Delta-aminolevulinate-induced host-parasite porphyric disparity for selective photolysis of transgenic Leishmania in the phagolysosomes of mononuclear phagocytes: a potential novel platform for vaccine delivery.
ABSTRACT Leishmania double transfectants (DTs) expressing the 2nd and 3rd enzymes in the heme biosynthetic pathway were previously reported to show neogenesis of uroporphyrin I (URO) when induced with delta-aminolevulinate (ALA), the product of the 1st enzyme in the pathway. The ensuing accumulation of URO in DT promastigotes rendered them light excitable to produce reactive oxygen species (ROS), resulting in their cytolysis. Evidence is presented showing that the DTs retained wild-type infectivity to their host cells and that the intraphagolysosomal/parasitophorous vacuolar (PV) DTs remained ALA inducible for uroporphyrinogenesis/photolysis. Exposure of DT-infected cells to ALA was noted by fluorescence microscopy to result in host-parasite differential porphyrinogenesis: porphyrin fluorescence emerged first in the host cells and then in the intra-PV amastigotes. DT-infected and control cells differed qualitatively and quantitatively in their porphyrin species, consistent with the expected multi- and monoporphyrinogenic specificities of the host cells and the DTs, respectively. After ALA removal, the neogenic porphyrins were rapidly lost from the host cells but persisted as URO in the intra-PV DTs. These DTs were thus extremely light sensitive and were lysed selectively by illumination under nonstringent conditions in the relatively ROS-resistant phagolysosomes. Photolysis of the intra-PV DTs returned the distribution of major histocompatibility complex (MHC) class II molecules and the global gene expression profiles of host cells to their preinfection patterns and, when transfected with ovalbumin, released this antigen for copresentation with MHC class I molecules. These Leishmania mutants thus have considerable potential as a novel model of a universal vaccine carrier for photodynamic immunotherapy/immunoprophylaxis.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: 5-aminolevulinic acid (ALA) and carnosine have important physiological and pathophysiological roles in the CNS. Both are substrates for the proton-coupled oligopeptide transporter PEPT2. The purpose of the current study was to determine the importance of PEPT2 in the uptake of ALA and carnosine in rat and mouse (PEPT2+/+ and PEPT2-/-) cultured neonatal astrocytes. Although neonatal astrocytes are known to express PEPT2, its quantitative importance in the transport of these compounds is not known. [14C]ALA uptake in neonatal rat astrocytes was inhibited by dipeptides, an alpha-amino containing cephalosporin (which is a PEPT2 substrate) but was not affected by a non-amino containing cephalosporin (which is not a PEPT2 substrate). Uptake was pH sensitive as expected from a proton-coupled transporter and was saturable (Vmax=715+/-29 pmol/mg/min, Km=606+/-14 microM). [3H]Carnosine uptake in neonatal rat astrocytes was inhibited by dipeptides but not by histidine (a substrate for the peptide/histidine transporters PHT1 and PHT2) and also showed saturable transport (Vmax=447+/-23 pmol/mg/min, Km=43+/-5.5 microM). Neonatal astrocytes from PEPT2-/- mice had a 62% reduction in [14C]ALA uptake and a 92% reduction in [3H]carnosine uptake compared to PEPT2+/+ mice. These results demonstrate that PEPT2 is the primary transporter responsible for the astrocytic uptake of ALA and carnosine.Brain Research 12/2006; 1122(1):18-23. · 2.88 Impact Factor
- The Journal of Immunology 01/1978; 119(6):2060-6. · 5.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Endogenous porphyrin accumulation after administration of 5-aminolevulinic acid is employed in photodynamic therapy of tumours. Due to its low membrane permeability, esterified 5-aminolevulinic acid derivatives less hydrophilic than the parental compound are under investigation. Knowledge of the mechanisms of 5-aminolevulinic acid derivatives uptake into target cells is essential to understand and improve photodynamic therapy and useful in the design of new derivatives with better affinity and with higher selectivity for tumour cells in specific tissues. The aim of this work was to assess the interaction of 5-aminolevulinic acid derivatives with the intestinal PEPT1 and renal transporter PEPT2 expressed in Pichia pastoris yeasts. We found that Undecanoyl, Hexyl, Methyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters and the dendron 3m-ALA inhibited (14)C-5-aminolevulinic acid uptake by PEPT2. However, only the Undecanoyl ester inhibited 5-aminolevulinic acid uptake by PEPT1. We have also found through a new developed colorimetric method, that Hexyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters display more affinity than 5-aminolevulinic acid for PEPT2 whereas none of the compounds surpass 5-aminolevulinic acid affinity for PEPT1. In addition, the Undecanoyl ester binds with high affinity to the membranes of PEPT2 and PEPT1-expressing yeasts and to the control yeasts. The main finding of this work was that some derivatives have the potential to improve 5-aminolevulinic acid-based photodynamic therapy by increased efficiency of transport into cells expressing PEPT2 such as kidney, mammary gland, brain or lung whereas in tissues expressing exclusively PEPT1 the parent 5-aminolevulinic acid remains the compound of choice.The International Journal of Biochemistry & Cell Biology 02/2006; 38(9):1530-9. · 4.15 Impact Factor
Delta-Aminolevulinate-Induced Host-Parasite Porphyric Disparity for
Selective Photolysis of Transgenic Leishmania in the Phagolysosomes
of Mononuclear Phagocytes: a Potential Novel Platform for
Sujoy Dutta,aCelia Chang,bBala Krishna Kolli,aShigeru Sassa,cMalik Yousef,bMichael Showe,bLouise Showe,band
Department of Microbiology/Immunology, Chicago Medical School/RFUMS, North Chicago, Illinois, USAa; The Wistar Institute, Philadelphia, Pennsylvania, USAb; and The
Rockefeller University, New York, New York, USAc
copresentationwithMHCclassImolecules.These Leishmania mutantsthushaveconsiderablepotentialasanovelmodelofa
phyrins, i.e., uroporphyrins (URO), coproporphyrins (COPRO),
generate cytolytic reactive oxygen species (ROS), ALA has been
used clinically to produce endogenous porphyrins for photolytic
todynamic therapy for skin tumors (21, 40). Although ALA-in-
duced elevation of porphyrins is often low and transient in mam-
malian cells, there are several advantages to this approach instead
of direct administration of porphyrins or other photosensitizers.
