Lepr Rev (2008) 79, 401– 409
The role of free-living pathogenic amoeba in the
transmission of leprosy: a proof of principle
RAMANUJ LAHIRI & JAMES L. KRAHENBUHL
Laboratory Research Branch, National Hansen’s Disease Programs,
Health Resources Service Administration, Baton Rouge, LA 70803
USA
Accepted for publication 28 August 2008
Summary
Objectives Leprosy transmission remains poorly understood, though, prolonged
skin contact and/or infection via nasal mucosa, are considered likely. Problematic in
any transmission hypothesis is the fastidious nature of Mycobacterium leprae outside
its host cell and the requirement for temporary survival in the environment, soil or
water. Experiments were carried out to test the hypothesis that free living pathogenic
amoeba might serve as host cells for M. leprae, protecting them from adverse
environmental conditions.
Design In this study we employed cultures of Acanthamoeba castellanii, a freeliving pathogenic soil amoeba, to determine whether these protozoa can ingest
M. leprae and whether the intracellular bacilli remain viable.
Results More than 90% of cultured amoeba ingested M. leprae at a 20:1 multiplicity
of infection while the infected amoebae thrived and multiplied normally. The
ingested M. leprae were not degraded and remained viable for at least 72 hours as
determined by their metabolic activity (radiorespirometry) and cell wall integrity
(viability staining). M. leprae isolated from infected amoebae multiplied at the same
rate as freshly harvested bacilli in the foot pads of nu/nu mice.
Conclusions These findings provide proof of principle that free-living pathogenic
amoebae are capable of ingesting and supporting the viability of M. leprae expelled
into the environment. Studies are underway to determine whether M. leprae-infected
A. castellanii and other pathogenic amoebae may also play a role in transporting
leprosy bacilli through broken skin or the nasal mucosa.
Introduction
The routes, methods and mechanisms of transmission of leprosy are poorly understood.
However, some of the pieces of the leprosy transmission puzzle have been partially defined
and support reasonable hypotheses. For example, it seems likely that the enormous numbers
of leprosy bacilli expelled into the environment in the nasal discharges of lepromatous
Correspondence to: James L. Krahenbuhl, PhD, Director, National Hansen’s Disease Programs, 1770 Physicians
Park Dr., Baton Rouge, LA 70816, USA (Tel: 225-756-3776; Fax: 225-578-9856; e-mail: [email protected])
0305-7518/08/064053+09 $1.00
q Lepra
401
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R. Lahiri and J. L. Krahenbuhl
patients1 is one source of infection and Desikan has demonstrated detectable viability of
M. leprae in nasal discharges.2 There is also evidence to support excretion of bacilli from skin
lesions.3 Other experimental evidence supports the entry of bacilli into a new host via one of
two (or both) portals; invasion and subsequent infection though the nasal mucosa4 or abraded
or punctured skin.5
Regardless of the hypothetical route of transmission, there remains an enigma. How does
M. leprae, an obligate intracellular pathogen that is so fastidious, it cannot be cultured after
130 þ years of effort, remain viable and infectious in a rather inhospitable environmental
niche between hosts? In the present hypothesis we explored whether M. leprae could be taken
up by free-living pathogenic amoebae in the soil or water and survive, sheltered
intracellularly in these protozoa serving, essentially as ‘feral macrophages’.
There is ample precedence for this hypothesis. A number of studies report that
microorganisms can survive endosymbiotically in free-living pathogenic amoebae.6 In 1980
an association between Acanthamoeba and Legionella pneumophila7 was reported by
Rowbotham who suggested that infected amoebae were the source of Legionaires Disease.
