Complex interactions of vaginal microorganisms with the genital tract epithelium shape mucosal innate immunity, which holds the key to sexual and reproductive health. Bacterial vaginosis (BV), a microbiome-disturbance syndrome prevalent in reproductive-age women, occurs commonly in concert with trichomoniasis, and both are associated with increased risk of adverse reproductive outcomes and viral infections, largely attributable to inflammation. To investigate the causative relationships among inflammation, BV and trichomoniasis, we established a model of human cervicovaginal epithelial cells colonised by vaginal Lactobacillus isolates, dominant in healthy women, and common BV species (Atopobium vaginae, Gardnerella vaginalis and Prevotella bivia).
Colonised epithelia were infected with Trichomonas vaginalis (TV) or exposed to purified TV virulence factors (membrane lipophosphoglycan (LPG), its ceramide-phosphoinositol-glycan core (CPI-GC) or the endosymbiont Trichomonas vaginalis virus (TVV)), followed by assessment of bacterial colony-forming units, the mucosal anti-inflammatory microbicide secretory leucocyte protease inhibitor (SLPI), and chemokines that drive pro-inflammatory, antigen-presenting and T cells.
TV reduced colonisation by Lactobacillus but not by BV species, which were found inside epithelial cells. TV increased interleukin (IL)-8 and suppressed SLPI, likely via LPG/CPI-GC, and upregulated IL-8 and RANTES, likely via TVV as suggested by use of purified pathogenic determinants. BV species A vaginae and G vaginalis induced IL-8 and RANTES, and also amplified the pro-inflammatory responses to both LPG/CPI-GC and TVV, whereas P bivia suppressed the TV/TVV-induced chemokines.
These molecular host–parasite–endosymbiont–bacteria interactions explain epidemiological associations and suggest a revised paradigm for restoring vaginal immunity and preventing BV/TV-attributable inflammatory sequelae in women.
TRICHOMONAS; VAGINAL MICROBIOLOGY; IMMUNOLOGY; BACTERIAL VAGINOSIS; WOMEN
Background: Trichomonas vaginalis lipoglycan (TvLG) mediates interactions between the parasite and human host.
Results: TvLG is composed of a polyrhamnose backbone with branches of poly-N-acetyllactosamine that are involved in attachment to host epithelium.
Conclusion: TvLG has a unique structure among solved parasite glycans.
Significance: This work provides a template to analyze TvLG from T. vaginalis with different binding properties.
The extracellular parasite Trichomonas vaginalis contains a surface glycoconjugate that appears to mediate parasite-host cell interaction via binding to human galectin-1. This glycoconjugate also elicits cytokine production from human vaginal epithelial cells, implicating its role in modulation of host immune responses. We have analyzed the structure of this glycoconjugate, previously described to contain the sugars rhamnose (Rha), N-acetylglucosamine (GlcNAc), galactose (Gal), xylose (Xyl), N-acetylgalactosamine (GalNAc), and glucose (Glc), using gas chromatograph mass spectrometry (GC-MS), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF), electrospray MS/MS, and nuclear magnetic resonance (NMR), combined with chemical and enzymatic digestions. Our data reveal a complex structure, named T. vaginalis lipoglycan (TvLG), that differs markedly from Leishmania lipophosphoglycan and Entamoeba lipopeptidophosphoglycan and is devoid of phosphosaccharide repeats. TvLG is composed of an α1–3 linked polyrhamnose core, where Rha residues are substituted at the 2-position with either β-Xyl or chains of, on average, five N-acetyllactosamine (-3Galβ1–4GlcNAcβ1-) (LacNAc) units and occasionally lacto-N-biose (-3Galβ1-3GlcNAcβ1-) (LNB). These chains are themselves periodically substituted at the Gal residues with Xyl-Rha. These structural analyses led us to test the role of the poly-LacNAc/LNB chains in parasite binding to host cells. We found that reduction of poly-LacNAc/LNB chains decreased the ability of TvLG to compete parasite binding to host cells. In summary, our data provide a new model for the structure of TvLG, composed of a polyrhamnose backbone with branches of Xyl and poly-LacNAc/LNB. Furthermore, the poly-LacNAc side chains are shown to be involved in parasite-host cell interaction.
Adhesion; Glycoconjugate; Glycolipid Structure; Host-Pathogen Interactions; Pathogenesis; Trichomonas; TvLG
Trichomonas vaginalis is one of the most common nonviral sexually transmitted human infections and, worldwide, has been linked to increased incidence of human immunodeficiency virus type 1 transmission, preterm delivery, low birth weight, cervical cancer, and vaginitis. The molecular pathways that are important in initiating host inflammatory and immune responses to T. vaginalis are poorly understood. Here we report interactions of human cervicovaginal epithelial cells with the most abundant cell surface glycoconjugate of the parasite, the T. vaginalis lipophosphoglycan (LPG). Purified LPG mediated the adhesion of parasites to human vaginal epithelial cells in a dose-dependent manner. Furthermore, T. vaginalis LPG (but not LPG from Tritrichomonas foetus, the causative agent of bovine trichomoniasis) induced a selective upregulation of chemotactic cytokines by human endocervical, ectocervical, and vaginal epithelial cells, which do not express Toll-like receptor 4/MD2. The T. vaginalis LPG triggered interleukin 8 (IL-8), which promotes the adhesion and transmigration of neutrophils across the endothelium, and macrophage inflammatory protein 3α, which is a chemoattractant for immune cells and is essential for dendritic cell maturation. These effects were dose dependent and sustained in the absence of cytotoxicity and IL-1β release and utilized, at least in part, a signaling pathway independent from the Toll-like/IL-1 receptor adaptor protein MyD88.
The extracellular protozoan parasite Trichomonas vaginalis causes the most prevalent non-viral sexually transmitted human infection, yet the pathogenesis of infection is poorly understood, and host cell receptors have not been described. The surface of T. vaginalis is covered with a glycoconjugate called lipophosphoglycan (LPG), which plays a role in the adherence and cytotoxicity of parasites to human cells. T. vaginalis LPG contains high amounts of galactose, making this polysaccharide a candidate for recognition by the galactose-binding galectin family of lectins. Here we show that galectin-1 (gal-1) is expressed by human cervical epithelial cells and binds T. vaginalis LPG. Gal-1 binds to parasites in a carbohydrate-dependent manner that is inhibited in the presence of T. vaginalis LPG. Addition of purified gal-1 to cervical epithelial cells also enhances parasite binding, while a decrease in gal-1 expression by small interfering RNA (siRNA) transfection decreases parasite binding. In contrast, the related galectin-7 (gal-7) does not bind T. vaginalis in a carbohydrate-dependent manner, and is unable to mediate attachment of parasites to host cells. Our data are consistent with the presence of multiple host cell receptors for T. vaginalis of which gal-1 is the first to be identified and highlight the importance of glycoconjugates in host–pathogen interactions.
An in vitro culture system of bovine vaginal epithelial cells (BVECs) was developed to study the cytopathogenic effects of Tritrichomonas foetus and the role of lipophosphoglycan (LPG)-like cell surface glycoconjugates in adhesion of parasites to host cells. Exposure of BVEC monolayers to T. foetus resulted in extensive damage of monolayers. Host cell disruption was measured quantitatively by a trypan blue exclusion assay and by release of 3H from [3H]thymidine-labeled host cells. Results indicated contact-dependent cytotoxicity of host cells by T. foetus. The cytopathogenic effect was a function of T. foetus density. Metronidazole- or periodate-treated T. foetus showed no damage to BVEC monolayers. A related human trichomonad, Trichomonas vaginalis, showed no cytotoxic effects, indicating species-specific host-parasite interactions. A direct binding assay was developed and used to investigate the role of a major cell surface LPG-like molecule in host-parasite adhesion. The results of competition experiments showed that the binding to BVECs was displaceable, was saturable, and yielded a typical binding curve, suggesting that specific receptor-ligand interactions mediate the attachment of T. foetus to BVECs. Progesterone-treated BVECs showed enhanced parasite binding. T. foetus LPG inhibited the binding of T. foetus to BVECs; the LPG from T. vaginalis and a variety of other glycoconjugates did not. These data imply specificity of LPG on host-parasite adhesion. Periodate-treated parasites showed no adherence to host cells, indicating the involvement of carbohydrate containing molecules in the adhesion process.
The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.