ALA is a natural metabolite that participates mainly in porphyrin
biosynthesis and is nontoxic even when used at a high concentra-
tion. It is also relatively stable, highly water soluble, and readily
taken up by cells. These properties of ALA present a favorable
Heme auxotrophs, e.g., the trypanosomatid protozoa, do not
convert exogenously supplied ALA to porphyrins when the heme
biosynthetic pathway is either absent or incomplete (26, 30, 36).
Among these single-cell parasites, Leishmania spp. are function-
ally missing at least 5 and very possibly 7 of the 8 enzymes needed
to complete heme biosynthesis (9, 30). The substantial loss of this
lution of parasitism in heme-rich environments, i.e., the gut of
blood-sucking insect vectors and the phagolysosomes of mono-
nuclear phagocytes in the mammalian reticuloendothelial system
xposure of aerobic eukaryotic cells to exogenous delta-amin-
olevulinate (ALA) is known to trigger overproduction of por-
(3, 4). The Leishmania defects in heme biosynthesis have been
partially defined functionally by genetic complementation of the
extracellular promastigote stage in vitro (9, 30). Transfection of
these cells with mammalian cDNAs that express the 2nd and 3rd
response to exogenously provided ALA, the product of the 1st
enzyme in heme biosynthesis (30). The immediate product ex-
pected is porphobilinogen (PBG), which in the absence of the
downstream enzyme, i.e., uroporphyrinogen cosynthase, con-
III (31). URO I is a nonmetabolizable by-product that is water
soluble and is released without reuptake by cells, including Leish-
mania (10). When exposed to light, uroporphyric promastigotes
and other leishmanolytic ROS (10).
Received 25 August 2011 Accepted 25 January 2012
Published ahead of print 3 February 2012
Address correspondence to Kwang-Poo Chang, kwangpoo.chang
This paper is dedicated to Shigeru Sassa in memory of his contributions and
Supplemental material for this article may be found at http://ec.asm.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
ec.asm.org1535-9778/12/$12.00Eukaryotic Cellp. 430–441
rinogenesis/photolysis of Leishmania double transfectants (DTs)
from the promastigotes to their intracellular amastigotes in 2 dif-
protein, when DTs were exposed as promastigotes to 1 mM ALA
by fluorescence microscopy and chromatography revealed that,
when used to pulse-expose preinfected host cells, ALA was taken
up and made available to their intraphagolysosomal/parasito-
preferentially, thereby sensitizing them for selective photolysis.
Photolysis of the intracellular DTs freed the major histocompati-
bility complex (MHC) class II molecules of macrophages from
Leishmania sequestration in phagolysosomes, released transgeni-
cally expressed ovalbumin for MHC class I copresentation, and
largely returned the expression profiles of their host cells to the
preinfection state. The functional recovery of the host cells by
phagolysosomes underscores the potential of these mutants to
serve as effective carriers of drugs/vaccines for efficient activation
and processing in these cells.
MATERIALS AND METHODS
Cells. Leishmania amazonensis (RAT/BA/74/LV78) clone 12-1 was rou-
tinely grown as promastigotes at 25°C in Medium 199 (Sigma) buffered
with 25 mM HEPES to pH 7.4 and supplemented with 10% heat-inacti-
vated fetal bovine serum (HIFBS). The uroporphyrinogenic mutants,
which were doubly transfected with pX-alad and p6.5-pbgd (DTs), were
heme biosynthetic pathway (30). Single transfectants (STs) with only one
or the other cDNA, i.e., pX-alad?p6.5 or pX?p6.5-pbgd (30), were in-
cluded as controls where appropriate in some experiments. All mutants
were briefly grown in drug-free medium to stationary phase before being
used for infection to avoid the potential cytotoxicity of the drugs being
carried over to the host cells (9, 10).
Mouse J774 macrophage (27, 34) and FSDC (15) lines were both
grown at 35°C in RPMI 1640 supplemented with 10% HIFBS. Peritoneal
tured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) plus 5%
HIFBS at 37°C in 5% CO2. The mouse immature dendritic cell (DC) line
DC2.4 was grown in HCM medium plus 10% HIFBS in 5% CO2(11).
Leishmania infection of macrophages and dendritic cells. Leishma-
nia infection of J774 cells and FSDC was initiated by mixing suspensions
of these cells and of stationary-phase promastigotes in RPMI 1640 plus
20% HIFBS at a host-parasite ratio of 1:5 to 1:10, i.e., 106host cells and
0.5 ? 107to 1 ? 107promastigotes/ml. The mixture was plated at 4
ml/25-cm2tissue culture (TC) flask or 2 ml/well in 6-well tissue culture
plates. Infected cultures were maintained at 35°C with daily medium re-
newal. They and control cultures were prepared in multiple sets for ex-
perimentation under various conditions and taken at different time
points for analyses. The total parasite load per culture was estimated by
microscopic counting as described previously, i.e., using the following
formula: total number of macrophages per flask ? (average number of
Leishmania amastigotes/host cell) ? percent infected cells (12).
