European Society of Mycobacteriology - Instituto Nacional de Saúde ...

OP-21
PRESENCE OF ESAT-6 AND CFP-10 GENES DOES NOT LEAD TO
PHAGOLYSOSOME TRANSLOCATION OF Mycobacterium szulgai
Jakko van Ingen 1,2 , Nicole van der Wel 3 , Richard Dekhuijzen 2 , Martin Boeree 2 , Dick van Soolingen 1
1 - National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven,
the Netherlands
2 - Department of Pulmonary diseases, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands
3 - Department of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands
Background
Bacterial virulence factors in nontuberculous mycobacteria are mostly unknown. In Mycobacterium tuberculosis complex
bacteria, the esat-6 and cfp-10 genes are important virulence factors, which facilitate translocation from the phagolysosome
to the cytosol of macrophages. Their presence and role among nontuberculous mycobacteria is largely unknown.
Methods
We assessed the presence of esat-6 and cfp-10 genes in all 5 M. kansasii subtypes based on 16S-23S internal transcribed
spacer sequencing (n=15), M. szulgai (4), M. marinum (4), M. avium (2), M. conspicuum (4), M. genavense (1), M. bohemicum
(2), M. interjectum (2), M. flavescens (5), M. xenopi (2), M. malmoense (2), “M. riyadhense” (1) and M. tuberculosis H37Rv by
PCR. We used Esa-12 CATGACAGAGCAGCAGTG and Esa-303 5’-GCCCTATGCGAACATCCC-3’ primers for esat-6
and opBR78 5’-GTAGCCCGGGATGGCAGAGATGAAGACCGATGCC-3’ and opBR103 5’-TCAGAAGCCCATTT-
GCGAGGACAGC-3’ primers for cfp-10. For PCR negatives, we performed Southern blotting with probes based on the
esat-6 gene of M. tuberculosis H37Rv.
One NTM species with esat-6 and cfp-10 genes was selected for macrophage infection. By cryo-immunogold electron microscopy
we tested whether these genes effect translocation from the phagolysosome to the cytosol of macrophages.
Results
We were able to amplify esat-6 and cfp-10 genes in M. tuberculosis H37Rv, all M. kansasii subtypes, M. szulgai, M. marinum
and “M. riyadhense”. All esat-6 and cfp-10 sequences among these nontuberculous mycobacteria were species-specific;
multisequence alignment revealed an average of 90% homology with the M. tuberculosis sequences. The remaining species
were repeatedly negative by both PCR and Soutern blotting. Mycobacterium szulgai was subsequently used for a
macrophage (THP-1) infection experiment, with an M. tuberculosis positive control. Seventy-two hours after infection, no
cytosolic M. szulgai bacteria were found, while 53% of the M. tuberculosis bacteria had translocated to the cytosol.
Conclusions
While some nontuberculous mycobacteria harbor esat-6 and cfp-10 genes, their products, at least for M. szulgai, do not
effect translocation of the bacteria from the phagolysosome to the cytosol in macrophages. ESAT-6 and CFP-10 protein
structure or secretion could be critical factors. While the presence of esat-6 and cfp-10 genes adds an interesting phylogenetic
marker, the pathogenesis of NTM infections remains unexplained.
European Society of Mycobacteriology | 30 th Annual Congress | July 2009 | Porto - Portugal
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