Tupanvirus-infected amoebas are induced to aggregate with uninfected cells promoting viral dissemination.

The discovery of giant viruses in the last years has fascinated the scientific community due to virus particles size and genome complexity. Among such fantastic discoveries, we have recently described tupanviruses, which particles present a long tail, and has a genome that contains the most complete set of translation-related genes ever reported in the known virosphere. Here we describe a new kind of virus-host interaction involving tupanvirus. We observed that tupanvirus-infected amoebas were induced to aggregate with uninfected cells, promoting viral dissemination and forming giant host cell bunches. Even after mechanical breakdown of bunches, amoebas reaggregated within a few minutes. This remarkable interaction between infected and uninfected cells seems to be promoted by the expression of a mannose receptor gene. Our investigations demonstrate that the pre-treatment of amoebas with free mannose inhibits the formation of bunches, in a concentration-dependent manner, suggesting that amoebal-bunch formation correlates with mannose receptor gene expression. Finally, our data suggest that bunch-forming cells are able to interact with uninfected cells promoting the dissemination and increase of tupanvirus progeny.

Itraconazole encapsulated PLGA-nanoparticles covered with mannose as potential candidates against leishmaniasis.

Leishmaniasis is a neglected disease threatening over 350 million people. Antimonials are first-line drugs due to resistance and side effects there is a demand for alternative chemotherapy. Itraconazole (ITZ) is an antimycotic. It was encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and covered with mannose. The NPs were 250 nm and -1.1 mV ± 0.7. PLGA-ITZ-mannose NPs presented a toxicity of 20.7% for J774 cells, and no toxicity for THP 1. The J774 cells were infected with three Leishmania promastigotes strains and treated with ITZ loaded PLGA NPs with/without mannose. The parasite percentage of L.(V.) panamensis intracellular amastigotes significantly (p < 0.01) decreased from 34.4% to 13.7% and 5.7% for PLGA-ITZ-mannose NPs and PLGA-ITZ NPs, respectively. For L.(L.) infantum there was a reduction (p < 0.001) from 18.1% to 4.8% and 8.3% for PLGA-ITZ-mannose NPs and PLGA-ITZ NPs, respectively. Further with L.(L.) braziliensis amastigotes there was a significant reduction (p < 0.001) from 54.9% to 28% and 21.1% for PLGA-ITZ-mannose NPs and PLGA-ITZ NPs, respectively. Adding mannose increased the efficacy PLGA-ITZ NPs against L.(L.) infantum, while it had no effect against L(V.) panamensis and L.(L.) braziliensis amastigotes. We recommend further investigation of PLGA-ITZ-mannose NPs in animal models to evaluate their potential. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000-000, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 680-687, 2019.

Purification of a thermostable antinociceptive lectin isolated from Andira anthelmia.

Andira anthelmia (tribe Dalbergieae), a plant from Brazilian Amazon, possesses a seed lectin that was purified by affinity chromatography in sepharose-mannose. This novel Dalbergieae lectin, named AAL, agglutinated rabbit erythrocytes treated with trypsin. The hemagglutinating activity of AAL was maintained after incubation at a wide range of temperature (40 to 70 °C) and pH, was shown to be dependent on divalent cations, and was inhibited by d-mannose and d-sucrose. AAL showed an electrophoretic profile in sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to other lectins of the tribe Dalbergieae, presenting a double band of molecular weight with approximately 20 kDa and other minor bands of 17, 15, and 13 kDa, being the smaller fragment glycosylated. AAL injected by intravenous route in mice showed antinociceptive activity in two behavioral tests (writhing and formalin). In the writhing test induced by acetic acid, AAL showed inhibitory effect at 0.01 mg/kg (68%), 0.1 mg/kg (46%) and 1 mg/kg (74%). In the formalin test, AAL (0.1 mg/kg) inhibited by 48% the licking time in the inflammatory phase, an effect that was recovered by the lectin association with mannose. In conclusion, AAL presents analgesic effect involving the lectin domain via peripheral mechanisms of inflammatory nociception. This activity highlights the importance of lectins as tools to be used for understanding the interaction of protein-carbohydrate in processes associated to inflammatory pain. Copyright © 2015 John Wiley & Sons, Ltd.

