Skip to main content
Download PDF

Lipophosphoglycan polymorphisms do not affect Leishmania amazonensis development in the permissive vectors Lutzomyia migonei and Lutzomyia longipalpis

Parasites & Vectors volume 10, Article number: 608 (2017) Cite this article

Abstract

Background

Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species.

Results

Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 β-glucose or 1 to 3 β-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi.

Conclusions

LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector.

Background

Leishmania amazonensis is the causative agent of localized cutaneous leishmaniasis (LCL) and anergic diffuse cutaneous leishmaniasis (ADCL). This species, found in the Amazon basin, is transmitted by Lutzomyia flaviscutellata (Diptera: Psychodidae), widely distributed in South America [1, 2].

The surface of Leishmania spp. is covered by several glycosylphosphatidylinositol (GPI)-anchored molecules. Those glycoconjugates are important for parasite survival either in vertebrate or invertebrate hosts. Among these, the most studied is lipophosphoglycan (LPG), a multivirulence factor covering the entire protozoan surface and flagellum. Structurally, LPGs have four domains: a conserved glycan core region of Gal(α1,6)Gal(α1,3)Galf(β1,3)[Glc(α1)-PO4]Man(α1,3)Man(α1,4)-GlcN(α1) linked to a 1-O-alkyl-2-lyso-phosphatidylinositol anchor, a region of repeat units Gal(β1,4)Man(α1)-PO4 and a small oligosaccharide cap [3].

Several biochemical analyses of LPG revealed intra and inter-species polymorphisms in the sequence and composition of sugars attached to repeat units. Early studies determined that these interspecies variations were important for attachment in the vector and for virulence in the vertebrate hosts [3, 4]. In the Old World species, intraspecific LPG variations were reported for Leishmania major [5, 6], Leishmania tropica [7, 8] and Leishmania donovani [9] and their ability to infect sand fly species (Phlebotomus papatasi, Phlebotomus sergenti/Phlebotomus arabicus and Phlebotomus argentipes) has been reported. In the New World, purified LPG from 14 strains of Leishmania infantum displayed important LPG polymorphisms (type I: no side-chains branching-off the repeat units; type II: one β-glucose residue branching-off the repeat units and; type III: 1–3 β-glucose as side-chains). However, those LPG variations did not affect the interaction with Lutzomyia longipalpis [10, 11]. Our preliminary data suggested qualitative LPG polymorphisms between two Le. amazonensis strains (PH8 and Josefa) based on antibody recognition, but they did not result in different activation in macrophages and/or sand fly infection [12].

Sand fly species can be divided into restrictive and permissive vectors depending on their ability to support development of various Leishmania species [13]. For example, Ph. papatasi, the restrictive vector, can sustain infection only with Le. major [4] and Leishmania turanica [14], two parasites with LPG terminated by β-galactosyl residues [15,16,17]. On the other hand, permissive vectors, like Lu. longipalpis and Lutzomyia migonei, can sustain infection of several Leishmania species; the former supports the development of Le. infantum, Le. amazonensis and Le. major (reviewed in [18]), whereas the latter supports the infection of Le. amazonensis, Leishmania braziliensis and Le. infantum [12, 19, 20].

The attachment of the parasite to a midgut receptor is a crucial event to avoid passage with the digested blood meal. The mechanisms of midgut attachment in restrictive vectors is LPG-dependent [21] and involves midgut galectin [22], while in permissive vectors it could be LPG-independent [23, 24] or may involve midgut mucin-like proteins [25].

The main vector of Le. amazonensis is Lu. flaviscutellata. However, established laboratory colonies of this species are not available. For this reason, Lu. migonei has been used as a successful model for interaction with Le. amazonensis [19]. Preliminary studies using Le. amazonensis strains (PH8 and Josefa) showed that they could survive for several days inside Lu. migonei [12]. However, their ability to reach the stomodeal valve and accomplish metacyclogenesis was not evaluated.

As a part of a wider project on the glycobiology of New World species of Leishmania, we described a detailed biochemical characterization of Le. amazonensis LPGs and found intraspecific differences. The interaction of Le. amazonensis strains with permissive and restrictive vectors was performed. Additionally, the ability of the Josefa strain, bearing terminal galactose residues in its LPG, to survive in the restrictive vector Ph. papatasi was evaluated.

