Role of Environmental Stresses on Pathogenicity of Listeria monocytogenes

1. Role of Environmental Stresses on Pathogenicity
of Listeria monocytogenes
Malvi Prakash Golwala (U53409030)
Cell, Molecular and Microbiology
09/30/2016

2. • Gram positive, rod shaped, non-sporulating,
facultative anaerobe.
• Motile via flagella at 30 °C and below
– but usually not at 37 °C, L. monocytogenes can
instead move within eukaryotic cells by
explosive polymerization of actin filaments
(known as comet tails or actin rockets).

3. Fig. 1: Listeria
monocytogenes polym
erizing host
cell actin into comet
tails, Dr. Matteo
Bonazzi, Paris
(http://www.nikonsmallworld.com
/galleries/entry/2008-
photomicrography-
competition/48)

4. Did you also know…
• Listeria is a resilient bacterium.
• Listeria monocytogenes is found in soil and
water. Vegetables can become contaminated
from the soil or from manure used as fertilizer.
• Listeria is killed by pasteurization, and heating
procedures used to prepare ready-to-eat
processed meats.

5. Why the Research?
• Listeria basically contaminates almost all
kinds of foods, processed or fresh.
• Due to its properties of withstanding low
temperatures, salt, pH, etc. it becomes a major
threat in food processing industries.
• A better knowledge of the adaptive
mechanisms of the bacteria to salt stress could
lead to a better control and prevention of this
pathogen in food processing plants. Thus, it is
significant.

7. The paper…
• The review paper focuses on all the recent
researches done in the recent years about how
Listeria is able to withstand these stresses. It
would help us in understanding those
mechanisms.
• The scientists have tried to find the proteins
that are induced or repressed when the bacteria
is under stress.

8. Background
• When bacteria are subjected to a sudden shift
in one/several parameters affecting their
growth or survival, a program of gene
expression is initiated, which is manifested as
an increased or decreased amount of a set of
proteins synthesized in response to stress.
– For instance, in Bacillus subtilis, salt stress
strongly stimulates the expression of a set of
proteins that probably allow the bacterium to
survive in the rapidly changing environment.

9. Background Contd…
• L. monocytogenes, shows elevated osmolarity
in the environment by the intracellular
accumulation of compatible solutes, called
osmolytes, through osmotic activation of their
transport from the medium rather than through
de novo synthesis.
• These osmolytes act in the cytosol by
counterbalancing the external osmolarity, thus
preventing water loss from the cell and
plasmolysis without adversely affecting
macromolecular structure and function(6).

10. How was it done?
• For that purpose, the two-dimensional (2-D)
electrophoresis method, an approach that has
often proved to be the appropriate tool to study
the general expression levels of proteins, was
used(29).
• Identification of salt shock proteins and salt
acclimation proteins was also undertaken by
mass spectrometry (MS) or N-terminal micro-
sequencing.
• Radiolabeling was used for detection.

11. What did they focus on?
• When cells are subjected to a severe stress,
they usually shut off most metabolic activity
and commit themselves to adaptive strategies.
• Consequently, they enter a physiological state
with very little protein synthesis, very different
from the normal growth physiology.
• So protein analysis was performed.

12. Background Contd…
• When salt was increased, Listeria in some
experiments was found to be less virulent. But,
when salt was increased it posed a threat on
people’s lives in terms of blood pressure.
• So, Na was replaced by Ca or K. CaCl2 and
KCl, both were found to maintain the sensory
characteristics in table olives in a research (23).

13. • In slow/ceased growth, the bacterium
undergoes the process of filamentation. It is
most effective at 4℃.
Filamentation of Listeria
Fig. 3: CFU recovered on
TSA and increases in the
relative length of the
longest 10% of cells
(L10, open symbols) of
cold adapted, log phase
cultures of Listeria
monocytogenes strains
FS2 (&), FS12 (N), and
CDC7762 (¤) incubated
in TSB at pH 6.0 with 4%
NaCl.

16. What did they find?
• They found proteins that were repressed as
well as induced due to salt stress.
• In the first 30mins, 26 proteins were modified,
20 of which were repressed and the rest were
over-expressed.
• In the first 60mins, 25 proteins varied, 11 of
which showed reduction in synthesis and 9
showed higher expression.

17. MALDI-TOF Identification
• Matrix-assisted laser desorption/ionization-
Time of Flight is a soft ionization technique
used in mass spectrometry, allowing the
analysis of biomolecules (biopolymers such
as DNA, proteins, peptides, sugars) and
large organic molecules (polymers,
dendrimers, other macromolecules), which
tend to be fragile and fragment when ionized
by more conventional ionization methods.