The primary macrophages were infected as a monolayer of adherent
cells with promastigotes in DMEM plus 5% HIFBS. Peritoneal cells were
first seeded at 2 ? 105cells/300 ?l/chamber in 8-chamber tissue culture
slides (Nunc). After incubation at 37°C in 5% CO2for 1 h, each chamber
before use. These macrophages were infected at a host/parasite ratio of
1:10 for 2 to 3 h at 37°C and then washed to remove uningested parasites.
The cultures were subsequently incubated at 35°C after infection and
treated with mouse gamma interferon (IFN-?) (Peprotech) at 100 U/ml
for 24 h for induction of MHC class II expression (23, 24).
Delta-aminolevulinate-induced porphyrinogenesis. Infected and
(10) daily for up to 3 days. This was done 2 to 3 days after infection was
well established, as indicated by the virtual absence of extracellular para-
sites and their appearance in the typical large parasitophorous vacuoles
(PVs) of the infected cultures. The cultures were washed and then chased
minimal light exposure until completion of the experiments.
Fluorescence microscopy and thin-layer chromatography of cellu-
lar porphyrins. Infected and noninfected cultures with and without ALA
treatment were withdrawn daily. A small aliquot from each sample was
examined first under phase-contrast microscopy and then for porphyrin
fluorescence by using a specific filter set (10). The remaining samples
(?5 ? 106macrophages each) were solvent extracted for analysis of por-
phyrins by thin-layer chromatography (TLC) using carboxymethylated
porphyrins (Porphyrin Products) as standards (9, 30).
Illumination of cultured cells. During medium renewal and micro-
scopic observations, cultures were exposed by necessity to light that was
either left unchanged, as usual for such routine operations, or minimized
by dimming the light sources and shortening the handling time. Cultures
were experimentally illuminated with white light at ?6.5 mW/cm2for
variable time periods and at porphyrin-excitable wavelengths, i.e., long-
wave UV light (?max? 366 nm), as described previously (9, 10, 30), or
are specified for individual experiments in the figure legends.
Localization of MHC class II and LAMP-1 by immunofluorescence
microscopy. Peritoneal cells cultured in 8-chamber microscope tissue
culture slides (Nunc) processed as described above were used. DT infec-
tion, ALA treatment, and light exposure of these cells, together with their
ent times up to 16 h after induction of MHC class II expression with
IFN-?. After fixation and permeabilization with Cytoperm-Cytofix re-
agent (BD Biosciences) for 15 min at 4°C, samples were treated with
anti-CD16/32 (1:100 dilution) in the presence of 5% bovine serum albu-
min (BSA) plus 5% normal rat and rabbit sera in Permwash buffer (BD
Biosciences) at 4°C for 30 min. After washing 3 times with this buffer, the
isothiocyanate (FITC)-labeled rat anti-mouse MHC class II-IgG (M5/
114.15.2; Biolegend) and rabbit anti-human LAMP-1 (6) (monoclonal
C-20 against the conserved C-terminal sequence; Santa Cruz Biotechnol-
ogy), both at a 1:100 dilution in 0.1% saponin plus 1% bovine serum
albumin in phosphate-buffered saline. After washing, the cells were fur-
at 1:1,000 for LAMP-1. The cells were finally washed and mounted in
per treatment, and the data presented are representative of the overall
MHC class I/OVA epitope copresentation by dendritic cells after
classI/ovalbumin(OVA)epitope copresentation by DCs after infection
with OVA-transfected DTs and their photolysis in situ is based on
monoclonal antibody immunodetection of the OVA-SIINFEKL
cell lines, as described previously (11). Briefly, DT promastigotes were
transfected with pX63hyg-ova, consisting of a truncated OVA (amino ac-
transfectants were selected and grown in the presence of 500 ?g/ml hy-
gromycin. OVA expression in the transfectants was verified by Western
Selective Porphyric Leishmania Photolysis in Host Cells
April 2012 Volume 11 Number 4ec.asm.org 431
blotting using anti-OVA rabbit antisera (Millipore; dilution, 1:1,000).
DT-infected DCs were assessed by immunofluorescence microscopy.
to the following materials: 100 pM SIINFEKL and 5 mg/ml OVA as re-
agent positive controls and medium alone as the negative control. OVA/
ALA ? light) treatments for uroporphyrinogenesis/photolysis were eval-
uated for comparison. Non-OVA transfectants were treated similarly
(DT ? ALA ? light) as an additional control. All cells were fixed and
permeabilized and then reacted at 4°C for 16 h with the monoclonal an-
tibody from the 25-D1.16 hybridoma culture supernatants, followed by
goat anti-mouse IgG-Alexa 488 (Molecular Probes; 1:500 dilution) to
assess the H-2Kb OVA(257–264) (SIINFEKL).
The filter sets used for image acquisition by fluorescence microscopy
were as follows: (i) DAPI, D365/10? (365-nm exciter), 400DCLP
40? (480-nm exciter), Q505LP (505-nm dichroic), and HQ 535/50 M
(535-nm emitter); (iii) Alexa 594, HQ 560/55? (560-nm exciter),
porphyrins, D405/10 (405-nm exciter), Q485DCXR (485-nm dichroic),
and RG610LP (610-nm emitter) (Chroma Tech Co., Brattleboro, VT).
a CoolSnap ES camera in conjunction with Metamorphosis image acqui-
sition software (version 6.2r6).
