Variable Surface Proteins Research Articles

Site-Specific O-Glycosylation Analysis of SARS-CoV-2 Spike Protein Produced in Insect and Human Cells.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence is difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S—insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

A202 ROLE OF EXTRACELLULAR VESICLES IN GIARDIA-MICROBIOTA INTERACTIONS.

Abstract Background Extracellular vesicles (EVs) have emerged as important mediators of host-parasite interactions. Studies in various parasites have shown that EVs have important roles in pathogenesis. The protozoan parasite Giardia duodenalis, which causes diarrheal disease, produces EVs in response to changes in the environment. However, their exact role in the pathogenesis of giardiasis remains unclear. In this study, we investigate the effects of Giardia EVs on the gut commensal bacteria. EVs mediated disruption of the microbiota represents a novel mechanism that has not been investigated before. Aims We hypothesize that Giardia EVs contain virulence factors that will modify the growth and behavior of commensal bacteria. Based on our hypothesis, the study has three aims: 1. Quantify and characterize the EVs produced by Giardia under varying conditions. 2. Examine the effects of Giardia EVs on commensal bacteria. 3. Identify proteins and virulence factors in Giardia EVs using proteomics. Methods G. duodenalis (isolate NF) was exposed to bile or left untreated and the EVs were isolated using Exo-Easy Maxi Kit (Qiagen). The isolated EVs were characterized using Nanosight track analysis (NTA) and transmission electron microscopy. To examine the effects of EVs on bacteria, we used a lab strain E.coli HB101 and human isolate E. cloacae. The bacteria were incubated with Giardia EVs and their growth rate was monitored using a bacterial kinetics assay. The swimming motility of bacteria exposed to Giardia EVs was assessed on a 0.3% agar. Additionally, the ability of the EVs treated bacteria to adhere to epithelial cells was also examined. Proteomic analysis of EVs was conducted using liquid chromatography with tandem mass spectrometry. Results Results indicate that bile treated Giardia trophozoites produce more EVs as compared to untreated trophozoites. Giardia EVs exerted bacteriostatic effects on E.coli HB101 and E. cloacae. Additionally, Giardia EVs significantly increased the swimming motility of both E.coli HB101 and E. cloacae as well as their adhesion to the intestinal epithelial cells. Finally, proteomic analysis of EVs revealed the presence of well-characterized Giardia virulence factors such as cysteine proteases, tenascins, variant surface proteins as well as metabolic enzymes such as arginine deaminase and ornithine carbamoyltransferase. Conclusions Our research highlights a novel mechanism of Giardia’s interaction with commensal bacteria. Our results indicate that Giardia can release EVs in response to changes in the environment. These EVs also contain virulence factors that can modify the growth and behavior of commensal bacteria. These effects may have significant implications in disease pathophysiology. The results of the study can also be applied to other parasitic diseases where the production of EVs has been shown to play a role in the pathogenesis process. Funding Agencies NSERC

Structure of the Plasmodium-interspersed repeat proteins of the malaria parasite

The deadly symptoms of malaria occur as Plasmodium parasites replicate within blood cells. Members of several variant surface protein families are expressed on infected blood cell surfaces. Of these, the largest and most ubiquitous are the Plasmodium-interspersed repeat (PIR) proteins, with more than 1,000 variants in some genomes. Their functions are mysterious, but differential pir gene expression associates with acute or chronic infection in a mouse malaria model. The membership of the PIR superfamily, and whether the family includes Plasmodium falciparum variant surface proteins, such as RIFINs and STEVORs, is controversial. Here we reveal the structure of the extracellular domain of a PIR from Plasmodium chabaudi We use structure-guided sequence analysis and molecular modeling to show that this fold is found across PIR proteins from mouse- and human-infective malaria parasites. Moreover, we show that RIFINs and STEVORs are not PIRs. This study provides a structure-guided definition of the PIRs and a molecular framework to understand their evolution.

Strain-specific joint invasion and colonization by Lyme disease spirochetes is promoted by outer surface protein C.

