Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion.
Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M(r) forms are found in C. psittaci-infected cells. This finding suggested that IncA may be post-translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of [32P]-orthophosphate-labelled infected HeLa cells with anti-IncA antisera demonstrated that the two highest M(r) IncA bands were phosphorylated. A vaccinia-virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background. IncA in lysates of these cells migrated identically to that seen in C. psittaci-infected cells, indicating the host cell was responsible for the phosphorylation of the protein. Microinjection of fluorescently labelled anti-IncA antibodies into C. psittaci-infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane. Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion.
Rockey
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Rocky Mountain Laboratories, NIH, NIAID Hamilton, Montana 59840, USA. rockey@ava.bcc.orst.edu
Grosenbach
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Hruby
D E
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Peacock
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Heinzen
R A
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Hackstadt
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T
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002691:Chlamydophila psittaci / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C094163:IncA protein, bacteria; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins