Figure 1.
Amoebae were cultured for 16 hr at 32°C at an M.O.I. of 10 with live fluorescently labeled (PKH26) M leprae freshly isolated from mouse footpads.
Cocultures were washed with 1/10 PYG and centrifuged at slow speed 3X to remove free M. leprae. Samples were treated with 1% paraformaldehyde in PBS (pH = 7.00) for 15 prior to mounting on slides. Microscopy slides were acid-fast stained and mounted with coverslips using a DAPI-containing medium to observe amoebae nuclei. Samples were viewed under visible light (panels A, B, and C) or TRITC-filtered fluorescent light to reveal fluorescent M. leprae within amoebae: A. castellanii (panel D), A. lenticulata (panel E) and A. polyphaga (panel F). Arrows indicate enveloped acid-fast bacilli (panels A, B and C) or fluorescent M. leprae and/or amoebae nuclei (panels D, E and F).
Figure 2.
Confocal microscopy (600X) of fluorescent (PKH26) stained live M. leprae phagocytosed by A. polyphaga.
Nine successive focal planes (A–I) from top through the bottom of the amoebae were imaged showing the bacillus to be located internally rather than simply being adsorbed on the protozoan exterior.
Figure 3.
M. leprae bacilli co-localizes with acid-rich regions of amoebae cytoplasm.
Axenic cultures of A. castellanii and A. polyphaga were infected with fresh viable M. leprae from nu/nu mouse footpads for 16 hr at 32°C in 1/10 PYG followed by pulsing with 100 µM Lysotracker Green DND for 2 hours.
Figure 4.
Axenic cultures of A. castellanii, and A. polyphaga, were infected with M. leprae isolated from either nu/nu footpads or armadillo tissue at various M.O.I. (1∶100 (black diamonds), 1∶50 (black inverted triangles), 1∶10 (black upright triangles), 1∶5 (black squares) and 1∶1 (black circles) [amoebae:M.leprae]) in 1/10 PYG at either 32°C or 4°C.
Aliquots were taken at the time of infection and each hr after 2 hr of incubation and analyzed by flow cytometry. Prior to flow cytometric analysis the aliquots were washed with 1/10 PYG and centrifuged 3X at 600×g to pellet and remove any cell-free bacilli. Samples were analyzed by flow cytometry and the mean fluorescence intensity (M.F.I.) was plotted per unit time (hr) in culture.
Figure 5.
Auramine/rhodamine staining of 6 month cocultures of M. leprae and amoebae that were induced to encyst and remained at 32°C under encystment conditions.
Arrows indicate acid-fast+ staining peripherally in amoebic cysts.
Figure 6.
Bacilli extracted from 45 day or 8-month cocultures of amoebal cysts and maintained membrane integrity by preferentially staining with Syto9 (green) and excluding propidium iodide (red) staining in the BacLight LIVE/DEAD assay system.
Trophozoites were infected with viable M. leprae at an M.O.I. of 5. 48 hours in 1/10 PYG, the cultures were washed and placed in a minimal encystment medium to form cysts. Cultures were maintained at 32°C in a humidified incubator for 45 days (Panels i–vi) or 8 months (Panels v–vii) then transferred to nutrient medium (PYG) for 48 hr and the bacilli from emergent trophozoites were extracted with 0.5% SDS. Samples were examined by fluorescence microscopy. Panel i, M. leprae cultured axenically for 2 weeks, Panel ii, bacilli extracted from 45-day cultures with A. lenticulata; Panel iii, A. castellanii; Panel iv, A. polyphaga; Panel iv. Panels v–vii, bacilli extracted from 8-month cocultures of A. lenticulata, v, A. castellanii, vi and A. polyphaga, vii.
Figure 7.
Acid-fast bacilli (arrows) are evident in 35-day old cyst cocultures that were induced to excyst as trophozoites.
Cultures of axenic amoebae or M. leprae-infected amoebae were infected at an M.O.I. of 5 and incubated for 33 days in encystment buffer followed by 48 hr induction to excyst in 1 X PYG for Acanthamoeba or modified PYNFH (ATCC medium 1034) medium for Hartmannella. Panels A, uninfected A. lenticulata; B, infected A. lenticulata; C, uninfected A. castellanii; D, infected A. castellanii; E, uninfected A. polyphaga; F, infected A. polyphaga; G, uninfected H. vermiformis str. ATCC; H, infected H. vermiformis str. ATCC 50237; I, uninfected H. vermiformis str. 172 and J, infected H. vermiformis str. 172. Slides were prepared for fluorescence microscopy as described and stained with DAPI for amoebae nuclei, with auramine/rhodamine for acid-fast bacilli (orange) and with Lysotracker Green-DND-26 for visualization of acid-rich cytoplasmic regions of amoebae (green).
Figure 8.
The 129 bp M. leprae-specific RLEP sequence is amplified from nucleic acid extracted from both M. leprae and 35 day cocultures of M. leprae + A. lenticulata, A. castellanii. A. polyphaga or H. vermiformis str 172.
The amplicon was not apparent from 35-day cultures of axenic M. leprae or uninfected cultures.
Figure 9.
Mice were challenged in the footpad with 107 viable M. leprae from nu/nu mouse FPs (Panel A, 1), or bacilli extracted from 35 day cultures of M. leprae alone in amoebae medium at 32°C (Panel A, 2), bacilli extracted from>30 day cultures with M. leprae +A. castellanii cysts at 32°C (Panel A, 3) and bacilli extracted from>30 day cultures with A. polyphaga cysts for 32°C (Panel A, 4) circled R denotes right unchallenged footpad; circled L denotes the left challenged footpad.
Two investigators in a double blind fashion measured footpads and the average thickness was determined and plotted on the graph. The timeframe used to follow the appearance of FP lesions from mice injected with coculture-extracted bacilli was based upon the time after the first injection. Inset pictures in the graphs are photographs of selected left footpads from each experimental group. Panel B shows photomicrographs of material obtained by FNA biopsy of the lesions. Samples were stained with the mycobacteria-specific, auramine/rhodamine and examined under a confocal microscope. Red staining bacilli are indicative of acid-fast organisms. Significant amounts of red-staining bacilli were observed in biopsies derived from the positive control samples (Panel B, i) as well as both amoebae cocultures (Panel B; iii and iv). No acid-fast bacilli were observed in biopsies from footpads challenged with M. leprae cultured alone without amoebae (Panel B, ii). Blue staining is DAPI+ nuclei fragments from mouse mononuclear cells.
Figure 10.
Tissue extracted from nu/nu mouse footpads by FNA 7 months after challenge either with 107 bacilli from passage mice (“107 M.leprae”) or extracted from of 35 day cocultures of M. leprae with either A. castellanii or A. polyphaga indicate 80% (4 out of 5) positive PCR signals for the M. leprae-specific repetitive element (RLEP).
The M. leprae specific amplicon was not detected in FNA material from mice challenged with M. leprae kept in amoebae medium alone.
Figure 11.
Comparative enumeration and quantification of M. leprae in mouse FPs that were either challenged with M. leprae collected from 35 day culture medium alone (PYG) or M. leprae extracted from 35 day cocultures with A. castellanii or A. polyphaga.
There was a 3–3.5 log increase in the number of bacilli recovered from FPs challenged with M. leprae extracted from amoebae coculture when compared to the amount detected from FPs challenged with M. leprae maintained in amoebae medium alone for 35 days. Error bars indicate the Mean and Standard Error Measurements (SEM) of 5 mice per group.