These few examples are all that is currently known about the molecular mechanisms underlying Brucella adhesion and internalization in eukaryotic cells. HeLa cells have extensively been used as a model to investigate the internalization of brucellae of epithelial cells during the colonization ITF2357 manufacturer of

the susceptible host [9, 10]. Here, we employed this cell line to evaluate the rate of invasion of B. melitensis at different growth phases. Our results indicate that cultures of B. melitensis in the late-log phase of growth were more invasive in non-professional phagocytic cells than cultures at mid-log and stationary growth phases. Using cDNA microarrays, we characterized the transcriptome of the most (late-log) and the least (stationary) invasive growth phases of B. melitensis cultures as a preliminary approach for identifying pathogen candidate genes involved in epithelial cell invasion process. Microarray analysis

revealed a greater number of genes up-regulated in these cultures than in stationary https://www.selleckchem.com/products/GDC-0449.html phase cultures. Consistent with the expected differences due to growth, there was a more active metabolism and invasiveness of cultures in late-log phase than cultures in stationary phase. Given the role that some of these genes have in pathogenesis in other bacterial species, we believe that these data may offer insight into potential growth-phase regulated Brucella virulence genes involved in the initial host:pathogen interactions. Results B. melitensis 16 M at late-log phase of growth were more invasive to epithelial cells than were bacteria at Celecoxib mid-log and stationary growth phases As described in the Methods section, B. melitensis was grown to mid-log growth phase, late-log growth phase, or stationary growth phase. At each of these growth phases, bacteria were enumerated, used to infect a representative epithelial cell

line (HeLa cells), and RNA was extracted and microarrays were performed to identify altered gene expression. Under our experimental conditions, there were 0.5 × 109 CFU/ml (OD = 0.18) at the mid-log growth phase, 2 × 109 CFU/ml (OD = 0.4) at late-log phase, and 5 × 109 CFU/ml (OD = 0.72) at stationary phase (Figure 1A). For invasion experiments, a consistent multiplicity of infection (MOI) factor of 1,000 B. melitensis cells per HeLa cell was used to normalize the number of bacteria used. The average number of intracellular bacteria recovered was 60 CFU at mid-log phase, 130 CFU at late-log phase of growth and 27 CFU at stationary growth phase per 103 cells inoculated (Figure 1B). These values represent the average of three C59 wnt price independent experiments. B. melitensis 16 M cultures grown to late-log phase and then co-incubated with HeLa cells for 30 min were therefore 2.2 (P < 0.05) and 4.8 (P < 0.01) times more invasive than were cultures at mid-log and stationary growth phases.