Elevated baseline expression of seven virulence factor RNA transcripts in visceralizing species of Leishmania: a preliminary quantitative PCR study.

Introduction: Leishmaniasis is a neglected tropical disease that manifests as three major disease phenotypes: cutaneous, mucocutaneous, and visceral. In this preliminary study, we quantified virulence factor (VF) RNA transcript expression in Leishmania species, stratified by geographic origin and propensity for specific disease phenotypes.
Methods: Cultured promastigotes of 19 Leishmania clinical and ATCC isolates were extracted for total cellular RNA, cDNA was reverse transcribed, and qPCR assays were performed to quantify VF RNA transcript expression for hsp23, hsp70, hsp83, hsp100, mpi, cpb, and gp63.
Results: Comparison of visceralizing species (Leishmania donovani, Leishmania chagasi, and Leishmania infantum) versus non-visceralizing species [Leishmania (Viannia) spp., Leishmania tropica, Leishmania major, Leishmania mexicana, and Leishmania amazonensis] revealed a significantly greater pooled transcript expression for visceralizing species (p = 0.0032). Similarly, Old World species demonstrated significantly higher VF RNA transcript expression than New World species (p = 0.0015). On a per-gene basis, species with a propensity to visceralize ubiquitously expressed higher levels of gp63 (p = 0.005), cpb (p = 0.0032), mpi (p = 0.0032), hsp23 (p = 0.0039), hsp70 (p = 0.0032), hsp83 (p = 0.0032), and hsp100 (p = 0.0032).
Conclusion: Here, we provide quantitative, preliminary evidence of elevated VF RNA transcript expression driven largely by the visceralizing causative species of Leishmania. This work highlights the extensive heterogeneity in pathogenicity mechanisms between Leishmania species, which may partly underpin the fatal progression of visceral leishmaniasis.

Introduction

Leishmaniasis is a vector-borne neglected tropical disease (NTD) that manifests as three major clinical syndromes: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and mucocutaneous/mucosal leishmaniasis (ML). Caused by over 20 species of protozoan parasites from the Leishmania genus, the disease presents with a wide array of symptoms that are influenced by both species-specific and host immunological characteristics.[1,2] Leishmaniasis is transmitted by the bite of over 90 species of female Phlebotomus and Lutzomyia sand flies, with over 70 animal species confirmed to be natural reservoir hosts. The World Health Organization (WHO) estimates an annual new case burden of 700,000–1 million, resulting in 20,000–30,000 deaths each year from all forms of leishmaniasis. Of that substantial global disease burden, VL is estimated to account for about 50,000–90,000 new cases per year.

Widely recognized as a disease of poverty, leishmaniasis is endemic to many subtropical and tropical parts of the world.[2,3] VL, in particular, is considered to be the most deadly form as parasites are able to evade host immune clearance and metastasize to internal organs.[1,4] Nearly 147 million people are at risk of contracting VL in Southeast Asia alone. VL is endemic to parts of Southern Europe, North Africa, and West and Central Asia, with the Indian subcontinent suffering the brunt of the disease burden in pediatric populations aged 1–4 years. VL has a wide but recognizable range of presenting symptoms including fever, anorexia, weight loss, abdominal distension, extreme weakness, and hepatosplenomegaly. Of note, Leishmania-HIV co-infection is known to increase the overall susceptibility to VL in endemic regions, as well as the rate of treatment failure and relapse.[1,2] Symptomatic VL, if left untreated, can lead to death within 2–3 years due to severe wasting, multiorgan dissemination, hemorrhagic diathesis, and secondary infections. As such, recent efforts by The Special Programme for Research and Training in Tropical Diseases, hosted by the WHO, have begun to shift focus onto regional VL elimination initiatives in the Indian subcontinent. Conversely, ML is exclusively found in Latin America with a significant burden in Brazil, Peru, and Bolivia.