Subsequently L. pneumophila was shown to resist phagosome-lysosome fusion and multiplies
in A. castellanii.8 A number of other organisms have been shown to be endosymbionts in
Acanthamoeba spp. including Pseudomonas,9 Chlamydia,10 Burkholderia11 and Listeria
monocytogenes.12 Several reports describe infection of Acanthamoebae with environmental13
or pathogenic mycobacteria such as M. avium-intracellularae,14,15 Mycobacterium
paratuberculosis,16 M. bovis and BCG.17
Infection of amoebae with M. leprae was suggested by Jadin in 1975 who demonstrated
the uptake of M. leprae by Acanthamoeba castellanii18 and by Grange et al.19 in 1987. Our
findings extend these reports by exploiting our routine access to highly viable and purified
nu/nu-derived M. leprae20,21 coupled with in vitro and in vivo assays for M. leprae viability.
We pose the question of whether the leprosy bacillus survives intracellularly in A. castellani.
Materials and Methods
A. CASTELLANII CULTURE
Frozen Acanthamoeba castellanii (Neff) culture was obtained from ATCC (Cat # 30010) and
maintained in T75 flasks (Corning) by twice weekly passage at room temperature (26 8C) in
ATCC 712 medium (www.atcc.org product information sheet 30010). A. castellanii was also
maintained in culture on a non-nutrient agar surface coated with killed Escherichia coli as
described by Neff.22 Briefly, 1·0 ml (1 £ 109) irradiated E. coli (106 Rad [Sheppard Model
484 60Co irradiator]) was spread evenly on 80 mm Petri plates of non-nutrient agar (Difco).
An A. castellanii suspension (5 £ 106 amoebae in 0·1 ml) was then spotted at the centre of
the plate and the cultures incubated at 33 8C. Over a 24 –48 hour period the amoebae ingested
the E. coli, spread radially and multiplied.
NUDE MOUSE DERIVED M. LEPRAE
The Thai-53 isolate of Mycobacterium leprae was maintained in the foot pads of athymic
nu/nu mice infected for 4 –6 months, and then harvested as described previously,20 washed by
centrifugation, resuspended in RPMI-1640 (Gibco) þ 10% (v/v) fetal calf serum [(FCS)
Gibco] and enumerated by direct count according to Shepard’s method.23 M. leprae
Pathogenic amoeba in the transmission of leprosy
403
suspensions were purified by NaOH treatment as described previously.24 Briefly, 1 £ 109
fresh M. leprae were suspended in 1·0 ml of 0·1N NaOH (Sigma) and incubated for 3 minutes
at room temperature, after which the bacteria were washed three times in Hanks balanced salt
solution and finally resuspended in the appropriate media. Freshly harvested viable bacilli
were always employed in experiments within 24 hours of harvest.
STAINING OF M. LEPRAE WITH THE VITAL RED STAIN PKH26
For confocal microscopy studies, freshly harvested and 0·1N NaOH treated M. leprae were
stained with fluorescent red PKH26 dye (Sigma) following a published protocol.24 Briefly,
M. leprae were stained for 2 minutes at room temperature with a 1:250 dilution of PKH26
dye. The suspension was washed three times in appropriate medium. The numbers of bacteria
were recounted following staining by Shepard’s direct count method.23
INFECTION OF A. CASTELLANII WITH LIVE M. LEPRAE
Monolayers of A. castellanii cultures in T75 flasks (Corning) containing 15 ml of ATCC 712
media were infected with viable M. leprae at an MOI of 20:1 and incubated overnight at
26 8C. Extracellular M. leprae were removed by decanting the media and washing 3 times
with phosphate buffered saline (PBS). For some experiments infected amoebae were
removed by vigorous shaking after chilling the cultures at 4 8C. For other experiments
intracellular M. leprae were released from amoebae by lysis with 0·1N NaOH (Sigma) at 24,
48, 72 and 96 hour post infection, and the M. leprae processed for radiorespirometry, viability
staining and inoculation into nu/nu mouse foot pads.
STAINING OF INFECTED A. CASTELLANII TROPHOZOITES
Briefly, A. castellanii cultures were stained with carbol fucshin for 20 minutes at room
temperature. After washing with acid alcohol the slides were counter stained with methyl
green.
CONFOCAL MICROSCOPY
A Leica SP2 confocal microscope was used to ascertain internalisation of live M. leprae by
A. castellanii trophozoites. Serial optical sections of the infected amoeba were taken at 0·2 nm
using 514 nm excitation laser and 560 ^ 20 nm emission filters.