Trichomonas vaginalis is the most common non-viral sexually transmitted pathogen. The infection is prevalent in reproductive age women and is associated with vaginitis, endometritis, adnexitis, pyosalpinx, infertility, preterm birth, low birth weight, bacterial vaginosis, and increased risk of cervical cancer, HPV, and HIV infection. In men, its complications include urethritis, prostatitis, epididymitis, and infertility through inflammatory damage or interference with the sperm function. The infection is often asymptomatic and recurrent despite the presence of specific antibodies, suggesting the importance of the innate immune defense. T. vaginalis adhesion proteins, cysteine proteases, and the major parasite lipophosphoglycan (LPG) play distinct roles in the pathogenesis and evasion of host immunity. LPG plays a key role in the parasite adherence and signaling to human vaginal and cervical epithelial cells, which is at least in part mediated by galectins. The epithelial cells respond to T. vaginalis infection and purified LPG by selective upregulation of proinflammatory mediators. At the same time, T. vaginalis triggers an immunosuppressive response in monocytes, macrophages, and dendritic cells. The molecular mechanisms underlying reproductive complications and epidemiologic risks associated with T. vaginalis infection remain to be elucidated.
Trichomonas vaginalis; lipophosphoglycan; cytokines; galectins; human vaginal epithelial cells
The flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T = 1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves −2, and not +1, ribosomal frameshifting, an uncommonly found mechanism to date.
Trichomonas vaginalis causes ~250 million new cases of sexually transmitted disease each year worldwide and is associated with serious complications, including premature birth and increased transmission of other pathogens, including HIV. It is an extracellular parasite that, in turn, commonly hosts infections with double-stranded RNA (dsRNA) viruses, trichomonasviruses, which appear to exacerbate disease through signaling of immunoinflammatory responses by human epithelial cells. Here we report the first three-dimensional structure of a trichomonasvirus, which is also the first such structure of any protozoan dsRNA virus; show that it has unusually wide channels at the capsid vertices, with potential for releasing the viral genome and promoting dsRNA-dependent responses by human cells; and provide evidence that it uses −2 ribosomal frameshifting, an uncommon mechanism, to translate its RNA polymerase in fusion with its capsid protein. These findings provide both mechanistic and translational insights concerning the role of trichomonasviruses in aggravating disease attributable to T. vaginalis.
Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In “selective” sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the “selective” fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both ‘poly-scGal’ and ‘null-scGal’ lines survived poorly relative to PpapJ-sympatric L. major FV1 and other ‘mono-scGal’ lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing ‘null-scGal’-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a ‘PpapJ-optimal’ scGal-LPG PAMP. Unexpectedly, these “L. major FV1-cloaked” L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific ‘mono-scGal’ pattern. However, failure of ‘mono-scGal’ L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is “selective” or “permissive”, with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania.
Phlebotomine sand flies are tiny blood-feeding insects that transmit Leishmania protozoan parasites, which cause diseases afflicting millions of people. The world-wide distribution of Leishmania is determined by the availability of transmission-competent vectors. In the laboratory, some vectors support many different Leishmania, while others are highly restricted. This is best exemplified by P. papatasi, which transmit only L. major despite a wide distribution in regions endemic for many Leishmania species. P. papatasi “selectivity” can be reproduced experimentally, and has been attributed to β1,3-linked galactose side chains decorating the abundant L. major surface lipophosphoglycan (LPG) adhesin, which mediate parasite attachment to the P. papatasi midgut to prevent elimination when the digested blood meal is excreted. As geographically diverse L. major display very different LPG galactosylation patterns (n = 0 - 8 βGals/side chain), we explored the consequences of this pattern diversity to survival in P. papatasi. Using natural isolates and L. major lines engineered to express a wide range of LPG galactosylation patterns, we showed L. major survival in P. papatasi PpapJ flies was optimized by expression of highly modified ‘mono-galactosylated’ LPG and extremely sensitive to LPG side chain length. Surprisingly, L. donovani lines engineered to express a “PpapJ-optimal” LPG mono-galactosylation pattern did not survive in PpapJ flies, suggesting that additional interactions are required. These studies reveal the fine specificity of Leishmania - sand fly interactions, and the nature of species- and strain-specific parasite molecules that have co-evolved to take advantage of midgut receptors specific to available sand fly vectors.
