Recent insights into the mucosal reactions associated with Giardia lamblia infections.

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.

Experimental infections of neonatal mice with cysts of Giardia lamblia clone GS/M-83-H7 are associated with an antigenic reset of the parasite.

Transmission of the protozoan parasite Giardia lamblia from one to another host individuum occurs through peroral ingestion of cysts which, following excystation in the small intestine, release two trophozoites each. Many studies have focused on the major surface antigen, VSP (for variant surface protein), which is responsible for the antigenic variability of the parasite. By using trophozoites of G. lamblia clone GS/M-83-H7 (expressing VSP H7) and the neonatal mouse model for experimental infections, we quantitatively assessed the process of antigenic variation of the parasite on the transcriptional level. In the present study, variant-specific regions identified on different GS/M-83-H7 vsp sequences served as targets for quantitative reverse transcription-PCR to monitor alterations in vsp mRNA levels during infection. Respective results demonstrated that antigenic switching of both the duodenal trophozoite and the cecal cyst populations was associated with a massive reduction in vsp H7 mRNA levels but not with a simultaneous increase in transcripts of any of the subvariant vsp genes analyzed. Most importantly, we also explored giardial variant-type formation and vsp mRNA levels after infection of mice with cysts. This infection mode led to an antigenic reset of the parasite in that a VSP H7-negative inoculum "converted" into a population of intestinal trophozoites that essentially consisted of the original VSP H7 type. This antigenic reset appears to be associated with excystation rather than with a selective process which favors expansion of a residual population of VSP H7 types within the antigenically diversified cyst inoculum. Based on these findings, the VSP H7 type has to be regarded as a predominant variant of G. lamblia clone GS/M-83-H7 which (re-)emerges during early-stage infection and may contribute to an optimal establishment of the parasite within the intestine of the experimental murine host.

Use of a novel DNA melting profile assay for the identification of PCR-amplified genomic sequences encoding for variant-specific surface proteins from the clonal GS/M-83-H7 line of Giardia lamblia.

During infections, Giardia lamblia undergoes a continuous change of its major surface antigens, the variant-specific surface proteins (VSPs). Many studies on antigenic variation have been performed using G. lamblia clone GS/M-83-H7, which expresses surface antigen VSP H7. The present study was focused on the identification and characterization of vsp gene sequences within the genome of the clonal G. lamblia GS/M-83-H7 line. For this purpose, we applied a PCR which specifically amplified truncated sequences from the 3'-terminal region of the vsp genes. Upon cloning, most of the vsp gene amplification products were shown to be approximately identical in size and thus could not be distinguished from each other by conventional gel electrophoresis. In order to pre-estimate the sequence complexity within the large panel of vsp clones isolated, we elaborated a novel concept which facilitated our large-scale genetic screening approach: PCR products from cloned DNA molecules were generated and then subjected to a DNA melting profile assay based on the use of the LightCycler Instrument. This high-throughput assay system proved to be well suited to monitor sequence differences between the amplification products from closely related vsp genes and thus could be used for the primary, sequence-related discrimination of the corresponding clones. After testing 50 candidates, vsp clones could be divided into five groups, each characterized by an individual DNA melting profile of the corresponding amplification products. Sequence analysis of some of these 50 candidates confirmed data from the aforementioned assay in that clones were demonstrated to be identical within, but different between, the distinct groups. The nucleotide and deduced amino acid sequences of five representative vsp clones showed high similarities both among each other and also with the corresponding gene segment of the variant-specific surface antigen (VSP H7) expressed by the original GS/M-83-H7 variant type. Furthermore, three of the genomic vsp sequences turned out to be identical to vsp sequences that represented previously characterized transcription products from in vivo- or in vitro-switched GS/M-83-H7 trophozoites. In conclusion, the DNA melting profile assay seems to be a versatile tool for the PCR-based genotyping of moderately or highly diversified sequence orthologues.

Capsule and fimbria interaction in Klebsiella pneumoniae

The capsular polysaccharide and type I fimbriae are two of the major surface-located virulence properties associated with the pathogenesis of Klebsiella pneumoniae. The capsule is an elaborate polysaccharide matrix that encases the entire cell surface and provides resistance against many host defense mechanisms. In contrast, type 1 fimbriae are thin adhesive thread-like surface organelles that can extend beyond the capsular matrix and mediate D-mannose-sensitive adhesion to host epithelial cells. These fimbriae are archetypical and consist of a major building block protein (FimA) that comprises the bulk of the organelle and a tip-located adhesin (FimH). It is assumed that the extended major-subunit protein structure permits the FimH adhesin to function independently of the presence of a capsule. In this study, we have employed a defined set of K. pneumoniae capsulated and noncapsulated strains to show that the function of type I fimbriae is actually impeded by the concomitant expression of a polysaccharide capsule. Capsule expression had significant effects on two parameters commonly used to define FimH function, namely, yeast cell agglutination and biofilm formation. Our data suggest that this effect is not due to transcriptional/translational changes in fimbrial gene/protein expression but rather the result of direct physical interference. This was further demonstrated by the fact that we could restore fimbrial function by inhibiting capsule synthesis. It remains to be determined whether the expression of these very different surface components occurs simply via random events of phase variation or in a coordinated manner in response to specific environmental cues.

