Phagocytosis of M. paratuberculosis fails to activate expression of NADH dehydrogenase and nucleolin-related protein in bovine macrophages

Abstract

Mycobacterium avium subspecies paratuberculosis ( M. paratuberculosis) is a facultative intracellular bacterium and causal agent of Johne’s disease in cattle. Following phagocytosis, M. paratuberculosis resides and replicates in macrophage phagosomes that fail to mature. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used as a high throughput initial screen to begin to test the hypothesis that macrophage gene expression patterns would be differentially affected by M. paratuberculosis when compared to readily degraded bacteria or non-degradable latex beads. Gene expression profiles from immortalized bovine macrophage cells (BOMAC) exposed to M. paratuberculosis were compared to gene expression profiles for BOMAC cells exposed to Escherichia coli, latex beads or PBS. Amplicons representing genes specifically activated or repressed during M. paratuberculosis phagocytosis were cloned for further investigation. Northern blot hybridizations preformed using DDRT-PCR-derived amplicons 3-1-4, 5-2-10, 5-4-2 and 4-1-6 confirmed stimuli dependent differential gene expression. Expression pattern observed for amplicon 3-1-4 represents genes that are up-regulated following phagocytosis of E. coli or latex beads, but not M. paratuberculosis. Amplicon 5-2-10 exhibited a pattern of expression representative of genes that are up-regulated strongly following phagocytosis of E. coli or latex beads but only moderately following M. paratuberculosis phagocytosis. Expression pattern of the gene for amplicon 5-4-2 was representative of genes that are specifically suppressed following M. paratuberculosis phagocytosis, while the amplicon 4-1-6 gene expression pattern represented genes that are generally suppressed following phagocytosis of any of the three stimuli. DNA sequencing and Genbank database analysis of these amplicons revealed that amplicon 3-1-4, whose expression failed to activate following M. paratuberculosis phagocytosis, had high levels of similarity to a Rattus norvegicus nucleolin-related protein (NRP). Amplicon 5-2-10, which increased expression moderately following M. paratuberculosis phagocytosis, was a near perfect match to bovine nicotinamide adenine dinucleotide dehydrogenase (FNADH dehydrogenase) subunit 1 (ND1). Failure to activate these two genes at levels observed following phagocytosis of either E. coli or latex beads may uncover new mechanisms for the survival of M. paratuberculosis within bovine macrophage cells.