Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis

Abstract

The trypanosomatids Leishmania amazonensis and Trypanosoma cruzi are excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellates L. amazonensis and T. cruzi lodge within acidified parasitophorous vacuoles (PVs). However, whereas L. amazonensis develops in spacious, phagolysosome-like PVs that may enclose numerous parasites, T. cruzi is transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation of T. cruzi, we constructed chimeric vacuoles by infection of L. amazonensis amastigote-infected macrophages with T. cruzi epimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate of T. cruzi in L. amazonensis PVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that although T. cruzi EPIs remained motile and conserved their morphology in chimeric vacuoles, T. cruzi MTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles of L. amazonensis are permissive to T. cruzi survival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude that T. cruzi differentiation can take place in Leishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol.

INTRODUCTION

Coinfections involving viruses and bacteria or parasites came to the fore after the post-WWI influenza pandemic and, more recently, with HIV epidemics; they also were extensively examined in laboratory animals and cell cultures (1, 2). The presence of more than one pathogen hosted by the same organism is a rule, not an exception, in nature. Although coinfections, or mixed infections, have been reported in all sorts of host eukaryotic organisms, and although humans and other mammals now are widely regarded as mixed, complex, interspecific organisms, few reports have dealt with in vitro mixed models of host-parasite interaction.

In addition, there are relatively few studies of coinfections involving two nonviral pathogens. Table 1 lists articles on coinfection models involving nonviral pathogens, highlighting their localization (extracellular or sharing or not sharing an intracellular compartment); most reports deal with pathogens of different species, but some studies include coinfections of different strains or mutants of the same species.

TABLE 1.

List of coinfection studies with nonviral pathogens published from 1960 to 2013

Speciesa	Shared compartment	Reference
1960–1969
Rickettsia prowazekii strains	Undetermined	3
1970–1979
Coxiella burnetii I, Coxiella burnetii II	Undetermined	4
Coxiella burnetii I, Coxiella burnetii II	Undetermined	5
Chlamydia trachomatis strains	Yes/vacuoles	6
1980–1989
Staphylococcus aureus, Pseudomonas aeruginosa	Extracellular	7
Trypanosoma cruzi, Toxoplasma gondii	No/vacuoles	8
Theileria parva strains	Undetermined	9
Theileria annulata, Theileria parva	Undetermined	10
Polymicrobial	Undetermined	11
Chlamydia trachomatis strains	Yes/vacuoles	12
1990–1999
Toxoplasma gondii, Mycobacterium avium	No/vacuoles	13
Chlamydia trachomatis, Chlamydia psittaci	Yes/vacuoles	14
Leishmania amazonensis, Coxiella burnetii	Yes/vacuoles	15
Coxiella burnetii, Chlamydia trachomatis	No/vacuoles	16
Leishmania mexicana, Listeria monocytogenes	Yes/vacuoles	17
Mycobacterium smegmatis, Streptococcus pyogenes	Undetermined	18
Leishmania amazonensis, Leishmania donovani, Leishmania infantum	Yes/vacuoles	19
Polymicrobial	Undetermined	20
Streptococcus pyogenes, Escherichia coli	Undetermined	21
Legionella pneumophila mutant	Yes/vacuoles	22
Mycobacterium avium, Coxiella burnetii	Yes/vacuoles	23
Mycobacterium avium, Mycobacterium tuberculosis, Coxiella burnetii	Yes/vacuoles	24
2000–2009
Chlamydia pneumoniae, Mycoplasma hominis-like	Undetermined	25
Mycobacterium smegmatis mutant	Undetermined	26
Coxiella burnetii, Toxoplasma gondii	No/vacuoles	27
Chlamydia trachomatis strains	Yes and no/vacuoles	94
Salmonella Typhimurium mutant	Undetermined	29
Chlamydia trachomatis, Chlamydia suis	Yes/vacuoles	30
Neisseria meningitidis, commensal Neisseria	Extracellular	31
Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium smegmatis	Yes/vacuoles	32
Salmonella Typhimurium mutant	Undetermined	33
Chlamydia trachomatis strains	Yes and no/vacuoles	34
Trypanosoma cruzi, Coxiella burnetii	Yes/vacuoles	35
Helicobacter pylori strains	Yes/vacuoles	36
Salmonella Typhimurium mutant	Undetermined	37
Haemophilus influenzae, Streptococcus pneumoniae	Undetermined	38
Legionella pneumophila, Coxiella burnetii	No/vacuoles	39
Listeria monocytogenes mutant	Yes/cytosol	40
Coxiella burnetii, Trypanosoma cruzi	Yes/vacuoles	41
Escherichia coli (ETEC), Escherichia coli (EPEC)	Undetermined	42
Salmonella Typhimurium mutant	Undetermined	43
Legionella pneumophila, Legionella longbeachae	Yes/vacuoles	44
Chlamydia trachomatis mutant	Yes/vacuoles	93
Coxiella burnetii, Salmonella Typhimurium, Escherichia coli	Yes/vacuoles	46
Coxiella burnetii, Trypanosoma cruzi	Yes/vacuoles	47
Chlamydia trachomatis, Chlamydia muridarum	No/vacuoles	48
Francisella tularensis mutants	Yes/cytosol	49
Salmonella Typhimurium strains	Undetermined	50
Chlamydia trachomatis strains	Yes/vacuoles	45
Polymicrobial	Undetermined	51
Escherichia coli mutant	Yes/cytosol	52
Trypanosoma cruzi strains	Yes/cytosol	53
Escherichia coli (EHEC) mutant	Undetermined	54
Toxoplasma gondii mutant	Undetermined	55
Porphyromonas gingivalis, Fusobacterium nucleatum	Undetermined	56
Helicobacter pylori, Escherichia coli (EPEC)	Undetermined	57
Porphyromonas gingivalis, Streptococcus gordonii	Undetermined	58
Polymicrobial	Undetermined	92
Chlamydia suis, Chlamydia trachomatis, Chlamydia muridarum	Yes and no/vacuoles	28
2010–2013
Trypanosoma cruzi strains	Undetermined	59
Escherichia coli (EPEC) mutants	Undetermined	60
Escherichia coli (EAEC), Citrobacter freundii	Extracellular	61
Leishmania amazonensis, Leishmania major	Yes and no/vacuoles	62
Leishmania infantum, Toxoplasma gondii	Undetermined	63
Neisseria lactamica, Neisseria meningitidis	Undetermined	64
Bartonella henselae mutants	Yes/vacuoles	65
Chlamydia muridarum, Neisseria gonorrhoeae	Undetermined	66
Yersinia enterocolitica mutants	Undetermined	67
Brucella suis mutants	Undetermined	68
Toxoplasma gondii, Chlamydia trachomatis	No/vacuoles	69
Toxoplasma gondii, Chlamydia trachomatis	No/vacuoles	70

^aPolymicrobial indicates studies that analyzed more than three microorganism species. The terms “mutant” and “strains” were used when the study compared mutants or strains of the same species. ETEC, enterotoxigenic E. coli; EPEC, enteropathogenic E. coli; EAEC, enteroaggregative E. coli.

The parasite Trypanosoma cruzi is the causative agent of Chagas' disease while Leishmania amazonensis is one of the causative agents of disseminated cutaneous leishmaniasis, and both are neglected tropical diseases with high morbidity (71). T. cruzi resides in a transient parasitophorous vacuole (PV) from which the parasite escapes into the host cell cytosol, where it then multiplies (72). L. amazonensis develops in spacious PVs containing dozens, even hundreds, of multiplying parasites that remain in these vacuoles until their transfer to other host cells (73, 74). Due to the morphology of large L. amazonensis PVs, it is possible to study coinfection between L. amazonensis and T. cruzi forms.

We have used multidimensional imaging to examine the dynamics of vacuolar cohabitation of these two trypanosomatid parasites in the macrophage cell line RAW 264.7 or in primary bone marrow-derived macrophages. Using spacious PVs formed by L. amazonensis after 48 h of in vitro infection in macrophages, we found that T. cruzi metacyclic forms are able to enter L. amazonensis PVs and, once within the new chimeric vacuole, can differentiate into amastigote-like forms capable of multiplication. By using an in vitro system of coinfection in which L. amazonensis PVs, easily assessed by microscopic approaches, were employed as recipient vacuoles for T. cruzi, we provide evidence that T. cruzi differentiation and multiplication take place inside phagolysosome-like structures.

MATERIALS AND METHODS

Ethical statement.

All animal procedures were conducted by abiding by international and regional policies on animal experimentation (UNIFESP CEUA 779916).

Host cells.

The RAW 264.7 macrophage-like cell line (ATCC, Manassas, VA, USA) was maintained by successive passages in RPMI 1640 medium (Gibco Cell Culture, Life Technologies, Carlsbad, CA, USA) supplemented with 10% inactivated fetal bovine serum (FBS) and 100 U ml⁻¹ penicillin–100 μg · ml⁻¹ streptomycin (complete medium) at 34°C in 5% CO₂. Cells were detached from 25-cm² culture flasks with 0.01 M phosphate-buffered saline (PBS), pH 7.2, supplemented with 20 mM HEPES and 1% EDTA for 20 min. After detachment and centrifugation at 300 × g for 10 min, cells were seeded into 24-well plates for epifluorescence microscopy or MatTek dishes (MatTek Corporation, Ashland, MA, USA) suitable for live-cell multidimensional imaging. Green fluorescent protein (GFP)-tagged RAW 264.7 cells were produced by the transduction of GIPZ lentivirus carrying a GFP reporter gene and a puromycin resistance gene (shRNA nonsilencing control; Thermo Scientific/GE Healthcare/Dharmacon, Buckinghamshire, United Kingdom). GFP-tagged RAW 264.7 cells were selected using 8 μg · ml⁻¹ puromycin. Bone marrow-derived macrophages (BMDMΦ) were obtained as previously described (17).