Purification and molecular characterization of a novel mannose-specific lectin from Dioclea reflexa hook seeds with inflammatory activity.

A novel lectin present in Dioclea reflexa seeds (DrfL) was discovered and described in this study. DrfL was purified in a single step by affinity chromatography in a Sephadex G-50 column. The lectin strongly agglutinated rabbit erythrocytes and was inhibited by α-methyl-D-mannoside, D-mannose, and D-glucose. The hemagglutinating activity of DrfL is optimum at pH 5.0-7.0, stable up to 50 °C, and dependent on divalent cations. Similar to other lectins of the subtribe Diocleinae, the analysis by mass spectrometry indicated that DrfL has three chains (α, ß, and γ) with masses of 25,562, 12,874, and 12,706 Da, respectively, with no disulfide bonds or glycosylation. DrfL showed inflammatory activity in the paw edema model and exhibited low cytotoxicity against Artemia sp.

Anticoagulant activity of native and partially degraded glycoglucuronomannan after chemical sulfation.

Heparin has great clinical importance as anticoagulant and antithrombotic agent. However, because of its risks of causing bleeding and contamination by animal pathogens, several studies aim to obtain alternatives to heparin. In the search for anticoagulant and antithrombotic agents from a non-animal source, a glycoglucuronomannan from the gum exudate of the plant Vochysia thyrsoidea was partially hydrolyzed, and both native and partially degraded polysaccharides were chemically sulfated, yielding VThS and Ph-VThS respectively. Methylation analysis indicated that sulfation occurred preferentially at the O-5 position of arabinose units in the VThS and at the O-6 position of mannose units in Ph-VThS. In vitro aPTT assay showed that VThS and Ph-VThS have anticoagulant activity, which could be controlled by protamine, and ex vivo aPTT assay demonstrated that Ph-VThS is absorbed by subcutaneous route. Like heparin, they were able to inhibit α-thrombin and factor Xa by a serpin-dependent mechanism. In vivo, VThS and Ph-VThS reduced thrombus formation by approximately 50% at a dose of 40 IU/kg, similarly to heparin. The results demonstrated that the chemically sulfated polysaccharides are promising anticoagulant and antithrombotic agents.

α-Glucosidase Inhibitors from a Xylaria feejeensis Associated with Hintonia latiflora.

Two new compounds, pestalotin 4'-O-methyl-ß-mannopyranoside (1) and 3S,4R-(+)-4-hydroxymellein (2), were isolated from an organic extract of a Xylaria feejeensis, which was isolated as an endophytic fungus from Hintonia latiflora. In addition, the known compounds 3S,4S-(+)-4-hydroxymellein (3), 3S-(+)-8-methoxymellein (4), and the quinone derivatives 2-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (5), 4S,5S,6S-4-hydroxy-3-methoxy-5-methyl-5,6-epoxycyclohex-2-en-1-one (6), and 4R,5R-dihydroxy-3-methoxy-5-methylcyclohexen-2-en-1-one (7) were obtained. The structures of 1 and 2 were elucidated using a set of spectroscopic and spectrometric techniques. The absolute configuration of the stereogenic centers of 1 and 2 was determined using ECD spectroscopy combined with time-dependent density functional theory calculations. In the case of 1, comparison of the experimental and theoretical (3)J6-7 coupling constants provided further evidence for the stereochemical assignments. Compounds 2 and 3 inhibited Saccharomyces cerevisiae α-glucosidase (αGHY), with IC50 values of 441 ± 23 and 549 ± 2.5 µM, respectively. Their activity was comparable to that of acarbose (IC50 = 545 ± 19 µM), used as positive control. Molecular docking predicted that both compounds bind to αGHY in a site different from the catalytic domain, which could imply an allosteric type of inhibition.

