Download Abstract: Listeria monocytogenes

Oxygen restriction increases the infection potential of Listeria monocytogenes –
verification of microarray chip data by quantitaive real-time PCR
Anders Bergström, Jens Bo Andersen, Bjarke Bak Christensen and Tine Rask Licht
Technical University of Denmark, Denmark; [email protected]
Abstract: Listeria monocytogenes has been implicated in several food borne outbreaks as well as sporadic cases of disease during the last two decades. Increased understanding of
the biology of this organism is important in the prevention of food borne listeriosis. This is highly relevant for safety assessment of this organism in food. We have previously shown
(Andersen et al., BMC Microbiology; 2007, 7:55) that the environmental conditions to which L. monocytogenes is exposed prior to ingestion are decisive for its in vivo infective potential in
the gastrointestinal tract after passage of the gastric barrier. Infection of Caco-2 cells revealed that Listeria cultivated under oxygen-restricted conditions were approximately 100 fold more
invasive than similar cultures grown without oxygen restriction. This means that not only the number of Listeria present in a given food item, but that also the physiological condition of these
bacteria is important for food safety. The in vitro and in vivo data suggest that an oxygen-restricted L. monocytogenes cell represents a significantly higher risk than a cell grown without
oxygen restriction. In order to identify transcriptional differences contributing to different invasiveness, microarrray gene chip technology was applied to cDNA created from RNA isolated
from oxygen restricted and non-restricted cultures. The analysis confirmed several relevant genes to be differentially transcribed in the two environmental conditions e.g. genes related to
virulence potential of Listeria monocytogenes. Quantitative PCR was used to verify the quantitative differences identified with the microarray chip for a selection of relevant and differentially
transcribed genes.
q-PCR validation
Experimental setup
Selected genes
Lmo0015 (qoxA): Quinol oxidase subunit III, involved in aerobic respiration
Lmo0200 (prfA): Virulence regulator PrfA
Lmo0202(hly): LLO toxin required for phagosomal escape during invasion.
Lmo0211(ctc): General stress protein
Lmo0433 (inlA): Surface protein Internalin A required for efficient invasion of epithelial cells in the small intestine
Lmo0355 : Fumerate reductase involved in anaerobic respiration
Lmo0895 (sigB): Sigma factor B involved in stress response
Lmo0943 (fri): Virulence factor, involved in early stages of infection
Lmo1014 (gbuA): Glycine betaine ABC transporter involved in osmotic tolerance
Lmo1956 (fur): Transcriptional regulator Fur regulating the fur regulon (iron uptake/metabolism)
Lmo2067 (bsh): Bile salt hydrolase, involved in bile salt tolerance (intestinal ”fitness”)
Lmo2182 : Fur regulated protein involved in iron uptake / metabolism
Lmo2185 (spvA): Fur regulated surface protein involved in late stages of infection
Lmo2459 (gap): Glyceraldehyde-3-phosphate dehydrogenase (glycolysis/gluconeogenesis), selected as housekeeping
(reference) gene throughout all the q-RT-PCR analysis.
q-PCR validation
(selected genes)
Transcriptome analysis
(full genome)
CFP tagged L. monocytogenes
ScottA cultivated in the presence
of oxygen prior to infection of
guinea pigs.
Mixed in a 1:1
ratio
- O2
Listeria monocytogenes transcriptional analysis
Log2 (Fold change -O2 / +O2)
YFP tagged L. monocytogenes
ScottA cultivated under oxygen
restricted conditions prior to
infection of guinea pigs.
10
Log2 (Fold Change -O 2/+O2)
+ O2
mRNA
mRNA
Oral co-infection of Guinea pigs
8
6
Microarray
4
1st replica
2
2nd replica
0
3rd replica
-2
-4
-6
-8
Spleen lysate
Jejunum content
Determine distribution of CFP tagged (+O2) and YFP tagged (-O2) bacteria in liverlysates, spleen-lysates and jejunum-content 4 or 7 days post infection.