Lyme disease, caused by Borrelia burgdorferi, B. afzelii and B. garinii, is a chronic, multi-systemic infection and the spectrum of tissues affected can vary with the Lyme disease strain. For example, whereas B. garinii infection is associated with neurologic manifestations, B. burgdorferi infection is associated with arthritis. The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner. Murine infection by isogenic B. burgdorferi strains differing only in their ospC coding region revealed that two OspC variants capable of binding dermatan sulfate promoted colonization of all tissues tested, including joints. However, an isogenic strain producing OspC from B. garinii strain PBr, which binds fibronectin but not dermatan sulfate, colonized the skin, heart and bladder, but not joints. Moreover, a strain producing an OspC altered to recognize neither fibronectin nor dermatan sulfate displayed dramatically reduced levels of tissue colonization that were indistinguishable from a strain entirely deficient in OspC. Finally, intravital microscopy revealed that this OspC mutant, in contrast to a strain producing wild type OspC, was defective in promoting joint invasion by B. burgdorferi in living mice. We conclude that OspC functions as an ECM-binding adhesin that is required for joint invasion, and that variation in OspC sequence contributes to strain-specific differences in tissue tropism displayed among Lyme disease spirochetes.

Giardia extracellular vesicles disrupt intestinal epithelial junctions and inhibit the growth of commensal bacteria while increasing their swimming motility

IntroductionExtracellular vesicles (EVs) have emerged as important mediators of host‐parasite interactions. The protozoan parasite Giardia duodenalis, the most common cause of parasitic diarrheal diseases worldwide, produces EVs in response to changes in the environment. The exact role of EVs in the pathogenesis of giardiasis remains unclear. In this study, we investigate the effects of EVs on the intestinal barrier such as the intestinal epithelium and the gut commensal bacteria. Disruption of the intestinal barrier has been implicated in the development of post‐giardiasis gut disorders and EVs may represent a novel mechanism of this disruption.AimsThe aim of this research is to characterize Giardia EVs and investigate their effect on the host epithelium and commensal gut bacteria.MethodsG. duodenalis (isolate NF) trophozoites were either incubated with 10X bile for 1 hour or left untreated and EVs were isolated using Qiagen Exo‐Easy Maxi Kit. The concentration and size of EVs were assessed using Nanosight track analysis (NTA). Proteomic analysis of EVs was conducted using liquid chromatography with tandem mass spectrometry. The effects of Giardia EVs on the host epithelium were assessed by exposing epithelial primary cell line (SCBN) to EVs for 24 hours and examining the tight junction proteins ZO‐1 and claudin‐4 by fluorescence microscopy. Antibacterial effect of Giardia EVs was assessed on gut commensals such as Escherichia coli strain HB101 and strain K12, Enterobacter cloacae (human isolate) and Enterococcus faecalis (human isolate) by measuring bacterial kinetic growth for 12 hours. Swimming motility of non‐invasive commensals was assessed on 0.3% agar after incubating with EVs for 24 hours and the halo sizes were measured as a marker of swimming behavior.ResultsOur findings indicate that bile treated Giardia trophozoites produce more EVs as compared to untreated trophozoites. NTA revealed that Giardia EVs range around 200 nm. Proteomic analysis of EVs revealed the presence of well characterized Giardia virulence factors such as cysteine proteases, tenascins, oxidative defense enzymes, variant surface proteins as well as metabolic enzymes such as arginine deaminase and ornithine carbamoyl transferase. Additionally, exposure of SCBN cells to EVs resulted in the disruption and/or rearrangement of tight junction proteins Zo‐1 and Claudin‐4. Moreover, our results also indicate that Giardia EVs exert bacteriostatic effects on E. coli strains HB101, K12, E. cloacae and E. faecalis. Finally, Giardia EVs also significantly increased the swimming motility of non‐invasive commensals E. coli strain HB101 and E. cloacae.ConclusionOur research highlights a novel mechanism of Giardia’s interaction with the intestinal barrier. Giardia EVs contain virulence factors that disrupt the host’s intestinal epithelium and modify the growth and motility of commensal bacteria. These effects may have significant implications in disease pathophysiology.Support or Funding InformationNatural Sciences and Engineering Research Council (NSERC)

The Organization and Clustering of Giardial Lipid Raft Domains After Treatment with Oseltamivir by Direct Stochastic Optical Resolution Microscopy