Lectin from Canavalia brasiliensis seeds (ConBr) is a valuable biotechnological tool to stimulate the growth of Rhizobium tropici in vitro.

To study the interactions between a Rhizobium tropici strain and lectins isolated from the seeds of Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr), a lectin fluorescence assay was performed. In addition, an experiment was designed to evaluate the effect of the two lectins on bacterial growth. Both lectins were found to bind to R. tropici cells, but the interactions were inhibited by D-mannose. Interestingly, only ConBr stimulated bacterial growth in proportion to the concentrations used (15.6-500 µg/mL), and the bacterial growth stimulation was inhibited by D-mannose as well. Structure/Function analyses by bioinformatics were carried out to evaluate the volume and carbohydrate recognition domain (CRD) configuration of ConA and ConBr. The difference of spatial arrangement and volume of CRD may indicate the variation between biological activities of both lectins. The results suggest that ConBr could be a promising tool for studies focusing on the interactions between rhizobia and host plants.

In vitro human immunodeficiency virus and sperm cell interaction mediated by the mannose receptor.

Leukocytes are considered to be the main source of HIV-1 infection in semen. However, HIV-1 interaction with spermatozoa has also been demonstrated, suggesting that both spermatozoa and leukocytes might play a role during sexual transmission of HIV-1. The purpose of the present study was to evaluate if HIV-1 particles interact with sperm cells through the mannose receptor (MR), and then to determine the ability of "infected" sperm cells to transmit the virus to susceptible targets. The expression of classical HIV-1 receptor and co-receptors and the MR by sperm cells was determined by flow cytometry; the interaction in vitro between sperm and HIV-1 was evaluated by fluorescence microscopy. Additionally, the in vitro interaction of sperm cells and HIV-1 was determined detecting viral nucleic acids by PCR. D-Mannose was used to block HIV-1-sperm cell interaction. Sperm cells preincubated with HIV-1 particles and activated mononuclear cells were co-cultured to determine viral transmission. The presence of viral RNA was detected in 28% of the samples in which sperm cells were preincubated with HIV-1 particles. Mannose was able to block interaction in 75% of the cases. Finally, we demonstrated that "infected" sperm cells were able to transmit the HIV-1 infection to susceptible targets. In conclusion, these results indicate that the MR is involved in sperm cell-HIV-1 interaction. Our results also suggest that sperm cells could be an important source of infection.

Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro.

Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (p<0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir formation in the llama UTJ oviductal segment.

Prodigiosin is not a determinant factor in lysis of Leishmania (Viannia) braziliensis after interaction with Serratia marcescens D-mannose sensitive fimbriae.

In this paper, the lytic activity of two variants of Serratia marcescens against promastigotes of Leishmania braziliensis was studied. In vitro assays showed that S. marcescens variant SM365 lyses L. braziliensis promastigotes, while the variant DB11 did not. Scanning electron microscopy (SEM) revealed that S. marcescens SM365 adheres to all cellular body and flagellum of the parasite. Several filamentous structures were formed and identified as biofilms. After 120min incubation, they connect the protozoan to the developing bacterial clusters. SEM also demonstrated that bacteria, adhered onto L. braziliensis promastigote surface, formed small filamentous structures which apparently penetrates into the parasite membrane. d-mannose protects L. braziliensis against the S. marcescens SM365 lytic effect in a dose dependent manner. SM365 variant pre cultivated at 37 degrees C did not synthesize prodigiosin although the adherence and lysis of L. braziliensis were similar to the effect observed with bacteria cultivated at 28 degrees C, which produce high concentrations of prodigiosin. Thus, we suggest that prodigiosin is not involved in the lysis of promastigotes and that adherence promoted by bacterial mannose-sensitive (MS) fimbriae is a determinant factor in the lysis of L. braziliensis by S. marcescens SM365.