VL, ML, and CL are each caused by different species in the Leishmania genus, with some species displaying multiple clinical presentations.[1,2] VL is often caused by Leishmania donovani, Leishmania infantum, and Leishmania chagasi; ML is caused by Leishmania Viannia braziliensis, L. V. panamensis, and L. V. guyanensis, largely found in Latin America; CL is caused by a large array of both New World (NW) and Old World (OW) species, some of which include Leishmania mexicana, Leishmania amazonensis, Leishmania tropica, and Leishmania major.[1,3]

Virulence factors (VFs) are components of a pathogen that facilitate its ability to inflict disease in a host but are often not requisite for general viability.[5,6] VFs can increase disease capability of the Leishmania parasite by increased expression, host cell invasion and stress tolerance, and modulation of the host immune system.[5,6] VFs have been identified across a wide range of Leishmania species, many of which are often ubiquitously expressed across species.[5,6] VF RNA transcripts have been shown to increase post-macrophage infection in a study focusing on NW species of the Viannia subgenus. To add, some Leishmania VFs include molecular chaperones such as heat-shock proteins (HSP), which aid in pathogen temperature tolerance;[5-11] cysteine peptidases (e.g. CPB), which are known to have immunomodulatory properties;[5,6,12-14] metallopeptidases (e.g. GP63), which protects the parasite against serum-mediated opsonization and cleaves antimicrobial peptides (AMP);[5,6,15] isomerases (e.g. MPI), which are required for high-demand glycoconjugate synthesis involved in the biosynthesis of other VFs such as GP63, lipophosphoglycan (LPG), and glycoinositolphospholipids (GIPL).[5,6,16] Endogenous VFs in Leishmania contribute to the pathogenicity of the parasite, but there are more immunopathogenic mechanisms as well.

Localized and organ-specific immunogenic ‘tropisms’ are a common theme in VL models.[17-19] Compared to vector and host determinants of VL development, parasite-specific determinants are often what differentiate VL from ML and CL. Parasite-specific determinants of VL development include differences in species-specific genes, polymorphisms, pseudogenes, and VF expression levels (both at the transcriptional and translational levels). Here, we quantify relative baseline VF RNA transcript abundance in both NW and OW Leishmania species, across the following VFs: gp63, cpb, mpi, hsp 23, hsp 70, hsp 83, and hsp 100, and compared across species by clinical phenotype propensities (VL versus non-VL), in order to elucidate emergent patterns in parasite-specific VL determinants.

Materials and methods

Cultured Leishmania spp

Cultured isolates of Leishmania were obtained from the American Type Culture Collection® (ATCC®) and the Public Health Ontario Laboratory (Toronto, Canada) Leishmania biobank. The following species of Leishmania were used: ATCC® strains of Leishmania Viannia braziliensis ATCC®50135™ (MHOM/BR/75/M2903), L. (V.) guyanensis ATCC®50126™ (MHOM/BR/75/M4147), L. (V.) panamensis ATCC®50158™ (MHOM/PA/71/LS94), L. amazonensis ATCC®50159™ (IFLA/BR/67/PH8), L. chagasi Cunha and Chagas ATCC®50133™ (MHOM/BR/74/PP75), L. donovani (Laveran and Mesnil) Ross ATCC®50212™ (MHOM/IN/80/DD8), L. infantum Nicolle ATCC®50134™ (MHOM/TN/80/IPT-1), L. major ATCC®50122™ (MHOM/IL/67/JERICHO II), L. mexicana (Biagi) Garnham ATCC®50157™ (MHOM/BZ/82/BEL21), and L. tropica (Wright) Luhe ATCC®50129™ (MHOM/SU/74/K27) and nine clinical strains including L. (V.) braziliensis (n = 1), L. (V.) panamensis (n = 4), L. tropica (n = 3), and L. infantum (n = 1) (Table 1). Age and sex of the host were collected, where possible. A full list of isolates and de-identified clinical characteristics is found in Table 1 (n = 19). Promastigotes were regularly subcultured in Tobie’s medium with Locke’s overlay at ambient room temperature on a weekly basis. For the purposes of RNA extraction standardization, cultures were counted using a hemocytometer.

Table 1.

Characteristics of cultured clinical and ATCC isolates of Leishmania..