Microarray analyses. Microarray analysis was done at a single time
point for different experimental and control groups when intraphagoly-
the experimental and control groups (?2 ? 106J774 cells each) and
analyzed to verify its integrity using a BioAnalyzer (Agilent). Each RNA
sample (2 ?g) was then amplified using the Arcturus RNA amplification
kit to produce a stock of amplified mRNA (aRNA) (18). The aRNA (1.6
?g) was33P labeled and hybridized simultaneously to 4 arrays spotted
with a total of 38,000 mouse cDNA clones carrying ?21,000 unique
carried, in addition to the mouse cDNAs, Leishmania genes, i.e., ndk,
?-tub, nagt, and dhfr (GenBank accession numbers L1648.07, M23441,
M96635, and L11705), encoding nucleoside diphosphate kinase b, beta-
tubulin, N-acetylglucosamine-1-phosphate transferase, and dihydrofo-
late reductase, respectively. nagt and dhfr are single-copy genes, while
?-tub and ndk are multicopy genes. The expression values were deter-
mined with ImaGene software (Biodiscovery) using the median pixel for
each spot and an area adjacent to the spot for background correction.
Average normalized median densities (nMD) were calculated in the four
determined. Data were exported to Excel for further analysis (18) and
transformed into corresponding z scores for clustering in TreeView (13).
Pairwise combinations were analyzed with the t test. The false-discovery
rate as a function of the P value was estimated by permuting the quadru-
plicate values under the two conditions being tested (37). Genes with
and then filtered to include only those genes that exhibited ?2-fold
change in expression.
Real-time PCR. To verify the changes in transcription profiles, dupli-
ate cDNAs with the GoScript Reverse Transcription system (Promega).
Multiplex real-time PCR was performed with the cDNAs produced using
TaqMan MGB gene expression probes for mouse il10 (part no. 4453320;
Applied Biosystems) and the housekeeping gene gapdh (part no.
4352339E) with 6-carboxyfluorescein (FAM) and VIC dyes, respectively,
according to the manufacturer’s recommendations, using the Applied
gapdh CTvalues from those of il10, which were converted into linear
values using the formula 2??CT.
Statistical analyses. All experiments pertaining to infection and ALA
exposure/illumination for porphyrinogenesis/photolysis were repeated
with samples in duplicate for microscopic counting of parasite loads and
in triplicate or quadruplicate for the rest. Data are presented as means
with standard errors calculated from multiple samples of representative
experiments. Student’s t test and one-way analysis of variance (ANOVA)
were used to calculate the statistical significance of data, as described.
infectivity to their host cells. Both porphyrinogenic (DT) and
nonporphyrinogenic (ST) mutants were found to infect host cells
A= to C=, phase-contrast), exactly as seen with their parental wild-
type clone (5). During the subsequent period of incubation for 9
days or longer, the intra-PV DTs showed kinetics of parasite bur-
den similar to those produced by the nonporphyrinogenic mu-
tants and their parental wild type (Fig. 1D, solid and open circles
versus open square), the kinetics of the host cell replication being
comparable among the 3 infected cultures (Fig. 1D=). The trans-
gene products of the DT mutants per se thus did not alter their
infectivity to the host cells examined.
posure of DT-infected cells to ALA. (i) Porphyrin fluorescence
dissipated rapidly in host cells but persisted in intra-PV DTs.
Cells were exposed to 1 mM ALA ?3 days after infection, when
intracellular. Porphyrin fluorescence was initially seen largely in
the cytoplasm of the host cells, irrespective of their infection with
DTs or STs in the presence of ALA (Fig. 1A and B, day 1 fluores-
cence), but not in its absence (Fig. 1C, day 1 fluorescence). These
fluorescent signals thus represent the porphyrins, which the host
ible by fluorescence microscopy. Porphyrin fluorescence dimin-
undetectable level and, concomitantly, emerged at variable inten-
sities in the intra-PV DTs (Fig. 1 A=, J ? DT ? ALA fluorescence
day 3), but not in those of the negative controls, i.e., intra-PV STs
in infected and ALA-exposed cells (Fig. 1 B=, J ? ST ? ALA fluo-
rescence day 3) or DTs of the infected cells not exposed to ALA
results of these studies indicate that exposure of DT-infected cells
to ALA induces host-parasite differential porphyrinogenesis in
time and porphyrin level.
(ii) URO persisted, while COPRO and PROTO dissipated
rapidly after a transient rise in DT-infected cells. DT-infected
J774 cells and controls, e.g., noninfected cells, were exposed to
ALA. Samples were withdrawn daily during the subsequent chase
for 3 days to evaluate porphyrin changes by TLC analysis (Fig. 2).
As expected, all 3 known naturally occurring porphyrins, i.e.,
URO, COPRO, and PROTO (in the order of their appearance in
the heme biosynthetic pathway), were present in day 1 samples of
both DT-infected and noninfected host cells (Fig. 2, J ? DT ?
ALA versus J ? 0 ? ALA, lane 1). PROTO was more obvious and
consistent than COPRO, but their fluorescence intensities were
comparable in DT-infected and noninfected cultures. URO was
highly dominant in relative intensity over the other species in
Dutta et al.
DT-infected versus uninfected cells (Fig. 2, J ? DT ? ALA versus
J ? 0 ? ALA, lane 1, URO). After further chase in ALA-free me-
dium, URO remained dominant in DT-infected cells (Fig. 2, J ?