A 401-residue-long protein, ST0452, has been identified from a thermophilic archaeon, Sulfolobus tokodaii strain 7, as a glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase) homolog with a 170-residue-long extra C-terminus portion. Functional analyses of the ST0452 protein have confirmed that the protein possessed dual sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) activities. The 24 repeats of a signature motif sequence which has been found in bacterial acetyltransferases, (L/I/V)-(G/A/E/D)-XX-(S/T/A/V)-X, were detected at the C terminus of the ST0452 protein. This observation prompted our group to investigate the acetyltransferase activity of the ST0452 protein. Detection of the release of coenzyme A (CoA) from acetyl-CoA and the production of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P) and UTP in the presence of the ST0452 protein revealed that this protein possesses the GlcN-1-P-specific acetyltransferase activity. In addition, analyses of substrate specificity showed that acetyltransferase activity of the ST0452 protein is capable of catalyzing the change of galactosamine-1-phosphate (GalN-1-P) to N-acetyl-d-galactosamine-1-phosphate (GalNAc-1-P) as well as GlcN-1-P and that its sugar-1-P NTase activity is capable of producing UDP-GalNAc from GalNAc-1-P and UTP. This is the first report of a thermostable bifunctional enzyme with GalN-1-P acetyltransferase and GalNAc-1-P uridyltransferase activities. The observation reveals that the bacteria-type UDP-GlcNAc biosynthetic pathway from fructose-6-phospate is utilized in this archaeon and represents a novel biosynthetic pathway for producing UDP-GalNAc from GalN-1-P in this microorganism.
In the protozoan parasite Leishmania, abundant surface
and secreted molecules such as lipophosphoglycan (LPG)1 and proteophosphoglycans (PPGs) contain extensive
galactose residues in the form of phosphoglycans (PGs) containing
[Gal-Man-PO4] repeating units. PGs are
synthesized in the parasite Golgi apparatus and require transport of cytoplasmic
nucleotide-sugar precursors to the Golgi lumen by nucleotide sugar transporters
(NSTs). GDP-Man transport is mediated by the LPG2 gene product,
and here we focused on transporters for UDP-Gal. Database mining revealed twelve
candidate NST genes in the L. major genome, including
LPG2, as well as a candidate endoplasmic reticulum
UDP-glucose transporter (HUT1L), and several pseudogenes. Gene
knockout studies established that two genes (LPG5A and
LPG5B) encoded UDP-Gal NSTs. While the single
lpg5B− mutants produced PGs, an
double mutant was completely deficient. PG synthesis was restored in the
mutant by heterologous expression of the human UDP-Gal transporter, and
heterologous expression of LPG5A and LPG5B
rescued the glycosylation defects of the mammalian Lec8 mutant, which is
deficient in UDP-Gal uptake. Interestingly, the LPG5A and
LPG5B functions overlap but are not equivalent, as the
lpg5A− mutant showed a partial
defect in LPG but not PPG phosphoglycosylation, while the
lpg5B− mutant showed a partial
defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs
in Leishmania will facilitate the dissection of glycoconjugate
synthesis and its role(s) in the parasite life cycle and further our
understanding of NSTs generally.
Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLeX), and related lectin ligands on effector leukocytes. Based on anti-sLeX antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLeX formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLeX (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLeX structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLeX on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.
Adhesion; Carbohydrate; Carbohydrate Structure; Glycoprotein Structure; Glycosylation Inhibitors; Lectin
Protozoan parasites of the genus Leishmania synthesize lipophosphoglycans (LPGs), phosphoglycans and proteophosphoglycans that contain phosphosaccharide repeat units of [−6)Gal(β1-4)Man(α1-OPO3H−]. The repeat structures are assembled by sequential addition of Manα1-OPO3H and β-Gal. In this study, an UDP-Gal-dependent activity was detected in L. donovani and L. major membranes using synthetic phospho-oligosaccharide fragments of lipophosphoglycan as acceptor substrates. Incubation of a microsomal preparation from L. donovani or L. major parasites with synthetic substrates and UDP-[6-3H]Gal resulted in incorporation of radiolabel into these exogenous acceptors. The [3H]galactose-labeled products were characterized by degradation into radioactive, low molecular mass fragments upon hydrolysis with mild acid and treatment with β-galactosidases. We showed that the activity detected with L. donovani membranes is the elongating β-d-galactosyltransferase associated with LPG phosphosaccharide backbone biosynthesis (eGalT). The eGalT activity showed a requirement for the presence of at least one phosphodiester group in the substrate and it was enhanced dramatically when two or three phosphodiester groups were present. Using the same substrates we detected two types of galactosyltransferase activity in L. major membranes: the elongating β-d-galactosyltransferase and a branching β-d-galactosyltransferase (bGalT). Both L. major enzymes required a minimum of one phosphodiester group present in the substrate, but acceptors with two or three phosphodiester groups were found to be superior.