Methods

Parasites, LPG extraction and purification

The Brazilian Le. amazonensis reference strain (IFLA/BR/1967/PH8) was isolated from the sand fly Lu. flaviscutellata from Pará State, whereas the Josefa strain (MHOM/BR/1975/Josefa) was isolated from a patient from Bahia State, Brazil. Molecular PCR identification was performed using primers for the HSP70 gene and kDNA minicircle [26, 27]. For immunoblotting, Le. donovani strain LD4 from Sudan (MHOM/SD/00/1S-2D) and the Le. donovani Mongi strain from India (MHOM/IN/1983/Mongi-142) were used as controls [9]. The strategy for purification and characterization of repeat units is depicted in Fig. 1a. Promastigotes were cultured in M199 medium supplemented with 10% fetal bovine serum (FBS). After the 6th day, stationary parasites were subjected to LPG extraction and purification as described elsewhere [28]. Purified LPGs (5 μg) were resolved by SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. Blots were probed with monoclonal antibody (mAb) CA7AE (1:1000), that recognizes the unsubstituted Gal(β1,4)Man repeat units [29] and LT22 (1:1000) that recognizes β-glucose/β-galactose side chains [30]. After washing in PBS (3 × 5 min), the membrane was incubated for 1 h with anti-mouse IgG conjugated with peroxidase (1:10,000) and the reaction was visualized using luminol substrate [31].

Fig. 1
figure 1

Procedures for the characterization of Le. amazonensis LPG repeat units and sand fly infections. a Purified LPGs were subjected to mild acid hydrolysis to depolymerize the repeat units and cap structures. Water-soluble fractions were partitioned using 1-butanol, treated with alkaline phosphatase (15 mM Tris buffer, pH 9.0, 1 unit, 16 h, 37 °C) and desalted by passage through a two-layered column of AG50W-X12 over AG1-X8. The desalted repeat units were subject to FACE analysis and enzymatic treatments with β-glucosidase, β-galactosidase to CE analysis. Additionally, the repeat units were subjected to strong acid hydrolysis (2 M trifluoroacetic acid, 3 h, 100 °C) and FACE assays to access monosaccharide composition. b Six combinations of Le. amazonensis strains (PH8 and Josefa) with Lu. longipalpis, Lu. migonei and Ph. papatasi were performed. Those were evaluated on days 1 and 5–6 post-infection (PI) for intensity, localization and morphometry

Full size image

Fluorophore-assisted carbohydrate electrophoresis (FACE) of polysaccharides and mono and capillary electrophoresis (CE)

LPGs were subjected to mild acid hydrolysis (0.02 M HCl, 5 min, 100 °C) to depolymerize the repeat units and cap structures (Fig. 1a). Water-soluble fractions were partitioned using 1-butanol and repeat units were purified as previously described [10]. The neutral oligosaccharides from PH8 and Josefa repeat units were labeled with 0.05 M ANTS (8-aminonaphthalene-1,3,6-trisulfate) fluorophore and 1 M cyanoborohydride (37 °C, 16 h). Oligoglucose ladders (G1-G7) were used as standards. Sugars were subjected to FACE and the gel was visualized by a UV imager [7]. To confirm the sugars branching-off the repeat units and the linkages, they were treated with E. coli β-galactosidase in 80 mM Na3PO4, (pH 7.3, 4 U, 16 h, 37 °C) and sweet almond β-glucosidase in 200 mM ammonium acetate buffer (pH 5.0, 1 U, 16 h, 37 °C) [31]. Samples were labeled with 0.02 M APTS (8-aminopyrene-1,3,6-trisulfonic acid trisodium salt) in 15% acetic acid in sodium cyanoborohydride buffer and incubated overnight at 37 °C. Samples were run on CE at 25 kV for 20 min using reverse phase chromatography started with 10 s and 5 psi pressure injection [11].

To access monosaccharide composition, LPGs from both strains were subjected to strong acid hydrolysis (2 M trifluoroacetic acid, 3 h, 100 °C) (Fig. 1a). Depolymerized and desalted monosaccharides were fluorescently labeled with 0.1 M AMAC (2-aminoacridone) in 5% acetic acid and 1 M cyanoborohydride. Labeled sugars were subjected to FACE and the gel was visualized under UV light. Monosaccharides (D-galactose, D-glucose and D-mannose) (Sigma, St. Louis, MO, USA) were used as standards [32].

Sand fly colonies

The Lu. migonei and Lu. longipalpis sand flies were initially captured from the Brazilian cities of Baturité (04°19′41"S, 38°53′05"W), Ceará State, and Jacobina (11°10′50"S, 40°31′06"W), Bahia State, respectively. Phlebotomus papatasi originate from South East Turkey. Laboratory colonies of the three sand fly species were maintained at Charles University, Prague, Czech Republic as previously described [33].

Experimental infections of sand flies

Sand fly females were infected through the chick-skin membrane on a mixture of promastigotes and heat-inactivated rabbit blood; the final concentration of parasites was 1 × 106 promastigotes/ml. The experiments were conducted with six sand fly-Leishmania combinations as depicted in Fig. 1b: Lu. migonei-PH8, Lu. migonei-Josefa, Lu. longipalpis-PH8 and Lu. longipalpis-Josefa, Ph. papatasi-PH8 and Ph. papatasi-Josefa. Terminal β-galactosyl residues were determinant for Le. major attachment to PpGalec in Ph. papatasi [22]. Since those sugars were also present in Josefa LPG, the ability of this strain to sustain infection in this vector was also checked compared to glucose-containing LPG from the PH8 strain.

