Listeria and Omics Approaches for Understanding Its Biology

Janet R. Donaldson, Ph.D.Mississippi State University

Department of Biological Sciencesdonaldson@biology.msstate.edu

Listeria monocytogenesOne of the deadliest

food-borne pathogensCantaloupe outbreak

in 2011 was the third most deadly outbreak since 1924; killed 30 people 1st: E. coli O104:H4

outbreak of 2011; Killed 53

2nd: Listeria outbreak in 1985; Killed 52

Soil bacteriumInfects both humans and animals

Low pH, intracellular growth, high bile salts5-10% of population are asymptomatic carriers

Grows in a wide range of temperatures (2°C to 39°C) and environmental conditions (salt, mildly acidic)

Major problems with ready to eat foods, such as smoked fish industry, deli meats, and cheese

Survives a variety of environments

It’s complicated…Many factors contribute to

the complications of studying Listeria monocytogenes13 different serotypes, 4

genetic lineagesMost food-borne outbreaks are related to serotype 4b, but 1/2a, 1/2b, and 4a are also important for human and animal health

Conversion to L-forms Formation of biofilms

Advances in molecular biology allowed for huge advancements to be made in terms of monitoring strain persistence in food processing plants

GOLDEN AGE OF OMICS

Advances in OmicsVast amount of

informationTranscriptomicsProteomicsSecretomicsMetabolomicsInteractomics

Functional Proteomics Approach to Answer Complicated Questions How do basic metabolic processes differ

between different serotypes?What are the mechanisms that allow for

survival of Listeria monocytogenes in stressful environments?

How do these strategies for survival differ between different serotypes of Listeria monocytogenes?

Proteomics AnalysisPreparation of protein samplesAnalyzed by HPLC coupled with nano-LTQ

linear ion trap mass spectrometerSpectra analyzed against trypsin digested

FASTA database with converted file Protein modifications need to be analyzed;

analysis run against decoy databaseProtein expression comparisons made

based on spectral intensitySignificantly expressed proteins

assigned to classification category based on ListiList

Variations in proteome expressionEGDe (1/2a

serotype) and F2365 (4b) proteomes differ in stationary phase

F2365total: 1427(350)

EGDtotal: 1754(677)

Common(1077)

Donaldson et al, 2009 Appl Environ Microbiol

F2365EGDe

Expression of 322 proteins was significantly increased in EGDe in comparison to F2365 Mostly in cell wall physiology and cell wall anchor

proteinsStress response proteins UvrC, UvrA, MutS, MutY,

RecN, GroEL, GroES, DnaKExpression of 91 proteins was significantly

decreased in F2365 in comparison to EGDeFlagellar biosynthesis protein Chemotaxis protein CheY Catalase Thiol peroxidase

Proteins Differentially Expressed

Donaldson et al, 2009 Appl Environ Microbiol

Functional Proteomics Approach to Answer Complicated Questions How do basic metabolic processes differ

between different serotypes?What are the mechanisms that allow for

survival of Listeria monocytogenes in stressful environments?

How do these strategies for survival differ between different serotypes of Listeria monocytogenes?

Mechanisms that allow for survival in stressful environmentsMinimal exposure to one stressor can

influence resistance to another stressorMethods used can increase resistance of

Listeria monocytogenes to other stressors encountered and also influence its persistence in facilities

Studies have suggested overlap in adaptive mechanisms used by L. monocytogenes to cold stress and osmotic stress

Cold Smoking ProcessProcess involves:

Freeze/ thaw of fish to remove parasitesBrining with salt Exposure to smoking conditions for less than

24 hPackaging and storage

Can exposure to cold influence survival under osmotic stress?

Functional Proteomics Approach to Answer Complicated Questions How do basic metabolic processes differ

between different serotypes?What are the mechanisms that allow for

survival of Listeria monocytogenes in stressful environments?

How do these strategies for survival differ between different serotypes of Listeria monocytogenes?

1 3 5 7

F2365

EGD-e

HCC23

Time (hr) post infection

Intr

acell

ula

r C

FU

/mL

Serotype variability in response to stressful environments

Responses to intracellular growthAvirulent: decrease in lipid metabolism,

increase in cell wall proteinsVirulent strains: increase in oxidative

repair proteins2x10

^7

1x10^5

Donaldson et al, 2011 J Proteomics

Other studies…Variations in the mechanism by which

avirulent and virulent strains of Listeria monocytogenes respond to bile salt environmentsEfficient repair mechanisms are essential for

long term survivalVariations in intracellular v. extracellular

growth between virulent and avirulent strains

Payne et al, 2013 J Med Microbiol

ConclusionsProteomics has advanced our understanding of

the biology of Listeria monocytogenesVariations between serotypes: provide insight

into ecological niches Mechanisms for survival in presence of stressors:

Cross adaptation between cold and osmotic stress: Proteins involved re-modeling of the cell wall and cell

membrane allow for the integrity and fluidity of the cell to be maintained in response to osmotic stress

Variations between different serotypes in response to stressorsAvirulent strains possess different mechanism to try

to survive in presence of bile salts than virulent strains

Thank You!MSU

Joseph PittmanKendrick Currie Jessica Grissett-WilsonGabe PosadasStephanie VamentaAngela PayneSusan BridgesChamali Thanthiriwatte Justin ThorntonMark LawrenceBindu NanduriTibor Pechan

Shane Burgess, Univ. AZKen Pendarvis, Univ. AZTodd Callaway, USDA-ARS Jeff Carroll, USDA-ARS Joe Buntyn, UNLTy Schmidt, UNL Tjakko Abee, Wageningen

University

Funding: MSU ORED

MSU Dept. of Biological SciencesDOE Sustainable Energy Research Center

MAFES Special Research InitiativesNIH (T35RR007071)