Lipid rafts contain combinations of glycosphigolipids, cholesterol, and protein receptors organized into lipid microdomains that form ordered structures. They play a role in signal transduction in order for cells to respond efficiently to changes in their environment and have the ability to undergo self-assembly and disassembly. Proteomic analysis reveals the presence of several proteins with biological functions in giardial lipid rafts (gLRs), including variable surface proteins, kinases, and glycosylphosphatidylinositol anchored proteins, giving rise to the idea that these lipid rafts could be a potential drug target. Previous data has shown that oseltamivir (Tamiflu), a neuraminidase inhibitor and FDA-approved drug, disrupts the growth and differentiation of Giardia by inhibiting formation of gLRs. Recalcitrant giardiasis is frequently contributed to drug resistance with treatment failures to the commonly prescribed drugs, metronidazole or albendazole, providing a need for new drug targets. We have determined the nanoscale clustering characteristics of GM1 ganglioside, a functionally important glycolipid in lipid rafts, using dSTORM, before and after treatment with oseltamivir. GM1 was labeled with CTxB-AF647 and Giardia were fixed in 4% agarose to prevent motion during acquisition for optimal resolution for dSTORM. In untreated Giardia, we observe clusters of GM1 with a mean radius of 19.3nm, with single probe density at 0.75E-3nm−2 and multi-probe density at 3.5E-5nm−2. After treatment with oseltamivir, we see a significant decrease in the size of GM1 clusters, with a mean radius of 15.1nm. Single probe density significantly increased to 1.5E-3nm−2, and multi-probe density significantly decreased to 1.25E-5nm−2. These results validate that at the nanoscale, oseltamivir disrupts gLR assembly and could be used as an anti-giardial drug, targeting specific raft clusters.

Transcriptomic and proteomic analyses of Giardia intestinalis: Intestinal epithelial cell interactions.

Giardia intestinalis is a unicellular protozoan parasite that infects the small intestines of humans and animals. Giardiasis, the disease caused by the parasite, occurs globally across socioeconomic boundaries but is mainly endemic in developing countries and particularly within young children, where pronounced effects manifests in a failure to thrive condition. The molecular pathogenesis of Giardia has been studied using in vitro models of human and rat intestinal epithelial cells (IECs) and parasites from the two major human genotypes or assemblages (A and B). High-quality, genome sequencing of representative isolates from assemblages A (WB) and B (GS) has enabled exploration of these host-parasite models using 'omics' technologies, allowing deep and quantitative analyses of global gene expression changes in IECs and parasites during their interactions, cross-talk and competition. These include a major up-regulation of immune-related genes in the IECs early after the start of interactions, as well as competition between host cells and parasites for nutrients like sugars, amino acids and lipids, which is also reflected in their secretome interactions. Unique parasite proteins dominate these interactions, with many major up-regulated genes being either hypothetical proteins or members of Giardia-specific gene families like the high-cysteine-rich membrane proteins (HCMPs), variable surface proteins (VSPs), alpha-giardins and cysteine proteases. Furthermore, these proteins also dominate in the secretomes, suggesting that they are important virulence factors in Giardia and crucial molecular effectors at the host-parasite interface.

Resistance formation to nitro drugs in Giardia lamblia: No common markers identified by comparative proteomics

In order to elucidate the question whether resistance to nitro drugs in G. lamblia is due to common resistance markers, trophozoites of three resistant G. lamblia strains, namely C4, 1062ID10, and 713M3 were grown in the presence of the two nitro drugs metronidazole and nitazoxanide and compared to their corresponding wild-types WBC6, 106, and 713 by mass spectometry shotgun analysis of their proteomes. Depending on the strain and the nitro drug, more than 200 to 500 differentially expressed proteins were identified, but there were no common patterns across strains and drugs. All resistant strains underwent antigenic variation with distinct surface antigens like variant surface proteins or cysteine rich proteins depending on strain and nitro compound. A closer look on enzymes involved in nitroreduction and detoxification of nitro radicals, NO or O2 suggested the existence of distinct strategies for each drug and each strain. Therefore, we conclude that resistance to nitro drugs in G. lamblia is not correlated with a specific pattern of differentially expressed proteins and therefore seems not to be the result of a directed process.

Giardia duodenalis: Role of secreted molecules as virulent factors in the cytotoxic effect on epithelial cells.