18. • Tryptic peptide masses on the plot showed
– heat shock protein (DnaK) : spot 13
– elongation factor (EF-Tu) : spot 84
– transporter of glycine betaine (GbuA) : spot 97
– catabolite control protein (CcpA) : spot 133
• Confirmation of correct identification was
done by analyzing the Z score.
MALDI-TOF Identification Contd…

19. • Other proteins found, similar to
– a phosphoglycerate mutase (Pgm): spot 36
– a pyruvate dehydrogenase (PdhD) : spot 44
– an inosine-5-monophosphate dehydrogenase (GuaB)
: spot 48-49
– an alanine dehydrogenase: spot 109
– a homolog of mannose-specific phosphotransferase
system (PTS) enzyme IIAB: spot 155
– general stress protein (Ctc): spot 206
MALDI-TOF Identification Contd…

20. Same Spot Proteins
• Using MS, spot 48 gave the same result as 49;
the same thing was observed for spots 105 and
106.
• Digestion of these proteins by trypsin gave
peptides having approximately the same
masses. It was therefore concluded that spots
48 and 49 were the same protein. Similarly,
spots 105 and 106 were the same protein.
Why was this was not surprising??

21. N-terminal Amino Acid Sequence
Determination
• Edman degradation (developed by Pehr
Edman) is a method of sequencing amino
acids in a peptide.
• In this method, the amino-terminal residue is
labeled and cleaved from the peptide without
disrupting the peptide bonds between other
amino acid residues.

22. N-terminal Amino Acid Sequence
Determination
• This technique enabled identification of the
spots 105-106, 113, and 173 as being similar to
– pyruvate dehydrogenase (PdhA)
– glyceraldehyde-3-phosphate dehydrogenase (Gap)
– CysK: spot 173
respectively,

23. Fig. 5: Two-
dimensional gel
electrophoresis
autoradiogram
of L.
monocytogenes
proteins labeled
during
exponential
growth showing
identified
proteins
synthesized in
response to salt
stress (3.5%
NaCl).

24. General Osmolyte Response of
Listeria
• Accumulated osmolytes (due to acid stress) and
regulon SigB have shown a major role in
preventing desiccation.
• SigB regulon is activated by stresses like, osmotic,
salt, hydrostatic pressure, etc. causes synthesis and
release of over 100 Gsps.
• Listeria has shown to survive desiccation on:
1. dust like particles at 10℃ for 191 days(25)
2. stainless steel for 91 days under simulated food
processing conditions(25).

25. General Osmolyte Response of
Listeria Contd…
Stabilityofcytoplasmicwaterand
turgor
Glycine betaine,
Carnitine (osmotic
stress)
Acetyl carnitine
Proline betaine and
Proline

26. General Osmolyte Response of
Listeria
• But, the research was concluded to the result that
SigB was synthesized generally for stress and the
desiccation survival process was independent of it.
It helps the bacterium survive in limited nutrient
PPS and MM substrates(25).

27. Fig. 6: Desiccation survival of L. monocytogenes 568; sigB (B) and
back complemented sigB (△) cells on stainless steel coupons incubated
at 15℃, 43% RH for 21 days in BHI (open symbols), TSB-glu (grey
symbols), PPS (black symbols). In a) cells were pre-cultured (15℃ for
3 days) and desiccated in the substrates with 0.5% NaCl while in cells
were pre-cultured and desiccated in the substrates with 5% NaCl.

28. Bsh contributes to survival in gut
• Bile salts are cholesterol end-products and are
synthesized in hepatic cells before conjugating to
taurine or glycine.
• Indigenous microflora has over the years
developed resistance for bile or have evolved
different protective mechanisms.
• The cytoplasmic enzyme is also known as
conjugated bile acid hydrolase (CBAH) and is
produced by many gram positive and negative
commensal bacteria (16), Clostridium (16) and
Enterococcus (16).

29. Bsh contributes to survival in gut
Contd…
• The bsh (bile salt hydrolase) gene is regulated by
PrfA (transcriptional activator of virulence
factors).
• The activity of this gene increases in O2 deficient
environments and thus its virulence in adverse
conditions.
• The scientists have shown that the bsh gene, inside
the bacterial genome helps it to protect itself from
the bile salts in the environment and thus acts as a
virulence factor (16).

30. Bsh contributes to survival in gut
Contd…
Fig. 7: Effect of BSH on
persistence in the guinea
pig gastrointestinal tract. L.
monocytogenes EGDe
(black), L. monocytogenes
EGDe Deficient bsh (white)
and back complemented L.
monocytogenes EGDe Dbsh
complemented (grey).
Listeria growth was
followed in the stools at 24,
48 and 72 h.

