Angana Ghoshal

Sialic acids acquired by Pseudomonas aeruginosa are involved in reduced complement deposition and siglec mediated host-cell recognition

The opportunism of Pseudomonas aeruginosa (PA) in immunocompromised hosts prompted us to explore the potential role of sialic acids (Sia) in this phenomenon. Culture of PA in the presence of exogenous Sia resulted in linkage‐specific incorporation of Sia which was associated with decreased complement deposition on the bacteria. Sia acquired by PA mediated enhanced binding of bacteria to recombinant‐CHO cells expressing human siglec‐7 or siglec‐9, as well as to human NK‐cells and monocytes naturally expressing these siglecs. Therefore, Sia may be acquired by PA in the host and contribute to bacterial pathogenicity and host‐cell interactions via reduction of complement deposition and siglec‐dependent recognition.

9- O -acetylated sialic acids enhance entry of virulent Leishmania donovani promastigotes into macrophages

SUMMARY Distribution of 9-O-acetylated sialic acids (9-O-AcSA) on Leishmania donovani has been previously reported. Considering their role in recognition, the differential distribution of sialic acids especially 9-O-acetylated sialic acids in avirulent (UR6) versus virulent (AG83 and GE1) promastigotes of Leishmania donovani and its role in entry into macrophages was explored. Fluorimetric-HPLC, fluorimetric determination and ELISA revealed 14-, 8- and 5-fold lower sialic acids in UR6 as compared to AG83. Interestingly, on UR6, flow cytometry indicated lower (alpha2-->6)-linked sialoglycoproteins along with minimal 9-O-acetylated sialoglycoproteins by Scatchard analysis. Further, UR6 demonstrated a 9- and 14.5-fold lower infectivity and phagocytic index than AG83. Additionally, de-O-acetylation and de-sialylation of AG83 demonstrated a 3- and 1.5-fold reduced phagocytic index. The role of 9-O-AcSA in entry was further confirmed by pre-blocking the macrophage surface with a cocktail of sugars followed by microscopic quantification. The phagocytic index of AG83 exclusively through 9-O-AcSA was significantly high. Interestingly, AG83 produced higher metacyclic promastigotes containing increased 9-O-AcSA as compared to avirulent UR6 supporting its virulent nature. Taken together; our results conclusively demonstrate the increased presence of 9-O-acetylated sialic acid on promastigotes of virulent Leishmania donovani as compared to avirulent UR6 and their subsequent role in entry within macrophages.

Leishmania cell surface prohibitin: role in host-parasite interaction

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95–100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host–parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild‐type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amino acid substitution at the GPI anchor site to prevent surface expression through GPI‐link show lesser surface expression and lower infective abilities. Furthermore, the presence of anti‐prohibitin antibodies during macrophage–Leishmania interaction in vitro reduces infection. The cognate binding partner for Leishmania prohibitin on the host cell appears to be macrophage surface HSP70, siRNA mediated downregulation of which abrogates the capability of the macrophage to bind to parasites. Leishmania prohibitin is able to generate a strong humoral response in visceral leishmaniasis patients. The above observations suggest that prohibitin plays an important role in events leading to Leishmania–host interaction.

Glycosylation of Erythrocyte Spectrin and Its Modification in Visceral Leishmaniasis