ID No.	Species	Source of isolate	Passage number	Sex of patient	Age of patient	VL or non-VL	Geographic location
1	L. chagasi	ATCC® 50133™	P2	Unknown	Unknown	VL	New World
2	L. infantum	Clinical 1	P2	Male	74	VL	Old World
3	L. infantum	ATCC® 50134™	P3	Unknown	Unknown	VL	Old World
4	L. donovani	ATCC® 50212™	P2	Unknown	Unknown	VL	Old World
5	L. major	ATCC® 50122™	P3	Unknown	Unknown	Non-VL	Old World
6	L. tropica	ATCC® 50129™	P6	Unknown	Unknown	Non-VL	Old World
7	L. tropica	Clinical 2	P0	Male	33	Non-VL	Old World
8	L. tropica	Clinical 3	P7	Male	10	Non-VL	Old World
9	L. tropica	Clinical 4	P0	Unknown	Unknown	Non-VL	Old World
10	L. V. braziliensis	ATCC® 50135™	P13	Unknown	Unknown	Non-VL	New World
11	L. V. braziliensis	Clinical 5	P12	Male	22	Non-VL	New World
12	L. V. panamensis	ATCC® 50158™	P12	Unknown	Unknown	Non-VL	New World
13	L. V. panamensis	Clinical 6	P12	Male	80	Non-VL	New World
14	L. V. panamensis	Clinical 7	P8	Male	17	Non-VL	New World
15	L. V. panamensis	Clinical 8	P9	Male	9	Non-VL	New World
16	L. V. panamensis	Clinical 9	P3	Male	71	Non-VL	New World
17	L. V. guyanensis	ATCC® 50126™	P20	Unknown	Unknown	Non-VL	New World
18	L. mexicana	ATCC® 50157™	P2	Unknown	Unknown	Non-VL	New World
19	L. amazonensis	ATCC® 50159™	P6	Unknown	Unknown	Non-VL	New World

VL, visceral leishmaniasis.

Leishmania species identification and confirmation

DNA was extracted using QIAamp DNA Mini Kit Blood (Qiagen, Germantown, MA, USA). Leishmania genus 18S real-time polymerase chain reaction (PCR) was performed as previously described. Species identification included analysis of the internal transcribed spacer 1 (ITS1), ITS2, CPB, HSP70, and MPI targets by PCR, restriction fragment length polymorphism (RFLP) analysis, and Sanger sequencing.[21,22] PCR-RFLP analysis of the ITS1 region can only differentiate L. V. braziliensis from the other species within the Viannia subgenus (L. V. guyanensis, L. V. peruviana, L. V. panamensis, L. V. lainsoni). Thus, PCR-RFLP and sequencing analysis of the CPB, HSP70, MPI, and ITS2 regions were required to differentiate species within the Leishmania Viannia subgenus and to provide a confirmation of the species identified in the initial ITS1 assay. Purified PCR product was used for Sanger sequencing as per Big Dye protocol (Life Technologies, Carlsbad, CA, USA). Sequence products were purified and analyzed using the Applied Biosystems 3130xl Genetic Analyzer. Data were standardized using the Sequencing Analyzer program and the BLAST search engine was used to analyze sequences.

RNA extraction, cDNA synthesis, and purification

RNA was extracted from 0.5 ml of baseline pure culture (up to 5 × 105 promastigotes) using QIAmp RNA Mini Kit (Qiagen, Germantown, MA, USA). In-column DNase treatment was included in all extractions as per manufacturer’s protocol. cDNA was synthesized using Superscript II Reverse Transcriptase and random hexamers (Life Technologies, Carlsbad, CA, USA), followed by purification with QIAquick PCR Purification Kit (Qiagen, Germantown, MA, USA) and eluted with 60 µl of nuclease-free water. cDNA aliquots were stored at −20°C until quantitative real-time polymerase chain reaction (qPCR) assays were conducted.

VF RNA transcript expression qPCR assays

In this study, VFs are defined as proteins associated with disease severity in the Leishmania literature.[5-16] Transcript expression of the following VFs: hsp23, hsp70, hsp90, hsp100, mpi, cpb, gp63, and 18S, was performed on the ABI 7900HT real-time instrument using previously published primers and probes. A real-time qPCR was set up in triplicate using 12.5 µl 2× Taqman Universal Master Mix (ThermoFisher Scientific, Carlsbad, CA, USA), 250 nM final concentration of forward and reverse primers, 10 nM probe, and 5 µl of cDNA from pure culture, in a total volume of 20 µl for each respective target. Amplification was performed with the following conditions: Uracil DNA-glycosylase (UDG) activation at 50°C for 2 min, polymerase activation at 95°C for 10 min, followed by 45 cycles of 95°C for 15 s, and 60°C for 1 min.