DT ? ALA, lanes 2 and 3), while COPRO and PROTO dissipated
to levels undetectable by TLC in all cells. The URO detected by
TLC in the DT-infected cells after chase originated from the in-
fluorescent when observed by fluorescence microscopy (Fig. 1A=,
day 3). Without exposure to ALA, neither DT-infected cells nor
noninfected cells (Fig. 2, J ? DT ? ALA and J ? 0 ? ALA) pro-
duced any TLC-detectable porphyrins, also consistent with the
fluorescence microscope observations (Fig. 1C and C=). Compar-
ison of DT- versus ST- or wild-type-infected cells produced the
same outcome under similar experimental conditions (not
The origin of the TLC-resolved URO from the intra-PV uro-
rophages, which were first infected with DT and then exposed to
ALA under the conditions used for the cell lines. Porphyrin fluo-
FIG 1 Host-parasite porphyric disparity after pulse-exposure of DT-infected cells to delta-aminolevulinate and equal infectivity of nonporphyric DT and ST
Leishmania mutants to host cells. (A to C and A= to C=) Phase-contrast (left) and porphyrin fluorescence (right) showing PVs and differences in the kinetics of
porphyrinogenesis between macrophages and their Leishmania DT mutants after ALA pulse-exposure (Day 1) and chase (Day 3). J ? DT ? ALA and J ? ST ?
as described above but not exposed to ALA. ST, transfectants with pX-alad and p6.5. Transfectants with another ST, i.e., pX and p6.5-pbgd, produced the same
results (not shown). Scale bar ? 10 ?m for all panels, except B=, in which a shorter scale is provided for 10 ?m. (D and D=) Kinetics of parasite loads (D) per
culture of J774 cells infected with the parental wild-type, uroporphyrinogenic double transfectants and nonporphyrinogenic single transfectants and of repli-
cation of the host cells in the respective cultures (D=). The error bars indicate standard errors.
Selective Porphyric Leishmania Photolysis in Host Cells
April 2012 Volume 11 Number 4 ec.asm.org 433
intracellular DT (see Fig. S1A in the supplemental material). TLC
analyses of the total porphyrins extracted from DT-infected peri-
toneal macrophages showed that URO was the sole porphyrin
species visible (see Fig. S1B in the supplemental material), clearly
corresponding to the fluorescent signals seen microscopically in
The results from all 3 types of host cells used indicate that the
porphyrin fluorescence seen to persist in their intra-PV DTs after
exposure to ALA corresponds to TLC-resolvable URO, but not
COPRO or PROTO. The last two made only a transient appear-
ance at a relatively low level in the host cell lines, irrespective of
infection or lack of infection and the Leishmania used for the
Selective photolysis of uroporphyric DTs in the phagolyso-
somes of DT-infected host cells by illumination. (i) Intra-PV
uroporphyric DTs were sensitive to dim light. The fact that in-
tra-PV uroporphyric DTs were sensitive to dim light was noted
initially in a preliminary study when DT-infected cultures were
handled as usual under normal laboratory lighting conditions.
tures were found to sharply decrease the intracellular amastigotes
ness decreased with time (see the legend to Fig. S2 in the supple-
mental material for details). By minimizing the exposure of DT-
infected cells to light, the intra-PV uroporphyric DTs no longer
decreased steeply, although they were still consistently fewer in
number than those of the control groups, which either remained
steady or increased over the period of incubation (Fig. 3A, DT ?
ALA versus ST ? A), while the host cells were not affected (Fig.
3A=). The susceptibility of the intra-PV uroporphyric DTs to am-
high photosensitivity, which was verified by experimental illumi-
nation (see below).
(ii) Experimental illumination of DT-infected cells exten-
sively photolysed their intra-PV uroporphyric DTs selectively.
By counting parasites and host cells, i.e., J774 macrophages and
FSDC of the DT-infected cultures after ALA treatment followed
trols, i.e., ALA alone plus incipient light (? ALA) and light alone
(Fig. 4), it was shown that experimental illumination of DT-in-
fected cells extensively photolysed their intra-PV uroporphyric
ALA ? light ? ? ALA ? light (equal to untreated [not shown])
(see legends to Fig. 4A and B for the experimental-illumination
conditions used). After exposure of the DT-infected cultures to
and never recovered throughout the period of observation up to
10 to 12 days. The parasite loads decreased progressively after the
combination treatments to a negligible level when the host/para-
J ? DT ? ALA, omission of the ALA treatments; J ? 0 ? ALA, omission of DT infection; J ? 0 ? ALA, J774 macrophages with neither DT infection nor ALA
treatment. Lanes 1 to 3, days 1 to 3 of chase after the ALA treatments. Porphyrins extracted from ?5 ? 106macrophages were loaded in each lane. Copro (C),
Proto (P), and Uro (U), carboxymethylated porphyrin markers corresponding to coproporphyrins, protoporphyrin IX, and uroporphyrins.
FIG 3 Depression of parasite loads in DT-infected, but not ST-infected, host
cells after ALA treatments under incipient light. Shown are the kinetics of
parasite loads (A) and host cell replication (A=) per culture of J774 cells in-
after exposure to ALA (? ALA). The arrows indicate daily ALA treatments
during medium renewals for 3 consecutive days from day 3 to day 5 after
infection for a total period of 9 days. Statistical significance, indicated by the
asterisks: P ? 0.01, calculated using the Student t test. The error bars indicate
Dutta et al.
site ratios used to initiate the infection were reduced from 1:10 to
1:2 (not shown). In contrast, the intra-PV DTs increased in num-
ber in the light-alone controls (Fig. 4A and B, solid squares) and
remained steady or decreased slightly in those treated with ALA
alone (Fig. 4A and B, open circles). Exposure of DT-infected J774
genesis of intraphagolysosomal DTs (cf. Fig. 3A; see Fig. S2 in the
supplemental material). None of the treatments noticeably af-
fected the host cells, as they replicated at comparable rates under
all conditions used (Fig. 4A= and B=).
from the DTs, resulting apparently from the leakiness of their
plasma membranes as a sign of cytolysis (Fig. 5, ? ALA ? light),
and disintegrated beyond recognition after further incubation
overnight (not shown). In contrast, the intra-PV DTs retained
their integrity in the control groups (Fig. 5, ? ALA ? light and ?
uroporphyric DTs are selectively susceptible to experimental illu-
mination for elimination from infected cells.