RADIORESPIROMETRY (RR)
Metabolism of suspensions of control and amoeba-derived M. leprae was measured by
evaluating the oxidation of 14C-palmitic acid to 14CO2 by RR as described previously.25
Briefly, 1 £ 107 M. leprae were suspended in 4·0 ml of acidified Middlebrook 7H12
BACTEC PZA media (Becton Dickinson) in a 5 ml glass vial with loosened cap which, in
turn was inserted into a wide mouth liquid scintillation vial lined with filter paper
impregnated with NaOH, 2,5-diphenyloxazole (Sigma) and Concentrate I (Kodak). When
read daily, captured 14CO2, determines the rate of 14C-palmitic acid oxidation. In the present
study the 7th day cumulative counts per minute (CPM) are reported. This measure of
404
R. Lahiri and J. L. Krahenbuhl
metabolic activity by suspensions of M. leprae correlates highly with viability as
demonstrated by growth in the mouse foot pad.20
FLUORESCENT VIABILITY STAINING (VS) OF M. LEPRAE
The membrane integrity of amoeba-derived M. leprae was evaluated with a LIVE/DEAD
BacLight Bacterial Viability Kit w (Molecular Probes) as described previously.21 Briefly,
M. leprae were washed in normal saline and incubated for 15 minutes at room temperature
with Syto9 and propidium iodide (PI). The bacteria were washed again and resuspended
in 10% (v/v) glycerol in normal saline. The dead and live bacteria were enumerated by
direct counting of fluorescent green and red bacilli using appropriate single bandpass filters.
The excitation/emission maxima are 480 nm/500 nm for Syto9 and 490 nm/635 nm for PI.
This VS method measures the cell wall integrity of individual bacilli and correlates highly,
both with RR and growth in the mouse foot pad.21
NUDE MOUSE FOOT PAD GROWTH OF M. LEPRAE
Athymic nu/nu mice, four in each group, were inoculated on the plantar surface of both
hind feet with 5 £ 106 M. leprae harvested 72 hour post infection from A. castellanii or
control M. leprae incubated in parallel at 26 8C. At 4- and 7-months both hind foot pads were
harvested from each of two mice, processed and the number of AFB enumerated using
Shepard’s technique.23
STATISTICAL ANALYSIS
The data are shown as means ^ standard deviation (SD) from a representative of three to four
experiments. The raw data were subjected to Student’s t test to determine whether the
observed differences between the means were significant. P , 0·05 was taken as significant.
Results
UPTAKE OF M. LEPRAE BY A. CASTELLANII
As shown by acid fast staining of infected amoebae in Figure 1A and confocal microscopy
of fluorescent stained M. leprae in Figure 1B, the amoebae were able to efficiently ingest
live M. leprae after overnight incubation at a MOI of 20:1. Greater than 90% of amoebae
were infected (Figure 1A). Ingestion of live M. leprae did not show any apparent adverse
effect on the amoeba as they divided normally over several days (data not shown) and did not
form cysts.
CULTURE OF A. CASTELLANII ON A SURFACE CONSISTING OF E. COLI OR M. LEPRAE
Figure 2B shows progress of spreading A. castellanii culture at 33 8C on non-nutrient agar
coated with irradiated E. coli, while amoeba did not advance on either non-nutrient agar alone
(2A) or on non-nutrient agar coated with irradiated M. leprae (2C).
Similar findings were seen at an incubation temperature of 37 8C while the radial advance
of amoebae on the E. coli plates was markedly reduced at 26 8C (data not shown). These
Pathogenic amoeba in the transmission of leprosy
405
Figure 1. Acid fast staining showing ingestion of live M. leprae by A. castellanii (A) and confocal microscopy
(600 £ ) of fluorescent (PKH26) stained live M. leprae inside A. castellanii (B) showing that the M. leprae are indeed
internalized.
results indicate that A. castellanii was not able to thrive solely on irradiated M. leprae as it
could efficiently on irradiated E. coli.
