Anders Bergström, Jens Bo Andersen, Bjarke Bak Christensen and Tine Rask Licht

1. q-PCR validation (selected genes) Transcriptome analysis (full genome) + O2 - O2 mRNA mRNA CFP tagged L. monocytogenes ScottA cultivated in the presence of oxygen prior to infection of guinea pigs. YFP tagged L. monocytogenes ScottA cultivated under oxygen restricted conditions prior to infection of guinea pigs. 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; adbe@food.dtu.dk 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. Mixed in a 1:1 ratio Oral co-infection of Guinea pigs Evaluation of infection potential Liver lysate Spleen lysate Jejunum content 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 transcription of the housekeeping gene lmo2459(gap) were used as an internal reference. The same biological samples were used for the micro-array analysis and the 1st q-RT-PCR replica (technical replicates), whereas the 2nd and the 3rd q-RT-PCR replicas were conducted with 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 Figure 2: From day 2 to day 5 after challenge faecal counts of L. monocytogenes were lower in the XOS and GOS groups than in the control group. Values are mean +/- SEM. * P < 0,05. Determine distribution of CFP tagged (+O2) and YFP tagged (-O2) bacteria in liver-lysates, spleen-lysates and jejunum-content 4 or 7 days post infection. L. monocytogenes mixed culture infections of Guinea pigs Conclusions Infection cycle of L. monocytogenes • 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 restric-ted Listeria monocytogenes appears to be reflected in the transcription profile present immediately prior to ingestion by guinea pigs. • The environmental conditions to which Listeria monocyto-genes 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 num-ber of Listeria monocytogenes but also the physiological condition of these bacteria are important parameters in food safety assessment. 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, follow-ing oral dosage of guinea pigs.