Localisation of mycobacterial DNA and mRNA in human tuberculous granulomas.
In situ hybridisation was used to detect the presence of Mycobacterium tuberculosis in paraffin-embedded lung tissue of nine patients diagnosed with tuberculosis (TB). Mycobacterial DNA was found in all nine patients and in 175 out of 191 granulomas examined. A combination of in situ hybridisation and immunohistochemistry techniques demonstrated that mycobacterial DNA was associated with CD68-positive cells with the morphology of macrophages and giant cells. Mycobacterial DNA was also found within the necrotic regions of some granulomas. mRNA for the mycobacterial RNA polymerase beta subunit (rpoB) was detected by RNA: RNA in situ hybridisation. The rpoB mRNA was also localised to CD68-positive cells with the morphology of macrophages and to giant cells of certain necrotic granulomas. No rpoB mRNA was found in the necrotic regions of granulomas. Mycobacterial DNA was detected in 92% of patient granulomas of which 8% were positive for rpoB mRNA. The ability to identify mycobacterial RNA transcripts within human tuberculous granulomas affords us the opportunity to analyse the interplay between pathogen gene expression and the human immune response and should provide valuable insight into the mechanisms used by M. tuberculosis to persist within the human host.
MRC Center for Molecular and Cellular Biology, University of Stellenbosch Medical School, Cape Town, South Africa. gfen@gerga.sun.ac.za
Gael
G
Fenhalls
Liesel
L
Stevens-Muller
Rob
R
Warren
Nora
N
Carroll
Juanita
J
Bezuidenhout
Paul
P
Van Helden
Philip
P
Bardin
D000328:Adult; D015703:Antigens, CD / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D004269:DNA, Bacterial / Q000032:analysis*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005260:Female; D006099:Granuloma / Q000382:microbiology*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008168:Lung / Q000382:microbiology*; D008297:Male; D012329:RNA, Bacterial / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000382:microbiology*; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases; D012329:RNA, Bacterial; D012333:RNA, Messenger; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C105078:RNA polymerase beta subunit