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Dynamics of stress protein expression in leishmania-infected macrophages: evidence for selective induction and recognition of leishmania heat shock protein 60

University of British Columbia

Heat shock protein (HSP) expression was examined in murine bone marrow-derived macrophages infected with stationary phase promastigotes of Leishmania donovani. Immunoblotting with a rabbit polyclonal antiserum raised against HSP60 from Heliothis virescens (moth), revealed the de novo appearance of proteins with subunit sizes of M r 65,000 and 67,000 in leishmania infected macrophages. Expression of the novel M r 65,000 and 67,000 proteins in infected cells was coordinately regulated and at 24 hr of infection reached maximal levels of 52-100% increases above initial levels at 3 hr. Proteins with identical electrophoretic mobilities and which were similarly regulated in response to heat were also detected in leishmania promastigotes. The appearance of these proteins in macrophages was specific to leishmania infection in that neither protein was detected in non-infected cells either in the basal state or following several treatments including: (i) infection with Y. pseudotuberculosis, (ii) phagocytosis of S. aureus, (iii) NaAsC>2, or (iv) heat shock. A monoclonal antibody that recognizes both mammalian HSP70 and HSP70 from Plasmodia detected single isoforms of both leishmania and murine HSP70 in infected cells and neither protein changed quantitatively during infection. Levels of host heat shock proteins 60, 70 and 90 did not change in response to infection, however, marked induction of the mammalian stress protein heme oxygenase-1 was observed under these conditions. Further evidence for selective expression of the M r 65,000 and 67,000 heat-regulated leishmania proteins was provided by the finding that as their concentration was increasing, the abundance of the leishmania surface protease gp63 in infected cells was noted to decrease. The prominence of leishmania HSP60 within infected macrophages suggested the possibility that it might be a target of the host response. To facilitate further studies concerned with the role of this protein in the host response and in the pathogenesis of the leishmaniases, the HSP60 gene of Leishmania major was cloned, sequenced and expressed. A XEMBL-3 L. major genomic library was screened with a PCR-generated DNA probe derived from a highly conserved region of the leishmania HSP60 gene. A single clone that hybridized strongly was selected and characterized. Sequence analysis revealed the presence of an open reading frame of 1770 bp encoding a putative polypeptide of 589 amino acids with a predicted size of M r 64,790 and with the highest degree of amino acid sequence similarity (56%) to HSP60 from T. cruzi. Less extensive amino acid sequence similarity (48%) was observed between the leishmania HSP60 and the corresponding human protein. Notably, regions of significant sequence dissimilarity between the leishmania and human proteins were identified and these were concentrated principally within the carboxy-terminal regions of the proteins. The entire coding region of the leishmania HSP60 gene was amplified by PCR, subcloned into the pET-3a vector and expressed in E. coli. Purified recombinant protein was used to examine sera from patients with tegumentary leishmaniasis from South America for the presence of antibodies to HSP60. Unlike sera from healthy, uninfected controls, sera from patients reacted strongly with recombinant leishmania HSP60. In contrast, these same sera showed little or no reactivity with recombinant mycobacterial HSP65. To examine the protective potential of LHSP60, immunization studies were done in susceptible Balb/c mice. Recombinant leishmania HSP60 alone appeared to confer early (within the first 5 weeks) resistance against infection with L. major as determined by reduced lesion sizes in HSP60 immunized mice. In contrast, long-lasting protection did not develop in rLHSP60 immunized mice as lesion sizes and parasite numbers in these animals at 7 weeks of infection did not differ from those observed in control animals. These findings provide evidence for the selective induction of leishmania HSP60 in infected macrophages and for the recognition of this protein in humans with leishmaniasis. Further studies of this protein should clarify its role in the host response to Leishmania and in disease pathogenesis.

Thesis/Dissertation

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2009-03-17

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http://hdl.handle.net/2429/6150

Microbiology and Immunology

University of British Columbia

UBCV

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