Study – Intracellular M.abscessus infection in Macrophages

Mycobacterium abscessus (M. abscessus or MABS) is a non-tuberculous mycobacterium and the major pathogen of the group of rapidly growing mycobacteria [1]. It is rod-shaped and reported to be 1.0-2.5 μm long and 0.5 μm wide [2]. The clinical spectrum of MABS has been well-described and broadly categorized as pulmonary and extrapulmonary disease [3]. MABS is also known to cause an extrapulmonary infection of the skin. Dissemination in immunosuppressed patients and direct inoculation are two possible causes of extrapulmonary infections [4]. There exist two clinically relevant morphotypes of MABS— Smooth(S) and Rough(R). It has been previously reported that while the rough variant can persist in the lungs of severe-combined-immunodeficient mice, can replicate in macrophages intracellularly, and forms cord-shaped, invasive microcolonies in fibroblast monolayers, the smooth morphotype demonstrates none of these characteristics [5].

Smooth (left) versus Rough MABS Colony, notice cording in the Rough morphotype | ©ankitkumar ©UPKIN, EPFL

The primary objective of this project was to study the interactions between M.abscessus and Macrophage, and perform timelapse study of the growth rate of intracellular MABS (S&R) with and without Interferon-gamma pre-activation. Although there were significant differences between intracellular and extracellular growth rates (see whisker plot below), there weren’t any observable differences between the smooth and rough morphotypes. Furthermore, the median intracellular growth rate decreased atleast by a factor of 1.5 after 24 hours of MABS intracellular residence, in all conditions, which shows that macrophages need some time to fully engage their defences.

Growth rates of intracellular MABS under different conditions. Note: Each dot represents intracellular bacteria in a single macrophage. | ©ankitkumar ©UPKIN, EPFL

Furthermore, Macrophage Extracellular Traps (METs) were found to be released by infection with Smooth MABS morphotype, with the traps being denser and more prominent when macrophages were pre-activated with IFn-gamma (see below).

Macrophage Extracellular Traps seen in green (Sytox Green nucleic acid stain) around the nucleus (center), and MABS seen in Orange (td-Tomato). Imaged using Confocal | ©ankitkumar ©UPKIN, EPFL

This work was done in McKinney Lab, EPFL School of Life Sciences, Switzerland under the supervision of Prof. John McKinney and Dr. Chiara Toniolo.

References

  1. David E. Griffith, William M. Girard, and Richard J. Wallace. Clinical features of
    pulmonary disease caused by rapidly growing mycobacteria: An analysis of 154 patients.
    American Review of Respiratory Disease, 147(5):1271–1278, 1993. PMID: 8484642.
  2. Shinji Kusunoki and Takayuki Ezaki. Proposal of mycobacterium peregrinum , and elevation of mycobacterium chelonae subsp. abscessus (kubica et al.) to species status: Mycobacterium abscessus comb. nov. International Journal of Systematic and Evolutionary Microbiology, 42(2):240–245, 1992.
  3. Howard, S. T., Byrd, T. F., and Lyons, C. R. (2002). A polymorphic region in Mycobacterium abscessus contains a novel insertion sequence element. Microbiology 148.
  4. Scholze, A., Loddenkemper, C., Grunbaum, M., Moosmayer, I., Offermann, G., and
    Tepel, M. (2005). Cutaneous Mycobacterium abscessus infection after kidney
    transplantation. Nephrol. Dial. Transplant. 20, 1764–1765.
  5. Byrd, T. F., and Lyons, C. R. (1999). Preliminary characterization of a Mycobacterium
    abscessus mutant in human and murine models of infection. Infect. Immun. 67.