To elucidate the roles of metalloproteinases and the Bcl-2 family of proteins in Trichovaginalis. vaginalis-induced apoptosis in human cervical cancer cells (SiHa cells) and vaginal epithelial cells (MS74 cells), SiHa cells and MS74 cells were incubated with live T. vaginalis, T. vaginalis excretory and secretory products (ESP), and T. vaginalis lysates, either with or without the specific metalloproteinase inhibitor 1,10-phenanthroline (1,10-PT), and examined apoptotic events and Bcl-2 signaling. The live T. vaginalis and the T. vaginalis ESP induced the release of cytochrome c into the cytosol, the activation of caspase-3 and caspase-9, and the cleavage of PARP. Additionally, the live T. vaginalis, but not the T. vaginalis lysate, induced the cleavage of the proapoptotic Bim protein. The live T. vaginalis and the T. vaginalis ESP, but not the T. vaginalis lysate, induced the dose-dependent cleavage of the antiapoptotic Bcl-xL and Mcl-1 proteins and decreased the association levels of Bcl-xL/Bim and Mcl-1/Bim complexes. We performed gelatin zymography and casein-hydrolysis assays on the live T. vaginalis and the T. vaginalis ESP to identify the apoptosis-inducing factor. Both the live T. vaginalis and the ESP contained high levels of metalloproteinases, of which activities were significantly inhibited by 1,10-PT treatment. Furthermore, the 1,10-PT blocked the cleavage of Bcl-xL, Mcl-1, PARP, caspase-3, and caspase-9, as well as the release of cytochrome c into the cytosol, and it significantly increased the association levels of the Bcl-xL/Bim and Mcl-1/Bim protein complexes, returning them to normal levels. Our results demonstrate that T. vaginalis induces mitochondria-dependent apoptosis in SiHa cells through the dissociation of Bcl-xL/Bim and Mcl-1/Bim complexes and that the apoptosis is blocked by the metalloproteinase inhibitor 1,10-PT. These results expand our understanding of the role of metalloproteinases in T. vaginalis-induced apoptosis and the signaling pathway in trichomoniasis of the cervicovaginal epithelial cells.
Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(β1,4)Man(α1-PO4)]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2− L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2−, instead resembling the L. major lipophosphoglycan-deficient lpg1− mutant. Metacyclic lpg5A−/lpg5B− promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A−/lpg5B− mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2− Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.
Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO4] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2− or lpg5A−/5B−) or LPG alone (lpg1−) along with their respective gene add-back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36–72 h after the infective feed, all parasites developed well except the lpg2− and lpg5A−/5B− mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2− in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2− promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage.
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.
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.
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).
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.
Leishmania enriettii; Glycoconjugates; Lipophosphoglycan; Glycoinositolphospholipids; Cavia porcellus; Macrophage; Innate immunity
Trichomonas vaginalis is a common sexually transmitted infection (STI) causing vaginitis. Microscopy has poor sensitivity but is used for diagnosis of trichomoniasis in resource-poor settings. We aimed to provide a more reliable diagnosis of trichomoniasis by investigating an association with leukorrhea. Women presenting for evaluation of vaginal discharge, STI exposure, or preventative gynecologic examination were evaluated for Trichomonas infection. Vaginal pH was determined and microscopy was performed by the provider, who recorded the number of polymorphonuclear leukocytes (PMNLs) per epithelial cell and the presence of clue cells, yeast, and/or motile trichomonads. Leukorrhea was defined as greater than one PMNL per epithelial cell. Culture and a nucleic acid amplification test (NAAT) were used to detect T. vaginalis. Patients were evaluated for Chlamydia trachomatis and Neisseria gonorrhoeae using NAATs and bacterial vaginosis using Gram stains. Two hundred ninety-four women were enrolled, and 16% were found to have Trichomonas (46/294). Trichomonas infection was more common in parous non-Hispanic, black women, who reported low rates of contraceptive use (33% versus 17%; P = 0.02) and a STI history (85% versus 55%; P = 0.002). These women were more likely to report vaginal discharge (76% versus 59%; P = 0.02) and have an elevated vaginal pH (87% versus 48%; P < 0.001) and gonorrhea infection (15% versus 4%; P = 0.002). Leukorrhea was associated with a 4-fold-increased risk of Trichomonas infection. Leukorrhea on microscopy was associated with Trichomonas vaginitis. Patients with leukorrhea should be evaluated with more-sensitive tests for T. vaginalis, preferably NAATs, if microscopy is negative.
Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization.
Trichomoniasis, the most common non-viral sexually transmitted disease worldwide, infects over 275 million people annually. Infection results from the colonization of the human urogenital tract by the parasite Trichomonas vaginalis. To establish and maintain infection the parasite adheres to host cells, a process that is poorly understood. Here, we show that T. vaginalis secretes small vesicles called exosomes that are capable of fusing with and delivering their contents to host cells. Parasite exosomes were found to induce changes in the host cell and to mediate the interaction of T. vaginalis with host by increasing the adherence of the parasite to host cells. Exosomes have been primarily studied in mammalian cells where they have been shown to mediate intercellular communication and have been implicated in processes including development, antigen presentation and cancer metastasis. Our data extend the function of exosomes to mediating host∶parasite interactions, cellular communication between two species and promoting colonization of an extracellular parasite. Research on T. vaginalis exosomes holds the potential for developing applications that would allow exosomes to be used in detecting and diagnosing trichomoniasis and for targeting drugs to the site of infection.
The life cycle of Leishmania parasites within the sand fly vector includes the development of extracellular promastigotes from a noninfective, procyclic stage into an infective, metacyclic stage that is uniquely adapted for transmission by the fly and survival in the vertebrate host. These adaptations were explored in the context of the structure and function of the abundant surface lipophosphoglycan (LPG) on Leishmania donovani promastigotes. During metacyclogenesis, the salient structural feature of L. donovani LPG is conserved, involving expression of a phosphoglycan chain made up of unsubstituted disaccharide-phosphate repeats. Two important developmental modifications were also observed. First, the size of the molecule is substantially increased because of a twofold increase in the number of phosphorylated disaccharide repeat units expressed. Second, there is a concomitant decrease in the presentation of terminally exposed sugars. This later property was indicated by the reduced accessibility of terminal galactose residues to galactose oxidase and the loss of binding by the lectins, peanut agglutinin, and concanavalin A, to metacyclic LPG in vivo and in vitro. The loss of lectin binding was not due to downregulation of the capping oligosaccharides as the same beta- linked galactose or alpha-linked mannose-terminating oligosaccharides were present in both procyclic and metacyclic promastigotes. The capping sugars on procyclic LPG were found to mediate procyclic attachment to the sand fly midgut, whereas these same sugars on metacyclic LPG failed to mediate metacyclic binding. And whereas intact metacyclic LPG did not inhibit procyclic attachment, depolymerized LPG inhibited as well as procyclic LPG, demonstrating that the ligands are normally buried. The masking of the terminal sugars is attributed to folding and clustering of the extended phosphoglycan chains, which form densely distributed particulate structures visible on fracture-flip preparations of the metacyclic surface. The exposure and subsequent masking of the terminal capping sugars explains the stage specificity of promastigote attachment to and release from the vector midgut, which are key events in the development of transmissible infections in the fly.
Francisella tularensis is a highly infectious pathogen that causes tularemia. Francisella lipid A contains an unusual galactosamine (GalN) unit, attached to its 1-phosphate moiety. Two genes, flmF2 and flmK, are required for the addition of GalN to Francisella lipid A, but the relevant enzymes and the GalN donor substrate have not been characterized. We now report the purification and identification of a novel minor lipid from Francisella novicida that functions as the GalN donor. Based on electrospray ionization mass spectrometry (ESI/MS) and NMR spectroscopy, we propose that this compound is undecaprenyl phosphate-β-D-GalN. Approximately 0.5 mg of pure lipid was obtained from 10 g of F. novicida by chloroform/methanol extraction, followed by DEAE-cellulose chromatography, mild alkaline hydrolysis, and thin layer chromatography. ESI/MS in the negative mode revealed a molecular ion [M-H]- at m/z 1006.699, consistent with undecaprenyl phosphate-GalN. 31P-NMR spectroscopy showed a single phosphorus atom in phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the sugar. 1H-NMR studies showed the presence of a polyisoprene chain and a sugar consistent with a β-D-GalN unit. Heteronuclear multiple quantum coherence (HMQC) analysis confirmed that nitrogen is attached to C-2 of the sugar. Purified undecaprenyl phosphate-β-D-GalN supports the in vitro modification of lipid IVA by membranes of E. coli cells expressing FlmK, an orthologue of E. coli ArnT, the enzyme that transfers 4- amino-4-deoxy-L-arabinose to lipid A in polymyxin-resistant strains. The discovery of undecaprenyl phosphate-β-D-GalN suggests Francisella modifies lipid A with GalN on the periplasmic surface of the inner membrane.
