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Invasion of HeLa 229 cells by virulent Bordetella pertussis
C A Ewanowich1, A R Melton, A A Weiss
1Department of Medical Microbiology and Infectious Diseases, University of Alberta, Edmonton, Canada.
Abstract:
Phase-dependent invasive behavior of Bordetella pertussis was demonstrated by recovery of viable organisms from gentamicin-treated HeLa cell monolayers and by transmission electron microscopy. Several mutants of B. pertussis with Tn5 or Tn5 lac inserted into various vir-regulated genes were evaluated for differences in their invasive abilities. Mutants lacking filamentous hemagglutinin, pertussis toxin, and two as yet uncharacterized vir-regulated products had levels of invasion significantly lower than that of the parent strain BP338. In contrast, invasion by mutants lacking adenylate cyclase toxin was significantly increased compared with that of wild-type B. pertussis. This increase in invasion was eliminated when concentrations of intracellular cyclic 3'-5' AMP were stimulated by treating HeLa cells with cholera toxin or forskolin. Entry of B. pertussis occurred through a microfilament-dependent phagocytic process, as evidenced by the marked reduction in uptake following treatment of HeLa cells with cytochalasin D. Invasion was inhibited with polyclonal anti-B. pertussis and anti-filamentous hemagglutinin antisera. In addition, a monoclonal antibody against lipooligosaccharide A reduced uptake by 65.5%. The preservation of HeLa cell integrity and the limited replication of intracellular bacteria suggest that invasion may represent a means by which B. pertussis evades an active host immune response.
Insights
Bordetella pertussis invasion of host cells depends on specific bacterial factors. Disrupting genes for filamentous hemagglutinin and pertussis toxin reduced invasion, while removing adenylate cyclase toxin increased it.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Bordetella pertussis causes whooping cough and possesses invasive capabilities.
- Understanding bacterial invasion mechanisms is crucial for developing effective treatments and vaccines.
Purpose of the Study:
- To investigate the phase-dependent invasive behavior of Bordetella pertussis.
- To identify specific bacterial virulence factors involved in host cell invasion.
Main Methods:
- Utilized gentamicin protection assays and transmission electron microscopy to assess bacterial invasion.
- Generated and analyzed mutants of B. pertussis with Tn5 insertions in vir-regulated genes.
- Investigated the role of intracellular cyclic AMP levels and host cell cytoskeleton in invasion.
- Evaluated the effect of specific antibodies on bacterial uptake.
Main Results:
- Mutants lacking filamentous hemagglutinin, pertussis toxin, or two uncharacterized products showed significantly reduced invasion.
- Mutants lacking adenylate cyclase toxin exhibited significantly increased invasion, which was reversed by stimulating intracellular cyclic AMP.
- B. pertussis entry is a microfilament-dependent phagocytic process, inhibited by cytochalasin D.
- Polyclonal and monoclonal antibodies targeting bacterial components reduced invasion.
Conclusions:
- Bordetella pertussis invasion is a regulated process involving multiple virulence factors.
- Adenylate cyclase toxin appears to negatively regulate invasion, possibly by modulating host cell cyclic AMP.
- Invasion may serve as a mechanism for B. pertussis to evade the host immune response.
- Specific bacterial components like filamentous hemagglutinin, pertussis toxin, and lipooligosaccharide A are critical for efficient invasion.