cytochalasin-d and Bordetella-Infections

Bordetella bronchiseptica causes respiratory disease in swine, yet there are no studies examining the interaction of B. bronchiseptica with swine alveolar macrophages. A swine isolate of B. bronchiseptica was able to adhere to, and survive intracellularly in, swine alveolar macrophages, but the relative ability of the bacteria to accomplish these functions was dependent on its phenotypic phase and culture conditions. More bacteria were observed extracellularly as well as intracellularly by immunofluorescent staining when B. bronchiseptica was cultured at 23 degrees C as compared to 37 degrees C. However, more bacteria cultured at 37 degrees C were found surviving intracellularly after the macrophages were cultured with polymyxin B to kill extracellular bacteria. Similar results were seen in experiments performed with an isogenic Bvg(-) phase-locked mutant of B. bronchiseptica cultured at 37 or 23 degrees C, indicating that another temperature dependent mechanism in addition to bvg may play a role in adhesion and intracellular survival. B. bronchiseptica was cytotoxic for swine alveolar macrophages in the Bvg(+) phase only. The cytotoxicity of B. bronchiseptica for alveolar macrophages, and its ability to survive phagocytosis, are no doubt important to escape from immune clearance mechanisms and establish infection, and could leave the host susceptible to secondary respiratory pathogens.

Topics: Animals; Antibodies, Bacterial; Bacterial Adhesion; Bacterial Outer Membrane Proteins; Bacterial Proteins; Benzenesulfonates; Bordetella bronchiseptica; Bordetella Infections; Cells, Cultured; Cytochalasin D; Fluorescent Dyes; Gene Expression Regulation; Immunoblotting; Macrophages, Alveolar; Mutation; Nucleic Acid Synthesis Inhibitors; Polymyxin B; Rabbits; Swine; Swine Diseases; Temperature; Transcription Factors; Virulence

Invasion and intracellular survival of Bordetella pertussis in HeLa 229 cells was studied by a new assay that utilizes polymyxin B instead of gentamicin to rapidly kill extracellular organisms. Invasion measured by this assay was time and temperature dependent and was inhibited by the microfilament drug cytochalasin D. The invasion process was also dependent on a functional vir locus (also known as bvg), the positive regulator of virulence gene expression in B. pertussis. Four spontaneous Vir- phase variants of B. pertussis and a mutant with a transposon insertion mutation in the vir locus did not invade. Cells that were environmentally modulated and thus did not express virulence determinants also did not invade. Two Vir- mutants, a vir-directed plasmid insertion mutant and a UV-light-induced mutant, were capable of invasion, although they did not produce other known virulence factors such as pertussis toxin and hemolysin but did produce small amounts of filamentous hemagglutinin (FHA) and the 69-kilodalton outer membrane protein. None of 70 Tn5 IS50L::phoA (TnphoA) insertion mutants of strain Bp18323 (including three mutants defective in FHA) tested showed any reproducible defect in invasion. A mutant carrying a site-directed deletion mutation in FHA was also capable of invasion in our assay. These data suggest that there is redundancy in the invasion functions of B. pertussis and that one or more of these are coordinately regulated with FHA and the 69-kilodalton outer membrane protein more tightly than with other vir-activated gene products.

Topics: Bacterial Proteins; Bordetella Infections; Bordetella pertussis; Cytochalasin D; Electrophoresis, Polyacrylamide Gel; Gene Expression; HeLa Cells; Hemagglutinins; Humans; Mutation; Phenotype; Plasmids; Polymyxin B; Temperature; Virulence; Virulence Factors