Sand fly species able to support the survival of the protozoan parasite Leishmania have been classified as permissive or specific, based upon their ability to support a wide or limited range of strains and/or species. Studies of a limited number of fly/parasite species combinations have implicated parasite surface molecules in this process and here we provide further evidence in support of this proposal. We investigated the role of lipophosphoglycan (LPG) and other phosphoglycans (PGs) in sand fly survival, using Leishmania major mutants deficient in LPG (lpg1−), and the phosphoglycan (PG)-deficient mutant lpg2−. The sand fly species used were the permissive species Phlebotomus perniciosus and P. argentipes, and the specific vector P. duboscqi, a species resistant to L. infantum development.
The lpg2− mutants did not survive well in any of the three sand fly species, suggesting that phosphoglycans and/or other LPG2-dependent molecules are required for parasite development. In vitro, all three L. major lines were equally resistant to proteolytic activity of bovine trypsin, suggesting that sand fly-specific hydrolytic proteases or other factors are the reason for the early lpg2− parasite killing. The lpg1− mutants developed late-stage infections in two permissive species, P. perniciosus and P. argentipes, where their infection rates and intensities of infections were comparable to the wild type (WT) parasites. In contrast, in P. duboscqi the lpg1− mutants developed significantly worse than the WT parasites.
In combination with previous studies, the data establish clearly that LPG is not required for Leishmania survival in permissive species P. perniciosus and P. argentipes but plays an important role in the specific vector P. duboscqi. With regard to PGs other than LPG, the data prove the importance of LPG2-related molecules for survival of L. major in the three sand fly species tested.
Phlebotomine sand flies are small blood-feeding insects, medically important as vectors of protozoan parasites of the genus Leishmania. Sand flies species can be divided roughly into two groups, termed specific or permissive, depending on their ability to support development of one or a few strains vs. a broad spectrum of these parasites. In this study, we explored the ability of two Leishmania major glycocalyx mutants to survive within these different types of vectors. The lpg1− mutant, which specifically lacks lipophosphoglycan (LPG), was able to survive normally in two permissive species, Phlebotomus argentipes and P. perniciosus, but was only able to survive within the specific species P. duboscqi for a limited time prior to dissolution of the peritrophic matrix. Consistent with its classification as a specific sand fly vector, P. duboscqi was not able to support development of L. infantum. The lpg2− L. major mutant, which is a broader mutant and lacks all phosphoglycans including LPG and proteophosphoglycans, was unable to survive in all the three vector species tested. This study extends the knowledge on the role of Leishmania major surface glycoconjugates to development in three important vector species and gives supporting evidence for the existence of an LPG-independent mechanism for survival in sand flies, as well as the importance of LPG2-dependent glycoconjugates in parasite survival.
A xylose-based glycosaminoglycan (GAG) core was recently identified at a Ser residue in the linker sequence of a recombinant Fc fusion protein. The linker sequence, G-S-G-G-G-G, and an upstream acidic residue were serving as a substrate for O-xylosyltransferase, resulting in a major glycan composed of Xyl-Gal-Gal-GlcA and other minor intermediates. In this paper, a portion of an unrelated protein was fused to the C-terminus of an IgG Fc domain using the common (G4S)4 linker repeat. This linker resulted in a heterogenous population of xylose-based glycans all containing at least a core Xyl. Commonly observed glycan structures include GAG-related di-, tri-, tetra-, and penta-saccharides (e.g., Xyl-Gal, Xyl-Gal-Gal, Xyl-Gal-Gal-GlcA, and Xyl-Gal-Gal-GlcA-HexNAc), as well as Xyl-Gal-Neu5Ac. Following alkaline phosphatase or sialidase treatment combined with CID fragmentation, low-level glycans with a mass addition of 79.9 Da were confirmed to be a result of phosphorylated xylose. A minute quantity of phosphorylated GAG pentasaccharides may also be sulfated (also 79.9 Da), possibly at the HexNAc moiety due to non-reactivity to alkaline phosphatase. The xylose moiety may be randomly incorporated in one of the three G-S-G sequence motifs; and the linker peptide shows evidence for multiple additions of xylose at very low levels.
