1. Growth analysis – Two types of M. marinum were used for this study: the parental 1218R strain and a derivative of this strain (LHM4) carrying a plasmid integrated into the genome that expresses resistance to kanamycin and encodes for expression of red fluorescent protein (RFP).
2. Gene expression – The M. marinum 1218R strain were used for these studies. The effect of growth in the HARV subjected to normal or simulated microgravity on gene expression was studied.
3. Resistance to hydrogen peroxide, acid and gamma radiation - These studies used M. marinum LHM4 grown and treated as described in Abshire et al NPJ Microgravity (2016) 2, 16038; doi: 10.1038/npjmgrav.2016.38..
The objective of this proposal was to determine whether waterborne pathogenic mycobacteria alter gene expression, become resistant to stress, and hence could be more infectious when grown under low shear simulated microgravity (MG). Mycobacterium marinum was the model organism used in this study. This is a bacterium that naturally infects fish and amphibians to produce a tuberculosis-like disease in a variety of organs. In humans, M. marinum can cause a disease known as swimming pool granuloma, which is restricted to the skin due to the lower temperature of body extremities. The pathology is similar to dermal Mycobacterium tuberculosis infection and the infection route is through cuts or abrasions. Infection is usually limited to the skin in humans since M. marinum grows at 28-35°C. M. marinum infects and survives inside immune cells called macrophage by the same mechanisms as M. tuberculosis and Mycobacterium avium. M. tuberculosis and M. avium cause serious lung infections in humans. M. marinum is therefore a good model to understand how these pathogenic mycobacteria survive and adapt to the hostile environment inside macrophage. Since M. marinum and M. avium exist in the environment on Earth and can form biofilms in water systems, it is important to understand the effect of microgravity on these pathogenic mycobacteria as they could exist in the water systems on space vehicles.
M. marinum strain 1218R and LHM4 (a derivative of M. marinum carrying a plasmid encoding kanamycin resistance and red fluorescent protein) were grown in high aspect ratio vessels (HARV) in a rotary cell culture system (RCCS) under normal gravity (NG) or MG. By following the optical density of the culture as well as the colony forming units per milliliter we determined that the growth of both strains was altered by growth under MG once the culture was in exponential phase. There was no difference in the growth for ~40 hrs (early exponential phase) but the growth of the MG culture was significantly slower after 40 hrs. The doubling time during early exponential was ~6 hrs and after 40 hrs this increased to ~10-11 hrs for the NG cultures and to ~15.5-16 hrs for the MG cultures. We hypothesized that the slowing of growth may have been due to an oxidative stress in the bacteria. Hydrogen peroxide can cross membranes and so we repeated the growth studies adding catalase to the culture. Any hydrogen peroxide exiting the bacteria and existing in the medium would be detoxified by the catalase. This did not however improve the growth of the M. marinum under MG.
To determine the effect of MG on the stress responses activated by the growth conditions, we used RNAseq to examine what genes were expressed. For RNAseq, the bacteria were harvested, RNA isolated and converted DNA (cDNA), and the cDNA sequenced using the MiSeq benchtop sequencer. Using bioinformatics, the amount of expression of the different M. marinum genes were compared between the NG and MG samples. To make sure that we were examining only gene expression changes due to MG, only bacteria in early exponential growth were used in the RNAseq studies. Triplicate NG and MG cultures were used to generate samples of bacteria grown for ~40 hrs. We also grew triplicate cultures for 4 days and then diluted them again and grew them for another ~40 hrs so we could examine gene expression from bacteria exposed for a longer time. From these studies it was obvious that stress response pathways were activated in the MG cultures and the stress responses were at a higher expression level the longer they were exposed to MG. Expression seemed to change to be similar to responses of M. tuberculosis inside macrophage. This suggests that the MG stress was similar to that of mycobacteria inside immune cells. Using the MG growth conditions may be useful in the future to test drugs designed to kill mycobacteria during infection. We are in the process of identifying regulator proteins that may be involved in aiding mycobacteria survival under MG.
Published studies indicate that other species of bacteria show altered resistance to hydrogen peroxide and acid (pH 3.5) once grown under MG. We tested our LHM4 strain and determined that as found for other bacteria M. marinum were more sensitive to killing by hydrogen peroxide but did not alter their resistance to acid conditions once grown under MG. M. marinum sensitivity to gamma radiation did not change also. This type of study may aid in designing disinfection techniques for future space travel.
In summary, growth of M. marinum under MG results in changes in growth, gene expression and sensitivity to stress conditions. SigH is a stress response protein that increases transcription at specific operons and this work suggests SigH may be involved in the response to growth under MG. We are in the process of generating an M. marinum sigH mutant that can be used in future studies to determine whether SigH is an important factor in the adaptation of M. marinum to the stress of growth under MG.
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