Leishmania (Leishmania) chagasi interactions with Serratia marcescens: ultrastructural studies, lysis and carbohydrate effects.

Studies on the lysis of L. chagasi caused by the bacteria Serratia marcescens were carried out. In vitro experiments demonstrated that S. marcescens variant SM 365, a prodigiosin pigment producer, lysed this species of Leishmania but variant DB11, a nonpigmented bacteria, was unable to lyse the parasite. High concentrations of d-mannose were found to protect L. chagasi markedly diminishing the lysis by S. marcescens SM 365. Promastigotes of L. chagasi bound the lectin Concanavalin A conjugated with FITC, the fluorescence was intensely found at the base of the flagellum (flagellar pocket). Scanning electron microscopy revealed that the bacteria adherence occurred mainly in the flagellar pocket. S. marcescens SM 365 formed filamentous structures, identified as biofilms, which connect the protozoan to the developing bacterial clusters, in low concentrations of bacteria after 30 min incubation time. We suggest that bacterial mannose-sensitive (MS) fimbriae are relevant to S. marcescens SM 365 in the lysis of L. chagasi.

Mannose-resistant Proteus-like and P. mirabilis fimbriae have specific and additive roles in P. mirabilis urinary tract infections.

Proteus mirabilis is an important uropathogen that can cause complicated urinary tract infections (UTI). It produces several types of fimbriae, including mannose-resistant Proteus-like (MR/P) fimbriae and P. mirabilis fimbriae (PMF). Previously, we determined that these fimbriae affect the ability of P. mirabilis to colonize the urinary tract. The objective of this study was to analyse the effect of the simultaneous lack of P. mirabilis MR/P and PMF fimbriae in UTI pathogenesis. A double mutant lacking both fimbriae was generated by allelic replacement mutagenesis. This mutant was characterized genetically and phenotypically, and tested using an in vitro uroepithelial cell adhesion assay and the ascending UTI murine model. In vitro adhesion to uroepithelial cells by the P. mirabilis pmfA/mrpA-D mutant was reduced when compared with the wild-type, although no significant differences were observed when it was compared with the single mrpA-D and pmfA mutants. However, in vivo assays showed that colonization of kidneys and bladders by the P. mirabilis pmfA/mrpA-D mutant was significantly reduced when compared with the wild-type and both single mutants. These results indicate that, although redundancy can occur, MR/P and PMF fimbriae have specific and additive roles in P. mirabilis UTI.

Fimbriae of uropathogenic Proteus mirabilis.

Proteus mirabilis is a common causative agent of cystitis and pyelonephritis in patients with urinary catheters or structural abnormalities of the urinary tract. Several types of fimbriae, which are potentially involved in adhesion to the uroepithelium, can be expressed simultaneously by P. mirabilis: mannose-resistant/Proteus-like (MR/P) fimbriae, P. mirabilis fimbriae (PMF), uroepithelial cell adhesin (UCA), renamed by some authors nonagglutinating fimbriae (NAF), and ambient-temperature fimbriae (ATF). Proteus mirabilis is a common cause of biofilm formation on catheter material and MR/P fimbriae are involved in this process. The considerable serious pathology caused by P. mirabilis in the urinary tract warrants the development of a prophylactic vaccine, and several studies have pointed to MR/P fimbriae as a potential target for immunization. This article reviews P. mirabilis fimbriae with regard to their participation in uropathogenesis, biofilm formation and as vaccine targets.

Inhibitory effects of d-mannose on trypanosomatid lysis induced by Serratia marcescens.