Selective photolysis of intra-PV DTs by pulse-exposure of
of intraphagolysosomal DTs released MHC class II sequestra-
tion by Leishmania. Mouse peritoneal macrophages were used
for photolysis of intraphagolysosomal DTs, since they were more
readily inducible with IFN-? to express MHC class II molecules
than the J774 cells. After infection of these macrophages with DT
(Fig. 6A, DT ? ALA ? light), MHC class II molecules (green)
were visualized only in discrete sections of the PV membrane,
FIG4 Significant clearance of parasite loads from DT-infected host cells after
ALA treatment followed by experimental illumination. J774 macrophages (A
The parasite loads (A and B) and the total numbers of host cells (A= and B=)
were estimated per infected culture at the time points indicated under various
conditions, as shown (? ALA ? light). The red and black arrows indicate the
time points of 1? ALA treatment and light exposure, respectively. Statistical
significance, denoted by the asterisks: P ? 0.001, calculated using one-way
ANOVA. The error bars indicate standard errors.
FIG 5 Selective photolysis of intra-PV uroporphyric DTs after experimental illumination of DT-infected host cells. Shown are phase-contrast (left) and
without 1 mM ALA exposure and/or white-light illumination for 15 min (12.15 J/cm2), as indicated (? ALA ? light). The cells were examined 2 h after
illumination. Scale bar ? 10 ?m.
Selective Porphyric Leishmania Photolysis in Host Cells
April 2012 Volume 11 Number 4ec.asm.org 435
which were delineated by the lysosomal marker, i.e., LAMP-1
(red). Adhesion of amastigotes to the PV membrane was evident
nuclei (blue). The merged image suggested that the MHC class II
molecules were often sequestered in the intervening region be-
were absent in the remaining portion of the large PV free of amasti-
gote attachment. When observed ?2 h after exposure of the DT-
infected macrophages to ALA ? light, PVs were found to become
smaller and contained disintegrating DTs (Fig. 6B, phase-contrast).
MHC class II (green) and LAMP-1 (red) molecules became dis-
incubation for up to 16 h (see Fig. S3 in the supplemental material),
tinguishable between the infected cultures after DT photolysis (see
Fig. S3, DT ? ALA ? light) and noninfected cells (see Fig. S3, con-
membrane-attached amastigotes (yellow) in large PVs (see Fig. S3,
DT ? ALA ? light). Photolysis of intra-PV porphyric DTs thus re-
leased the sequestered MHC class II molecules and restored their
(ii) Expression profiles of the host cells returned to normal
after photolysis of their intra-PV DTs. The cDNA microarray
analyses of DT-infected J774 cells versus the control groups
showed that photolysis of the intra-PV DTs reversed the effect of
their infection on the host cells more generally than just releasing
scription profile of the host cells became more similar to their
preinfection state after photolytic suppression of the infection.
FIG 6 Recruitment of MHC class II molecules to LAMP-1-positive PVs after infection of mouse peritoneal macrophages with DTs and their return to normal
to the conditions for selective photolysis of intra-PV uroporphyric DTs, i.e., treatment with 1 mM ALA overnight (? ALA) followed by illumination (400 nm)
at 7.5 J/cm2(? light). The remaining set of infected (A) and uninfected (D) cells served as porphyrin-free and thus nonphotolytic controls. A monolayer of all
nucleus (blue) by immunofluorescence microscopy. See Materials and Methods for experimental details. Scale bar ? 10 ?m.
Dutta et al.
infection of macrophages was found to alter the expression of the
3,102 genes at a P value of ?0.05, i.e., ?15% of the ?21,000
unique clones. The list was reduced to 247 when it was filtered to
include only those with a P value of ?0.01 and with ?2-fold
change. Changes in expression levels ranged from up 900-fold to
down 200-fold (Fig. 7A, J ? DT versus J). Table S1 in the supple-
mental material lists the most significant 100 genes (by P value)
that changed after infection. The effects of treatments with ALA
assessed by one-way ANOVA within the 3,102 genes (P ? 0.05)
that had their expression levels altered by the infection. Figure 7A
shows hierarchical clustering of the 318 genes that had their ex-
Light alone (J ? DT ? light) had some effect on gene expression
and especially ALA ? light, resulted in a more significant reversal
of the infection-produced changes in gene expression of the mac-
rophages (see Table S2 in the supplemental material for a gene
list). These changes before and after uroporphyrinogenesis/pho-
tolysis were verified by reverse transcription (RT)-PCR for inter-
FIG 7 Macrophage and Leishmania expression profiles of DT-infected cells after ALA-induced porphyrinogenesis and selective photolysis of intra-PV uropor-
nm) for 10 min; J ? DT ? ALA, treatment of J ? DT with 1 mM ALA for 16 h; J ? DT ? ALA ? light, exposure of J ? DT to both ALA and light, followed by
chase in ALA-free medium for 3 days. All samples were subjected to microarray analysis (?21,000 mouse genes plus 4 Leishmania genes) (see Materials and
Methods for details). (A) Hierarchical clustering of genes showing their expression patterns in DT-infected cells versus uninfected cells and subsequent changes
that occur upon induction of Leishmania photolysis. A total of 3,102 macrophage genes showed altered expression (P ? 0.05) compared to uninfected (J) and
to the preinfection expression patterns for these genes. The color scale indicates the change for each gene based on median intensity (pale green) from
downregulated (dark blue to light blue) to upregulated (yellow to red) for each condition using units of Z (standard deviations). Gene clustering is indicated at
left, with two principal families: red, those which start low, increase, and then return to low initial values; and green, those which start high, decrease, and then
rise to initial values. (B) Progressive decrease of Leishmania DT-specific gene expression from ALA-induced uroporphyrinogenesis of DTs and their photolysis
in infected host cells. The four Leishmania genes spotted in triplicate on the arrays were used to assess the effects of various treatments on the protozoan
replicate experiments were used to plot the expression levels. Leishmania ndk, nagt, and dhfr genes are plotted on the left-hand scale, and beta-tubulin is on the
right-hand scale (1st bar of the 4 for each treatment group). The beta-tubulin value is higher, presumably resulting from cross-hybridization of the Leishmania
probe with sequence-conserved murine beta-tubulin messages inherently abundant in macrophages. The error bars indicate standard errors.
