Long-term Survival and Virulence of Mycobacterium

leprae in Amoebal CystsWilliam 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

Overview

• Background

• M. leprae and you

• Leprosy

• Immunity and pathogenesis

• Free-living Amoeba

• BRIEF Look at methods

• Figures

Some Terms Before We Begin

• Granuloma

• Cyst

• Schwann cells

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

Leprosy

• Tuberculoid and Lepromatous types

• Very old disease, strong social stigma

• Treatment has come a long way

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

Immune Response to M. leprae

• Tuberculoid leprosy

• Restricted growth

• Th1 cells and type 1 cytokines (IL-2 and IFN- ) predominate 𝛄in 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

Schwann Cell Reprogramming

Schwann Cell Reprogramming

Reprogramming adult Schwann cells to stem cell-like cells by

leprosy bacilli promotes dissemination of infection.

Masaki et al

Leprosy Persistence

• Considered major health problem until the 90’s

• Improved control but transmission still occurs

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

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

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.

Figure 1- Phagocytosis?

Figure 1 - Yes, Phagocytosis

Conclusion: FLAs can phagocytose M. leprae… probably

Figure 2 - Intracellular?

Figure 2 - Yes, Intracellular

M.l. actually made it into the cell

Figure 3 - Where in the Cell?

Figure 3 - In the Lysosome

Similar to DCs, Schwann cells and macrophages, M.l. were localized in acid-rich lysosomal regions

Figure 4 - Viability and Temperature

Figure 4 - Viability and Temperature

Uptake of M.l. is best at physiological temperatures with viable bacteria

Figure 5 - Per-cyst-ence

Figure 5 - Per-cyst-ence

M.l. remain intact (with

rounder morphology) in

cysts for at least 6 months post

encystment

Figure 6 - I Will Survive

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.

Figure 8 - M. leprae RLEP

Figure 8 - M. leprae RLEP

Strong amplicon signal recovered from co-cultured amoeba. M.l.

genome was degraded on its own.

Figure 7 - What about the children?

Figure 7 - What about the children?

M.l. were in the lysosomal regions of trophozoites of spores that were allowed to excyst and reproduce

Figure 9 - Footpad Infection

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.

Figure 10 - But was it leprae?

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

Figure 11 - Replication Ability

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.

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

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

Thanks for Listening

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