Date of Award
Doctor of Philosophy
Norman F. Cheville
John P. Kluge
Fluorescence microscopy and quantitative bacteriology were used to study penetration and intracellular growth of Brucella spp. in non-phagocytic cells in vitro. B. abortus infected all established cell lines tried; Vero cells proved to be the most reproducible system. Rough strains of B. abortus adhered to and entered a greater number of Vero cells, however smooth strains replicated within a larger percentage of cells. Differences in adhesiveness and invasiveness were correlated to hydrophobicity of the organism, as measured by hydrocarbon adherence. Ultrastructurally, intracellular smooth and rough brucellae were within cisternae of the rough endoplasmic reticulum (RER). The results suggest that transfer to the RER is the limiting step in the infection of Vero cells by B. abortus;Infection of Vero cells by B. abortus was suppressed when inhibitors of energy metabolism, receptor-mediated endocytosis, or endosomal acidification were added to the inoculum. No inhibition was observed when these drugs were added after the inoculation period. Infection of Vero cells could be modulated by altering intracellular cyclic nucleotides. Uptake of B. abortus by Vero cells was not prevented by colchicine, but was abolished by cytochalasin B. Uptake of heat-killed B. abortus and non-invasive E. coli was similar to that of viable brucellae. Intracellular growth of B. abortus was not affected by cycloheximide. These results suggest that (i) B. abortus is internalized by receptor-mediated phagocytosis, (ii) transfer of B. abortus to RER requires endosomal acidification, and (iii) replication of B. abortus within RER does not depend on host-cell protein synthesis;Studies using fluorescence and electron microscopy showed that all species of Brucella entered Vero cells. Intracellular brucellae were first within phagosomes and phagolysosomes. Subsequent localization and replication within cisternae of the RER was seen with all species, except B. canis. These results suggest that Brucella-containing phagosome follows one of two routes: (i) it fuses with cisternae of the RER (in which unrestricted bacterial replication takes place), or (ii) it fuses with lysosomes, forming phagolysosomes (in which Brucella spp. fail to replicate). Strains of Brucella differ in their ability to induce their own transfer to the RER.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Philippe Gabriel Detilleux
Detilleux, Philippe Gabriel, "Penetration and intracellular growth of Brucella spp in Vero cells " (1989). Retrospective Theses and Dissertations. 9032.