During the course of giardiasis in humans and experimental models, G. duodenalis trophozoites express and secrete several proteins (ESPs) affecting structural, cellular and soluble components of the host intestinal milieu. These include the toxin-like molecules CRP136 and ESP58 that induce intestinal hyper-peristalsis. After the completion of the Giardia genome database and using up-to date transcriptomic and proteomic approaches, secreted 'virulence factors' have also been identified and experimentally characterized. This repertoire includes arginine deiminase (ADI) that competes for arginine, an important energy source for trophozoites, some high-cysteine membrane proteins (HCMPs) and VSP88, a versatile variant surface protein (VSP) that functions as an extracellular protease. Another giardial protein, enolase, moonlights as a metabolic enzyme that interacts with the fibrinolytic system and damages host epithelial cells. Other putative Giardia virulence factors are cysteine proteases that degrade multiple host components including mucin, villin, tight junction proteins, immunoglobulins, defensins and cytokines. One of these proteases, named giardipain-1, decreases transepithelial electrical resistance and induces apoptosis in epithelial cells. A putative role for tenascins, present in the Giardia's secretome, is interfering with the host epidermal growth factor. Based on the roles that these molecules play, drugs may be designed to interfere with their functions. This review presents a comprehensive description of secreted Giardia virulence factors. It further describes their cytotoxic mechanisms and roles in the pathophysiology of giardiasis, and then assesses their potential as targets for drug development.

VirB10 vaccination for protection against Anaplasma phagocytophilum

BackgroundHuman granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the etiologic agent Anaplasma phagocytophilum. HGA was designated a nationally notifiable disease in the United States in 1998. Currently there are no vaccines available against HGA. Conserved membrane proteins that are subdominant in Anaplasma species, such as VirB9 and VirB10, may represent better vaccine targets than the variable immunodominant surface proteins. VirB9 and VirB10 are constituents of the Type 4 secretion system (T4SS) that is conserved amongst many intracellular bacteria and performs essential functions for invasion and survival in host cells.ResultsImmunogenicity and contribution to protection, provided after intramuscular vaccination of plasmid DNA encoding VirB9-1, VirB9-2, and VirB10 followed by inoculation of homologous recombinant proteins, in a prime-boost immunization strategy was evaluated in a murine model of HGA. Recombinant VirB9-1-, VirB9-2-, and VirB10-vaccinated mice developed antibody responses that specifically reacted with A. phagocytophilum organisms. However, only the mice vaccinated with VirB10 developed a significant increase in IFN-γ CD4+ T cells and partial protection against challenge with A. phagocytophilum.ConclusionsThis work provides evidence that A. phagocytophilum T4SS VirB10 is partially protective in a murine model against infection in an IFN-γ-dependent fashion and suggests that this protein may be a potential vaccine candidate against this and possibly other pathogenic bacteria with a T4SS.

Capsular Type, Sequence Type and Microbial Resistance Factors Impact on DNase Activity of Streptococcus agalactiae Strains from Human and Bovine Origin.

Extracellular deoxyribonucleases (DNases) contribute to the spread of pathogenic bacteria through the evasion from host innate immunity. Our main objective was to evaluate the production of extracellular DNases by human and bovine Streptococcus agalactiae clinical strains and perform a correlation of genetic lineages and DNase activity with capsular type, genetic determinants, clinical origin (colonization and infection), and host (human or bovine). DNase activity was evaluated by qualitative and quantitative assays for a collection of 406 human (n = 285) and bovine (n = 121) strains. All (121/121) bovine were isolated from mastitis and revealed to be DNase (+), indicating a putative pathogenic role in this clinical scenario. From the human S. agalactiae strains, 86% (245/285) showed DNase activity, among which all strains belonging to capsular types, namely, Ia, Ib, III-2, and IV. All CC17 strains (n = 58) and 56/96 (58.3%) of the CC19 displayed DNase activity. DNase (–) strains belonged to the CC19 group. However, the subcharacterization of CC19 S. agalactiae strains through multiple-locus variable number tandem repeat analysis (MLVA), antibiotic resistance, mobile elements, and surface proteins did not provide any distinction among DNase producers and non-producers.The production of DNases by all human CC17 strains, about two-fifths of human CC19, and all bovine strains, suggest an important contribution of DNases to hypervirulence.

Molecular Characterization of Trypanosoma evansi Mevalonate Kinase (TeMVK).