Expression quantification and statistical analysis

Transcript expression was quantified using the 2–ΔCt method (a modified version of the 2–ΔΔCt method, i.e., relative abundance of target versus reference housekeeping gene 18S).[7,23] Expression values of all seven VFs were added together to give a ‘pooled VF’ quantification. Transcript expression values were grouped into four categories, for which categorical hypothesis testing was performed: VL versus non-VL, and OW versus NW (Table 1). Before statistical testing, all groupings were screened for normality via the D’Agostino and Pearson Normality Test. Normally distributed groupings underwent parametric unpaired t-tests and, non-normal groupings underwent non-parametric Mann–Whitney U-test, on a per-gene and pooled basis. A log transformation was performed to better graphically visualize the data (Figures 1 and 2). All groupings for categorical statistical analysis are outlined in Table 1. All statistical analyses were conducted using GraphPad Prism v5.04 software (GraphPad Software Inc, La Jolla, CA). Significance was set at p < 0.01.

Figure 1.

VF RNA transcript expression in visceralizing versus non-visceralizing species.

Log transformed, and 2–ΔCt (i.e. relative abundance of VF target against reference 18S) quantified VF RNA transcripts represented as box plots (median with maximum and minimum whiskers) between VL and non-VL groupings of species compared by t-test. For the VL versus non-VL comparison, with the following VFs: (a) pooled (p = 0.0032), (b) gp63 (p = 0.005), (c) cpb (p = 0.0032), (d) mpi (p = 0.0032), (e) hsp23 (p = 0.0039), (f) hsp70 (p = 0.0032), (g) hsp83 (p = 0.0032), and (h) hsp100 (p = 0.0032). *p < 0.01. VF, virulence factor; VL, visceral leishmaniasis.

Figure 2.

VF RNA transcript expression in old world versus new world species of Leishmania.

Log transformed, and 2–ΔCt (i.e. relative abundance of VF target against reference 18S) quantified VF RNA transcripts represented as box plots (median with maximum and minimum whiskers) between OW and NW groupings of species compared by t-test. For the NW versus OW comparison, with the following VF transcripts: (a) pooled (p = 0.0015), (b) gp63 (p = 0.2149), (c) cpb (p = 0.0093), (d) mpi (p = 0.302), (e) hsp23 (p = 0.9626), (f) hsp70 (p = 0.0015), (g) hsp83 (p = 0.0057), and (h) hsp100 (p = 0.002). *p < 0.01. VF, virulence factor; VL, visceral leishmaniasis.

Results

Clinical and demographic characteristics

Where available, median age was 22 years old (range, 7–80 years), with isolates being derived from predominantly male hosts (Table 1). Three of 19 (16%) isolates were derived from pediatric patients (age <18), 5 of 19 (26%) isolates were derived from adult patients (age >18), and 10 of 19 (53%) ATCC® isolates were from an unknown host origin (Table 1). Eight of 19 (42%) patients were male, and the rest of the isolates were derived from an unknown host sex (Table 1).

VF RNA transcript expression: VL versus non-VL species

A significant difference was found between VL and non-VL species on a per-gene and pooled basis among all VFs. On a pooled VF basis, all VL species consistently and significantly expressed higher VF RNA transcript levels than non-VL species (p = 0.0032) (Figure 1(a)); moreover, the mean pooled VF transcript expression for all VL species was 159-fold higher than the mean pooled VF transcript expression for all non-VL species. Across all analyzed VF transcripts, VL species expressed significantly greater RNA transcripts than non-VL species: gp63 (79.2-fold; p = 0.005) (Figure 1(b)), cpb (556-fold; p = 0.0032) (Figure 1(c)), mpi (78.5-fold; p = 0.0032) (Figure 1(d)), hsp23 (12.9-fold; p = 0.0039) (Figure 1(e)), hsp70 (178-fold; p = 0.0032) (Figure 1(f)), hsp83 (107-fold; p = 0.0032) (Figure 1(g)), and hsp100 (84.5-fold; p = 0.0032) (Figure 1(h)). The following VFs could not be quantified in the following species: gp63 could not be amplified from L. tropica specimens, and hsp23 could not be amplified from either L. mexicana or L. amazonensis.

VF RNA transcript expression, Old World versus New World species

A significant difference was found between OW and NW species on a per-gene and pooled basis among most VF transcripts. On a pooled VF basis, OW species significantly expressed higher VF RNA transcript levels than NW species (6.24-fold; p = 0.0015) (Figure 2(a)). Across most analyzed VFs, OW species expressed significantly greater RNA transcripts than NW species: gp63 (17.8-fold; p = 0.2149) (Figure 2(b)), cpb (4.29-fold; p = 0.0093) (Figure 2(c)), mpi (9.01-fold; p = 0.3020) (Figure 2(d)), hsp23 (2.83-fold; p = 0.9626) (Figure 2(e)), hsp70 (6.84-fold; p = 0.0015) (Figure 2(f)), hsp83 (5.26-fold; p = 0.0057) (Figure 2(g)), and hsp100 (4.45-fold; p = 0.0020) (Figure 2(h)). Much of these differences are largely driven by the VL-causative species, when taking both Figures 1 and 2 into account.

