Persistence of Mycobacterium ulcerans

in Acanthamoeba

Mycobacterium ulcerans is an environmental mycobacterium andenvironmental mycobacterium and the cause of a necrotizing skin disease in humans and other animals called Buruli ulcer.animals called Buruli ulcer.It is associated with fish, frogs, insects and plants.The natural host and reservoir ofThe natural host and reservoir of this M. ulcerans is unknown.

• Acanthamoeba are free-living amoeba that normally livein soil and water

• They can infect humans– infection after swimming in contaminated water– infection after swimming in contaminated water– can cause severe damage to the eye

A th b t h t f i t ll l• Acanthamoeba can act as a host for some intracellularbacterial pathogens.

– Legionella, Chlamydia,g , y ,– Vibrio cholera, Listeria, Aeromonas,– Edwardsiella, Francisella,– Escherichia coli O157:H7 and Mycobacterium spEscherichia coli O157:H7, and Mycobacterium sp,such as M.marinum.

Wh t b t M l ?• What about M. ulcerans ?

Mycobacterium ulcerans project

Environmental isolationCollection of samples f diff t

ATCCIsolated strains of

M. ulcerans,M. marinum

M tifrom different sources ponds, puddles and possum feces.

M. smegmatis

Isolate and separate

Cultivation and transformation with RFP andIsolate and separate

Amoeba, protozoa, worms, etc…

with RFP and GFP protein

Incubation with Fixation Extractionlaboratory grown Acanthamoeba(A. Iugdunensis) at RT and 30°c

Fixation of isolated protozoa on the lid d

Extraction and purification of DNA for PCR using diff tslide and

IFA test different methods.

Fluorescent Confocal and

Lysing protozoa and cultivation of

Specific PCR reaction for detection of M.u

Flowcytometry

andElectron microscopy

cultivation of remaining material and colony count of M. ulcerans or

f i Q

Results and analysis

performing Q-PCR.

Publication of article

Fluorescence and confocal microscopy

• Centrifuged 1 ml of mixture of bacteria and amoeba at 300 400 g for 10 minutes after 2hrs 24hrs 48hrs 72300-400 g for 10 minutes after 2hrs, 24hrs, 48hrs, 72 hrs,1, 2, 3 and 4 weeks of mixing.

Fl t d f l i i ti f• Fluorescent and confocal microscopy examination of the pellet to monitor the fate of bacteria inside amoeba.

M d f h ll ll d fi d h lid• Made smears of the cell pellet and fixed them on slides using alcohol/ acetone.

• Detected bacteria inside amoeba using a specific monoclonal Ab’s against the small 18kDa Hsp of M. ulcerans conjugated with fluorescein.

M. ulcerans RFP within Acanthamoeba sp.

Cyst after two weeks Trophozoite after72 hrs

M. ulcerans RFP within Acanthamoeba sp. Also stained with anti-18kDa small Hsp monoclonal Ab after 24hrs.

A D

B E

C F

M. Marinum GFP within the cyst of Acanthamoeba sp. After Two weeks.

M. Ulcerans (parkin) GFP within Acanthamoeba sp.

A B C DA B C D

I

II

IIIIII

48h 72h4h 48h 72h 7 days cyst

M.ulcerans GFP inside acanthamoeba cyst after 22 daysM.ulcerans GFP inside acanthamoeba cyst after 22 days

Confocal microscopy image of M. ulcerans GFP within Acanthamoeba after 24 hrs.

iFlowcytometry experiment1. 1 ml of bacterial and amoeba mixture centrifuged

at 300-400 g for 10 minutes for each time points.

2. Supernatant discarded and FACS solution added to the sediment and washed 3 times.

3. Final 1 ml solution transferred and examined by Flowcytometer.

4. Non fluorescent solution of the same bacteria prepared and used at the same conditions with same number of amoeba along with fluorescent same number of amoeba along with fluorescent bacterial exp. (control)

M1

M. smegmatis non GFP and GFP histogram inside amoeba after 48 hrs

Fluorescence levels of M.ulcerans (parkin)in Acanthamoeba over time

600

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ence 500

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fluor

esce

400

Incr

ease

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Hours

3 24 48 72 168

336

504

672

0

Fluorescence levels of M.ulcerans (malaysian)in Acanthamoeba over time

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ence 500

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e flu

ores

ce

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Incr

ease

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Hours

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Fluorescence levels of M.ulcerans 30 degrees (parkin)in Acanthamoeba over time