Blood-engorged females were separated, maintained at 26 °C and dissected on days 1 and 5–6 post-infection (PI). Individual guts were placed into a drop of saline and examined microscopically for the localization and intensity of Leishmania infections. Parasite loads were graded according to [23] as light (< 100 parasites per gut), moderate (100 to 1000 parasites per gut) and heavy (> 1000 parasites per gut). The experiments were repeated two times using the same vector/strain combinations. Data were evaluated statistically by means of the Fisher’s exact or Chi-square (χ 2) tests using SPSS statistics version 23 software.

Morphometry

Parasite smears from midguts of the three sand fly species infected with Le. amazonensis strains were obtained on days 1 and 5–6 PI. The midguts were carefully dissected using fine needles and each part was separated and respective parasites counted. Slides were fixed with methanol, stained with Giemsa, examined under a light microscope with an oil-immersion objective and photographed with an Olympus D70 camera. For morphometry, body length, body width and flagellar length of 240 randomly selected promastigotes from six midgut smears were measured for each sand fly species and time interval using Image-J software. The morphological forms were distinguished based on the criteria of Sádlová et al. [34] and Rogers et al. [35]: (i) short nectomonads: body length < 14 μm and flagellar length < 2 times body length; (ii) long nectomonads: body length ≥ 14 μm; (iii) metacyclic promastigotes: body length < 14 μm and flagellar length ≥ 2 times body length. Data were evaluated statistically by analysis of variance using SPSS statistics version 23 software.

Results

Characterization of LPG repeats units

Purified LPGs from Le. amazonensis strains were differentially recognized by the mAbs CA7AE and LT22 (Fig. 2a-d). As shown in Fig. 2a-b, the LPG from the PH8 strain and respective controls (LD4 and Mongi) were recognized by CA7AE and LT22. However, a different recognition profile was observed for the Josefa strain since its LPG was recognized by LT22 (Fig. 2d) but not by CA7AE (Fig. 2c), indicating the presence of side-chains branching-off the repeat units.

Fig. 2
figure 2

Immunoblotting of purified lipophosphoglycan (LPG). Purified LPG (10 μg per lane) from promastigotes of Le. amazonensis PH8 (a and b) and Josefa (Jos) (c and d) strains were incubated with the antibody CA7AE (1:1000) (a and c) and LT22 (1:1000) (b and d). The LPGs purified from Le. donovani LD4 and Mongi strains were used as positive controls

Full size image

Confirming the previous immunoblotting profiles, both Le. amazonensis strains displayed a distinct oligosaccharide profile of their neutral repeat units (Fig. 3a). The repeat units of the PH8 strain exhibited higher disaccharide content, represented by Gal-Man (G2 position) that is common to all LPGs, which explains their reactivity against CA7AE (Fig. 2a). The FACE analysis also revealed up to 1 and 2 side-chains in their structures (Fig. 3a). On the other hand, the repeat units of the Josefa strain showed up to 1 to 3 side-chains, and no Gal-Man disaccharide was detected (Fig. 3a). This profile elucidates the observed lack of recognition by CA7AE (Fig. 2c) and the positive reaction against LT22 (Fig. 2d), suggesting that most, if not all, repeating units contained side-chains.

Fig. 3
figure 3

Fluorophore-assisted carbohydrate electrophoresis (FACE) of lipophosphoglycan (LPG) repeat units and monosaccharides of Le. amazonensis (PH8 and Josefa strains). a FACE analysis of dephosphorylated repeat units of PH8 and Josefa strain. Lane Std1: oligoglucose ladder represented by G1-G7; Lane PH8: repeat unit of PH8 strain; Lane Josefa: repeat unit of Josefa strain. b FACE of monosaccharides from LPG repeat units after strong acid hydrolysis. Lane Std2: monosaccharide standards (Man = mannose, Glc = glucose, Gal = galactose); Lane PH8: PH8 strain; Lane Jos: Josefa strain

Full size image

The monosaccharide profile of the PH8 strain revealed galactose and mannose, common to all LPGs, and high content of glucose (Fig. 3b). These data confirmed the presence of glucose as side-chains, as previously indicated by immunoblotting. The Josefa strain, however, exhibited only galactose and mannose as monosaccharides (Fig. 3b).

Confirming the FACE analysis, the dephosphorylated repeat units from the PH8 strain consisted of a di- (67%), a tri- (30%) and a tetrasaccharide (3%) (Fig. 4a). The trisaccharide and tetrasaccharide were susceptible to treatment with β-glucosidase (Fig. 4b). These data are consistent with the structure of the trisaccharide as Glc(β)Gal(β1,4)Man and the tetrasaccharide as Glc2(β)Gal(β1,4)Man. The LPG of the Josefa strain showed a small amount of di- (9%), an abundance of tri- (29%) and tetra- (57%), and again a small amount of pentasaccharide (5%) (Fig. 4c). The minimal disaccharide content supports the non-reactivity by CA7AE (Fig. 2c). Interestingly, this LPG was not susceptible to β-glucosidase treatment (Fig. 4d), indicating that another sugar is terminating the repeat units. After β-galactosidase treatment, we observed the disappearance of side-chains confirming the presence of β-galactoses (Fig. 4e). These results confirmed that both strains possess intraspecific polymorphisms (Fig. 5).

Fig. 4
figure 4

Capillary electrophoresis (CE) analyses of dephosphorylated LPG repeat units from Le. amazonensis strains (PH8 and Josefa). Designations above the peaks are retention times in minutes as well as di, disaccharide; tri, trisaccharide, etc. a LPG PH8 strain repeat units. b LPG PH8 strain treated with β-glucosidase. c LPG Josefa strain repeat units. d LPG Josefa strain treated with β-glucosidase. e LPG Josefa strain treated with β-galactosidase

Full size image
Fig. 5
figure 5

Schematic diagram of Le. amazonensis LPG structures of promastigotes from PH8 and Josefa strains. The repeat units contain the Gal(β1,4)Man(α1)-PO4 as backbone structure. For PH8 strain, the LPG bears sugar branch substitutions of one or two β-glucose residues, while in Josefa strain most of the side chains are terminated in β-galactoses. The precise locations of the sugar side chains in the repeat unit domain are not known

Full size image

Sand fly infections and morphometry

The development of Le. amazonensis strains was studied in Lu. migonei, Lu. longipalpis and Ph. papatasi on days 1 and 5–6 PI. During the early phase of infection (on day 1 PI), the infection rates were fully comparable in all parasite-vector combinations; more than 82% of females were infected and moderate or heavy intensities of infections were observed in majority of females. Parasites were in the endoperitrophic space within the blood meal surrounded by the PM. On days 5–6 PI, both Le. amazonensis strains could establish heavy late stage infections with the colonization of the stomodeal valve in permissive vectors Lu. longipalpis and Lu. migonei. In contrast, in Ph. papatasi the infections were lost during defecation of bloodmeal remnants (Figs. 6 and 7).

Fig. 6
figure 6

Experimental infections of Le. amazonensis (PH8 and Josefa strains) in Lu. longipalpis (L. lon.), Lu. migonei (L. mig.) and Ph. papatasi (P. pap.). Rates and intensities of infections were evaluated microscopically on days 1 and 5–6 post-infection (PI), and were classified into three categories: light (< 100 parasites/gut), moderate (100–1000 parasites/gut), or heavy (> 1000 parasites/gut). Data are representative of two experiments

Full size image
Fig. 7
figure 7

Localization of Le. amazonensis (PH8 and Josefa strains) in Lu. longipalpis (L. lon.), Lu. migonei (L. mig.) and Ph. papatasi (P. pap.). Localization of infections in vectors (stomodeal valve, SV; abdominal midgut, AMG; thoracic midgut, TMG; cardia and endoperithrophic space) was evaluated microscopically on days 1 and 5–6 post-infection (PI). Data are representative of two experiments

Full size image

Since no infection was developed in Ph. papatasi after defecation, morphological analysis was performed on Le. amazonensis parasites derived from Lu. migonei and Lu. longipalpis infections on days 1 and 5–6 PI. Both strains accomplished metacyclogenesis in both vectors. Interestingly, Lu. longipalpis infections with PH8 strains resulted in a higher percentage of metacyclic forms compared to Lu. migonei by days 5–6 PI (P < 0.0001) (Fig. 8).

Fig. 8
figure 8

Morphological forms (metacyclics, short promastigotes, elongated nectomonads and procyclic promatigotes) of Le. amazonensis (PH8 and Josefa strains) during development in Lu. longipalpis (L. lon.) and Lu. migonei (L. mig.). Morphological forms of Leishmania parasites in vectors were evaluated microscopically on days 1 and 5–6 post-infection (PI). Data are representative of two experiments and were evaluated statistically by analysis of variance. P < 0.05 was considered statistically significant

Full size image

Discussion

Leishmania amazonensis, a member of the Leishmania mexicana complex, is one of the most studied Leishmania species. It is an excellent model for immunology, molecular biology and chemotherapy. This species is often associated with treatment failure, being naturally resistant to the first line chemotherapy drugs [36]. However, studies involving Le. amazonensis glycoconjugates and their interaction with sand fly vectors are still scarce.

The LPGs are implicated in a variety of functions in the sand flies including attachment to a microvilli receptor in Old World Ph. papatasi (restrictive) [37]. Early in vitro studies reported the role of interspecies LPG polymorphisms in the specificity during sand fly-Leishmania interactions [4]. In this study, Ph. papatasi midguts were recognized only by Le. major phosphoglycan (PG), whereas those from Ph. argentipes (permissive) were recognized by PGs from several species. Later, several in vitro studies demonstrated that those models are consistent while using natural vector-Leishmania pairs not only in Old World but also in New World vectors. For example, the interaction with PGs from New World Le. infantum and Le. braziliensis adversely affected parasite attachment in permissive Lu. longipalpis and restrictive Lutzomyia intermedia/Lu. whitmani [10, 38,39,40]. However, in vitro system limitations appear while using unnatural sand fly-Leishmania combinations including the attachment of Le. braziliensis (New World) promastigotes to Ph. papatasi (Old World) midguts [41]. To circumvent such limitations, in this study we have performed experiments using in vivo models with previously tested permissive sand flies known to become infected with Le. amazonensis [12, 19, 20].

In the New World species of Leishmania, LPG variations were only studied in Le. infantum. In this species, polymorphisms in the glucose levels were not important for in vivo infectivity to Lu. longipalpis [11]. However, this sugar was important for in vitro interaction of Le. infantum (PP75 strain) with the midguts of Lu. longipalpis [10].

Consistent with our previous observations, a more detailed biochemical analysis of LPG using FACE and CE from Le. amazonensis strains revealed important intraspecific polymorphisms. The LPG of the PH8 strain displayed β-glucose residues as side-chains whereas the Josefa strain had β-galactose residues (Fig. 5). The β-glucose residues are commonly found in the New World species of Leishmania including Le. mexicana [42], Le. infantum [10] and Le. braziliensis [31]. Those β-glucose residues in Le. infantum procyclic were downregulated after metacyclogenesis resulting in loss of in vitro interaction with the midgut of Lu. longipalpis [10]. Surprisingly, the occurrence of β-galactose residues as side-chains in the Josefa strain of Le. amazonensis was detected for the first time in a New World Leishmania species. This sugar is often observed in the Old World Leishmania species including Le. major [15], Le. tropica /Leishmania aethiopica [5, 7] and Le. turanica [17]. Unlike Le. major and Le. turanica, Le. amazonensis Josefa strain with β-galatosylated LPG could not survive inside Ph. papatasi. This phenomenon could be explained by other LPG-independent mechanisms, probably related to the distance of the sand fly-Leishmania pair used (Old vs New World) or other molecules involved. Together with LPG, glycoprotein 63 (GP63) was also evaluated in Le. mexicana/Le. amazonensis - Lu. longipalpis attachment in vivo [43] or in vitro [44]. A recent study demonstrated that both glycoconjugates could be determinant for inhibiting in vitro parasite attachment in Lu. longipalpis and Lu. intermedia [40].

Several studies have already described the differentiation of Le. amazonensis, Le. braziliensis and Le. infantum in New World sand fly species including Lu. longipalpis and Lu. migonei [12, 19, 20, 45]. Consistent with those observations, both Le. amazonensis strains could survive defecation of permissive vectors Lu. longipalpis and Lu. migonei, establish late-stage infections, and colonize anterior midgut reaching the stomodeal valve. This is a strong indication that the parasite could attach and migrate towards the mouth parts for subsequent transmission. More importantly, both strains accomplished metacyclogenesis. This morphological transformation was more pronounced in Lu. longipalpis infected with the PH8 strain, probably due to the higher permissiveness of this species. This sand fly can sustain a wide variety of pathogens, including viruses, non-Leishmania protozoans and even helminths (reviewed in [18]).

Conclusions

In combination with previous studies, the LPG polymorphism in Le. amazonensis did not affect infection of the three sand fly species tested. To our knowledge, the biochemical data obtained from the Josefa strain represents the first description of a galactosylated LPG in a New World Leishmania species. However, these residues were not sufficient for survival in Ph. papatasi.

Abbreviations

ADCL:

Anergic diffuse cutaneous leishmaniasis

CE:

Capillary electrophoresis

FACE:

Fluorophore-assisted carbohydrate electrophoresis

GPI:

Glycosylphosphatidylinositol

LCL:

Localized cutaneous leishmaniasis

LPG:

Lipophosphoglycan

PI:

Post-infection

References

  1. Carvalho BM, Rangel EF, Ready PD, Vale MM. Ecological niche Modelling predicts southward expansion of Lutzomyia (Nyssomyia) flaviscutellata (Diptera: Psychodidae: Phlebotominae), vector of Leishmania (Leishmania) amazonensis in South America, under climate change. PLoS One. 2015;30:10(11).

    Google Scholar 

  2. Marques FA, Soares RP, Almeida GG, Souza CC, Melo MN, Pinto SA, et al. Parasitology international effectiveness of an immunohistochemical protocol for Leishmania detection in different clinical forms of American tegumentary leishmaniasis. Parasitol Int. 2017;66:884–8.

    Article  PubMed  Google Scholar 

  3. De Assis RR, Ibraim IC, Nogueira PM, Soares RP, Turco SJ. Glycoconjugates in new world species of Leishmania: polymorphisms in lipophosphoglycan and glycoinositolphospholipids and interaction with hosts. Biochim Biophys Acta. 2012;1820:1354–65.

    Article  PubMed  Google Scholar 

  4. Pimenta PF, Saraiva EM, Rowton E, Modi GB, Garraway L a, Beverley SM, et al. Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmania is controlled by structural polymorphisms in the surface lipophosphoglycan. Proc Natl Acad Sci USA 1994;91:9155–9159.

  5. McConville MJ, Schnur LF, Jaffe C, Schneider P. Structure of Leishmania lipophosphoglycan: inter- and intra-specific polymorphism in old world species. Biochem J. 1995;310:807–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dobson DE, Kamhawi S, Lawyer P, Turco SJ, Beverley SM, Sacks DL. Leishmania major survival in selective Phlebotomus papatasi sand fly vector requires a specific SCG-encoded lipophosphoglycan galactosylation pattern. PLoS Pathog. 2010;11;6(11):e1001185.

  7. Soares RPP, Barron T, McCoy-Simandle K, Svobodova M, Warburg A, Turco SJ. Leishmania tropica: intraspecific polymorphisms in lipophosphoglycan correlate with transmission by different Phlebotomus species. Exp Parasitol. 2004;107:105–14.

    Article  CAS  PubMed  Google Scholar 

  8. Svobodova M, Votypka J, Dvorak J, Nasereddin A, Baneth G, Sztern J, et al. Distinct transmission cycles of Leishmania tropica in 2 adjacent foci, northern Israel. Emerg Infect Dis. 2006;12:1860–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Mahoney AB, Sacks DL, Saraiva E, Modi G, Turco SJ. Intra-species and stage-specific polymorphisms in lipophosphoglycan structure control Leishmania donovani - sand fly interactions. Biochemistry. 1999;38:9813–23.

    Article  CAS  PubMed  Google Scholar 

  10. Soares RPP, Macedo ME, Ropert C, Gontijo NF, Almeida IC, Gazzinelli RT, et al. Leishmania chagasi: Lipophosphoglycan characterization and binding to the midgut of the sand fly vector Lutzomyia longipalpis. Mol Biochem Parasitol. 2002;121:213–24.

    Article  CAS  PubMed  Google Scholar 

  11. Coelho-Finamore JM, Freitas VC, Assis RR, Melo MN, Novozhilova N, Secundino NF, et al. Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts. Int J Parasitol. 2011;41:333–42.

    Article  CAS  PubMed  Google Scholar 

  12. Nogueira PM, Assis RR, Torrecilhas AC, Saraiva EM, Pessoa NL, Campos MA, et al. Lipophosphoglycans from Leishmania amazonensis strains display immunomodulatory properties via TLR4 and do not affect sand fly infection. PLoS Negl Trop Dis. 2016;10:e0004848.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Volf P, Myskova J. Sand flies and Leishmania: specific versus permissive vectors. Trends Parasitol. 2007;23:91–2.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Chajbullinova A, Votypka J, Sadlova J, Kvapilova K, Seblova V, Kreisinger J, et al. The development of Leishmania turanica in sand flies and competition with L. major. Parasit Vectors. 2012;5:219.

    Article  PubMed  PubMed Central  Google Scholar 

  15. McConville MJ, Turco SJ, Ferguson MA, Sacks DL. Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious. EMBO J. 1992;11(10):3593–600.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Butcher BA, Turco SJ, Hilty BA, Pimenta PF, Panunzio M, Sacks DL. Deficiency in b1,3-galactosyltransferase of a Leishmania major lipophosphoglycan mutant adversely influences the Leishmania -sand fly interaction. J Biol Chem. 1996;271:20573–9.

    Article  CAS  PubMed  Google Scholar 

  17. Volf P, Nogueira PM, Myskova J, Turco SJ, Soares RP. Structural comparison of lipophosphoglycan from Leishmania turanica and L. major, two species transmitted by Phlebotomus papatasi. Parasitol Int. 2014;63:683–6.

    Article  CAS  PubMed  Google Scholar 

  18. Soares RP, Turco SJ. Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae): a review. Ann Acad Bras Cienc. 2003;75:301–30.

    Article  Google Scholar 

  19. Nieves E, Pimenta PFP. Development of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the sand fly Lutzomyia migonei (Diptera: Psychodidae). J Med Entomol. 2000;37:134–40.

    Article  CAS  PubMed  Google Scholar 

  20. Guimarães VCFV, Pruzinova K, Sadlova J, Volfova V, Myskova J, Filho SPB, et al. Lutzomyia migonei is a permissive vector competent for Leishmania infantum. Parasit Vectors. 2016;9:159.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Pimenta PF, Turco SJ, McConville MJ, Lawyer PG, Perkins PV, Sacks DL. Stage-specific adhesion of Leishmania promastigotes to the sandfly midgut. Science. 1992;256:1812–5.

    Article  CAS  PubMed  Google Scholar 

  22. Kamhawi S, Ramalho-Ortigao M, Van MP, Kumar S, Lawyer PG, Turco SJ, et al. A role for insect galectins in parasite survival. Cell. 2004;119:329–41.

    Article  CAS  PubMed  Google Scholar 

  23. Myskova J, Svobodova M, Beverley SM, Volf PA. Lipophosphoglycan-independent development of Leishmania in permissive sand flies. Microbes Infect. 2007;9:317–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Svárovská A, Ant TH, Seblová V, Jecná L, Beverley SM, Volf P. Leishmania major glycosylation mutants require phosphoglycans (lpg2-) but not lipophosphoglycan (lpg1-) for survival in permissive sand fly vectors. PLoS Negl Trop Dis. 2010;4:1–7.

    Article  Google Scholar 

  25. Myšková J, Dostálová A, Pěničková L, Halada P, Bates PA, Volf P. Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment. Parasit Vectors. 2016;9:413.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Garcia L, Kindt A, Bermudez H, Llanos-Cuentas A, De Doncker S, Arevalo J, et al. Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol. 2004;42:2294–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rocha MN, Margonari C, Presot IM, Soares RP. Evaluation of 4 polymerase chain reaction protocols for cultured Leishmania spp. typing. Diagn Microbiol Infect Dis. 2010;68:401–9.

    Article  CAS  PubMed  Google Scholar 

  28. Tavares NM, Araújo-Santos T, Afonso L, Nogueira PM, Lopes UG, Soares RP, et al. Understanding the mechanisms controlling Leishmania amazonensis infection in vitro: the role of LTB4 derived from human neutrophils. J Infect Dis. 2014;210:1–11.

    Article  Google Scholar 

  29. Tolson DL, Turco SJ, Beecroft RP, Pearson TW. The immunochemical structure and surface arrangement of Leishmania donovani lipophosphoglycan determined using monoclonal antibodies. Mol Biochem Parasitol. 1989;35:109–18.

    Article  CAS  PubMed  Google Scholar 

  30. Ilg T, Harbecke D, Wiese M, Overath P. Monoclonal antibodies directed against Leishmania secreted acid phosphatase and lipophosphoglycan. Partial characterization of private and public epitopes. Eur J Biochem. 1993;217:603–15.

    Article  CAS  PubMed  Google Scholar 

  31. Soares RPP, Cardoso TL, Barron T, Araújo MSS, Pimenta PFP, Turco SJ. Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis. Int J Parasitol. 2005;35:245–53.

    Article  CAS  PubMed  Google Scholar 

  32. Assis RR, Ibraim IC, Noronha FS, Turco SJ, Soares RP. Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: modulation of innate immune system and variations in carbohydrate structure. PLoS Negl Trop Dis. 2012;6:1–11.

    Article  Google Scholar 

  33. Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011;

  34. Sádlová J, Price HP, Smith BA, Votỳpka J, Volf P, Smith DF. The stage-regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector, Phlebotomus papatasi. Cell Microbiol. 2010;12:1765–79.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Rogers ME, Chance ML, Bates PA. The role of promastigote secretory gel in the origin and transmission of the infective stage of Leishmania mexicana by the sandfly Lutzomyia longipalpis. Parasitology. 2002;124(Pt 5):495–507.

    CAS  PubMed  Google Scholar 

  36. Rocha MN, Nogueira PM, Demicheli C, de Oliveira LG, da Silva MM, Frézard F, Melo MN, Soares RP. Cytotoxicity and in vitro antileishmanial activity of antimony (V), bismuth (V), and tin (IV) complexes of Lapachol. Bioinorg Chem Appl. 2013;2013:961783.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends Parasitol. 2006;22:439–45.

    Article  PubMed  Google Scholar 

  38. Kamhawi S, Modi GB, Pimenta PF, Rowton E, Sacks DL. The vectorial competence of Phlebotomus sergenti is specific for Leishmania tropica and is controlled by species-specific, lipophosphoglycan-mediated midgut attachment. Parasitology. 2000;121:25–33.

    Article  CAS  PubMed  Google Scholar 

  39. Soares RP, Margonari C, Secundino NC, MacÊdo ME, Da Costa SM, Rangel EF, et al. Differential midgut attachment of Leishmania (Viannia) braziliensis in the sand flies Lutzomyia (Nyssomyia) whitmani and Lutzomyia (Nyssomyia) intermedia. J Biomed Biotechnol. 2010;2010:439174.

    Article  PubMed  Google Scholar 

  40. Soares RP, Altoé ECF, Ennes-Vidal V, da Costa SM, Rangel EF, de Souza NA, et al. In vitro inhibition of Leishmania attachment to sandfly midguts and LL-5 cells by divalent metal chelators, anti-gp63 and phosphoglycans. Protist. 2017;168:326–34.

    Article  CAS  PubMed  Google Scholar 

  41. Wilson R, Bates MD, Dostalova A, Jecna L, Dillon RJ, Volf P, et al. Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay. PLoS Negl Trop Dis. 2010;4:e816.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Ilg T, Etges R, Overath P, McConville MJ, Thomas-Oates J, Thomas J, et al. Structure of Leishmania mexicana lipophosphoglycan. J Biol Chem. 1992;267(10):6834–40.

    CAS  PubMed  Google Scholar 

  43. Hajmová M, Chang KP, Kolli B, Volf P. Down-regulation of gp63 in Leishmania amazonensis reduces its early development in Lutzomyia longipalpis. Microbes Infect. 2004;6:646–9.

    Article  PubMed  Google Scholar 

  44. Jecna L, Dostalova A, Wilson R, Seblova V, Chang K-P, Bates P. A, et al. the role of surface glycoconjugates in Leishmania midgut attachment examined by competitive binding assays and experimental development in sand flies. Parasitology. 2013;140:1026–32.

    Article  CAS  PubMed  Google Scholar 

  45. Freitas VC, Parreiras KP, Duarte APM, Secundino NFC, Pimenta PFP. Development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis. Am J Trop Med Hyg. 2012;86:606–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

RPS, PMN, RRA and ACG are supported by Conselho Nacional de Pesquisa e Desenvolvimento (CNPq PAPES VI 407438/2012-2) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (PPM-00102-16). PV, JS, JM, KP and JH are supported by Charles University, project UNCE 204017/2012. ACT is supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP 2016–01917-3).

Availability of data and materials

All data generated or analyzed during this study are included in this article. The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Author information

Author notes

    Authors and Affiliations

    1. Instituto René Rachou/FIOCRUZ, Belo Horizonte, MG, Brazil

      Paula M. Nogueira, Rafael R. Assis & Rodrigo P. Soares

    2. Departamento de Parasitologia, UFMG, Belo Horizonte, MG, Brazil

      Paula M. Nogueira & Agna C. Guimarães

    3. Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic

      Jovana Sadlova, Jitka Myskova, Katerina Pruzinova, Jana Hlavackova & Petr Volf

    4. Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY, USA

      Salvatore J. Turco

    5. Laboratório de Imunologia Celular e Bioquímica de Fungos e Protozoários, Departamento de Farmácia, UNIFESP, São Paulo, SP, Brazil

      Ana C. Torrecilhas

    Authors
    1. Paula M. Nogueira
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Agna C. Guimarães
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Rafael R. Assis
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Jovana Sadlova
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Jitka Myskova
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Katerina Pruzinova
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Jana Hlavackova
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Salvatore J. Turco
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Ana C. Torrecilhas
      View author publications

      You can also search for this author in PubMed Google Scholar

    10. Petr Volf
      View author publications

      You can also search for this author in PubMed Google Scholar

    11. Rodrigo P. Soares
      View author publications

      You can also search for this author in PubMed Google Scholar

    Contributions

    PMN, ACG, RRA, RPS and PV conceived and designed the study. PMN, ACG, JS, JM, JH and KP carried out the data analysis. All authors read and approved the final manuscript.

    Corresponding author

    Correspondence to Paula M. Nogueira.

    Ethics declarations

    Ethics approval and consent to participate

    All experiments were approved by the Committee on the Ethics of Laboratory Experiments of the Charles University in Prague and were performed under the Certificate of Competency (Registration Number: CZ 03069). Rabbit blood was commercially purchased.

    Consent for publication

    Not applicable.

    Competing interests

    The authors declare that they have no competing interests.

    Publisher’s Note

    Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

    Rights and permissions

    Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Nogueira, P.M., Guimarães, A.C., Assis, R.R. et al. Lipophosphoglycan polymorphisms do not affect Leishmania amazonensis development in the permissive vectors Lutzomyia migonei and Lutzomyia longipalpis . Parasites Vectors 10, 608 (2017). https://doi.org/10.1186/s13071-017-2568-8

    Download citation

    • Received:

    • Accepted:

    • Published:

    • DOI: https://doi.org/10.1186/s13071-017-2568-8

    Keywords

    Parasites & Vectors

    ISSN: 1756-3305

    Contact us