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Infection and Immunity, July 2002, p. 3816-3823, Vol. 70, No. 7
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.7.3816-3823.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Inhibition of Fusion of Chlamydia trachomatis Inclusions at 32°C Correlates with Restricted Export of IncA

K. A. Fields,1 E. Fischer,2 and T. Hackstadt1*

Host-Parasite Interactions Section, Laboratory of Intracellular Parasites,1 Microscopy Branch, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana 598402

Received 30 January 2002/ Returned for modification 21 March 2002/ Accepted 1 April 2002

Chlamydia trachomatis is an obligate intracellular bacterium that develops within a parasitophorous vacuole termed an inclusion. The inclusion is nonfusogenic with lysosomes but intercepts lipids from a host cell exocytic pathway. Initiation of chlamydial development is concurrent with modification of the inclusion membrane by a set of C. trachomatis-encoded proteins collectively designated Incs. One of these Incs, IncA, is functionally associated with the homotypic fusion of inclusions. Inclusions also do not fuse when cultures are multiply infected with C. trachomatis and cultivated at 32°C. We obtained evidence linking these experimental observations by characterizing IncA localization in 32°C cultures. Analysis of inclusions by light and transmission electron microscopy confirmed that HeLa cells infected with multiple C. trachomatis elementary bodies and cultivated at 32°C for 24 h contained multiple, independent inclusions. Reverse transcriptase PCR and immunoblot analyses of C. trachomatis-infected HeLa cells demonstrated the presence of IncA at 24 h in 32°C cultures. When parallel cultures were probed with IncA-specific antibodies in indirect immunofluorescence assays, IncA was detectable in intracellular chlamydiae but not within the inclusion membrane. In addition, analysis of purified reticulate bodies from 37 and 32°C cultures showed that bacterium-associated pools of IncA are enriched in cultures grown at 32°C. Microscopic observation of infected cells revealed that some vacuoles had fused by 48 h postinfection, and this finding was correlated with the detection of IncA in inclusion membranes by immunofluorescence microscopy. The data are consistent with a requirement for IncA in fusions of C. trachomatis inclusions and suggest that the effect of incubation at 32°C is manifested by restricted export of IncA to the inclusion membrane.

* Corresponding author. Mailing address: Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT 59840. Phone: (406) 363-9308. Fax: (406) 363-9253. E-mail: Ted_Hackstadt{at}NIH.GOV.

Editor: D. L. Burns

Infection and Immunity, July 2002, p. 3816-3823, Vol. 70, No. 7
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.7.3816-3823.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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