HeLa cells (Instituto Adolfo Lutz, São Paulo, Brazil) were cultivated by successive passages in RPMI medium (Gibco) supplemented with 10% inactivated FBS and 100 U · ml⁻¹ penicillin–100 μg · ml⁻¹ streptomycin. This cell line was transfected with a plasmid containing red fluorescent protein (RFP)-tagged Rab7 GTPase (kindly donated by Norma Andrews, Maryland University, USA) using FuGene HD transfection reagent (Roche Applied Science, Penzberg, Germany).

Parasites.

Parasite cultures employed in this study were wild-type (WT) Leishmania amazonensis M2269 (MHOM/BR/1973/M2269) and a Discosoma sp. red fluorescent protein (DsRed)-transfected or GFP-transfected Trypanosoma cruzi CL strain. Leishmania amastigotes were isolated from BALB/c mouse footpad lesions by following protocols established previously (75). T. cruzi epimastigote (EPI) forms were maintained in liver infusion tryptose (LIT) medium supplemented with 10% FBS and 10% glucose at 26°C. T. cruzi metacyclic (MT) forms were obtained by the cultivation of epimastigotes in Grace's medium (Gibco) at 26°C for 7 to 8 days and were isolated using a DEAE-cellulose column as described previously (76). The transfection and selection of DsRed-tagged or GFP-tagged T. cruzi were performed according to reference 77.

Infection of host cells.

L. amazonensis amastigotes were incubated with BMDMΦ or RAW 264.7 cells at a multiplicity of infection (MOI) of 5 or 20 parasites per cell, respectively; infected cells were incubated at 34°C in 5% CO₂ in complete medium overnight prior to washing noninternalized amastigotes with Hanks' saline (Sigma-Aldrich). Infected cells were maintained for 48 h at 34°C in 5% CO₂ to allow the formation of spacious L. amazonensis PVs, which are easily observed under optical microscopes.

L. amazonensis-infected RAW 264.7 cells then were superinfected with DsRed- or GFP-tagged T. cruzi MT or EPI forms at an MOI of 20:1. After 2 h of interaction pulse, noninternalized parasites were washed away with PBS and cells containing parasites were incubated in complete medium at 34°C in 5% CO₂. Motile forms of T. cruzi inside L. amazonensis PVs were considered evidence of chimeric vacuoles, being regularly observed and quantified with an Olympus IX-70 inverted epifluorescence microscope (Olympus Corporation, Tokyo, Japan) coupled to a preheated stage adjusted to 34°C. The time of points analyzed were 2, 24, and 72 h postinfection. To evaluate longer periods of coinfection, L. amazonensis-infected BMDMΦ plated onto glass coverslips were superinfected with DsRed-tagged T. cruzi MT forms at an MOI of 20:1. After 24 h of interaction pulse, noninternalized parasites were washed away with PBS. The coverslips then were fixed with 4% paraformaldehyde in PBS after 1, 3, 4, 5, and 7 days postinfection. After the fixation, the coverslips were washed with PBS, incubated with DAPI (4′,6-diamindino-2-phenylindol; 1 μM; Invitrogen), and analyzed under epifluorescence microscopy (Olympus BX51; Olympus Corporation, Tokyo, Japan).

Transmission and scanning electron microscopy.

Suspensions of RAW 264.7 cells infected for 24 h were fixed with modified Karnovsky solution (1% paraformaldehyde, 3% glutaraldehyde in 0.07 M sodium cacodylate buffer, pH 7.2) at room temperature for 30 min, postfixed with 1% osmium tetroxide, and then processed for transmission electron microscopy (TEM) according to procedures described elsewhere (78). Cells were observed in a JEOL 1200 EXII electron microscope (JEOL Ltd., Peabody, MA, USA). For scanning electron microscopy (SEM), superinfected RAW 264.7 cells on coverslips were fixed with 2.5% glutaraldehyde, washed in 0.1 M cacodylate solution, pH 7.2, postfixed with 1% osmium tetroxide, treated with tannic acid, dehydrated with ethanol, and then dried in a CPD 030 critical point dryer (Bal-Tec AG, Balzers, Liechtenstein). To expose the content of chimeric PVs, samples were fractured by peeling with the aid of scotch tape (79) and then coated with a gold layer using a sputtering method. Samples were observed in a field emission FEI Quanta FEG 250 SEM (Hillsboro, OR, USA).

Confocal live cell imaging.

For multidimensional imaging, we employed a Leica TCS SP5 confocal system (Leica Microsystems, Wetzlar, Germany) coupled to microincubators and a motorized stage, which allowed for the acquisition of 20 to 30 z-stack images of live samples from five different microscopic fields at 5- to 15-min intervals between each acquisition. The system was set to a resonant scanner modality (8,000-Hz frequency). RAW 264.7 macrophage live cells coinfected with L. amazonensis amastigotes and DsRed-tagged T. cruzi MT forms at an MOI of 20:1 were monitored by this methodology for 48-h periods of image acquisition. L. amazonensis-infected BMDMΦ were superinfected with GFP-tagged T. cruzi MT forms at an MOI of 20:1. After 96 h of coinfection, cells were monitored by the methodology described above for an additional 48 to 72 h of image acquisition.