Using a lectin, Achatinin-H, having preferential specificity for glycoproteins with terminal 9-O-acetyl sialic acid derivatives linked in α2-6 linkages to subterminal N-acetylgalactosamine, eight distinct disease-associated 9-O-acetylated sialoglycoproteins was purified from erythrocytes of visceral leishmaniaisis (VL) patients (RBCVL). Analyses of tryptic fragments by mass spectrometry led to the identification of two high-molecular weight 9-O-acetylated sialoglycoproteins as human erythrocytic α- and β-spectrin. Total spectrin purified from erythrocytes of VL patients (spectrinVL) was reactive with Achatinin-H. Interestingly, along with two high molecular weight bands corresponding to α- and β-spectrin another low molecular weight 60 kDa band was observed. Total spectrin was also purified from normal human erythrocytes (spectrinN) and insignificant binding with Achatinin-H was demonstrated. Additionally, this 60 kDa fragment was totally absent in spectrinN. Although the presence of both N- and O-glycosylations was found both in spectrinN and spectrinVL, enhanced sialylation was predominantly induced in spectrinVL. Sialic acids accounted for approximately 1.25 kDa mass of the 60 kDa polypeptide. The demonstration of a few identified sialylated tryptic fragments of α- and β-spectrinVL confirmed the presence of terminal sialic acids. Molecular modelling studies of spectrin suggest that a sugar moiety can fit into the potential glycosylation sites. Interestingly, highly sialylated spectrinVL showed decreased binding with spectrin-depleted inside-out membrane vesicles of normal erythrocytes compared to spectrinN suggesting functional abnormality. Taken together this is the first report of glycosylated eythrocytic spectrin in normal erythrocytes and its enhanced sialylation in RBCVL. The enhanced sialylation of this cytoskeleton protein is possibly related to the fragmentation of spectrinVL as evidenced by the presence of an additional 60 kDa fragment, absent in spectrinN which possibly affects the biology of RBCVL linked to both severe distortion of erythrocyte development and impairment of erythrocyte membrane integrity and may provide an explanation for their sensitivity to hemolysis and anemia in VL patients.

Sialoglycosylation of RBC in Visceral Leishmaniasis Leads to Enhanced Oxidative Stress, Calpain-Induced Fragmentation of Spectrin and Hemolysis

Visceral leishmaniasis (VL) caused by the intracellular parasite Leishmania donovani accounts for an estimated 12 million cases of human infection. It is almost always associated with anemia, which severely complicates the disease course. However, the pathological processes leading to anemia in VL have thus far not been adequately characterized to date. In studying the glycosylation patterns of peripheral blood cells we found that the red blood cells (RBC) of VL patients (RBC(VL)) express eight 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) that are not detected in the RBC of healthy individuals (RBC(N)). At the same time, the patients had high titers of anti-9-O-AcSGP IgG antibodies in their sera. These two conditions appear to be linked and related to the anemic state of the patients, as exposure of RBC(VL) but not RBC(N) to anti-9-O-AcSGPs antibodies purified from patient sera triggered a series of responses. These included calcium influx via the P/Q-type but not L-type channels, activation of calpain I, proteolysis of spectrin, enhanced oxidative stress, lipid peroxidation, externalization of phosphatidyl serine with enhanced erythrophagocytosis, enhanced membrane fragility and, finally, hemolysis. Taken together, this study suggests that the enhanced hemolysis is linked to an impairment of membrane integrity in RBC(VL) which is mediated by ligand-specific interaction of surface 9-O-AcSGPs. This affords a potential explanation for the structural and functional features of RBC(VL) which are involved in the hemolysis related to the anemia which develops in VL patients.

Sialic acids in different Leishmania sp., its correlation with nitric oxide resistance and host responses

The presence of different derivatives of sialic acids (SA) on Leishmania donovani instigated us to investigate their status on different strains of Leishmania sp. causing different forms of the disease. Leishmania tropica (K27), Leishmania major (JISH118) and Leishmania mexicana (LV4) responsible for cutaneous, Leishmania braziliensis (L280) and Leishmania amazonensis (LV81) causing diffuse and Leishmania infantum (MON29) responsible for visceral leishmaniasis were included in this study. The strains showed a differential distribution of SA in spite of their close resemblance in pathogenesis. K27, JISH118, L280 and MON29 were categorized as high SA-containing strains having enhanced 9-O-acetyl sialic acid (9-O-AcSA(high)) whereas LV4 and LV81 evidenced considerably reduced SA. Interestingly, 9-O-AcSA(high) promastigotes showed significant viability as compared to their de-O-acetylated forms after exposure to NaNO(2) suggesting the involvement of 9-O-AcSA in conferring nitric oxide (NO) resistance. Enhanced intracellular survivability was demonstrated following infection of human macrophages with 9-O-AcSA(high) promastigotes in contrast to their de-O-acetylated forms indicating their contribution in bestowing a survival benefit. Additionally, reduced accumulation of NO, interleukin-12 and interferon-gamma in the supernatant of macrophages infected with 9-O-AcSA(high) promastigotes indicated suppression of leishmanicidal host responses. However, LV4 and LV81 with least 9-O-AcSA, before and after de-O-acetylation, showed unaltered NO resistance, multiplicity and host responses signifying the probable involvement of other determinants which may be a function of their inherent parasitic attribute. Hence, enhanced levels of 9-O-AcSA serve as one of the potential determinants responsible for increased NO resistance and survivability of parasites by inhibition of host responses.