Selective Porphyric Leishmania Photolysis in Host Cells
April 2012 Volume 11 Number 4 ec.asm.org 437
leukin 10 (IL-10) (see Fig. S4 in the supplemental material). Pho-
many downregulated and upregulated genes to baseline unin-
fected levels. This is more obvious for the genes upregulated than
for those downregulated by infection.
Photolytic suppression of DTs in infected macrophages is in-
included in the arrays (beta-tubulin, nagt, ndk, and dhfr) upon
infection and then as a function of the treatments (Fig. 7B). The
but were reduced by ALA treatment (J ? DT ? ALA) and subse-
quently returned to the background of no infection when further
experimentally illuminated with light (J ? DT ? ALA ? light).
Transcription of intra-PV DTs was reduced with uroporphyrino-
to experimental illumination for photolysis.
The results of the microarray analyses support the conclusion
that the intra-PV DTs are selectively affected, reversing the inhi-
bition of their host cells by the infection.
Uroporphyrinogenesis/photolysis of DTs released antigens
in dendritic cells for MHC class I copresentation. OVA released
ysis in the DCs was apparently processed correctly by these anti-
gen-presenting cells (APCs) to present the known MHC class I-
specific SIINFEKL epitope. This is indicated by the positive
reaction of this MHC class I-epitope complex with a specific
monoclonal antibody, 25-D1.16 (see Fig. S5 in the supplemental
material). The positive immunoreaction products (green or pale
blue when overlapped over DAPI-stained nuclei) were present in
DCs infected with DT OVA after their photolysis in situ (see Fig.
S5, [? DT OVA] ? ALA ? light), but not with these mutants
without photolysis (see Fig. S5, [? DT OVA] ? ALA ? light) or
the positive reactivity, however, was weaker than for those ex-
posed to SIINFEKL or OVA (see Fig. S5, left, ? SIINFEKL pep-
tides, ? OVA).
The present study provides the first demonstration of the new
induces the neogenesis and accumulation of URO to an excep-
ously, a partial rescue of Leishmania’s natural defects in heme
biosynthesis by forward genetics was shown to make this possible
phyrinogenesis is triggered by exposure of these promastigotes to
DT-infected macrophages to ALA also readily elicits uroporphy-
of the latter. The ALA, which is imported into the PVs of the host
cells via their pinocytic activity (unpublished data), appears to
provide the substrate that is used to initiate this response by the
DT parasite. This is strongly supported by the exclusion of the
likely alternative ALA sources from the cells, since Leishmania
lacks the ALA synthase needed to produce this substrate (9), and
the host cells, i.e., macrophages and dendritic cells, as noneryth-
rocytic cells, stringently regulate ALA synthase as a rate-limiting
enzyme in their heme biosynthetic pathway (17, 32). While the
exact mechanism for the uptake of exogenous ALA by DT-in-
fected host cells awaits further study, it is apparently made avail-
DTs is not unexpected, considering the stochastic cellular events
of uroporphyrinogenesis, as noted previously with ALA-exposed
DT promastigotes (10).
The most significant finding of the present study is the host-
the intra-PV amastigotes after pulse-exposure of the DT-infected
cells to ALA. The host cells clearly utilize this exogenous ALA
to overproduce porphyrins of the expected species, i.e., URO,
COPRO, and PROTO, rendering them “visible” microscopically
and chromatographically (Fig. 1 and 2). Other mammalian cells
are known to undergo similar porphyrinogenesis by taking up
exogenous ALA via their plasma membrane peptide transporters
(28, 39). On removal of the exogenous ALA, the rapid loss of
accounted for in part by their release (16) and largely by their
comes in all samples except the DT-infected cells, in which URO
persists in the intra-PV DTs (Fig. 1A= and 2), consistent with the
observation of ALA-exposed DT promastigotes (10, 30). There is
no apparent host-parasite transfer or exchange of the porphyrins
overproduced by the respective cells. This is least likely for URO,
since it is known to be released without reuptake by both Leish-
mania and mammalian cells (10). Any significant transfer of
COPRO and/or PROTO from the host cells to intra-PV DTs
would be expected to result in their persistence, since Leishmania
has no enzymes to utilize these host-derived porphyrins. Taken
together, the results suggest that the host cells and their DTs un-
dergo independent porphyrinogenesis, almost certainly by using
Significantly, the host-parasite differential porphyrinogenesis
after ALA pulse-exposure of the DT-infected cells makes it possi-
ble to achieve a selective accumulation of URO in the DTs, ac-
There has been no report of such a strategy to selectively lyse
intracellular parasites. The photosensitivity of the intra-PV uro-
porphyric DTs is such that their cytolysis is notable even when
exposed to dim light (Fig. 3; see Fig. S2 in the supplemental ma-
terial) and is extensive after experimental illumination under all
Another finding of equal importance in the present study is
that the host cells remain viable and functional after ROS-medi-
ated photolysis of their intra-PV uroporphyric DTs. This is not
unexpected, considering that Leishmania normally resides in the
phagolysosomes of macrophages, which are resistant to the ROS
generated by their own respiratory burst (29). Indeed, the integ-
rity and viability of the uroporphyric-DT-infected host cells is
unaffected by the illumination conditions used. Timely illumina-
PVs/phagolysosomes, i.e., ?3 days after infection, produces opti-
mal selectivity of DT photolysis. Although how the host cells de-
toxify the singlet oxygen expected to form immediately after light
excitation of URO is unknown (10), they clearly return largely to
Dutta et al.