Live cell multidimensional images (three dimensional [3D] plus time and fluorescent channels) were processed by Imaris software (Bitplane AG, Andor Technology, Belfast, Great Britain), which allowed the construction of isosurfaces, three-dimensional, graphical interpretations of the fluorescent signals obtained by the microscope. These isosurfaces were employed for the precise localization of T. cruzi forms within L. amazonensis PVs.

Statistics.

All results are representative of at least three independent experiments. Excel (Microsoft Corp.) and SPSS (IBM) were employed for data plotting and statistical analysis. Statistical tests included Student's t test and analysis of variance (ANOVA), and a statistical threshold of a P value of <0.05 was used.

RESULTS

Chimeric vacuoles are formed after coinfection with L. amazonensis and T. cruzi.

To investigate the formation of chimeric vacuoles containing L. amazonensis and T. cruzi, a double infection methodology was established consisting of L. amazonensis amastigote infection for 48 h, followed by the addition of T. cruzi EPIs or MTs to RAW 264.7 cells. Transmission and scanning electron microscopy of superinfected host cells show the coexistence of round and elongated flagellated forms within the same spacious PVs 24 h after the second infection (Fig. 1). The presence of motile, DsRed-T. cruzi EPIs or MTs inside L. amazonensis PVs also was observed in live infected macrophages under epifluorescence microscopy, allowing us to quantify the percentage of chimeric vacuoles among L. amazonensis PVs in double-infected macrophages. Regardless of the T. cruzi form (EPI or MT) or time after double infection (2, 24, and 72 h), around 15% of L. amazonensis PVs also harbored at least one T. cruzi parasite (Fig. 2A).

FIG 1.

Formation of chimeric vacuoles containing flagellated and round forms of intracellular parasites in RAW 264.7 macrophages by electron microscopy. Micrographs were obtained from macrophages infected with L. amazonensis lesion-derived amastigotes for 48 h and then superinfected with epimastigote (A to C) or metacyclic (D) forms of T. cruzi. (A to C) Transmission electron micrographs showing round amastigote forms (arrows) cohabiting spacious PVs with flagellated forms (arrowheads) after 24 h of infection. (D) The same round and flagellated forms inhabiting spacious PVs also were observed by scanning electron microscopy. Flagellated forms were digitally colorized in blue; green color was attributed to round forms. Bar, 5 μm.

FIG 2.

Transfer and morphological differentiation of metacyclic trypomastigotes within chimeric vacuoles. RAW 264.7 macrophages were infected with L. amazonensis amastigotes for 48 h and then infected with T. cruzi CL strain epimastigotes (EPIs) or metacyclics (MTs). After 2, 24, and 72 h of coinfection, 100 L. amazonensis vacuoles were observed by epifluorescence microscopy and the number and morphology of T. cruzi cells within chimeric PVs were evaluated. (A) The bar graph shows the means ± standard errors of the percentage of chimeric PVs among scored L. amazonensis PVs in two independent experiments performed in triplicate. Statistical analysis was performed by ANOVA (ns, nonsignificant). (B) A possible T. cruzi MT form (red; marked by an arrowhead) transferred to the L. amazonensis PV to generate a chimeric vacuole after 3 h of acquisition of multidimensional images. The times shown are relative to the time of image acquisition in days (d) hours:minutes. The upper row shows the differential interference contrast (DIC) channel merged with the red fluorescent channel; the lower row shows the merged red fluorescent channel for parasite DsRed and the green fluorescent channel for GFP expressed by RAW 264.7 cells. The precise localization of the transferred MT was accessed by 3D rendering using Imaris software (Bitplane), a blend filter, and isosurfaces reconstructed based on the green fluorescent channel (green isosurface representing GFP signal expressed by the macrophage) and the red fluorescent channel (red isosurface representing the DsRed signal expressed by T. cruzi). Bar, 10 μm. (C) Section graphs show T. cruzi morphological classification within the chimeric PVs after infection for 24 or 72 h with metacyclic (MTs; upper graphs) or epimastigote (EPIs; lower graphs) T. cruzi forms. The morphology of the parasites was classified as typical MT, typical EPI, or indeterminate/intermediate form (IF), exemplified in the images on the right (bar, 10 μm).

Metacyclic forms of T. cruzi are transferred to L. amazonensis PVs before their differentiation into amastigotes.