Discussion

The immense mechanistic diversity with which the Leishmania parasite infects its hosts, along with the large range of clinical presentations, makes the pathogenesis quite complex.[1,5,15] For the development of VL, parasites must (1) traverse from the skin to visceral organs and (2) survive the host environment. Some parasitological factors for the development of VL are as follows: species-specific genes, pseudogenes, gene mutations and polymorphisms, and most importantly, the expression of VFs. Our preliminary data suggest that higher baseline levels of VF RNA transcripts are significantly correlated with L. donovani, L. infantum, and L chagasi, which may partly explain the aggressive and fatal development of VL. Recent evidence suggests that even within a given species, geographically distinct populations have extremely divergent genomic and metabolomic pathogenesis mechanisms, particularly in the Indian subcontinent (ISC1) L. donovani population.

The ability to tolerate host temperature differentials is crucial for survival and infection of Leishmania.[5-11] Molecular chaperones, such as HSPs, are crucial for maintaining cellular homeostasis in infective thermotolerance.[5-11] Under heat stress, which invariably occurs during the process of mammalian infection, HSP23 and HSP70 co-localize, upregulating their expression to meet host temperature requirements.[7-9] HSP70 is the most conserved protein in all organisms, and plays essential roles in prevention of peptide denaturation in Leishmania, which is especially important in the high-temperature mammalian host environment.[5,9] HSP83 (an HSP90 homolog) facilitates both virulence via drug resistance and thermotolerance.[9,11] HSP100 is involved in the refolding of misfolded proteins, particularly under environmentally stressed conditions.[9,10] Our preliminary study quantified the RNA transcripts for hsp 23, 70, 83 (90), and 100 to better understand species-specific expression correlates, which may well elucidate clinical presentation propensities in the diverse pathogenesis of leishmaniasis. Our preliminary data suggest that for all four HSPs, VL-causative species of Leishmania consistently and significantly express larger quantities of baseline VF RNA transcripts. The substantial and significant 13-, 178-, 107-, and 85-fold higher levels of hsp23, 70, 83, 100, respectively, noted in the VL species investigated herein corroborates this differential. Furthermore, in this study, VL-causative species drove much of the difference between the OW and NW groupings. Therefore, there is potentially some speculative mechanistic underpinning to this correlation: L. donovani, L. infantum, and L. chagasi have such high baseline expression of all four HSPs to perhaps better infect thermally challenging environments in the internal organs of mammalian host organisms. For example, HSP23 plays a crucial role in adaptation to mammalian host temperatures in L. donovani, as it is significantly upregulated in amastigotes, as evidenced in both RNA and protein studies. For HSP100, the expression levels correlate to stage-specific roles, whereas HSP70 is often constitutively kept at high levels. Therefore, HSPs show a dual role in Leishmania pathogenesis: selective or universal. Hence, initially high levels of HSPs may expedite and enhance parasite propensity to produce the VL phenotype.

Cysteine peptidases, such as CPB, are VFs in Leishmania that are known to have significant cytokine immunomodulatory effects as well as parasite–host interaction regulation.[5,6,12-14] Three distinct genes exist: CPA, CPB, and CPC, all belonging to the same group designated Clan CA, Family C1. CPB has also been implicated in the regulation of other VFs’ expression, such as GP63. Zinc-dependent metalloprotease GP63 is a surface antigen expressed on all Leishmania species at the promastigote stage. It is involved in parasite adherence to macrophages and circumvention of complement-mediated lysis.[12,15] GP63 activates protein tyrosine phosphatases to reduce NO production, thus facilitating parasite persistence in the macrophage vacuole. Our preliminary data suggest that VL-causative species express 79- and 556-fold higher quantities of gp63 and cpb RNA transcripts at baseline, respectively. Since CPB is required for GP63-mediated parasite persistence in macrophages, our preliminary data’s similar correlation pattern for these two VFs supports their mechanistic contribution to pathogenesis in VL-causative species. Finally, MPI is an enzyme involved in the reversible conversion of fructose-6-phosphate and mannose-6-phosphate required for biosynthesis of various glycoconjugates. MPI is involved in constructing other VFs such as GP63, LPG, and GIPL.[5,6,16] Again, our preliminary data demonstrate mpi expression in 79-fold greater quantities in VL species than in other isolates, implying a possibly high demand for pre-infection glycoconjugate production. This increased baseline necessity for glycoconjugates may tie into the increased expression of gp63 transcripts.

