Presented at 2013 Arkansas Association for Food Protection annual conference. Janet R. Donaldson, Ph.D. Mississippi State University Department of Biological Sciences

1. Janet R. Donaldson, Ph.D.
Mississippi State University
Department of Biological Sciences
donaldson@biology.msstate.edu

2. Listeria monocytogenes
 One of the deadliest food-
borne pathogens
 Cantaloupe 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

3.  Soil bacterium
 Infects both humans and animals
 Low pH, intracellular growth, high bile salts
 5-10% 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

4. It’s complicated…
 Many factors contribute to the
complications of studying
Listeria monocytogenes
 13 different serotypes, 4 genetic
lineages
 Most 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

5. GOLDEN AGE
OF OMICS

6. Advances in Omics
 Vast amount of information
 Transcriptomics
 Proteomics
 Secretomics
 Metabolomics
 Interactomics

7. 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?

8. Proteomics Analysis
 Preparation of protein samples
 Analyzed by HPLC coupled with nano-LTQ linear ion
trap mass spectrometer
 Spectra analyzed against trypsin digested FASTA
database with converted file
 Protein modifications need to be analyzed; analysis
run against decoy database
 Protein expression comparisons made
based on spectral intensity
 Significantly expressed proteins assigned
to classification category based on ListiList

9. Variations in proteome expression
 EGDe (1/2a serotype)
and F2365 (4b)
proteomes differ in
stationary phase
F2365
total: 1427
(350)
EGD
total: 1754
(677)
Common
(1077)
Donaldson et al, 2009 Appl Environ Microbiol
F2365 EGDe

10.  Expression of 322 proteins was significantly increased in
EGDe in comparison to F2365
 Mostly in cell wall physiology and cell wall anchor proteins
 Stress response proteins
UvrC, UvrA, MutS, MutY, RecN, GroEL, GroES, DnaK
 Expression of 91 proteins was significantly decreased in
F2365 in comparison to EGDe
 Flagellar biosynthesis protein
 Chemotaxis protein CheY
 Catalase
 Thiol peroxidase
Proteins Differentially Expressed
Donaldson et al, 2009 Appl Environ Microbiol

11. 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?

12. Mechanisms that allow for survival
in stressful environments
 Minimal exposure to one stressor can influence
resistance to another stressor
 Methods 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

13. Cold Smoking Process
 Process involves:
 Freeze/ thaw of fish to remove parasites
 Brining with salt
 Exposure to smoking conditions for less than 24 h
 Packaging and storage
Can exposure to cold influence
survival under osmotic stress?

14. 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?

16. Other studies…
 Variations in the mechanism by which avirulent and
virulent strains of Listeria monocytogenes respond to
bile salt environments
 Efficient repair mechanisms are essential for long term
survival
 Variations in intracellular v. extracellular growth
between virulent and avirulent strains
Payne et al, 2013 J Med Microbiol

17. Conclusions
 Proteomics has advanced our understanding of the biology
of Listeria monocytogenes
 Variations 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
stressors
 Avirulent strains possess different mechanism to try to
survive in presence of bile salts than virulent strains

18. Thank You!
 MSU
 Joseph Pittman
 Kendrick Currie
 Jessica Grissett-Wilson
 Gabe Posadas
 Stephanie Vamenta
 Angela Payne
 Susan Bridges
 Chamali Thanthiriwatte
 Justin Thornton
 Mark Lawrence
 Bindu Nanduri
 Tibor Pechan
 Shane Burgess, Univ. AZ
 Ken Pendarvis, Univ. AZ
 Todd Callaway, USDA-ARS
 Jeff Carroll, USDA-ARS
 Joe Buntyn, UNL
 Ty Schmidt, UNL
 Tjakko Abee, Wageningen
University
Funding:
MSU ORED
MSU Dept. of Biological Sciences
DOE Sustainable Energy Research Center
MAFES Special Research Initiatives
NIH (T35RR007071)