The presence of motile, flagellated forms of T. cruzi within L. amazonensis PVs suggested that the formation of chimeric vacuoles takes place prior to MT differentiation into T. cruzi amastigote forms. To investigate this hypothesis further, multidimensional live imaging of superinfected macrophages was performed to track T. cruzi MTs before and after their transfer to L. amazonensis PVs. Figure 2B shows a sequence of images of the same infected, GFP-tagged macrophage in which a DsRed-tagged T. cruzi MT is transferred to a L. amazonensis PV as an intermediate form. The morphology of these intermediate forms is characterized by the presence of a flagellum and by an increase in the sphericity of the parasite. The presence of intermediate forms of T. cruzi within L. amazonensis PVs and their morphological features were confirmed by 3D rendering of multidimensional images (Fig. 2B; also see Movie S1 in the supplemental material). Using epifluorescence microscopy, we quantified the number of MT-like, EPI-like, and intermediate forms of T. cruzi within chimeric vacuoles after superinfection with EPIs or MTs (Fig. 2C). Typical epimastigotes (EPI-like) represented the main morphological group found within chimeric PVs for up to 72 h after superinfection with epimastigotes. The preservation of epimastigotes within chimeric PVs suggests that the compartment is less hostile to these developmental forms, which are known to be killed after macrophage internalization. This feature could be due to the macrophage cell line or the nature of L. amazonensis PVs (62). When infection was performed using MTs, we observed an increase in the number of intermediate forms among T. cruzi organisms inhabiting chimeric PVs. The data suggest that metacyclic T. cruzi forms are transferred to L. amazonensis PVs prior to their full differentiation into amastigote forms.

Motile T. cruzi forms differentiate into amastigote-like forms within phagolysosomal vacuoles.

To investigate whether L. amazonensis PVs would support the differentiation of T. cruzi intermediate forms into amastigotes, we tracked DsRed-tagged T. cruzi intermediate forms, found inside chimeric PVs, by multidimensional imaging (Fig. 3). The tracking was performed with reference to two different periods after infection: early tracking comprised the first 48 h postinfection and confirmed a morphological change of MTs to an intermediate form within chimeric PVs (Fig. 3A; also see Movie S2 in the supplemental material). Late multidimensional tracking of these intermediate forms, comprising image acquisition from 72 h postinfection, allowed for the recording, in a few cases, of the multiplication of round T. cruzi organisms within an L. amazonensis PV (Fig. 3B; also see Movie S3). These round forms are a late developmental stage of intermediate forms, and their capacity of multiplication, although limited, indicates differentiation from metacyclics into amastigotes within phagolysosomal-like vacuoles, such as L. amazonensis PVs. We investigated whether T. cruzi differentiation into amastigotes also occurs in a phagolysosome-like compartment in monoinfected host cells. In these experiments, HeLa cells were transfected with GTPase Rab7 previously tagged with RFP prior to infection with GFP-tagged T. cruzi MT (Fig. 4; also see Movie S4). Rab7 is a marker of late endosomes and phagolysosomes, which can be observed in the membrane of PVs formed by L. amazonensis or T. cruzi in the first few hours postinfection in vitro (47, 73). At the beginning of our recordings, MT forms could be seen surrounded by Rab7; a flagellum also could be inferred from Rab7 contours (Fig. 4, arrow). The parasite differentiated into a round form still surrounded by Rab7 for up to 10 h of intracellular infection; after this time period, Rab7 surrounding the parasite disappeared. The parasite shown in Fig. 4 was tracked for 12 additional hours without further clear association or colocalization with the phagolysosomal marker.

FIG 3.

Multidimensional images showing the differentiation and multiplication of T. cruzi within L. amazonensis PVs. (A and B) RAW 264.7-GFP macrophages (green) were infected with L. amazonensis and superinfected with T. cruzi-DsRed metacyclics (MTs) (red). The acquisition of images started after 2 h of superinfection with T. cruzi, and the time shown refers to image acquisition periods represented as days (d) hours:minutes. The upper row shows the DIC channel merged with the red fluorescent channel; the lower row shows the merged green and red fluorescent channels. (A) T. cruzi MT (red; marked by an arrowhead) forms differentiate into rounded shapes, similar to amastigotes (time point 0 d, 14 h, 0 min) within the L. amazonensis vacuole. Bar, 10 μm. (B) After its differentiation into a rounded form, T. cruzi was observed in a binary fission event, dividing as rounded forms (time point 3 d, 12 h, 0 min) retained within a chimeric PV (arrowhead). Bar, 5 μm.

FIG 4.

Differentiation of a T. cruzi MT form (arrowhead) is triggered within Rab7-positive PVs in monoinfected cultures of HeLa cells. The sequential images show T. cruzi CL strain metacyclics (MTs) expressing GFP (green), internalized by HeLa cells transfected with Rab7-RFP (red). A typical MT form of T. cruzi-GFP (flagella indicated by arrows) was observed within a Rab7-positive PV and tracked overnight by multidimensional image acquisition. The parasite remained within this vacuole during its morphological differentiation into a rounded form, inferred as an amastigote (time point 09 h, 55 min). At time point 10 h, 35 min, the Rab7 signal surrounding the parasite was lost. The sequential images are arranged by differential interference contrast (DIC) channel (first row), gray-scaled green channel (GFP, second row), gray-scaled red channel (Rab7-RFP, third row), and merged green and red channel (GFP merged with Rab7-RFP signal). The acquisition of images started after 2 h of monoinfection with T. cruzi, and the time shown refers to image acquisition periods represented as hours:minutes. Bar, 5 μm.

