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Chapter 16 : Morphological and Physiological Evidence for a Developmental Cycle in

Authors: Rafael A. Garduño1, Paul S. Hoffman1, Elizabeth Garduño2, Margot Hiltz2, David Allan2
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Affiliations: 1: Department of Medicine, Dalhousie University, Halifax, Nova Scotia B3H-4H7, Canada; 2: Department of Microbiology-Immunology, Dalhousie University, Halifax, Nova Scotia B3H-4H7, Canada
Content Type: Monograph
Publication Year: 2002

Category: Bacterial Pathogenesis

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Morphological and Physiological Evidence for a Developmental Cycle in , Page 1 of 2

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Abstract:

This chapter focuses on the cyclical nature of some morphological and physiological changes that take place in infected HeLa cells and during growth in vitro. Coverslip cultures of HeLa cells were infected with purified mature intracellular forms (MIFs), and changes in Gimenez staining were followed for up to 3 days. The bacterial strains used throughout these studies were Lp1-SVir and 2064, two virulent strains previously reported and characterized. Cultures with 100% green forms (after Gimenez staining) were identified upon growth of on buffered charcoal-yeast extract (BCYE) plates. Rowbotham reported earlier that infection of amoebae resulted in the formation of small, highly motile, and infectious legionellae that are believed to be equivalent to the HeLa-derived MIFs reported. Purified MIFs were placed in buffered yeast extract (BYE) and their growth was followed through viable cell counts. Researchers concluded that the green forms arising in the BYE cultures were replicating bacteria that developed from the MIFs originally used as inoculum. In summary, undergoes cyclical morphological and physiological changes that suggest the existence of a developmental program.

Citation: Garduño R, Hoffman P, Garduño E, Hiltz M, Allan D. 2002. Morphological and Physiological Evidence for a Developmental Cycle in , p 82-85. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch16

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Morphological and Physiological Evidence for a Developmental Cycle in Legionella pneumophila
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Citation: Garduño R, Hoffman P, Garduño E, Hiltz M, Allan D. 2002. Morphological and Physiological Evidence for a Developmental Cycle in , p 82-85. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch16
/content/10.1128/9781555817985.chap16.fig16-1
FIGURE 1

Electron micrograph showing the typical ultrastructural features of the intermediate forms that arise after MIFs of the Lp1-SVir are inoculated into BYE broth. IV, intraperiplasmic vesicles; MC, membrane coils. Bar represents 0.5

10.1128/9781555817985/fig16-1_thmb.gif
10.1128/9781555817985/fig16-1.gif
Image of FIGURE 1
FIGURE 1

Electron micrograph showing the typical ultrastructural features of the intermediate forms that arise after MIFs of the Lp1-SVir are inoculated into BYE broth. IV, intraperiplasmic vesicles; MC, membrane coils. Bar represents 0.5

Citation: Garduño R, Hoffman P, Garduño E, Hiltz M, Allan D. 2002. Morphological and Physiological Evidence for a Developmental Cycle in , p 82-85. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch16
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/content/10.1128/9781555817985.chap16.fig16-2
FIGURE 2

An ∼20-kDa protein is prominent in MIFs obtained from HeLa cells infected with Lp1-Vir. Autoradiogram (A) and immunoblot (B) from an SDS-PAGE gel run with whole cell lysates from stationary-phase bacteria grown in vitro ( ) or MIFs ( ). For autoradiography, bacteria were placed in distilled water for 4 h and then pulsed with radiolabeled methionine for 1 h. The blot was immunostained with a hyperimmune rabbit serum against the purified 20-kDa protein and a second antibody conjugated with alkaline phosphatase and then developed with NBT-BCIP. Numbers on the left of panel A indicate the position of molecular weight standards (× 1,000) and the arrowheads on the right of the two panels point at the protein band of interest.

10.1128/9781555817985/fig16-2_thmb.gif
10.1128/9781555817985/fig16-2.gif
Image of FIGURE 2
FIGURE 2

An ∼20-kDa protein is prominent in MIFs obtained from HeLa cells infected with Lp1-Vir. Autoradiogram (A) and immunoblot (B) from an SDS-PAGE gel run with whole cell lysates from stationary-phase bacteria grown in vitro ( ) or MIFs ( ). For autoradiography, bacteria were placed in distilled water for 4 h and then pulsed with radiolabeled methionine for 1 h. The blot was immunostained with a hyperimmune rabbit serum against the purified 20-kDa protein and a second antibody conjugated with alkaline phosphatase and then developed with NBT-BCIP. Numbers on the left of panel A indicate the position of molecular weight standards (× 1,000) and the arrowheads on the right of the two panels point at the protein band of interest.

Citation: Garduño R, Hoffman P, Garduño E, Hiltz M, Allan D. 2002. Morphological and Physiological Evidence for a Developmental Cycle in , p 82-85. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch16
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References

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1. Cirillo, J. D.,, S. L. G. Cirillo,, L. Yan,, L. E. Bermudez,, S. Falkow,, and L. S. Tompkins. 1999. Intracellular growth in Acanthamoeba castellani affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila. Infect. Immun. 67: 4427– 4434.
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9. Miyamoto, H.,, S. Yoshida,, H. Taniguchi,, M. H. Qin,, H. Fujio,, and Y. Mizuguchi. 1993. Protein profiles of Legionella pneumophila Philadelphia-1 grown in macrophages and characterization of a gene encoding a novel 24 kDa Legionella protein. Microb. Pathog. 15: 469– 484.
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