glycosaminoglycans; xylose; Fc-fusion protein; linkers; serine; phosphorylation; sulfation
Metronidazole, the U.S. Food and Drug Administration-approved drug against trichomoniasis, is nonspermicidal and thus cannot offer pregnancy protection when used vaginally. Furthermore, increasing resistance of Trichomonas vaginalis to 5-nitro-imidazoles is a cause for serious concern. On the other hand, the vaginal spermicide nonoxynol-9 (N-9) does not protect against sexually transmitted diseases and HIV in clinical situations but may in fact increase their incidence due to its nonspecific, surfactant action. We therefore designed dually active, nonsurfactant molecules that were capable of killing Trichomonas vaginalis (both metronidazole-susceptible and -resistant strains) and irreversibly inactivating 100% human sperm at doses that were noncytotoxic to human cervical epithelial (HeLa) cells and vaginal microflora (lactobacilli) in vitro. Anaerobic energy metabolism, cell motility, and defense against reactive oxygen species, which are key to survival of both sperm and Trichomonas in the host after intravaginal inoculation, depend crucially on availability of free thiols. Consequently, molecules were designed with carbodithioic acid moiety as the major pharmacophore, and chemical variations were incorporated to provide high excess of reactive thiols for interacting with accessible thiols on sperm and Trichomonas. We report here the in vitro activities, structure-activity relationships, and safety profiles of these spermicidal antitrichomonas agents, the most promising of which was more effective than N-9 (the OTC spermicide) in inactivating human sperm and more efficacious than metronidazole in killing Trichomonas vaginalis (including metronidazole-resistant strain). It also significantly reduced the available free thiols on human sperm and inhibited the cytoadherence of Trichomonas on HeLa cells. Experimentally in vitro, the new compounds appeared to be safer than N-9 for vaginal use.
Glucosamine (GlcN), which has previously been shown to rescue fruiting body formation, lysis, and sporulation in a developmental mutant (G. Janssen and M. Dworkin, Dev. Biol. 112:194-202, 1985), induced lysis in vegetative and developing wild-type cells and inhibited fruiting body formation. It also resulted in a transient, intracellular increase in the concentration of glycerol, a known sporulation inducer, and sporulation of the surviving cells. Phospholipase activity, which was shown to be normally developmentally regulated, increased 7.6-fold after treatment of vegetative cells with 50 mM GlcN. Likewise, autocidal activity, which normally increased 18 to 24 h after the initiation of development, increased 20% when vegetative or developing cells were exposed to GlcN. Two mutants resistant to GlcN-induced lysis (MD1021 and MD1022) were isolated and showed neither an increase in autocide production nor an increase in phospholipase activity in response to added GlcN. MD1021 was developmentally deficient, and GlcN rescued fruiting body formation as well as phospholipase activity and autocide production. We propose that GlcN exerts its lytic effect by regulating the activity of phospholipase enzymes that release autocides, compounds that are believed to be responsible for developmental autolysis. GlcN-induced sporulation was found to depend on several factors: the initial cell density, the amount of lysis induced by GlcN, and the presence of tan-phase variants. An initial cell density of greater than 2 x 10(5) cells per ml was required to support GlcN-induced sporulation, and sporulation did not occur unless 50 to 75% of these cells had lysed. Mutants that were resistant to GlcN-induced lysis also did not sporulate in the presence of GlcN. The effects of GlcN on developing cells depended on the concentration of GlcN added; the addition of low concentrations of GlcN resulted in enhancement of sporulation, while higher concentrations resulted in the inhibition of sporulation. The ultrastructure of GlcN-induced spores resembled that of spores induced by the exogenous addition of glycerol, in contrast to spores isolated from mature fruiting bodies. A model by which GlcN may regulate both lysis and sporulation is presented.