T. cruzi amastigotes within chimeric PVs increase in number during coinfection with L. amazonensis.

In order to quantify the number of T. cruzi amastigote forms present within chimeric vacuoles at longer time periods after coinfection, we employed the same coinfection protocol using BMDMΦ that can be cultured for several days in vitro. Taking into account only coinfected macrophages, we quantified the number of metacyclic, intermediary, amastigote, and trypomastigote forms inside and outside L. amazonensis PVs for up to 7 days after coinfection with T. cruzi GFP-tagged metacyclic forms (Fig. 5A). Considering the number of T. cruzi forms found within chimeric PVs (Fig. 5A, upper graph), a statistically significant increase in T. cruzi amastigote population in parallel with the decrease in metacyclic numbers was observed after 4 days of coinfection, which infers that T. cruzi differentiation took place in L. amazonensis PVs. A transient increase in intermediary forms at day 3 postcoinfection also suggests that the increase in T. cruzi amastigote numbers inside L. amazonensis PVs is due to differentiation within this compartment. There is no statistical significance in the numbers of T. cruzi amastigotes between days 4, 5, and 7 postcoinfection, suggesting a limited amastigote multiplication inside chimeric PVs and also a limited entrance of these amastigotes into L. amazonensis PVs at later time points.

FIG 5.

Differentiation and multiplication of T. cruzi-GFP within chimeric PVs developed in bone marrow-derived macrophages (BMDMΦ). (A) Quantification of the number of amastigote, intermediary, metacyclic, or trypomastigote forms of T. cruzi present in coinfected BMDMΦ according to their location. The upper graph shows the number of T. cruzi amastigotes found inside chimeric PVs, while the lower graph shows the quantification of T. cruzi found outside them. There is an increase in the number of T. cruzi amastigotes inside or outside L. amazonensis PVs after 4 days of coinfection (*, P < 0.05 by ANOVA), although the differences found after that time point are not statistically significant. (B) Multidimensional live imaging of BMDMΦ coinfected for 4 days and imaged for an additional 2 days. The upper row shows a sequence of images of the same coinfected BMDMΦ at different time points; pictures are a composite of GFP signal from T. cruzi and DIC. The lower row shows only the GFP signal from the same monitored BMDMΦ. Arrowheads indicate a dividing amastigote, and arrows point to trails and vesicles released by T. cruzi amastigotes The acquisition of images started after 4 days of coinfection and the time shown refers to image acquisition periods represented as hours:minutes. Bar, 5 μm.

Similar dynamics of T. cruzi amastigote population found outside chimeric PVs was observed (Fig. 5A, lower graph): there is an increase in T. cruzi amastigote numbers in parallel with a decrease in metacyclic numbers at day 4 postcoinfection, and from this time point on, the increase in amastigote numbers is not statistically significant.

To check whether multiplication or transfer of T. cruzi amastigotes occurs within chimeric PVs in these long-term coinfected BMDMΦ, we followed the fate of T. cruzi-GFP inside L. amazonensis PVs by live imaging, started after 4 days of T. cruzi metacyclic administration and performed for an additional 2 days (Fig. 5B; also see Movie S5 in the supplemental material). The multidimensional imaging of coinfected BMDMΦ revealed no apparent destruction of T. cruzi within chimeric PVs and a very limited flux of entry/exit of T. cruzi into/from L. amazonensis PVs (data not shown). However, it was possible to observe T. cruzi amastigote multiplication within chimeric PVs (Fig. 5B, arrowhead; also see Movie S5), as also demonstrated in RAW 264.7 macrophage-like cells. Interestingly, T. cruzi amastigotes clearly appear to release trails and vesicles (80) inside the Leishmania PV (Fig. 5B arrows; also see Movie S5). Amastigote multiplication also was inferred by the increase in fluorescence intensity (GFP or DsRed) and in volumetric dimensions presented by some T. cruzi amastigotes in around 10% of chimeric vacuoles. These features can be interpreted as division processes (fully or partially accomplished) without the complete separation of multiplying amastigotes. The observation is compatible with the slow kinetics of growth in macrophages (primary and RAW 264.7) of the fluorescence-tagged T. cruzi strain employed in this study.

DISCUSSION

Coinfection with nonviral pathogens provides an additional tool to investigate monoinfection paradigms. Given the numerous available intracellular pathogens and the vast number of questions to be answered regarding their interaction with host cells, how is one to select the most promising pair for coinfection? Thanks to their exceptionally large, fusogenic, and hospitable pathogen-containing PVs, the bacterium Coxiella burnetii and the protozoan L. amazonensis have been extremely useful in experiments involving chimeric vacuoles.