Studies were carried out on the effects of different carbohydrates on the lysis of Trypanosoma cruzi, Trypanosoma rangeli and erythocytes caused by the bacteria Serratia marcescens variants SM 365 and RPH. High concentrations of d-mannose were found to protect T. cruzi and T. rangeli markedly diminishing the lysis caused by S. marcescens. However, this carbohydrate is unable to interfere with the hemolysis induced by SM 365 and RPH variants. These results showed that the trypanolytic effect induced by S. marcescens SM 365 and RPH variants is dependent on d-mannose and distinct from the hemolytic activity, strongly suggesting that bacterial fimbriae are relevant to S. marcescens in lysis of parasites.

Phagocytosis by Trichomonas vaginalis: new insights.

BACKGROUND INFORMATION: The parasitic protozoan Trichomonas vaginalis is the causative agent of trichomoniasis, a sexually transmitted disease. The phagocytic activity of this parasite has not been completely elucidated. In order to better understand the mechanisms of trichomonal phagocytosis, we have studied the in vitro capacity of T. vaginalis to phagocytose and degrade Saccharomyces cerevisiae cells. RESULTS AND CONCLUSIONS: To analyse the phagocytic ability and capacity, two isolates of T. vaginalis presenting different virulence grades were used. Complementary techniques, such as fluorescence microscopy, computer-based fluorescence analysis, scanning and transmission electron microscopy and the use of drugs that interfere with the actin microfilaments, were used in order to follow the behaviour of the actin cytoskeleton during phagocytosis of yeast cells by T. vaginalis. It was concluded that: (1) T. vaginalis changes its shape rapidly and engulfs the yeast cells, which are almost as large as the parasite; (2) long-term and fresh cultures are able to phagocytose, although the low-virulence strain JT demonstrated a lower activity when compared with the highly virulent T016 isolate; (3) the T016 strain exhibited an amoeboid morphology during the internalization of yeast cells in contrast with the JT strain; (4) attachment of yeast cells to the parasite occurs via the whole cell surface, including both anterior and recurrent flagella; (5) two forms of phagocytosis were observed: a 'sinking' process without any apparent participation of plasma membrane extensions and the classical phagocytosis where pseudopodia are extended toward the target cell; (6) the internalized S. cerevisiae are digested in lysosomes; (7) competitor sugars D-mannose or L-fucose inhibit the phagocytosis, and inhibition was 1.67 times higher in long-term cultured JT than that of the parasites from fresh isolate T016; (8) a thick layer of actin microfilaments was present underlying the plasma membrane, and especially in the pseudopodia and around the phagocytosed particles; (9) a dramatic change in the distribution pattern of fibrillar actin occurred during phagocytosis; (10) cytochalasin D depressed the phagocytosis; (11) a non-specific recognition and phagocytosis of yeast cells by T. vaginalis is mediated by a mannose receptor present on the parasite surface; (12) the phagocytic process may occur simultaneously during mitosis of the parasite.

Characterization of a nonfimbrial mannose-sensitive hemagglutinin (MSH) produced by Salmonella enterica serovar enteritidis.

A nonfimbrial mannose-sensitive hemagglutinin (MSH) with adhesive properties produced by Salmonella enterica serovar Enteritidis was characterized. The MSH was characterized as glycoprotein and consisted of three noncovalently bound subunits of M(r) 28, 33 and 40 kDa determined by SDS-PAGE. The hemagglutinin was heat-stable and resistant to alkaline (high) or acid (low) pH, however, it was inhibited by proteolytic enzymes, by EDTA and by sodium periodate. Mouse antiserum raised against MSH reacted with the 28 kDa band in immunoblotting, and also inhibited hemagglutination and bacterial adherence to HeLa cells. Electron microscope examinations showed that MSH is not a fimbriae-like structure. MSH and anti-MSH IgG competitively inhibited bacterial adherence to HeLa cells. The immunofluorescence test, using MSH on HeLa cells and specific anti-MSH IgG, supported the view that MSH contributes to adherence of the organism. These results indicate that MSH is a nonfimbrial putative adhesive factor that may mediate the adherence of Salmonella enteritidis to eucaryotic cells.