Infection cycle of L. monocytogenes
Un-restricted
Prevalence
Oxygen-restricted
Mean
Prevalence
Log(CFU/g)
Liver
Spleen
Jejunum
2/24 (8%)
4/24 (17%)
2/24 (8%)
85
( sp
vA
)
82
lm
o2
1
( bs
67
lm
o2
1
h)
r)
56
( fu
lm
o2
0
14
lm
o1
9
( gb
uA
)
( fr
i)
43
lm
o1
0
95
lm
o0
8
lm
o0
9
( si
gB
)
lA)
33
( in
55
q-RT-PCR and micro array determined transcription ratios (fold changes) of selected Listeria
monocytogenes genes following cultivation under oxygen restricted and non-restricted
conditions. Data are presented as mean + SEM of log2-transformed fold changes, where the
fold changes were calculated as (Transcription level under oxygen restricted conditions) /
(transcription level under non-restricted conditions). The q-RT-PCR results are average
results from two independent experiments, and throughout all q-RT-PCR experiments
Figure
2: From day 2 to day 5 after challenge faecal counts of L.
transcription of the housekeeping gene lmo2459(gap) were used as an internal reference. The
monocytogenes
were lower
in the
XOS
and GOS
groups
than
in
same biological samples
were used
for the
micro-array
analysis
and the
1st q-RT-PCR
replica
whereas
themean
2nd and
theSEM.
3rd q-RT-PCR
replicas were conducted with
the(technical
controlreplicates),
group. Values
are
+/* P < 0,05.
other biological samples. There is a high level of reproducibility and a statistical comparison
of the 4 groups of transcription ratios by one-way ANOVA is highly insignificant (p = 0,98).
Transcription ratios from the micro array and 1st replica show the highest level of similarity,
but also the two other biological replicates have fold changes very similar to fold changes
found by miro array analysis
L. monocytogenes mixed culture
infections of Guinea pigs
Organ
lm
o0
4
11
lm
o0
2
Genes
Evaluation of infection potential
Liver lysate
lm
o0
3
( ct
c)
y)
( hl
lm
o0
2
02
( pr
00
lm
o0
2
lm
o0
0
15
( qo
xC
)
fA
)
-10
1,5 (+/- 0,21)
1,9 (+/- 0,76)
2,2 (+/- 0,21)
Mean
Log(CFU/g)
18/24 (75%)
12/24 (50%)
14/24 (58%)
1,9 (+/- 0,32)
2,1 (+/- 0,58)
3,1 (+/- 0,65)
Numbers of guinea pigs where L. monocytogenes was recovered from liver and spleen after
oral dosage with a 1:1 mixture og un-restricted and oxygen-restricted L. monocytogenes.
Samples were taken either 4 or 7 days post infection. Mean log (CFU/g) designate the mean
levels found in animals positive for Listeria in the given organs, followed by standard
deviations..
• Oxygen restricted Listeria monocytogenes is significantly
more invasive than its non-restricted counterpart, following oral dosage of guinea pigs.
Conclusions
• The q-RT-PCR analysis validate the transcribtion ratios
determined in the micro array analysis.
• Transcription of genes previously demonstrated to play
crucial roles in the early stages of the infection process,
such as inlA, hly, fri and bsh, is significantly up-regulated
in oxygen deprived Listeria monocytogenes. Hence, the
observed increased infection potential of oxygen restricted Listeria monocytogenes appears to be reflected in the
transcription profile present immediately prior to ingestion
by guinea pigs.
• The environmental conditions to which Listeria monocytogenes is exposed to prior to ingestion can be decisive for
its in vivo infection potential in the intestinal tract after
passage of the gastric barrier. Thus, not merely the number of Listeria monocytogenes but also the physiological
condition of these bacteria are important parameters in
food safety assessment.