31. Bsh contributes to survival in gut
Contd…
• Resistance to Glycol-Conjugated Bile Acid
(GDCA) Supports Virulence in Listeria
-Considering the fact that bile has
antimicrobial activity, a research done by
Sleator, R., et al., 2005, concludes that Listeria
is resistant to the bile in murine gall bladder
and especially to one compound, glycol-
conjugated bile acid (GDCA) (19).

32. Surface Proteins and Enzyme
Interactions
• The genome of Listeria monocytogenes contains
331 transport proteins including 19 of those
involved in phosphotransferase sugar-uptake
systems.
• Because it has so many surface secreted proteins,
it becomes easier for it to interact with so many
environmental stresses.
• Almost 30 of the 133 genes code for surface
proteins (17).

34. Surface Proteins and Enzyme
Interactions Contd…
2 significant
genes
srtA
Covalently bind
proteins to CM by
binding to InlA
Encode for sortases and
transamidases
Help escape
macrophages
srtB
Encode proteins that anchor
proteins with C-terminal NXXTN
motif (SvpA)
Encode for sortases and
transamidases

35. Signal Transduction
• The mechanism of signal transduction is made of
two components:
- Histidine kinase (HK)
- Response regulator (RR)
- both are highly conserved

36. Signal Transduction
• In adverse environmental conditions, histidine gets
auto-phosphorylated and that phosphate is
transferred to the aspartic acid in the response
regulator
• When conditions change, this arrangement is
adjusted in order to adjust to the environmental
alterations (18).

37. • Six Ssp proteins were induced:
– two general stress proteins (Gsp), DnaK (17) and Ctc
(18, 36),
– DnaK (heat shock protein) is required for stress
tolerance (stabilizes cellular proteins)
– Ctc is induced in B. subtilis in response to various
stresses, such as salt stress, but its basic function is
unknown
Let’s Discuss!!

38. Let’s Discuss!!
• Phosphoglycerate mutase (Pgm) (enzyme of
glycolysis and gluconeogenesis in B. subtilis
(24), only one of the 20 proteins repressed during
the first 30 min following salt stress).
– This decreased synthesis could be related to the
increasing synthesis of Gap (in order to
compensate for the increasing amount of
phosphoglycerate produced, but still unclear).

39. Let’s Discuss!!
• 11 Sap proteins were detected, and 7 of them
were identified
– GbuA (a subunit of the glycine betaine transport
system GbuABC) (osmoprotectant transporter
accumulated in response to salt stress by L.
monocytogenes and many bacteria, such as B.
subtilis (12)).

40. Let’s Discuss!!
– elongation factor (EF-Tu) (whose synthesis rate
increased in Mycobacterium tuberculosis in the
presence of a high iron concentration (38); might be
implicated in protein folding and/or protection
from stress in Escherichia coli (5))
– homolog of mannose-specific PTS enzyme IIAB
– two pyruvate dehydrogenase subunits (PdhA and
PdhD)

41. Conclusion
• GbuA (overexpressed in the presence of salt) is
directly connected to the salt stress response.
• General stress response, as two Gsp proteins
(DnaK and Ctc) induced after salt stress.
• In addition, because the synthesis of Gap,
PdhA, PdhD, and Pgm is modified in the
presence of salt, it appears that glycolysis is
important after this stress.

42. Conclusion
• SigB protects from all kinds of stresses but
desiccation survival is independent of this
particular regulon.
• Glycine betaine (osmotic stress), proline,
acetyl carnitine, carnitine (osmotic stress),
proline betaine are the most abundantly
released osmolytes.

43. Conclusion
• bsh gene, inside the bacterial genome helps it
to protect itself from the bile salts in the
environment and thus acts as a virulence
factor.
• It shows resistance to GDCA.

44. Conclusion
• Many surface secreted proteins help it to
interact with many environmental stresses.
- srtA and srtB
• Signal transduction mechanisms help it to
survive changing environments which is
common in food processing industries.

45. Well, I think….
• The bacteria is very efficient and smart, which
is kind of fascinating.
• A lot needs to be done for a better
understanding of these processes and
mechanisms especially those of signal
transduction, before we can start devising
techniques to prevent the bacteria.
INTERESTING…!!!

46. Acknowledgments
• Dr. Diane TeStrake, for mentoring me through
the paper and presentation.
• CMMB Dept., for giving me an opportunity to
stand here and present this topic.
• Fellow classmates, friends and family.