Novel Concepts of Mycobacterial Evasion Mechanisms

Tuberculosis is caused by bacterium Mycobacterium tuberculosis, and it is currently the deadliest infectious disease worldwide. The standard antimicrobial regimen is a cocktail of antimicrobials taken for several months, which still does not guarantee a successful eradication of the bacteria. Treatment dropouts and the use of antimicrobials that are inefficient in eradicating the infection have led to emerged drug-resistance. Also, the lack of safe and effective vaccine makes the prevention of tuberculosis difficult. One reason for the challenges in both prevention and treatment of tuberculosis is the ability of mycobacterium to cause chronic infection and protect itself from environmental hazards such as active immune responses and antimicrobials.

The aim of this study was to examine mycobacterial evasion mechanisms against antimicrobials and immune responses and propose new intervention strategies. Mycobacterium marinum and zebrafish were used to study the interaction of the host and the pathogen. Mycobacterium can actively suppress immune responses and delay the onset of adaptive responses, and to prevent this immune evasion, an immunomodulation was tested. In the adult zebrafish population, around 10% of the population is able to naturally retain the M. marinum load below the detection limit of qPCR. This proportion could be increased up to 25% by priming with heat- killed Listeria monocytogenes prior the mycobacterial infection. The induced protective immune response significantly reduced mycobacterial loads after four weeks of infection and induced clearance in both wildtype and rag1-/- mutant fish showing the important role of innate immune responses in the sterilizing response. The protective immune response was characterized with an increased expression of mpeg1, tnf and nos2b and decreased expression of sod2. These results indicate early clearance mediated via pro-inflammatory responses and enhanced killing of mycobacteria. Importantly, the immunomodulation was ineffective if the infection was already established. However, a similar approach could be used as a prophylactic treatment in high burden areas.

Another mycobacterial evasion strategy is the formation of biofilms. After suppressing immune responses and establishing the infection, mycobacteria form bacterial communities in granulomas and produce extracellular matrix that gives structure and protection for the bacterial population. The fast-growing M. marinum with a bioluminescent cassette was used to study the biofilm maturation and composition in vitro. The results showed that the biofilm maturation did not alter the minimum inhibitory concentration (MIC) but increased minimum bactericidal concentration (MBC) 63 times compared to planktonic cells within two days of biofilm culturing. It was further confirmed with repeated antimicrobial exposures that the increased tolerance of biofilm cultures was not due to genetic resistance. Degrading any of the major extracellular matrix components in combination with rifampicin reduced the number of live bacteria in a biofilm, demonstrating the important role of the biofilm matrix in the antimicrobial tolerance.

Time-kill curve analysis with a quick bioluminescence read-out was established to specifically measure the subpopulation of persister mycobacteria. In the analysis, the killing kinetics of a bacterial population was followed after the exposure to high concentration of a bactericidal antimicrobial, here rifampicin, to measure the persister subpopulation that can tolerate the drug for a prolonged time. For maturing

M. marinum biofilm, the killing kinetics were significantly different after one week of culturing compared to planktonic cells, and the rifampicin concentration was saturated at 400 μg/ml after which increasing the antimicrobial concentration did not alter the killing kinetics. This method has a potential in screening for treatments that specifically target mycobacterial persisters.

Finally, sybodies against biofilm extracellular matrix proteins GroEL1 and GroEL2 were used to specifically target M. marinum and M. tuberculosis biofilms in vitro and in ex vivo zebrafish granulomas. Sybodies are synthetic molecules mimicking the binding domain of antibodies. The binding properties of the sybodies can be easily modified and screened against the wanted target in sybody libraries. The potential sybodies acquired from the screens were assessed by confocal imaging. Fluorescence labelled anti-GroEL sybodies were able to bound GroEL on both in vitro and ex vivo biofilms making sybodies an attractive molecular carrier to target mycobacterial biofilms. A treatment that specifically targets mycobacterial persisters in biofilms inside granulomas could enhance the efficacy of antimicrobial therapy and shorten the current treatment time. By understanding better, the mycobacterial evasion mechanisms, future treatments can more effectively be targeted against Mtb.