The mevalonate pathway is an essential part of isoprenoid biosynthesis leading to production of a diverse class of >30,000 biomolecules including cholesterol, heme, and all steroid hormones. In trypanosomatids, the mevalonate pathway also generates dolichols, which play an essential role in construction of glycosylphosphatidylinositol (GPI) molecules that anchor variable surface proteins (VSGs) to the plasma membrane. Isoprenoid biosynthesis involves one of the most highly regulated enzymes in nature, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which catalyzes the conversion of HMG-CoA to mevalonic acid. The enzyme mevalonate kinase (MVK) subsequently converts mevalonic acid to 5-phosphomevalonic acid. Trypanosoma evansi is a flagellate protozoan parasite that causes the disease “Surra” in domesticated large mammals, with great economic impact. T. evansi has only a trypomastigote bloodstream form and requires constant modification of the variant surface glycoprotein (VSG) coat for protection against the host immune system. We identified MVK of T. evansi (termed TeMVK) and performed a preliminary characterization at molecular, biochemical, and cellular levels. TeMVK from parasite extract displayed molecular weight ~36 kDa, colocalized with aldolase (a glycosomal marker enzyme) in glycosomes, and is structurally similar to Leishmania major MVK. Interestingly, the active form of TeMVK is the tetrameric oligomer form, in contrast to other MVKs in which the dimeric form is active. Despite lacking organized mitochondria, T. evansi synthesizes both HMGCR transcripts and protein. Both MVK and HMGCR are expressed in T. evansi during the course of infection in animals, and therefore are potential targets for therapeutic drug design.

Gap analysis of Mycoplasma bovis disease, diagnosis and control: An aid to identify future development requirements.

There is a worldwide problem of disease caused by Mycoplasma (M.) bovis in cattle; it has a significant detrimental economic and animal welfare impact on cattle rearing. Infection can manifest as a plethora of clinical signs including mastitis, pneumonia, arthritis, keratoconjunctivitis, otitis media and genital disorders that may result in infertility and abortion. Current diagnosis and control information are reviewed and analysed to identify gaps in knowledge of the causative organism in respect of the disease pathology, diagnosis and control methods. The main considerations are as follows: no vaccines are commercially available; antimicrobial resistance is increasing; diagnostic and antimicrobial sensitivity testing needs to be improved; and a pen-side test would facilitate more rapid diagnosis and implementation of treatment with antimicrobials. More data on host susceptibility, stress factors, immune response and infectious dose levels are required. The impact of asymptomatic carriers, M.bovis survival in the environment and the role of wildlife in transmitting the disease also needs investigation. To facilitate development of vaccines, further analysis of more M.bovis genomes, its pathogenic mechanisms, including variable surface proteins, is required, along with reproducible disease models.

Expression and secretion of the Giardia duodenalis variant surface protein 9B10A by transfected trophozoites causes damage to epithelial cell monolayers mediated by protease activity

Giardia duodenalis is the protozoan parasite responsible for most cases of parasitic diarrhea worldwide. The pathogenic mechanisms of giardiasis have not yet been fully characterized. In this context parasite's excretory/secretory products have been related to the damage induced by the parasite on enterocytes. Among these is the Variable Surface Proteins (VSPs) family involved in antigenic variation and in the induction of protective response. In proteomic analyses carried out to identify the proteases with high molecular weight secreted by Giardia trophozoites during the initial phase of interaction with IEC-6 cell monolayers we identified the VSP9B10A protein. In silico bioinformatics analyses predicted a central region in residues 324–684 displaying the catalytic triad and the substrate binding pocket of cysteine proteases. The analysis of the effect of the VSP9B10A protein on epithelial cell monolayers using trophozoites that were transfected with a plasmid carrying the vsp9b10a gene sequence under the control of a constitutive promoter showed that transfected trophozoites expressing the VSP9B10A protein caused cytotoxic damages on IEC-6 and MDCK cell monolayers. This was characterized by loss of cell-cell contacts and cell detachment from the substrate while no damage was observed with trophozoites that did not express the VSP9B10A protein. The same cytotoxic effect was detected when IEC-6 cell monolayers were incubated only with supernatants from co-cultures of IEC-6 cell monolayers with VSP9B10A transfected trophozoites and this effect was not observed when transfected trophozoites were incubated with a monospecific polyclonal antibody anti-VSP9B10A previous to interaction with IEC-6 monolayers. These results demonstrate that the VSP9B10A protein secreted upon interaction with epithelial cells caused damage in these cells. Thus this protein might be considered as a conditional virulence factor candidate. To our knowledge this is the first report on the proteolytic activity from a Giardia VSP opening new research lines on these proteins.