Coxiella burnetii is an obligate intracellular bacterium that replicates within a spacious, pathogen-containing vacuole (PcV) with phagolysosomal features (16). The accommodating characteristics of the Coxiella vacuole drove Veras et al. to coinfect cells already harboring C. burnetii with L. amazonensis, which also lives inside an expanded, phagolysosome-like acidified vacuole. Other examples of C. burnetii coinfection models involve different bacterial partners, such as Mycobacterium avium, Mycobacterium tuberculosis, Legionella pneumophila, and Salmonella enterica serovar Typhimurium (23, 24, 39, 46, 81), as well as protozoan partners, such as Toxoplasma gondii and Trypanosoma cruzi. In regard to coinfections of C. burnetii with protozoan parasites, the coinfection of Toxoplasma gondii and Coxiella contrasted with that of L. amazonensis and Coxiella (15), since the former did not result in vacuole fusion (27). Conversely, T. cruzi displayed different fusogenic capacities with Coxiella vacuoles, depending on the parasite developmental form analyzed. Kinetic studies on the transfer of T. cruzi trypomastigote forms to Coxiella vacuoles indicated that the PVs of metacyclic trypomastigotes are more fusion prone to C. burnetii vacuoles than tissue culture-derived trypomastigote vacuoles (41). Based on the fusogenic characteristic of T. cruzi metacyclic vacuoles, an additional study investigated the fate of metacyclic trypomastigotes within C. burnetii vacuoles, revealing that after 12 h postinfection, metacyclic trypomastigotes began to differentiate into amastigotes that replicated and became the prevailing form within C. burnetii vacuoles (35). In addition, a study comparing the ability of two T. cruzi strains to invade cells harboring C. burnetii and escape from their vacuoles suggested that despite a contrasting difference in their invasion capabilities, these strains behaved similarly once inside the host cell (47).

Besides the wide use of C. burnetii in coinfection assays, other pathogen combinations also have been tested. The protozoan parasites T. cruzi and T. gondii were used to coinfect cells; however, in the few cells that were found to be coinfected, none displayed pathogen colocalization in the same vacuole (8). In addition, although the C. burnetii vacuole was able to fuse with M. avium and L. amazonensis vacuoles in independent coinfection assays, colocalization was rarely found between M. avium and L. amazonensis vacuoles when these pathogens were used to coinfect cells (23). Another study reported that phagosomes containing live Listeria monocytogenes rapidly fused with Leishmania mexicana vacuoles, while those containing latex beads or heat-killed L. monocytogenes failed to do so. This study reported that the absence of fusion between these species correlated with the acquisition of annexin I on the phagosome membrane (17), shedding some light on the mechanism driving the formation of chimeric vacuoles. Moreover, coinfection with two Leishmania species revealed that the fusion of PVs containing Leishmania major and L. amazonensis did not occur if cells were infected with amastigotes but did occur when cells were infected by L. major promastigotes. Although L. major promastigotes remained motile and multiplied in these chimeric vacuoles, they did not differentiate into amastigotes (62). These results indicate that vacuoles customized by L. major amastigotes or promastigotes differ in their ability to fuse with L. amazonensis vacuoles (62). More importantly, it was demonstrated that a species-specific PV is required for the normal differentiation of infective promastigotes into amastigotes.

However, this did not seem to apply to T. cruzi hosted within L. amazonensis PVs. Although we have previously shown that living, motile trypomastigotes could be found within cohabiting L. amazonensis PVs, we did not record their differentiation within chimeric vacuoles at the time (82). Now, using multidimensional imaging and parasites constitutively expressing fluorescent proteins, we were able to follow not only the passage of metacyclic forms of T. cruzi into L. amazonensis PVs but also their differentiation into round forms suggestive of amastigotes. Interestingly, during image acquisition, we observed the multiplication of these amastigote-like forms in some cases and we did not record their escape from L. amazonensis PVs into macrophage cytosol, which suggests that this particular feature of T. cruzi intracellular infection depends on a parasite-specific PV.

Although we were able to demonstrate the multiplication of T. cruzi amastigotes within a phagolysosome-like vacuoles developed by L. amazonensis, the precise quantification of multiplying T. cruzi amastigotes is still a challenging task. T. cruzi amastigotes are not synchronized in their division cycles, and in the large majority of recorded chimeric PVs, amastigotes could be observed for up to 72 h of image acquisition without dividing. Additionally, after binary fission, amastigotes remain close to each other for several hours, which could lead to the underestimation of the quantification of multiplying parasites. The separation and clear distinction of dividing amastigotes are easily observed in T. cruzi singly infected cells, especially adherent cells with a flatter cytosol. However, in the midst of spacious L. amazonensis PVs, dividing T. cruzi amastigotes could remain associated for longer time periods, moving along wide three-dimensional coordinates, making them difficult to track and even to classify them as dividing parasites. We restricted our classification of dividing T. cruzi amastigotes to those fluorescent parasites which clearly separate from each other after binary fission, a feature observed in few multidimensional images.