Neutrophil migration in mice induced by a mannose-binding lectin isolated from Annona coriacea seeds.

A novel 14-kDa lectin from Annona coriacea seeds (ACLEC) with hemagglutinating activity on erythrocytes has been recently described. Since plant lectins are known to present inflammatory activity, this study aimed to investigate the leukocyte migration induced by ACLEC, and inflammatory mediators involved in this phenomenon. Male Swiss mice were intraperitoneally injected with ACLEC (3-100 microg/cavity), and at 4-96 h thereafter the leukocyte counts in peritoneal lavage fluid were evaluated. ACLEC induced a dose-dependent neutrophil accumulation, reaching maximal responses at 16 h after injection (approximately 40-fold increase for 30 microg/cavity). Significant accumulation of mononuclear cells was observed at 72 h (2.7-fold increase). The carbohydrate mannose nearly abolished the neutrophil influx, whereas sucrose, glucose and galactose had no effect. Dexamethasone, the cyclooxygenase-2 (COX-2) inhibitor celecoxib and the Platelet activating factor (PAF) receptor antagonist PCA4248 significantly reduced ACLEC-induced neutrophil influx. The tachykinin NK(1) antagonist SR140333, the tachykinin NK(2) antagonist SR48968, the non-selective NO inhibitor L-NAME, the selective inducible NOS inhibitor aminoguanidine and the lypoxygenase inhibitor AA861 all failed to modify the ACLEC-induced responses. In conclusion, ACLEC is able to attract neutrophils into the mice peritoneal cavity by mechanisms involving interactions of the lectin with cell-specific mannose recognition, leading to the release of COX-2-derived mediators and PAF.

In vitro evaluation of the binding capacity of Saccharomyces cerevisiae Sc47 to adhere to the wall of Salmonella spp.

Saccharomyces cerevisiae Sc47 (Biosaf) is a commercially available baker's yeast strain (Lesaffre, France) that has been used as a probiotic in animal nutrition. It has been previously reported that animals fed with the yeast showed an improved resistance to several enteric infectious diseases. Some of the S. cerevisiae strains adhere potentially pathogenic bacteria such as Escherichia coli and Salmonella spp. This could be a mechanism through which animals fed with the yeast may become more resistant to infections caused by these microorganisms. In this paper, the adhesion of forty-five Salmonella spp. isolates to Sc47 was assessed by sedimentation and agglutination tests, and by light and electron microscopy. Results showed that 57.7% (26/45) of the isolates and 66.6% (6/9) of the Salmonella serovars tested adhered to the Sc47 cell wall.

Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells.

Pseudallescheria boydii is an emerging fungal pathogen that has a worldwide distribution. Virulence mechanisms of P. boydii are largely unknown. We studied the interaction between P. boydii and HEp2 cells and demonstrated that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane. Recently, our group characterized a peptidorhamnomannan (PRM) antigen on the cell surface of P. boydii. In order to better understand the role played by this surface glycoconjugate during cell adhesion and endocytosis, inhibition assays were performed using intact PRM and anti-PRM polyclonal antibody. When HEp2 cells were pre-treated with whole PRM molecule, the adhesion and endocytic indices were, respectively, 50% and 60% lower than in non-treated epithelial cells. Moreover, when the conidial cells were pre-incubated with anti-PRM antibodies, the adherence and endocytosis processes were inhibited in a dose-dependent manner. As PRM influenced the conidia P. boydii-HEp2 cell interaction, we also performed inhibition assays in order to observe which PRM moieties could be involved in this process. Treatment of PRM with proteinase K promoted a slight inhibition of adhesion. However, the de-O-glycosylated PRM molecule as well as the monosaccharide mannose was able to efficiently inhibit the adhesion and endocytic processes. In addition, our results indicate for the first time that P. boydii PRM binds to a polypeptide of 25 kDa on the HEp2 cell surface.

The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck).

A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides.