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Parasit Vectors
2015 Jan 17;8:31. doi: 10.1186/s13071-015-0633-8.
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Leishmania enriettii: biochemical characterisation of lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) and infectivity to Cavia porcellus.
Paranaíba LF
,
de Assis RR
,
Nogueira PM
,
Torrecilhas AC
,
Campos JH
,
Silveira AC
,
Martins-Filho OA
,
Pessoa NL
,
Campos MA
,
Parreiras PM
,
Melo MN
,
Gontijo Nde F
,
Soares RP
.
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BACKGROUND: Leishmania enriettii is a species non-infectious to man, whose reservoir is the guinea pig Cavia porcellus. Many aspects of the parasite-host interaction in this model are unknown, especially those involving parasite surface molecules. While lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of Leishmania species from the Old and New World have already been described, glycoconjugates of L. enriettii and their importance are still unknown.
METHODS: Mice peritoneal macrophages from C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production were performed. MAPKs (p38 and JNK) and NF-kB activation were evaluated in J774.1 macrophages and CHO cells, respectively.
RESULTS: LPGs were extracted, purified and analysed by western-blot, showing that LPG from L88 strain was longer than that of Cobaia strain. LPGs and GIPLs were depolymerised and their sugar content was determined. LPGs from both strains did not present side chains, having the common disaccharide Gal(β1,4)Man(α1)-PO4. The GIPL from L88 strain presented galactose in its structure, suggestive of type II GIPL. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not previously observed. Mice peritoneal macrophages from C57BL/6 and knock-outs (TLR2 -/- and TLR4 -/-) were primed with IFN-γ and stimulated with glycoconjugates and live parasites. No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO, IL-6, IL-12 and TNF-α preferably via TRL2. However, in CHO cells, only GIPLs were able to activate TRL2 and TRL4. In vivo studies using male guinea pigs (Cavia porcellus) showed that only strain L88 was able to develop more severe ulcerated lesions especially in the presence of salivary gland extract (SGE).
CONCLUSION: The two L. enriettii strains exhibited polymorphisms in their LPGs and GIPLs and those features may be related to a more pro-inflammatory profile in the L88 strain.
Figure 1.
Experimental procedures scheme. Promastigotes of L. enriettii were used for infection in C. porcellus in the presence/absence of Salivary Gland Extract (SGE). LPG and GIPLs were extracted with organic solvents and purified using Phenyl-Sepharose. LPG purification was confirmed by western-blot. The LPG was depolymerased using mild acid hydrolysis (0.02 N HCl, 5 min, 100°C) and the repeat units were dephosphorylated using alkaline phosphatase. The profiles were analysed by Fluorophore-assisted carbohydrate electrophoresis (FACE). Purified GIPLs were subjected to strong acid hydrolysis (2 N trifluoroacetic acid, 100°C, 3 hours) and monosaccharides were analyzed by FACE. Purified LPGs and GIPLs were incubated with murine peritoneal macrophages, CHO cells and J774.A1 cells for NO, cytokines, MAPKs and NF-kB activation.
Figure 2.
Western blot of purified LPG (10 μg per lane) of
L. infantum
(BH46 strain) and
L. enriettii
(L88 and Cobaia strains) in the presence of CA7AE antibody (1:1000). Upper and lower arrow indicates the smears of L. enriettii LPGs (L88 and Cobaia strains), respectively.
Figure 3.
Carbohydrate profile of LPGs (repeat units) and GIPLs (core) from
L. enriettii
(L88 and Cobaia strains). STD, standard represented by oligoglucose ladder (G1-G7). (A) Oligosaccharide profile from LPG repeat units. (B) Monosaccharide profile of L. enriettii (L88 and Cobaia strains). STD, standard represented by the monosaccharide sugars (100 μg/mL). Man, mannose, Glc, glucose, Gal, galactose.
Figure 6.
GIPLs purified from two strains of
L. enriettii
(L88 and Cobaia) induce translocation of NF-kB through TLRs. CHO cells expressing TLR2 (TLR2+), TLR4 (TLR4+), or neither (TLR2-/TLR4-) were either untreated (black line) or exposed (gray line) to LPS, S. aureus (SA), L. infantum LPG, L. braziliensis LPG, L. enriettii strain L88 LPG (LPG L88), L. enrietti strain Cobaia LPG (LPG Cobaia), L. enriettii strain L88 GIPL (GIPL L88) or L. enriettii strain Cobaia GIPL (GIPL Cobaia), as indicated. CD25 expression was measured by flow cytometry 18 h after stimulation. Percentage = percentage of CD25 expression on stimulated cells minus percentage of CD25 expression on non-stimulated cells.
Figure 7.
Activation of MAPKs (p38 and JNK) by
L. enriettii
glycoconjugates (LPG and GIPLs) from both strains (L88 and Cobaia) in J774A.1 peritoneal macrophages. Macrophages were stimulated for 5, 15, 30 and 45 min with 10 μg/mL of LPG and GIPLs from L. enriettii L88 (A, C) and L. enriettii Cobaia (B and D). Dually phosphorylated MAPKs (JNK and p38) were detected by western blot. C-, negative control; Total p38 content was used as the normalizing protein.
Figure 8.
Lesions in
C. porcellus
infected by
L. enriettii
strains in the presence/absence of salivary gland extract (SGE) from
Lutzomyia longipalpis. Male C. porcellus were inoculated with 1x105 parasites of L. enriettii strains (L88 and Cobaia). (A) non-infected C. porcellus; (B)
C. porcellus infected with L88 strain (4 weeks of infection); (C)
C. porcellus infected with L88 strain (5 weeks infection); (D)
C. porcellus infected with L88 strain + SGE (7 weeks of infection); (E)
C. porcellus infected with L88 strain (8 weeks of infection) and (F)
C. porcellus infected with Cobaia strain (4 weeks of infection).
Figure 9.
Development of lesions (mm
2
) in
C. porcellus
after inoculation with
L. enriettii
(L88 strain) in the presence/absence of salivary gland extract (SGE) from
L. longipalpisv. Dark circles, C. porcellus infected with L. enriettii strain L88 and dark squares, C. porcellus infected with L. enriettii strain L88 + SGE. Male C. porcellus were inoculated with 1x105 parasites of L. enriettii (L88 strain). Lesion size (mm2) was followed weekly.
Figure 10.
Schematic diagram of
L. enriettii
LPGs. The central portion of the structure is Gal(α1,6)Gal(α1,3)Galf(α1,3)[Glc(α1-PO4)-6]Man(α1,3)Man(α1,4)GlcN(α1:6), attached lipid anchor to alkyl-2-lyso-1-O phosphatydylinositol (PI). The repeat units are 6-Gal(β1,4)Man(α1)-PO4. The precise numbers of the repeat units in the L. enriettii LPGs and the CAP constitution are not known. P = phosphate.
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