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Open Access Highly Accessed Research article

Laser microdissection coupled with RNA-seq analysis of porcine enterocytes infected with an obligate intracellular pathogen (Lawsonia intracellularis)

Fabio A Vannucci1*, Douglas N Foster2 and Connie J Gebhart1

Author Affiliations

1 Department of Veterinary and Biomedical Science , College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA

2 Department of Animal Science, College of Food, Agricultural and Natural Resource Science, University of Minnesota, St. Paul, MN, USA

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BMC Genomics 2013, 14:421  doi:10.1186/1471-2164-14-421

Published: 24 June 2013

Abstract

Background

Lawsonia intracellularis is an obligate intracellular bacterium and the etiologic agent of proliferative enteropathy. The disease is endemic in pigs, emerging in horses and has been described in various other species including nonhuman primates. Cell proliferation is associated with bacterial replication in enterocyte cytoplasm, but the molecular basis of the host-pathogen interaction is unknown. We used laser capture microdissection coupled with RNA-seq technology to characterize the transcriptional responses of infected enterocytes and the host-pathogen interaction.

Results

Proliferative enterocytes was associated with activation of transcription, protein biosynthesis and genes acting on the G1 phase of the host cell cycle (Rho family). The lack of differentiation in infected enterocytes was demonstrated by the repression of membrane transporters related to nutrient acquisition. The activation of the copper uptake transporter by infected enterocytes was associated with high expression of the Zn/Cu superoxide dismutase by L. intracellularis. This suggests that the intracellular bacteria incorporate intracytoplasmic copper and express a sophisticated mechanism to cope with oxidative stress.

Conclusions

The feasibility of coupling microdissection and RNA-seq was demonstrated by characterizing the host-bacterial interactions from a specific cell type in a heterogeneous tissue. High expression of L. intracellularis genes encoding hypothetical proteins and activation of host Rho genes infers the role of unrecognized bacterial cyclomodulins in the pathogenesis of proliferative enteropathy.

Keywords:
Microdissection; RNA-seq; Obligate intracellular; Lawsonia intracellularis; Proliferative enteropathy