Combined Transcriptome Sequencing of Mycoplasma hyopneumoniae and Infected Pig Lung Tissue Reveals Up-Regulation of Bacterial F1-Like ATPase and Down-Regulation of the P102 Cilium Adhesin in vivo

Tjerko Kamminga; Nirupama Benis; Vitor Martins Dos Santos; Jetta J E Bijlsma; Peter J Schaap

doi:10.3389/fmicb.2020.01679

Combined Transcriptome Sequencing of Mycoplasma hyopneumoniae and Infected Pig Lung Tissue Reveals Up-Regulation of Bacterial F1-Like ATPase and Down-Regulation of the P102 Cilium Adhesin in vivo

Front Microbiol. 2020 Jul 20:11:1679. doi: 10.3389/fmicb.2020.01679. eCollection 2020.

Authors

Tjerko Kamminga^{1

2}, Nirupama Benis¹, Vitor Martins Dos Santos¹, Jetta J E Bijlsma³, Peter J Schaap¹

Affiliations

¹ Laboratory of Systems and Synthetic Biology, Department of Agrotechnology and Food Sciences, Wageningen University and Research, Wageningen, Netherlands.
² Bioprocess Technology and Support, MSD Animal Health, Boxmeer, Netherlands.
³ Discovery and Technology, MSD Animal Health, Boxmeer, Netherlands.

Abstract

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes enzootic pneumonia in pigs but it is still largely unknown which host-pathogen interactions enable persistent infection and cause disease. In this study, we analyzed the host and bacterial transcriptomes during infection using RNA sequencing. Comparison of the transcriptome of lung lesion tissue from infected pigs with lung tissue from non-infected animals, identified 424 differentially expressed genes (FDR < 0.01 and fold change > 1.5LOG2). These genes were part of the following major pathways of the immune system: interleukin signaling (type 4, 10, 13, and 18), regulation of Toll-like receptors by endogenous ligand and activation of C3 and C5 in the complement system. Besides analyzing the lung transcriptome, a sampling protocol was developed to obtain enough bacterial mRNA from infected lung tissue for RNA sequencing. This was done by flushing infected lobes in the lung, and subsequently enriching for bacterial RNA. On average, 2.2 million bacterial reads were obtained per biological replicate to analyze the bacterial in vivo transcriptome. We compared the in vivo bacterial transcriptome with the transcriptome of bacteria grown in vitro and identified 22 up-regulated and 30 down-regulated genes (FDR < 0.01 and fold change > 2LOG2). Six out of seven genes in the operon encoding the mycoplasma specific F1-like ATPase (MHP_RS02445-MHP_RS02475) and all genes in the operon MHP_RS01965-MHP_RS01990 with functions related to nucleotide metabolism, spermidine transport and glycerol-3-phoshate transport were up-regulated in vivo. Down-regulated in vivo were genes related to glycerol uptake, cilium adhesion (P102), cell division and myo-inositol metabolism. In addition to providing a novel method to isolate bacterial mRNA from infected lung, this study provided insights into changes in gene expression during infection, which could help development of novel treatment strategies against enzootic pneumonia caused by M. hyopneumoniae.

Keywords: F1-like ATPase; Mycoplasma hyopneumoniae; P102 cilium adhesin; RNA sequencing; host-pathogen interaction; infection; pathogen enrichment.