ABSTRACT
<p><b>BACKGROUND</b>Isocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage.</p><p><b>METHODS</b>MTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique.</p><p><b>RESULTS</b>RT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS.</p><p><b>CONCLUSIONS</b>MTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.</p>
Subject(s)
Animals , Apoptosis , Genetics , Physiology , Cell Line , In Situ Nick-End Labeling , Interferon-gamma , Metabolism , Isocitrate Lyase , Genetics , Metabolism , Macrophages , Cell Biology , Metabolism , Microbiology , Microbial Viability , Microscopy, Fluorescence , Mycobacterium smegmatis , Genetics , Mycobacterium tuberculosis , Genetics , Nitric Oxide , Metabolism , Plasmids , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Transformation, GeneticABSTRACT
<p><b>OBJECTIVE</b>To review recent developments in therapeutic DNA vaccines against tuberculosis.</p><p><b>DATA SOURCES</b>The data used in this review were obtained mainly from the studies of therapeutic DNA vaccines against tuberculosis reported from 2000 to 2006.</p><p><b>STUDY SELECTION</b>Relevant articles about studies of therapeutic DNA vaccines against tuberculosis were selected.</p><p><b>DATA EXTRACTION</b>Data were mainly extracted from the 32 articles listed in the reference section of this review.</p><p><b>RESULTS</b>Some DNA vaccines which previously showed to induce protective immunity against infection by Mycobacterium tuberculosis in a prophylactic manner are also surprisingly effective when used therapeutically, including persistent Mycobacterium tuberculosis and multidrug-resistant tuberculosis which are refractory to immune system and antibacterial chemotherapy alone. When used in combination with antibacterial drugs, therapeutic DNA vaccines could effectively eliminate residual bacteria in infected animals and shorten the therapy course of conventional chemotherapy. Detailed studies demonstrated that therapeutic effects of DNA vaccines may at least partly be due to the restoration of the Th(1)/Th(2) balance. Some problems have also emerged along with these exciting results.</p><p><b>CONCLUSIONS</b>Therapeutic DNA vaccine is a promising strategy against tuberculosis, however developing an ideal DNA vaccine for therapy of tuberculosis will require further development.</p>