Comparative Genomics of Mycoplasma bovis Strains Reveals That Decreased Virulence with Increasing Passages Might Correlate with Potential Virulence-Related Factors.

Mycoplasma bovis is an important cause of bovine respiratory disease worldwide. To understand its virulence mechanisms, we sequenced three attenuated M. bovis strains, P115, P150, and P180, which were passaged in vitro 115, 150, and 180 times, respectively, and exhibited progressively decreasing virulence. Comparative genomics was performed among the wild-type M. bovis HB0801 (P1) strain and the P115, P150, and P180 strains, and one 14.2-kb deleted region covering 14 genes was detected in the passaged strains. Additionally, 46 non-sense single-nucleotide polymorphisms and indels were detected, which confirmed that more passages result in more mutations. A subsequent collective bioinformatics analysis of paralogs, metabolic pathways, protein-protein interactions, secretory proteins, functionally conserved domains, and virulence-related factors identified 11 genes that likely contributed to the increased attenuation in the passaged strains. These genes encode ascorbate-specific phosphotransferase system enzyme IIB and IIA components, enolase, L-lactate dehydrogenase, pyruvate kinase, glycerol, and multiple sugar ATP-binding cassette transporters, ATP binding proteins, NADH dehydrogenase, phosphate acetyltransferase, transketolase, and a variable surface protein. Fifteen genes were shown to be enriched in 15 metabolic pathways, and they included the aforementioned genes encoding pyruvate kinase, transketolase, enolase, and L-lactate dehydrogenase. Hydrogen peroxide (H2O2) production in M. bovis strains representing seven passages from P1 to P180 decreased progressively with increasing numbers of passages and increased attenuation. However, eight mutants specific to eight individual genes within the 14.2-kb deleted region did not exhibit altered H2O2 production. These results enrich the M. bovis genomics database, and they increase our understanding of the mechanisms underlying M. bovis virulence.

Strain-specific antibodies reduce co-feeding transmission of the Lyme disease pathogen, Borrelia afzelii.

Vector-borne pathogens use a diversity of strategies to evade the vertebrate immune system. Co-feeding transmission is a potential immune evasion strategy because the vector-borne pathogen minimizes the time spent in the vertebrate host. We tested whether the Lyme disease pathogen, Borrelia afzelii, can use co-feeding transmission to escape the acquired immune response in the vertebrate host. We induced a strain-specific, protective antibody response by immunizing mice with one of two variants of OspC (A3 and A10), the highly variable outer surface protein C of Borrelia pathogens. Immunized mice were challenged via tick bite with B. afzelii strains A3 or A10 and infested with larval ticks at days 2 and 34 post-infection to measure co-feeding and systemic transmission respectively. Antibodies against a particular OspC variant significantly reduced co-feeding transmission of the targeted (homologous) strain but not the non-targeted (heterologous) strain. Cross-immunity between OspC antigens had no effect in co-feeding ticks but reduced the spirochaete load twofold in ticks infected via systemic transmission. In summary, OspC-specific antibodies reduced co-feeding transmission of a homologous but not a heterologous strain of B. afzelii. Co-feeding transmission allowed B. afzelii to evade the negative consequences of cross-immunity on the tick spirochaete load.

Transcriptional profiling of Giardia intestinalis in response to oxidative stress

Giardia intestinalis is a microaerophilic parasite that infects the human upper small intestine, an environment that is fairly aerobic with reactive oxygen species being produced to fight off the parasite. It is quite perplexing how Giardia, lacking conventional eukaryotic antioxidant machinery (e.g. catalase, superoxide dismutase and glutathione peroxidase), can cope with the oxidative stress in this environment. We used transcriptomics (RNA sequencing and quantitative PCR) to study giardial gene expression changes in response to oxygen (O2; 1h) and hydrogen peroxide (H2O2; 150μM, 500μM and 1mM for 1h). The results showed phenotypic and transcriptional differences between Giardia isolates of different genotypes (WB, assemblage A and GS, assemblage B), with GS being more tolerant to H2O2 and exhibiting higher basic transcript levels of antioxidant genes (e.g. NADH oxidase lateral transfer candidate, peroxiredoxin 1 (Prx1) and thioredoxin (Trx)-like proteins). Cysteine is a major antioxidant in Giardia and its role in oxidative defense could be highlighted here by the up-regulation of gene transcripts encoding the cysteine-rich variable surface proteins (VSPs) and high cysteine membrane proteins (HCMPs). Genes in the thioredoxin system (Prx1, Trx and Trx reductase) occupied a central role in the gene expression response to oxidative stress, together with genes encoding metabolic (NADPH-producing enzymes, glutathione and glycerol biosynthetic enzymes) and O2-consuming nitric oxide detoxification enzymes (e.g. nitroreductase, flavohemoprotein and a flavodiiron protein). This study reveals the intricate network of genes associated with the oxidative stress response in Giardia, and provides a stepping-stone towards future studies at the protein level.