IN VITRO TESTS FOR VIABILITY OF M. LEPRAE ISOLATED FROM INFECTED
A. CASTELLANII
Both RR and VS results show negligible loss of viability in M. leprae ingested by amoebae
even after 96 hour (RR P ¼ 0·802, VS P ¼ 0·783) (Figure 3A and 3B).
It was difficult to perform the assays beyond 96 hours due to the multiplication of the
amoebae and the consequent dilution of the infected population. In a representative
Figure 2. The migration on agar assay showing A. castellanii on non-nutrient agar alone (A) non-nutrient agar
layered over with irradiated E. coli (B) and non-nutrient agar layered over with irradiated M. leprae (C).
406
R. Lahiri and J. L. Krahenbuhl
Figure 3. Radiorespirometry (A) and viability staining (B) of in vitro ingested M. leprae obtained by lysis of
A. castellanii trophozoites at different time points after infection.
experiment where 90% of the amoebae were infected at 0 hours, 49% were infected at 24
hours, 15% at 72 hours and , 5% at 96 hours.
GROWTH IN THE NUDE MOUSE FOOT PAD OF M. LEPRAE ISOLATED FROM
A. CASTELLANII
M. leprae harvested from A. castellanii 72 hours after ingestion were used to inoculate both
hind foot pads of athymic nu/nu mice. In both groups foot pads were visibly enlarged at
4 months. At 4 and 7 months post inoculation the growth in the nude mouse foot pad of
M. leprae harvested from A. castellanii 72 hour post-infection was indistinguishable from
(P ¼ 0·894) that of the control (Figure 4).
Discussion
Our laboratory has devoted considerable effort to defining the biophysical optima of
M. leprae,20 quantifying ‘viability’ in vitro in this uncultivable organism25,21 and developing
Figure 4. Growth of M. leprae in nu/nu mouse foot pad. Growth of M. leprae derived from A. castellanii 72 hr after
ingestion is compared with growth of a control suspension of M. leprae, held at 26 8C for 72 hr.
Pathogenic amoeba in the transmission of leprosy
407
routine access (weekly) to large numbers of fresh viable M. leprae from the foot pads of
athymic (nu/nu) mice.21,24 These techniques and this valuable research resource were critical
to carrying out the present study.
We chose for the present studies Acanthamoebae castellanii as a putative amoebic ‘host
cell’ for M. leprae because these protozoa have been shown to harbour and support the
growth of a number of other bacteria, including environmental mycobacteria;13 and there is
ample precedence for Acanthamoeba being a host cell for several pathogenic mycobacteria;
M. avium-intraclllularae,14 M. paratuberculosis,16 BCG17 and M. bovis.17 Acanthamoeba
may play a role in transmission of mycobacterial infection to man.14
The present studies demonstrated the avid uptake of M. leprae by A. castellanii in a dose
dependant manner. Even with a high MOI where virtually all of the amoebae phagocytosed
M. leprae, there appeared to be no ill effect on the multiplication of the infected protozoa
(data not shown). Time course observations (data not shown) revealed that with time (24 –48
hours) individual intracellular bacilli, distributed throughout the cytoplasm at 0 hours,
appeared to be packaged by the host amoeba into a single large vacuole.
Whereas the amoebae could survive and multiply on a simple diet of killed E.coli,22
M. leprae alone did not appear to provide the necessary nutrients and were not digested or
degraded. In fact, M. leprae survived for at least 4 days as shown by their metabolic activity
(RR) and cell wall integrity (VS), in vitro assays for viability.21 More importantly, viability
after 3 days of infection of amoebae was undiminished when evaluated by inoculation into the
foot pads of athymic (nu/nu) mice. We consider these findings as proof of principle
supporting the hypothesis that A. castellanii and perhaps other soil or aquatic free-living
pathogenic amoebae might perform as ‘feral macrophages’ by facilitating the survival of the
leprosy bacillus in the environment when expelled from their human host.
