Primary transcriptomes of Mycobacterium avium subsp. paratuberculosis reveal proprietary pathways in tissue and macrophages

Abstract

Background: Mycobacterium avium subsp. paratuberculosis (MAP) persistently infects intestines and mesenteric lymph nodes leading to a prolonged subclinical disease. The MAP genome sequence was published in 2005, yet its transcriptional organization in natural infection is unknown. While prior research analyzed regulated gene sets utilizing defined, in vitro stress related or advanced surgical methods with various animal species, we investigated the intracellular lifestyle of MAP in the intestines and lymph nodes to understand the MAP pathways that function to govern this persistence.

Results: Our transcriptional analysis shows that 21%, 8% and 3% of the entire MAP genome was represented either inside tissues, macrophages or both, respectively. Transcripts belonging to latency and cell envelope biogenesis were upregulated in the intestinal tissues whereas those belonging to intracellular trafficking and secretion were upregulated inside the macrophages. Transcriptomes of natural infection and in vitro macrophage infection shared genes involved in transcription and inorganic ion transport and metabolism. MAP specific genes within large sequence polymorphisms of ancestral M. avium complex were downregulated exclusively in natural infection.

Conclusions: We have unveiled common and unique MAP pathways associated with persistence, cell wall biogenesis and virulence in naturally infected cow intestines, lymph nodes and in vitro infected macrophages. This dichotomy also suggests that in vitro macrophage models may be insufficient in providing accurate information on the events that transpire during natural infection. This is the first report to examine the primary transcriptome of MAP at the local infection site (i.e. intestinal tissue). Regulatory pathways that govern the lifecycle of MAP appear to be specified by tissue and cell type. While tissues show a "shut-down" of major MAP metabolic genes, infected macrophages upregulate several MAP specific genes along with a putative pathogenicity island responsible for iron acquisition. Many of these regulatory pathways rely on the advanced interplay of host and pathogen and in order to decipher their message, an interactome must be established using a systems biology approach. Identified MAP pathways place current research into direct alignment in meeting the future challenge of creating a MAP-host interactome.

Figure 1

MAP infection in subclinically infected animals. (A) Section of bovine ileum infected with MAP: Longitudinal section of ileum showing inflammation and corrugated appearance of inner mucosal layer from a dairy cow with subclinical Johne's disease.(B) Histopathology of bovine ileum with MAP: Acid fast stain (400×) (left) and hematoxylin and eosin stain (100×) (right) of an ileal section of subclinical JD cow in Fig. 1A showing MAP organisms.

Figure 2

Classification of differentially expressed MAP genes into Clusters of orthologous genes (COG) groups. Differentially expressed genes in the tissues or infected macrophages were grouped based on clusters of orthologous genes (COG) classification. Significantly enriched COGs under each condition are represented in the Venn diagram. Shown in the parenthesis is the code for each COG category.

Figure 3

Comparisons of fold changes of selected genes by microarray and real time RT PCR. (A) Selected MAP genes that were differentially regulated (up or down) after subtraction with broth culture (data in linear scale). (B) These genes were validated for their expression pattern by real-time PCR to demonstrate similar trends in gene expression (data in logarithmic scale).