C/EBP delta and C/EBP gamma bind the CCAAT-box in the human beta-globin promoter and modulate the activity of the CACC-box binding protein, EKLF

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBP beta and C/EBP gamma as candidate regulators of beta-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBP delta as an additional beta-globin CCAAT-box binding protein. In reporter assays, we show that C/EBP delta can co-operate with EKLF, a CACC-box binding protein, to activate the beta-globin promoter, whereas C/EBP gamma inhibits the transcriptional activity of EKLF in this assay. (c) 2005 Elsevier B.V. All rights reserved.

A novel plasma etching and emission monitoring system (PEEMS) to assess protein and lipid spoilation for hydrogel contact lenses

The work presents a new method that combines plasma etching with extrinsic techniques to simultaneously measure matrix and surface protein and lipid deposits. The acronym for this technique is PEEMS - Plasma Etching and Emission Monitoring System. Previous work has identified the presence of proteinaceous and lipoidal deposition on the surface of contact lenses and highlighted the probability that penetration of these spoilants will occur. This technique developed here allows unambiguous identification of the depth of penetration of spoilants to be made for various material types. It is for this reason that the technique has been employed in this thesis. The technique is applied as a 'molecular' scalpel, removing known amounts of material from the target. In this case from both the anterior .and posterior surfaces of a 'soft' contact lens. The residual material is then characterised by other analytical techniques such as UV/visible .and fluorescence spectroscopy. Several studies have be.en carried out for both in vivo and in vitro spoilt materials. The analysis and identification of absorbed protein and lipid of the substrate revealed the importance of many factors in the absorption and adsorption process. The effect of the material structure, protein nature (in terms of size, shape and charge) and environment conditions were examined in order to determine the relative uptake of tear proteins. The studies were extended to real cases in order to study the. patient dependent factors and lipoidal penetration.

Cell surface analysis of the basidiomycete yeast cryptococcus neoformans

Cell surface properties of the basidiomycete yeast Cryptococcus neoformans were investigated with a combination of novel and well proven approaches. Non-specific cell adhesion forces, as well as exposed carbohydrate and protein moieties potentially associated with specific cellular interaction, were analysed. Experimentation and analysis employed cryptococcal cells of different strains, capsular status and culture age. Investigation of cellular charge by particulate microelectrophoresis revealed encapsulated yeast forms of C. neoformans manifest a distinctive negative charge regardless of the age of cells involved; in turn, the neutral charge of acapsulate yeasts confirmed that the polysaccharide capsule, and not the cell wall, was responsible for this occurrence. Hydrophobicity was measured by MATH and HICH techniques, as well as by the attachment of polystyrene microspheres. All three techniques, where applicable, found C. neoformans yeast to be consistently hydrophilic; this state varied little regardless of strain and culture age. Cell surface carbohydrates and protein were investigated with novel fluorescent tagging protocols, flow cytometry and confocal microscopy. Cell surface carbohydrate was identified by controlled oxidation in association with biotin hydrazide and fluorescein-streptavidin tagging. Marked amounts of carbohydrate were measured and observed on the cell wall surface of cryptococcal yeasts. Furthermore, tagging of carbohydrates with selective fluorescent lectins supported the identification, measurement and observation of substantial amounts of mannose, glucose and N-acetyl-glucosamine. Cryptococcal cell surface protein was identified using sulfo-NHS-biotin with fluorescein-streptavidin, and then readily quantified by flow cytometry. Confocal imaging of surface exposed carbohydrate and protein revealed common localised areas of vivid fluorescence associated with buds, bud scars and nascent daughter cells. Carbohydrate and protein fluorescence often varied between strains, culture age and capsule status of cells examined. Finally, extension of protein tagging techniques resulted in the isolation and extraction of two biotinylated proteins from the yeast cell wall surface of an acapsulate strain of C.neoformans.

A comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines:characterisation, environmental scanning electron microscopy and immunisation studies in mice

Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell-mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non-ionic surfactants, such as 1-monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,6′-dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (T c) of DDA-based vesicles by ∼12°C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi-lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP-Chol-DDA-TDB (16:16:4:0.5 μmol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4°C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP-Chol or TDB was shown to inhibit this instability. Alternatively, at 25°C only the MP-based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in-vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85b-ESAT-6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MP- and DDA-based systems induced antibody responses at comparable levels whereas the DDA-based systems induced more powerful cell-mediated immune responses. © 2006 The Authors.

