant proteins for use as biomarkers is assessed using the immunogenicepitope prediction algorithm SVMTriP.

In Chapter 5, the methods developed for analysis of the spore insolublefraction are applied to a CotE knockout mutant strain. As CotE is requiredfor proper formation of the exosporium layer, the knockout strain lacks anintact exosporium layer. Several proteins normally located in this outermostexosporium layer are reported to be absent or of lower abundance in sporeinsoluble fraction isolates of the CotE knockout mutant. As such, informationon protein localisation on a proteome wide scale is obtained and potentialinteractions between proteins are indicated.

As a result of these investigations we are able to identify robust, abundant andaccessible spore protein biomarkers which may be implemented in a biosensor platform used to detect spores of food pathogenic Bacilli.

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SamenvattingKwantitatieve proteomics van voedselpathogene Bacillen– een zoektocht naar biomarkers

Voedselproducenten hebben verschillende methoden tot hun beschikking omte verzekeren dat voedsel microbieel veilig is. Echter, er ontstaat een steedsgrotere vraag vanuit consumenten naar producten die minimaal bewerkt en vanhoge sensorische kwaliteit zijn. Als gevolg hiervan zijn de gebruikte methodenvaak mild wat een probleem naar voren brengt in de vorm van bacteriën vanhet genus Bacillus die deze methoden kunnen overleven. Dit genus bestaat uitsporenvormende, Grampositieve, aerobe of facultatief anaerobe bacteriën enbevat een groep pathogenen bekend onder de naam Bacillus cereus sensu lato. Binnen deze groep staan Bacillus cereus en Bacillus weihenstephanensis bekendals notoire voedselpathogenen die vooral in zuivel en kant-en-klaar maaltijdenvoor problemen kunnen zorgen. Beide zijn potentieel in staat om het cereulidetoxine alsmede enkele enterotoxines te produceren. Ingestie van deze toxinesof sporen van deze bacteriën resulteert in braken respectievelijk diarree. Sporenzijn een metabool slapende, extreem resistente overlevingsvorm die extremeomgevingscondities gemakkelijk overleven. De milde behandelingen die men inde voedselindustrie gebruikt zijn daarom niet in staat om sporen te doden of inactiveren.

ISO richtlijnen (ISO 7932, ISO 21871) voor detectie van Bacillus cereus zijn gebaseerd op het uitplaten en incuberen van isolaten gedurende 24 uur of langer. Ontwikkeling van een platform voor directe detectie van sporen zou danbetere, snellere en uiteindelijk goedkopere methoden moeten opleveren. Omdeze ontwikkeling mogelijk te maken is het belangrijk een goede biomarker

systeem moeten zijn, abundant genoeg zijn om een betrouwbare interactie metde sensor aan te gaan, en toegankelijk moeten zijn voor het receptormolecuulvan de sensor waar het mee moet binden. Sporen verkrijgen hun uniekeresistentie-eigenschappen door hun karakteristieke complexe beschermingslagen. De buitenste van deze lagen, de sporencoat en het exosporium, bestaan voor het grootste deel uit eiwitten. Als zodanig hebben we het proteoom van sporenvan voedselpathogenen van het genus Bacillus

In Hoofdstuk 2 is het genoom van de type strain van Bacillus weihenstephanensisgesequenced met als doel het toegankelijk maken van deze soort voor verderestudies. Annotatie van de verkregen genoomsequentie maakte het mogelijk omhet volledige proteoom te voorspellen en vervolgens te onderzoeken met behulp

30°C is met elkaar vergeleken waarbij enkele honderden (ongekarakteriseerde)

zijn enkele processen aangetoond afhankelijk te zijn van groeitemperatuur zoals

wordt een posttranslationele regulatoire functie voor acetylering voorgesteld. Productie van toxines was vrijwel onafhankelijk van groeitemperatuur.

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In Hoofdstuk 3 wordt gekeken naar het proteoom van sporen van B. weihenstephanensis hetgeen een indruk geeft van hoe reproduceerbaar hetsporenproteoom is bij verschillende sporulatieomstandigheden en hoe robuust

bleken er 47 afhankelijk te zijn van sporulatietemperatuur. Enkele variatiesin het proteoom (N-acetylmuramoyl-L-alanine amidase, N-acetylmuramoyl-L-alanine amidase family protein, GerD, SpoVAD) zijn gecorreleerd aan

dipicolinezuur in de sporekern. In deze eerste directe studie van hetsporenproteoom van B. weihenstephanensis zijn 23 eiwitten voor het eerst

In Hoodfstuk 4 wordt de abundantie van 21 sporeneiwitten van B. cereus bepaald

van sporeneiwitten alsook de eerste keer dat een QconCAT wordt toegepast ineen heterogeen systeem. Op deze manier is 5.66% ±0.51 van het totaalgewicht

anderzijds. De bepaalde stoichiometrie verschaft ook inzicht in hoe bekende of potentieel interacterende eiwitten zich tot elkaar verhouden. Als laatste is van demeest abundante eiwitten een voorspelling gedaan wat betreft hun immunogeneregio’s hetgeen van belang is voor hun toepasbaarheid als biomarker.

In Hoofdstuk 5 zijn de ontwikkelde methodes voor analyse van de onoplosbarefractie van sporen toegepast op een CotE knock-out mutant. Gezien CotEvan essentieel belang is voor het vormen van een intact exosporium is dezelaag zwaar aangedaan in de mutant. Eiwitten die normaal in het exosporiumgelokaliseerd zijn worden of helemaal niet of in mindere mate teruggevondenin isolaten van de onoplosbare fractie van sporen van de knock-out mutant. Alszodanig is het mogelijk om informatie te verkrijgen over de locatie van eiwittenop een proteoom breed niveau alsmede over potentiele interacties tusseneiwitten.

Als gevolg van dit onderzoek is het mogelijk geworden om robuuste, abundante

kunnen worden in een biosensor platform ter detectie van sporen vanvoedselpathogene Bacillen.

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AcknowledgementsYears of work, hundreds of papers read, uncountable bytes of data generatedand only one last page to write… what hopefully won’t be the most read sectionof this book. I would like to thank all the people who have been a part of this

lengths of time spent strategizing, analysing samples, discussing results, and notto mention maintenance of our precious FTMS. You have been instrumental tothe success of my PhD research. Of course, I would like to thank my promotorsChris and Stanley for their support and trust. It’s been an absolute pleasure

the combination of proteomics and spore research is headed for great things.

spent reading this thesis and participating in the proceedings.

A special thanks to my paranymphs Reuben and Pascal who’ve helped me enjoymy life these past four years, not only at the university but out in the real worldas well. I’m curious to see how (and if?) the three of us will keep implementingthe knowledge obtained during our PhD’s and look forward to our continuedfriendship.

Throughout my PhD I’ve had the privilege to work, share labs, or at least havelunch together with many wonderful colleagues. Special mention to the ones

Luitzen. It’s been an absolute pleasure and I hope my penchant for always having

hasn’t bothered you too much. That also goes for the technical assistance side, of which I’ve required my fair share. Henk and Winfried, without you our dear MassSpec group would be lost.

time with one than the other. Still, a warm thanks to “the people upstairs” of the MBMFS group for your time and attention spent trying to stay awake whilstlistening to my presentations during work discussions. One particular person Imust call out though; Frans, your insights have repeatedly advanced my research

possible without your solutions to my aggregation problems.

of support and the fun I’ve had alongside my working life. You’ve almost made thisPhD feel easy. Last but most certainly not least, my dearest Klazien, thank you for

these four years of my life… For Science!