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Attachment and entry of Legionella pneumophila in Hartmannella vermiformis
B S Fields1, S R Fields, J N Loy
1Respiratory Diseases Branch (Division of Bacterial and Mycotic Diseases), Centres for Disease Control and Prevention, Atlanta, Georgia 30333.
Abstract:
Legionella pneumophila is an intracellular parasite of Hartmannella vermiformis. Attachment to the amebae and entry of L. pneumophila were studied by two quantitative assays: One used plate counts to measure the number of bacteria attaching to amebae at 4 degrees C; the other determined the number of intracellular bacteria by use of transmission electron microscopy (TEM). The attachment assay showed that L. pneumophila are inefficient in attachment to amebae. About 0.05% of the bacteria were bound after 1 h with a 10- to 40-fold increase over the next 11 h. Attachment of both virulent and avirulent strains of L. pneumophila occurred at a similar rate. Uptake of L. pneumophila was measured by counting intracellular bacteria using TEM. Limited numbers of virulent L. pneumophila were found intracellularly before 4 h, but the numbers increased logarithmically after this time. The number of amebae containing virulent L. pneumophila increased linearly during the 12-h co-incubation. Avirulent L. pneumophila were rarely detected within amebae throughout the 12-h incubation. Results indicate that entry, not attachment, of virulent L. pneumophila is the limiting step in infection of axenically grown H. vermiformis.
Insights
Legionella pneumophila attachment to Hartmannella vermiformis is inefficient. Entry into the amoeba, not attachment, limits virulent L. pneumophila infection, as shown by quantitative assays and TEM.
Area of Science:
- Microbiology
- Cell Biology
- Parasitology
Background:
- Legionella pneumophila is an intracellular parasite.
- Hartmannella vermiformis serves as the host amoeba.
- Understanding L. pneumophila-H. vermiformis interactions is crucial for parasitology research.
Purpose of the Study:
- To quantitatively assess the attachment and entry mechanisms of L. pneumophila into H. vermiformis.
- To differentiate between attachment and entry as potential limiting steps in L. pneumophila infection.
- To compare the infectivity of virulent and avirulent L. pneumophila strains.
Main Methods:
- Quantitative attachment assay using plate counts at 4°C.
- Transmission electron microscopy (TEM) to determine intracellular bacterial numbers.
- Co-incubation of L. pneumophila with axenically grown H. vermiformis for 12 hours.
Main Results:
- L. pneumophila exhibited inefficient attachment to H. vermiformis, with only 0.05% bound after 1 hour.
- Attachment rates were similar for both virulent and avirulent L. pneumophila strains.
- Intracellular numbers of virulent L. pneumophila increased logarithmically after 4 hours, while avirulent strains were rarely detected intracellularly.
Conclusions:
- Bacterial entry, rather than attachment, is the rate-limiting step for virulent L. pneumophila infection of H. vermiformis.
- Virulent L. pneumophila demonstrate a distinct intracellular proliferation pattern compared to avirulent strains.
- TEM and quantitative assays provide valuable insights into host-pathogen dynamics at the cellular level.