9-O-acetylated sialoglycoproteins are important immunomodulators in Indian visceral leishmaniasis.

ABSTRACT Overexpression of disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) on peripheral blood mononuclear cells (PBMC) of visceral leishmaniasis (VL) patients (PBMCVL) compared to their levels of expression in healthy individuals has been demonstrated using a lectin, achatinin-H, with specificity toward 9-O-acetylated sialic acid derivatives α2-6 linkage with subterminal N-acetylgalactosamine (9-O-AcSAα2-6GalNAc). The decreased presence of disease-associated 9-O-AcSGPs on different immune cells of parasitologically cured individuals after successful treatment relative to the levels in patients with active VL prior to treatment was demonstrated. However, their contributory role as immunomodulatory determinants on PBMCVL remained unexplored. Accordingly, 9-O-AcSGPs on PBMCVL were sensitized with achatinin-H, leading to their enhanced proliferation compared to that observed with different known mitogens or parasite antigen. This lymphoproliferative response was characterized by evaluation of the TH1/TH2 response by intracellular staining and enzyme-linked immunosorbent assay for secreted cytokines, and the results were corroborated by their genetic expression. Sensitized PBMCVL evidenced a mixed TH1/TH2 cellular response with a predominance of the TH1 response, indicating the ability of 9-O-AcSGPs to modulate the host cell toward a favorable response. Interestingly, the humoral and cellular responses showed a good correlation. Further, high levels of anti-9-O-AcSGP antibodies with an order of distribution of immunoglobulin M (IgM) > IgG1 = IgG3 > IgG4 > IgG2 > IgE could be explained by a mixed TH1/TH2 response. A good correlation of enhanced 9-O-AcSGPs with both the cell-mediated (r = 0.98) and humoral (r = 0.99) response was observed. In summary, it may be concluded that sensitization of 9-O-AcSGPs on PBMCVL may provide a basis for the modulation of the hosts immune response by their controlled expression, leading to a beneficial immune response and influencing the disease pathology.

Sialoglycotherapeutics in protozoal diseases.

The manipulation of glycosylation, mainly sialylation, holds enormous potential for understanding the biological functions of glycoproteins and glycolipids to treat many diseases. The existing knowledge in the field of glycobiology is exploited by glycotherapeutics for combating protozoan diseases. This review focuses on the development of novel glycobiological therapeutic strategies in the field of protozoan infections.

A Perspective on the Emergence of Sialic Acids as Potent Determinants Affecting Leishmania Biology

Leishmaniasis caused by Leishmania sp. has a wide range of manifestations from cutaneous to the deadly visceral form. They shuttle between the invertebrate and vertebrate hosts as promastigotes and amastigotes having adaptations for subverting host immune responses. Parasite-specific glycoconjugates have served as important determinants influencing parasite recognition, internalization, differentiation, multiplication, and virulence. Despite the steady progress in the field of parasite glycobiology, sialobiology has been a less traversed domain of research in leishmaniasis. The present paper focuses on identification, characterization, and differential distribution of sialoglycotope having the linkage-specific 9-O-acetylated sialic acid in promastigotes of different Leishmania sp. causing different clinical ramifications emphasizing possible role of these sialoglycotopes in infectivity, virulence, nitric oxide resistance, and host modulation in Leishmania spp. asserting them to be important molecules influencing parasite biology.

RETRACTED: Sialic acids, important constituents and selective recognition factors of Pseudomonas aeruginosa, an opportunistic pathogen.

This article has been retracted.