An interesting corollary to this hypothesis would be the potential role of dormant
encysted amoeba in protecting M. leprae during adverse environmental conditions such as
desiccation, changes in temperature and pH.26 In preliminary experiments with M. lepraeinfected A. castellanii, where encystment was induced by raising NaCl concentration we
observed, under phase microscopy, that as the trophozoites condensed in size and formed
thickened cell walls they appeared to expel their particulate contents, including M. leprae
(unpublished observation). However, a report by Steinert et al. employed electron
microscopy to demonstrate the presence of M. avium within the outer walls of the double
walled Acanthamoeba cyst when infected trophozoites were encouraged to encyst.15
Additional studies are being carried out with M. leprae.
Finally, the demonstration that M. leprae will survive in a pathogenic free-living amoeba
will allow us to move this hypothesis forward and determine if M. leprae infected free-living
pathogenic amoebae could play a role in the actual transmission of leprosy by facilitating the
invasion of human tissue. First we will need to confirm these in vitro findings in other
members of the genus Acanthamoeba, A. culbertsoni, and A. polyphaga, both of which have
already been shown to support the growth of pathogenic mycobacteria.27 Various species of
Acanthamoeba, A. castellani, A. culbertsoni and A. polyphaga are the agents of human
diseases27 including Granulomatous Amoebic Encephalitis (GAE) a slowly progressive CNS
infection, cutaneous acanthamoebiasis (CA), and amoebic keratitis (AK). GAE and CA are
diseases seen in immunosuppressed patients while (AK) is a sight-threatening corneal disease
that can occur in immunocompetent individuals and is acquired from contact lens cases or
cleaning solutions. The route of infection for GAE and CA are not clearly understood but the
nasal passages and/or cutaneous lesions are considered likely routes.27
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R. Lahiri and J. L. Krahenbuhl
In preparation for the next (in vivo) stage in these studies we will also be concerned with
Naeglaria fowleri, a notorious human pathogen.28 N. fowleri is the causative agent of primary
amoebic meningoencephalitis, (PAM) a rare but rapidly fatal human disease.28 N. fowleri is
found in warm fresh water ponds and lakes worldwide, inadequately chlorinated swimming
pools, and moist soil.28 The portal of entry for humans appears to be the nasal mucosa with
subsequent invasion of the olfactory nerve plexus and rapid travel (24 hours) of the amoebae
up the olfactory nerves, through the cribiform plate and into olfactory bulb and spread to
other areas of the CNS.29
The crux of this second hypothesis is that the vast majority of infections with free-living
pathogenic amoebae are of no consequence; that human disease is an extremely rare outcome.
Hundreds of millions have likely been exposed to N. fowleri by diving, swimming or
splashing in infected fresh water, yet there are less than 200 recorded cases of the rapidly fatal
fulminate PAM, suggesting a high level of resistance or that infection is usually
asymptomatic.30 Mice immunised with killed N. fowleri are highly resistant to a lethal
intravenous challenge with amoebae31 and serologic studies in endemic areas show a high
prevalence of protective antibodies in humans suggesting exposure without disease.32 One
estimate of the probability of contracting PAM, once exposed to Naegleria is 1 in 100 million
exposures.30
The earliest response of the host to amoebae consists of an influx of polymorphonuclear
neutrophils to the site of infection, followed by an influx of macrophages33 which have been
shown in vitro to have a deleterious effect on target amoebae.34 This is the exact sequence of
events, culminating in uptake of M. leprae by its preferred host cell, the macrophage that
would complete the hypothesis for a role of free-living amoebae in facilitating leprosy
transmission. We intend to exploit already described mouse models for N. fowleri29,31,35 – 36
and Acanthomoeba sp.34 infection to determine if M. leprae infected amoebae transport the
bacilli through the nasal mucosa or through intact or abraded skin.
Acknowledgements
We gratefully acknowledge the excellent technical support of Baljit Randhawa and Greg
McCormick. These studies were support by a grant from The German Leprosy Relief
Association, Würzburg, Germany.
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