Integrative bioinformatics for functional genorne annotation:trawling for G protein-coupled receptors

G protein-coupled receptors (GPCR) are amongst the best studied and most functionally diverse types of cell-surface protein. The importance of GPCRs as mediates or cell function and organismal developmental underlies their involvement in key physiological roles and their prominence as targets for pharmacological therapeutics. In this review, we highlight the requirement for integrated protocols which underline the different perspectives offered by different sequence analysis methods. BLAST and FastA offer broad brush strokes. Motif-based search methods add the fine detail. Structural modelling offers another perspective which allows us to elucidate the physicochemical properties that underlie ligand binding. Together, these different views provide a more informative and a more detailed picture of GPCR structure and function. Many GPCRs remain orphan receptors with no identified ligand, yet as computer-driven functional genomics starts to elaborate their functions, a new understanding of their roles in cell and developmental biology will follow.

Direct effect of Plasmodium vivax recombinant vaccine candidates AMA-1 and MSP-1(19) on the innate immune response

The recombinant apical membrane antigen 1 (AMA-1) and 19-kDa fragment of merozoite surface protein (MSP-1(19)) are the lead candidates for inclusion in a vaccine against blood stages of malaria due to encouraging protective studies in humans and animals. Despite the importance of an efficacious malaria vaccine, vaccine-related research has focused on identifying antigens that result in protective immunity rather than determining the nature of anti-malarial immune effector mechanisms. Moreover, emphasis has been placed on adaptive rather than innate immune responses. In this study, we investigated the effect of Plasmodium vivax vaccine candidates Pv-AMA-1 and Pv-MSP-1(19) on the immune response of malaria-naive donors. Maturation of dendritic cells is altered by Pv-AMA-1 but not Pv-MSP-1(19), as observed by differential expression of cell surface markers. In addition, Pv-AMA-1 induced an increased production of MIP-1 alpha/CCL3 and decreased production of TARC/CCL17 levels in both dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs). Finally, a significant pro-inflammatory response was elicited by Pv-AMA-1-stimulated PBMCs. These results suggest that the recombinant vaccine candidate Pv-AMA-1 may play a direct role on innate immune response and might be involved in parasite destruction. (C) 2007 Elsevier Ltd. All rights reserved.

Plasmodium vivax apical membrane antigen-1: comparative recognition of different domains by antibodies induced during natural human infection

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria. (C) 2008 Elsevier Masson SAS. All rights reserved.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund`s Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria. (C) 2010 Elsevier Ltd. All rights reserved.

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety. (C) 2008 Elsevier Ltd. All rights reserved.

Changes in vancomycin-resistant Enterococcus faecium causing outbreaks in Brazil

Enterococci have been implicated in severe human infections as a consequence of associated determinants of virulence and antimicrobial resistance. The majority of vancomycin-resistant Enterococcus faecium (VRE(fm)) connected to outbreaks worldwide pertains to the clonal complex 17 (CC17). In Brazil, the majority of VRE(fm) involved in outbreaks reported so far are not related to CC17. VRE(fm) strains responsible for an outbreak and sporadic cases in hospitals located in the city of Campinas, Brazil, were compared to other VRE(fm) strains in the country. Twenty-two out of 23 E. faecium were vancomycin-resistant and harboured the vanA gene. One vancomycin-susceptible E. faecium (VSE(fm)) strain was included in this study because it was isolated from a patient who one week later harboured a VRE(fm). All strains, except VSE, showed the same alteration in the VanA element characterised by deletion of the left extremity of the transposon and insertion of IS1251 between the vanS and vanH genes. Genes codifying virulence factors such as collageneadhesin protein, enterococcal surface protein and hyaluronidase were detected in the VRE(fm) and VSE(fm) studied. Both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) revealed that VRE(fm) and VSE(fm) strains have a clonal relationship. New sequence types (STs) were identified by MLST as ST447, ST448, ST478 and ST412 but all belonged to the CC17. The present study revealed that VRE(fm) outbreaks in Brazil were caused by strains that did not share a common evolutionary history, and that VRE(fm) strains belonging to CC17 could be predominant in Brazil as in other countries. (C) 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Wolbachia infections of phlebotomine sand flies (Diptera: Psychodidae)

Old and New World phlebotomine sand fly species were screened for infection with Wolbachia, intracellular bacterial endosymbionts found in many arthropods and filarial nematodes. Of 53 samples representing 15 species, nine samples of four species were found positive for Wolbachia by polymerase chain reaction amplification using primers for the Wolbachia surface protein (wsp) gene. Five of the wsp gene fragments from four species were cloned, sequenced, and used for phylogenetic analysis. These wsp sequences were placed in three different clades within the arthropod associated Wolbachia (groups A and B), suggesting that Wolbachia has infected sand flies on more than one occasion. Two distantly related sand fly species, Lutzomyia (Psanthyromyia) shannoni (Dyar) and Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho), infected with an identical Wolbachia strain suggest a very recent horizontal transmission.