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Long-term Survival and Virulence of Mycobacterium leprae in Amoebal Cysts
William H. Wheat, Amy L. Casali, Vincent Thomas, John S. Spencer, Ramanuj Lahiri, Diana L. Williams, Gerald E. McDonnell, Mercedes Gonzalez-Juarrero, Patrick J. Brennan, Mary Jackson
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Overview Background M. leprae and you Leprosy
Immunity and pathogenesis Free-living Amoeba BRIEF Look at methods Figures
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Some Terms Before We Begin
Granuloma Cyst Schwann cells
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Mycobacterium leprae Causative agent of leprosy
Very hard to study in lab Intracellular LONG doubling time Growth in vivo in animals Closely related to M. tuberculosis Large amount of pseudoogenes Unusual cell wall
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Leprosy Tuberculoid and Lepromatous types
Very old disease, strong social stigma Treatment has come a long way
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Immune Response to M. leprae
Cell-mediated immunity TLR2 mediates early innate response to M. leprae Nerve damage likely occurs via activation of TLR2 with the N- terminal lipopeptide from M. leprae TLR2 mutations interfere with TNF-α production NF-κB cannot be activated
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Immune Response to M. leprae
Tuberculoid leprosy Restricted growth Th1 cells and type 1 cytokines (IL-2 and IFN-𝛄) predominate in lesions Granulomatous leprosy Pathogen growth not contained; poor granuloma formation Th2 cells and type 2 cytokines (IL-4 and IL-10) IL-10 polymorphisms associated with resistance to M. leprae IL-12 is key to eliciting proper response
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Schwann Cell Reprogramming
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Schwann Cell Reprogramming
Reprogramming adult Schwann cells to stem cell-like cells by leprosy bacilli promotes dissemination of infection. Masaki et al
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Leprosy Persistence Considered major health problem until the 90’s
Improved control but transmission still occurs
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Free-living Amoeba Acanthamoeba can cross the blood-brain barrier
Usually only opportunistic pathogens Ubiquitous Acanthamoeba has been isolated from soil (fresh/brackish), sea water, sewage, swimming pools, contact lens equipment, dialysis machines, heating systems, ventilating systems, air conditioning systems, mammalian cell cultures, vegetables, human nostrils and throats, and human and animal brain, skin, and lung tissues >50% human exposure
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Hypothesis M. leprae is able to use free-living amoebas (FLAs) to be transmitted between hosts FLAs can help M. leprae enter unnoticed into the mammalian host immune system M. leprae can evolve and acquire survival traits from FLAs, using them as practice for our macrophages, DCs and Schwann cells FLAs (especially those that are encysted) can protect M. leprae from challenges both in the external environment and the immune system
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General Methods Bacteria grown in footpads of immunocompromised mice and in southwest armadillos 5 different strains of MLAs (3 Acanthamoeba, 2 Hartmannella) Immunohistochemical staining, PCR, flow cytometry etc.
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Figure 1- Phagocytosis?
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Figure 1 - Yes, Phagocytosis
Conclusion: FLAs can phagocytose M. leprae… probably
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Figure 2 - Intracellular?
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Figure 2 - Yes, Intracellular
M.l. actually made it into the cell
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Figure 3 - Where in the Cell?
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Figure 3 - In the Lysosome
Similar to DCs, Schwann cells and macrophages, M.l. were localized in acid-rich lysosomal regions
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Figure 4 - Viability and Temperature
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Figure 4 - Viability and Temperature
Uptake of M.l. is best at physiological temperatures with viable bacteria
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Figure 5 - Per-cyst-ence
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Figure 5 - Per-cyst-ence
M.l. remain intact (with rounder morphology) in cysts for at least 6 months post encystment
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Figure 6 - I Will Survive
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Figure 6 - I Will Survive M.l. stays viable for at least 8 months in cysts, with little decrease in viability. It fares poorly on its own.
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Figure 8 - M. leprae RLEP
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Figure 8 - M. leprae RLEP Strong amplicon signal recovered from co-cultured amoeba. M.l. genome was degraded on its own.
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Figure 7 - What about the children?
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Figure 7 - What about the children?
M.l. were in the lysosomal regions of trophozoites of spores that were allowed to excyst and reproduce
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Figure 9 - Footpad Infection
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Figure 9 - Footpad Infection
Co-culture extracted M.l. were infectious. They produce leprosy symptoms and were present in tissue. Those cultured alone were not infectious.
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Figure 10 - But was it leprae?
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Figure 10 - But was it leprae?
RLEP amplicon was present in footpads infected with viable cells and co-cultured cells, but not in footpads from uninfected mice or mice infected with M.l. cultured alone or
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Figure 11 - Replication Ability
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Figure 11 - Replication Ability
The number of M.l. recovered per footpad is greater when using bacteria derived from protozoan co-cultures. These bacteria were able to reproduce in the host.
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Big Takeaways M. leprae can enter FLAs by phagocytosis, and reside in acid-rich lysosomal regions of the cytoplasm M. leprae survives much better in FLAs than on its own (extracellularly), and seems to stay viable for up to 8 months in encysted amoebas M. leprae gets passed to trophozoite offspring when FLAs multiply M. leprae that resides in FLAs is more infectious and replicative than M. leprae residing in cultures in a mouse footpad model
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Other Observations M. leprae is an admirable foe of the human immune system, and a generally frightening bacteria whose long-term survival and FLA manipulation should give pause, while producing a tinge of panic and admiration It would have been nice to see them inject the FLAs themselves into the mice to see how/if M. leprae stay viable, reproductive, and pathogenic
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