Another interesting finding of the present coinfection model was the demonstration that a phagolysosomal-like intracellular compartment is where the differentiation of metacyclic forms into amastigote forms of T. cruzi occurs. However, the exact developmental form of T. cruzi that escapes from the vacuole and whether differentiation takes place in the transient (83) vacuole or in the cytosol remain controversial. It has been demonstrated that trypomastigotes of T. cruzi were able to induce the formation of membrane pores on host cells. This was due possibly to either a secreted, membrane pore-forming factor, which increases parasite internalization via host cell membrane repair machinery (84), and/or another secreted factor, active at low pH, that could be responsible for the disruption of a transient phagolysosome-like vacuole (72). Evidence suggests that the latter factor is secreted by trypomastigotes and amastigotes at low pH, and this indicates that vacuolar disruption and escape to the cytosol occurs prior to differentiation into amastigote forms. However, disrupted phagolysosome-like vacuoles can be observed surrounding rounded and intermediate forms, suggesting that T. cruzi escapes from PVs as amastigote forms (83).

We have demonstrated that trypomastigotes can differentiate into amastigotes inside vacuoles because of the following: (i) metacyclic trypomastigotes differentiated into round parasites lacking visible flagella once inside the acidic phagolysosome-like PV formed by L. amazonensis, and (ii) differentiation into round forms was observed dynamically within individualized Rab7-positive vacuoles in the first hours of T. cruzi single infection prior to the disappearance of this lysosomal marker surrounding parasites (suggestive of vacuole disruption), corroborating previous results in the literature (83). Considering that around 15% of chimeric vacuoles are found in the first 2 h of infection and that this percentage does not change during further scored time periods, we have indirect evidence that T. cruzi parasites are transferred to L. amazonensis PVs by vacuolar fusion.

The nature of the T. cruzi vacuoles formed after coinfection is also relevant for understanding their fusogenic capabilities and the fate of this parasite inside phagolysosome-like vacuoles. In experiments employing tissue culture trypomastigotes, T. cruzi lysosome-dependent internalization induces host cell membrane damage, which is repaired via exocytosis and/or recruitment of lysosomes to the plasma membrane; this lysosome-mediated membrane repair promotes the accumulation of ceramide at the sites of invasion, facilitating trypomastigote internalization by an inward vesicle formation of host cell plasma membrane (84, 85). The exocyst complex, exocytosis machinery involved in membrane fusion between post-Golgi vesicles and the plasma membrane, also was detected at the sites of membrane damage and in nascent trypomastigote PVs (around 20 min postinfection) (86).

Recently, Cortez et al. demonstrated, using HeLa cells as host cells, that lysosome availability, either by biogenesis or scattering, increased the lysosome-dependent invasion of T. cruzi metacyclic forms, which are lodged in intracellular PVs that display phagolysosomal markers (87). The harboring of metacyclic forms within phagolysosome-like transient vacuoles also could facilitate the homotypic fusion with phagolysosome-like L. amazonensis PVs before T. cruzi escape to the cytosol; such homotypic fusion is promoted by effectors, such as Rab7, present in both donor and recipient vacuole membranes (88, 89). The presence of exocyst complex in early metacyclic T. cruzi PVs as a consequence of host cell repair in the process of invasion and its participation in their fusion with L. amazonensis PVs remains to be elucidated.

It is possible, however, to speculate that the depletion of secondary lysosomes (90) and acidic vesicles after 14 h (73) in BMDMΦ infected with L. amazonensis impairs not only T. cruzi metacyclic lysosome-dependent infection but also the consequent biogenesis of metacyclic PVs and their fusogenic properties. The macrophage phagocytic machinery and low availability of lysosomes in L. amazonensis-infected macrophages would induce T. cruzi metacyclic internalization by a lysosome-independent pathway. In this case, the parasite is internalized by invagination of the plasma membrane in a process mediated by the accumulation of phosphatidylinositol triphosphate (PIP3), which will delay fusion events between the PV and lysosomes compared to that with lysosome-dependent internalization (91). This delay in PV maturation could render metacyclic-containing PVs more fusogenic to L. amazonensis PVs, considering that chimeric PV formation is restricted to the first hours of coinfection. Although these considerations highlight the complexity in interpreting coinfection models, they provide novel insights for experimentally approaching the T. cruzi requirement of lysosomes and phagolysosome-like environments for establishment within host cells.

Basic, in vitro studies are important in understanding the functions of individual molecules and their interactions, thus clarifying mechanisms that would be obscure in more complicated, in vivo systems. In vitro coinfections can increase our understanding of the interactions of different pathogens within the complex setting of a larger host. As our technological capabilities expand, our ability to deal with complex interactions between living organisms should follow the same trend, and we should be able to graduate from studies of one-to-one relationships to combinational and web-like interactions, slowly progressing toward a broad, physiologically accurate understanding. Here, we present and review in vitro coinfection models, highlighting their potential to contribute to understanding the biology of the host cell-pathogen relationship. Thus, we provide a robust experimental example of how coinfection (taking advantage of spacious vacuoles formed by one of the partners) allowed for the demonstration of T. cruzi amastigote multiplication inside a phagolysosome-like vesicle. The precise localization of the site where T. cruzi differentiates intracellularly is crucial for the development of new, optimized drug delivery systems for the treatment of Chagas' disease, which emphasizes the usefulness and importance of in vitro coinfections for the parasitology research field.