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ence 500

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fluor

esce

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Incr

ease

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F lu o r e s c e n c e le v e ls o f M .s m e g m a t is inA c a n t h a m o e b a o v e r t im e

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1 0

scen

ce

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e Fl

uore

s

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Incr

ease

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H o u r s

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Fluorescence levels of M. marinumin acanthamoeba over time (exp 1)

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100

in acanthamoeba over time (exp.1)

cenc

e

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90ed

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ores

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eas

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0 50 100 150 200 250 300 350 400 450 500 550 600 6500

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Hours

Q-PCR to determine the number of Q PCR to determine the number of M. ulcerans within Acanthamoeba

1. 1ml of cell suspensions from co-culture withdrawn at each time points.

2. Acanthamoeba lysed with 0.5 % sodium dodecyl sulfate (SDS) 2. Acanthamoeba lysed with 0.5 % sodium dodecyl sulfate (SDS) in PBS.

3. The lysate passed through a 27-gauge needle several times to insure breakage of the amoeba.insure breakage of the amoeba.

4. The lysate was centrifuged at 5000 g for 5 minutes; the pellet of bacteria was resuspended in 500 μl of PBS.

l 2 l f i d f Q C5. For M. ulcerans 2 µl of suspension used for Q-PCR.6. For M. smegmatis and M.marinum dilutions were plated on

7H10 agar containing 25µg/ml kanamycin to determine CFU.

M. ulcerans persistence within Acanthamoeba sp.

Q-PCR results for M. ulcerans in Acanthamoeba sp

106

alen

ts 105

ome

equ

iva

30oC22oC

104

103

10

Gen

o

102

1

10

0 100 200 300 400 500 600 700 800

Time (hrs)

Fluorescence levels of M.smegmatis inAcanthamoeba over time M smegmatis colony countAcanthamoeba over time

9

10

M.smegmatis colony count

ria 4.0

4.5

5.0

Fluo

resc

ence

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mbe

r of

bac

ter

104 )

2.5

3.0

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4.0

Incr

ease

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5

Prob

able

num ( X

1.0

1.5

2.0

Hours

3 24 48 72 168

336

504

672

1

2

Hours

3 24 48 72 168

336

504

672

P0.0

0.5

Hours Hours

Colony count of M.marinum in Acanthamoeba (Exp.2)

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1 2

mbe

r

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roba

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(x 1

05 )

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Mos

t pr

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0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 40

1

D a y s

Electron microscopyElectron microscopy1 1 l f ll i f lt ithd t diff t 1. 1ml of cell suspensions from co-culture withdrawn at different

time points and centrifuged at 300-400g for 10 min.2. Each pellet of infected amoeba was fixed with 2.5 % glutar

ld h d i 0 1 M di d l t b ff ( H 7 3) f 2h t aldehyde in 0.1 M sodium cacodylate buffer ( pH 7.3) for 2h at room temperature.

3. The samples were then washed three times in the same buffer d fi d i 1 % d d i t t id f 1 h and fixed in 1 % reduced osmium tetroxide for 1 h.

4. The specimens were centrifuged into pellets, dehydrated in graded ethanol (30-100 %) and embedded in an Epon-Araldite

i t mixture. 5. Ultra thin sections were stained with uranyl acetate and lead

citrate and examined with a transmission electron microscope

Uninfected Acanthamoeba

Infected Amoeba 3hInfected Amoeba 3h

Infected 24h

Infected 48h

Uninfected cyst Infected cyst

C t fi th i l f PCR to confirm the survival of M.ulcerans inside amoeba

1. Transferred the cysts of Acanthamoeba after mixing experiment on NNA plates spread by E.coli.

2. Washed the surface of agar and separated the vegetative amoeba after 3-4 days.

3 E t t d th i DNA d f d 3. Extracted the genomic DNA and performed PCR to detect M.ulcerans.

KS III KRA

Unanswered questions, need further Unanswered questions, need further investigation.

1 Is the same phenomena happening in the nature?1. Is the same phenomena happening in the nature?2. Are there any other protozoa acting same as

Acanthamoeba?3. What are the mechanisms involved in resistance

of these kind of protozoa against bacterial toxin and visa versa?

4. Finding these resistant factors may help us to find possible drug or vaccine to cure the disease without using invasive methods. without using invasive methods.

5. Can intra-amoebal cycle increase the pathogenicity of M.ulcerans?