Mycobacterium tuberculosis persisters at the host-pathogen interface
Zimvo Maqeda, Liezel Smith, Jomien Mouton, Samantha Sampson,
DSI/NRF Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research, Stellenbosch University, South Africa
Treatment failure remains a major roadblock to tuberculosis control. This is partly attributable to formation of persisters, a bacterial population that survives longer inside the host, despite antibiotic treatment. Persisters are difficult to study, partly because they are present in very low numbers. To address this, a dual fluorescence reporter system, able to measure Mycobacterium tuberculosis (Mtb) replication, is a potential tool to monitor persister formation in vivo. Here, we investigated (i) whether theophylline administration affects host and disease progression and (ii) whether theophylline induction of the replication reporter can be achieved in vivo.
BALB/c mice were intravenously infected with the Mtb reporter strain. Groups (n=5/group) received different doses of theophylline (250, 300 and 350 mg/kg) in drinking water for 2 and 4 (0, 125, 250, 300, 350 mg/kg) weeks post-infection. Lungs and spleens were harvested following infection and plated for colony-forming-unit (CFU) determination. A portion of intact lung tissue was reserved for histopathological analysis and a portion of spleen homogenate was prepared for fluorescence activated cell sorting to isolate all cells containing viable bacteria (GFP positive). Subsequently the Amnis® ImageStream® was exploited to determine the optimal theophylline concentration that induces maximal expression of TurboFP635.
As expected, organ bacterial burden increased over time accompanied by the recruitment of inflammatory cells and the development of small lesions in lung tissue at D28. There was no difference between treated and untreated groups. Amongst the different theophylline treated groups, 250 and 300 mg/kg theophylline concentration induced maximum expression of the reporter in vivo, after 2 weeks of theophylline treatment.
Administration of theophylline does not affect the host and disease progression. Theophylline induction of the replication reporter was achieved in vivo. These results indicate that the reporter system can be used to probe Mtb persister formation in vivo.