Diverse expression of VF RNA transcripts is found within species with different propensity to cause a particular type of leishmaniasis. VL-causative species tend to express significantly greater amounts of all quantified VFs, when compared with both all non-VL species and NW species. L. donovani, L. infantum, and L. chagasi are distinct in how much VFs they express, even in a preinfection, pure culture, baseline model, such as ours. Moreover, only 0.2–5% of the Leishmania functional genome is differentially expressed between the amastigote and promastigote life stages; consequently, the Leishmania genome is largely constitutively expressed with a limited set of genes showing stage-specific expression patterns.[25,26] Some genomic evidence suggests that Leishmania amastigotes are pre-adapted for intracellular survival and experience little alterations in gene expression at the RNA level. Our preliminary data partially suggest this pre-adaption phenomena to be especially present in the VL species, L. donovani, L. infantum, and L. chagasi. However, this small number of differentially expressed genes may still be significant in predicting the clinical outcome of infection. Recently, Shadab et al. performed RNA-seq comparison in a murine macrophage model of virulent and avirulent L. donovani infection. This preliminary analysis demonstrated the vast differences in transcriptomic expression profiles between parasites, particularly macrophages infected with virulent L. donovani strains showed marked anti-inflammatory responses and dysregulation of protein synthesis. Furthermore, virulent L. donovani were shown to globally upregulate virulence and parasite survival factors.

Therefore, a proposed novel model for VL development, as compared with CL or ML, is that VL species (L. donovani, L. infantum, and L. chagasi) tend to ‘start off’ their preinfection (baseline) status at a significantly more ‘virulent’ state with large quantities of available VFs, ready for usage when a host is present, whereas non-VL species tend not to be in a ‘triggered’ virulence state and perhaps not as parasitologically primed for visceral infection. It may well be that the Viannia subgenus species are more prone toward disease modulation via more host immunological responses. This elevated level of ubiquitous VFs in VL-causative species may help ‘prime’ the parasite for a more efficient and effective visceral infection of the host organism. Seemingly, VFs are expressed in a species and stage-specific manner, as previously demonstrated by increased RNA transcripts pre- and post-macrophage infection. Hence, the uniquely severe pathogenesis of VL may partly be explained by elevated expression of VFs. This preliminary study requires extension and broader interrogation with larger sets of Leishmania species.

Limitations and next steps

Some confines for this work include the small number of cultured isolates and the fact that not all possible VF transcripts from all possible species were assessable (e.g. Gp63 for L. tropica and hsp23 for L. mexicana and L. amazonensis). Moreover, given limitations in biobank availability, some isolates were evaluated at different times of passage, which could have impacted our analysis and interpretation. When selecting VFs to target, those that tied directly to mechanistic virulence methods and were the most evidentially pathogenic were selected. This limitation may have biased some part of our interpretation of our data, but with likely minimal overall effect on our conclusions. We evaluated VF RNA transcript expression at baseline, but did not quantify protein expression; thus, it is unknown whether or not transcript abundance would correlate to protein abundance. This is especially crucial as much of the regulation in Leishmania occurs post-transcriptionally and even post-translationally.[23,25,26] Further validation of our hypothesis is required with a comprehensive genome-wide VF screen, macrophage, or other model, while taking the many immunomodulatory features of the host organism into account. Importantly, a more robust evaluation of multiple isolates from different species at the exact same time of passaging would be crucial for corroborating our findings from this preliminary work. It would also be interesting to characterize the energy requirements of high preinfection VF expression in VL Leishmania species in order to compare by propensity for particular clinical presentations.

In conclusion, we herein provide quantitative, preliminary evidence of elevated VF RNA transcript expression driven largely by the visceralizing causative species of Leishmania, which underscores the extensive heterogeneity in pathogenicity mechanisms between Leishmania species, and which may partly explain the fatal progression of visceral leishmaniasis.