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Physiopathology
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User name	Wolf-Dietrich Hardt
Email	hardt@micro.biol.ethz.ch
Phone / Fax number	+41446325143 / +41446321129
Location	ETH-HÃ¶nggerberg (Biology - Institut f. Mikrobiologie) - Wolfgang-Pauli-Str. 10 - 8093 ZÃ¼rich, SWITZERLAND

Scientific description

Non-typhoidal Salmonella (NTS) infections are a major cause of diarrhea in humans. Acute symptoms cease rapidly, but some patients remain infectious by fecal pathogen-shedding for weeks or months (= 'asymptomatic excretor' state). The factors determining "asymptomatic pathogen excretion" are unclear. We analyzed the parameters involved in clearance of an acute NTS infection in mice. S. typhimurium (S.tmatt, sseD) recapitulated all key phases of the human disease, i.e. acute gut inflammation, cessation of symptoms, appearance of "asymptomatic excretors" and an adaptive, O-antigen-specific sIgA response protecting against gut inflammation in challenge infections. Mice lacking sIgA (TCRÎ²Î´-/-, JHT, IgA-/-, pIgR-/-) and "LCM-mice" harboring a defined microbiota permitting long-term S.tm excretion, revealed that the sIgA-response had little effect on pathogen-shedding by "asymptomatic excretors". In contrast, when exposed to a normal complex microbiota, LCM-mice efficiently limited pathogen-excretion. Thus, the microbiota is a critical factor in terminating NTS-infections and may offer new treatment approaches in humans.

Technical description

Total RNA was analyzed on ABI Mouse Whole Genome Arrays v2.0 (Applied Biosystems) as previously described (Eilebrecht et al. 2008). One single round of linear amplification was performed from 1mg of total RNA. cDNA synthesis, in vitro transcription, amplification, fragmentation, hybridization, staining, and scanning were performed as directed by the supplier. Raw data, after having removed the signals stemming from the different control spots as well as failed measurements, were once more median normalized in log2-space. Pairwise normalization and subtraction profile calculation in an everyone-against-everyone scheme was achieved using the NeONORM method with sensitivity parameter k set to 0.2 (Noth et al., 2006a). Significance of logarithmic base two foldchanges ("log2Q", "L") were determined based on a mixture lognormal distribution hypothesis (Noth et al., 2006b) of signal intensities using ANOVA. Multiple probes for a single gene, cross-reactivity of a single probe to several genes, as well as the resolution of probe-ID annotations was done according to the standards defined previously (Noth and Benecke, 2005). Gene Ontology (GO) annotations were analyzed using the Panther Protein Classification System (Thomas et al., 2003) (http://www.pantherdb.org) to identify functional annotations that were significantly enriched in the differentially expressed gene set when compared to the whole set of genes present on the ABI microarray.

References

Eilebrecht S*, Pellay FX*, OdenwÃ¤lder P, Brysbaert G, Benecke BJ, Benecke A (2008) EBER2 RNA-induced transcriptome changes identify cellular processes likely targeted during Epstein Barr Virus infection. BMC Res. Notes. 1:100. 

Noth S, Brysbaert G, Pellay FX, Benecke A (2006b) High-sensitivity transcriptome data structure and implications for analysis and biologic interpretation. Genomics, Proteomics & Bioinformatics. 4:212-229. 

Noth S, Brysbaert G, Benecke A (2006a) Normalization using weighted negative second order exponential error functions (NeONORM) provides robustness against asymmetries in comparative transcriptome profiles and avoids false calls. Genomics, Proteomics & Bioinformatics. 4:90-109. 

Noth S, Benecke A (2005) Avoiding inconsistencies over time and tracking difficulties in Applied Biosystems AB1700(TM) / PANTHER(TM) probe-to-gene annotations. BMC Bioinformatics. 22;6:307. 

Thomas, P. D., Campbell, M. J., Kejariwal, A., Mi, H., Karlak, B., Daverman, R., Diemer, K., Muruganujan, A., and Narechania, A. (2003). PANTHER: A Library of Protein Families and Subfamilies Indexed by Function. Genome Res 13, 2129-2141.

Reference

Endt K, Stecher B, Chaffron S, Slack E, Tchitchek N, et al. (2010) The Microbiota Mediates Pathogen Clearance from the Gut Lumen after Non-Typhoidal
Salmonella Diarrhea. PLoS Pathog 6(9): e1001097. doi:10.1371/journal.ppat.1001097