FIG. 5.

Role of Ca²⁺, monocyte adherence, and the bacterial virB9 gene cluster in uptake and intracellular survival of Brucella. (A and B) Freshly isolated PBM were challenged with nonopsonized GFP-expressing wild-type (wt) B. suis or a virB9 mutant for 20 min and chased for another 30 min. Results are expressed as means ± standard deviations for duplicate samples from one out of three experiments with monocytes from different donors which gave comparable results. (A) Uptake. Counts of fluorescent bacteria internalized by nonadherent (non-adh.) or adherent PBM in the presence (+ Ca/Mg) or absence (− Ca/Mg) of external Ca²⁺ and Mg²⁺ are shown. (B) Survival. Counts of CFU of bacteria reisolated from osmotically lysed PBM following uptake in the presence or absence of external Ca²⁺ and Mg²⁺ are shown. (C) Electron micrograph of a PBM treated with 2 μM thapsigargin for 5 min before challenge with wt B. suis (15-min pulse, 60-min chase) in the presence of external Ca²⁺ and Mg²⁺. Thapsigargin-induced depletion of the internal Ca²⁺ stores did not affect the uptake of Brucella but did affect the centripetal transport of the phagosomes, as indicated by the predominantly peripheral localization of the phagosomes. The walls of the phagosomes show no tight apposition. Bar, 7 μm.