In Silico Study of Variable Surface Proteins in Plasmodium Species: Perspectives in Drug Design.

The variable surface proteins expressed by P. falciparum and P. vivax are transported to the surface of infected erythrocyte and are exposed to the host immune system. The possibility of using variable surface proteins as a common drug target has been analyzed in both the Plasmodium species. Sequence analysis of variable surface proteins showed a low-level conservation within as well as between the species. Amino acid composition analysis revealed higher frequency of hydrophilic amino acids as compared with that of hydrophobic residues. In order to gain more insight into their diverse functional role, the three-dimensional structure was predicted using comparative modeling approach. These models were evaluated and validated by checking stereochemistry of underlying amino acids. Structural alignment of variable surface proteins by superimposing them shows less conservation. Due to differences at sequence as well as structural level, the variable surface proteins are expected to show difference in their degree of invasiveness. These differences were also cross-examined by evolutionary study, and the results obtained were in accordance with the aforesaid study. The existence of structural differences noticed in the present study showed that the variable surface proteins could not be used as a common drug target in both the malarial species. Therefore, species-specific strategy may be followed for drug targeting against variable surface proteins of P. falciparum and P. vivax.

Host defences against Giardia lamblia

Giardia spp. is a protozoan parasite that inhabits the upper small intestine of mammals and other species and is the aetiological agent of giardiasis. It has been demonstrated that nitric oxide, mast cells and dendritic cells are the first line of defence against Giardia. IL-6 and IL-17 play an important role during infection. Several cytokines possess overlapping functions in regulating innate and adaptive immune responses. IgA and CD4(+) T cells are fundamental to the process of Giardia clearance. It has been suggested that CD4(+) T cells play a double role during the anti-Giardia immune response. First, they activate and stimulate the differentiation of B cells to generate Giardia-specific antibodies. Second, they act through a B-cell-independent mechanism that is probably mediated by Th17 cells. Several Giardia proteins that stimulate humoral and cellular immune responses have been described. Variant surface proteins, α-1 giardin, and cyst wall protein 2 can induce host protective responses to future Giardia challenges. The characterization and evaluation of the protective potential of the immunogenic proteins that are associated with Giardia will offer new insights into host-parasite interactions and may aid in the development of an effective vaccine against the parasite.

Quantitative proteomic analysis of Giardia duodenalis assemblage A: A baseline for host, assemblage, and isolate variation.

Giardia duodenalis is a gastrointestinal protozoan parasite of vertebrates and is a species complex comprised of eight assemblages, with the zoonotic assemblage A one of two subtypes infective for humans. With increasing genomic and transcriptomic data publicly available through the centralized giardiaDB.org, we have quantitatively analyzed the proteomes of eight G. duodenalis assemblage A strains (seven A1 and one A2) to provide a proteomic baseline to complement the available data. A nonredundant total of 1197 subassemblage A1 proteins and 719 subassemblage A2 proteins were identified with an average of 770 proteins in each strain. The eight strains were also searched against both assemblage A genome sequences (subassemblage A1 and A2 genomes) and demonstrated subassemblage specific differences in protein identifications, especially for variable gene families. Substantial differences were observed in the numbers and abundance in the variable surface protein family, and two different variable surface protein expression profiles that were independent of host origin, subassemblage, or geographic origin. We hypothesize that this variation in surface antigen switching events may be related to karotype and chromosomal variation, which would indicate an assemblage-independent mechanism of diversity generation in G. duodenalis. All MS data have been deposited in the ProteomeXchange with identifier PXD001272 (http://proteomecentral.proteomexchange.org/dataset/PXD001272).