their preinfection state, judging from their ability to maintain
their integrity as a monolayer and to replicate subsequently (Fig.
of the ?300 genes in the macrophages that are most affected by
DT infection become increasingly similar to those of the unin-
fected host cells, consistent with decreasing levels of the parasite
transcripts after uroporphyrinogenesis/photolysis (Fig. 7). Fur-
FIG 8 Schematic presentation of the cellular events involved in the use of Leishmania DT mutants as potential delivery vehicles of vaccines for photodynamic
Leishmania, leading to the establishment of intraphagolysosomal parasitism (cf. Fig. 1). (e to h) Pulse-exposure of DT-infected cells to ALA for selective
accumulation of URO in the intra-PV DTs and their intralysosomal photolysis (cf. Fig. 2 to 5). (i to j) Drugs/vaccines (dots) hypothetically released from
photolysed Leishmania cells (cf. Fig. 6 and 7).
Selective Porphyric Leishmania Photolysis in Host Cells
April 2012 Volume 11 Number 4 ec.asm.org 439
ther analyses of samples at additional time points may reveal a
more complete reversal and the potential significance of the up-
S1 and S2 in the supplemental material).
Most significantly, evidence is presented indicating that the
mal in macrophages after DT photolysis (Fig. 6; see Fig. S3 in the
supplemental material). Infection of macrophages with L. ama-
zonensis is known to sequester their MHC class II molecules in
their residential phagolysosomes/PVs, thereby diminishing the
capacity of these APCs for antigen presentation as a strategy of
immune evasion (20, 23). Our observation is consistent with the
previous report that these molecules are sequestered in the com-
partment between the parasite and the vacuolar membranes (2).
Selective photolysis of the intra-PV uroporphyric DTs clearly re-
stores the normal distribution of these molecules, suggestive of
releasing these host cells from Leishmania immunosuppression.
In situ photolysis of uroporphyric DTs was shown to release
antigens in dendritic cells for presentation via the MHC class I
pathway. Transfection of DTs to express OVA as a model antigen
makes it possible to demonstrate this antigen-presenting capacity
by using a monoclonal antibody that recognizes an OVA SIIN
unless OVA-DTs were photolysed in situ, indicative of antigen
clonal antibody is not as intense as those with the peptide and
pacity of this vaccine delivery system. Work is under way to opti-
mize the experimental conditions for OVA antigen presentation
to peptide-specific T cells along the line, as shown with phthalo-
cyanine-loaded Leishmania as an alternative photolytic vaccine
delivery vehicle (11).
phyrinogenic DT is uniquely suitable as a carrier for drug/vaccine
delivery (30). Figure 8 schematically summarizes this model to
illustrate its potential advantages in photodynamic vaccination
(22). First, the 2 transgenic products of the DTs produce no
change in their ability to home to the phagolysosomes of APCs
(Fig. 8a to d); hence, they are fully capable of protecting their
potential payloads of drugs/vaccines en route to the desirable des-
compromised Leishmania parasites that were used previously for
the same purpose after drug treatments (7) or after knocking out
the DTs are signaled for lysis to release their payloads only after
taking up residence in the relatively ROS-resistant phagolyso-
somes of infected APCs. This is made possible by induction of
uroporphyrinogenesis for their selective photolysis (Fig. 8e to i),
leaving the host cells viable and functional. Such treatments thus
differ from the use of cytotoxic nucleoside analogues to kill Leish-
mania transfectants with suicide genes after immunization (8, 14,
25). Optimal recovery of the host cells, as shown in our case, may
well be crucial, enabling them not only to be free of Leishmania-
imposed immunosuppression, but also to adequately process an-
to elicit effective immunity (Fig. 8i to j). This may account for the
immune clearance of the few surviving DTs and for the prophy-
lactic protection of animals against wild-type parasite challenges
Work is ongoing to enhance the applicability of the DTs to
vaccination. For example, transfection of DTs with luciferase was
found to mediate the emission of luminescence in the vicinity of
the cytosolic URO for their complete photolysis (unpublished
data). Also, additional transfection of DTs with ALA synthase has
the potential to render exogenous application of ALA unneces-
sary. Expression of these genes in constructs for amastigote stage-
specific expression is also under way. As these transfectants reach
macrophage phagolysosomes, their differentiation from promas-
tigotes into amastigotes is thus expected to trigger uroporphyri-
thereby eliminating the need for externally supplied ALA and il-
intraphagolysosomal uroporphyric DTs observed is not unex-
pected, as noted for the promastigote, due to the stochasticity of
ALA-induced uroporphyrinogenesis (10). Previously, we pre-
after illumination in situ. Although their persistence elsewhere
cannot be ruled out, the immunized animals developed a solid
prophylactic immunity that was adoptively transferable to naïve
animals (22). Whether the development of this immunity also
cleared the residual DTs is unknown. More recently, prephotoin-
nines was found to effectively deliver this model antigen to den-
dritic cells (DC4.2) for presentation of OVA to activate OVA
peptide-specific CD8?T cells in vitro (11). Similar approach for
with novel phthalocyanines represents alternative strategies,
which are under study, to produce effective but nonproliferating
and nonviable vaccine carriers to ensure their safety margin. To
serve as a universal platform, they could be engineered to express
any of a variety of peptides for therapy/prophylaxis against other
drugs/vaccines may be enhanced by the adjuvant and other im-
munogenic activities of Leishmania endogenous molecules
against diseases that are otherwise refractory to conventional ap-
proaches to prophylaxis and therapy.
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