Temporal Gene Expression Analysis of the intracellular pathogen Brucella melitensis following bovine Peyer's patch infection.

In a time course study, we characterized global gene expression profile of B. melitensis infection in bovine Peyer patches in the first 4 h p.i. Statistical analysis of microarray results reveal that 2,356 B. melitensis genes were detected as differentially expressed (z-score p < 0.025) compared with control between 15 min and 4 h post-infection. A group of 1,740 genes that were differentially expressed in at least 4 out of 5 time points was considered the core set of genes that reflect the major changes in B. melitensis gene expression during the early in vivo bovine Peyer’s patch infection and therefore important in understanding key events in the modulation of host response. From this set of 1,740 differentially expressed genes, 925 (53%) were up-regulated and 815 (47%) were down-regulated compared with the in vitro grown culture. These results reflect significant Brucella metabolic modifications and adaptation from an extracellular to an intracellular lifestyle. Specific genes and biological processes identified in this study will further help elucidate how both host and Brucella interact during the early infectious process to the eventual benefit of the pathogen and to the detriment of the naïve host. Microarrays were used to examine the transcriptional profiles of wild type Brucella melitensis 16M intraluminally inoculated in bovine ileal loops across five time points (15 min, 30 min, 1, 2 and 4 hours). To account for the possibility of the cross-hybridization from the surgery samples, the original total RNA from B. melitensis-infected bovine Peyer’s patches (i.e. non-enriched non-amplified) (n = 20) were also hybridized against B. melitensis gDNA on the B. melitensis oligo-arrays, and those oligospots with signal were considered non-specific and eliminated from all analyses to avoid false positive pathogen gene detection. The intracellular B. melitensis gene expression at every time point was compared to the gene expression of the inoculum (i.e. in vitro-grown cultures of B. melitensis at late-log phase of growth) (n = 4). Experiments and controls were performed in quadruplicate (bovine ligated ileal loops surgeries were performed with four calves), generating a total of 44 arrays.