Variable Surface Proteins Research Articles

UV irradiation responses in Giardia intestinalis

The response to ultraviolet light (UV) radiation, a natural stressor to the intestinal protozoan parasite Giardia intestinalis, was studied to deepen the understanding of how the surrounding environment affects the parasite during transmission. UV radiation at 10 mJ/cm2 kills Giardia cysts effectively whereas trophozoites and encysting parasites can recover from UV treatment at 100 mJ/cm2 and 50 mJ/cm2 respectively. Staining for phosphorylated histone H2A showed that UV treatment induces double-stranded DNA breaks and flow cytometry analyses revealed that UV treatment of trophozoites induces DNA replication arrest. Active DNA replication coupled to DNA repair could be an explanation to why UV light does not kill trophozoites and encysting cells as efficiently as the non-replicating cysts. We also examined UV-induced gene expression responses in both trophozoites and cysts using RNA sequencing (RNA seq). UV radiation induces small overall changes in gene expression in Giardia but cysts show a stronger response than trophozoites. Heat shock proteins, kinesins and Nek kinases are up-regulated, whereas alpha-giardins and histones are down-regulated in UV treated trophozoites. Expression of variable surface proteins (VSPs) is changed in both trophozoites and cysts. Our data show that Giardia cysts have limited ability to repair UV-induced damage and this may have implications for drinking- and waste-water treatment when setting criteria for the use of UV disinfection to ensure safe water.

Global phylogeography of the avian malaria pathogen Plasmodium relictum based on MSP1 allelic diversity

Knowing the genetic variation that occurs in pathogen populations and how it is distributed across geographical areas is essential to understand parasite epidemiology, local patterns of virulence, and evolution of host‐resistance. In addition, it is important to identify populations of pathogens that are evolutionarily independent and thus ‘free’ to adapt to hosts and environments. Here, we investigated genetic variation in the globally distributed, highly invasive avian malaria parasite Plasmodium relictum, which has several distinctive mitochondrial haplotyps (cyt b lineages, SGS1, GRW11 and GRW4). The phylogeography of P. relictum was accessed using the highly variable nuclear gene merozoite surface protein 1 (MSP1), a gene linked to the invasion biology of the parasite. We show that the lineage GRW4 is evolutionarily independent of GRW11 and SGS1 whereas GRW11 and SGS1 share MSP1 alleles and thus suggesting the presence of two distinct species (GRW4 versus SGS1 and GRW11). Further, there were significant differences in the global distribution of MSP1 alleles with differences between GRW4 alleles in the New and the Old World. For SGS1, a lineage formerly believed to have both tropical and temperate transmission, there were clear differences in MSP1 alleles transmitted in tropical Africa compared to the temperate regions of Europe and Asia. Further, we highlight the occurrence of multiple MSP1 alleles in GRW4 isolates from the Hawaiian Islands, where the parasite has contributed to declines and extinctions of endemic forest birds since it was introduced. This study stresses the importance of multiple independent loci for understanding patterns of transmission and evolutionary independence across avian malaria parasites.

Genomic characterization of variable surface antigens reveals a telomere position effect as a prerequisite for RNA interference-mediated silencing in Paramecium tetraurelia.

ABSTRACTAntigenic or phenotypic variation is a widespread phenomenon of expression of variable surface protein coats on eukaryotic microbes. To clarify the mechanism behind mutually exclusive gene expression, we characterized the genetic properties of the surface antigen multigene family in the ciliate Paramecium tetraurelia and the epigenetic factors controlling expression and silencing. Genome analysis indicated that the multigene family consists of intrachromosomal and subtelomeric genes; both classes apparently derive from different gene duplication events: whole-genome and intrachromosomal duplication. Expression analysis provides evidence for telomere position effects, because only subtelomeric genes follow mutually exclusive transcription. Microarray analysis of cultures deficient in Rdr3, an RNA-dependent RNA polymerase, in comparison to serotype-pure wild-type cultures, shows cotranscription of a subset of subtelomeric genes, indicating that the telomere position effect is due to a selective occurrence of Rdr3-mediated silencing in subtelomeric regions. We present a model of surface antigen evolution by intrachromosomal gene duplication involving the maintenance of positive selection of structurally relevant regions. Further analysis of chromosome heterogeneity shows that alternative telomere addition regions clearly affect transcription of closely related genes. Consequently, chromosome fragmentation appears to be of crucial importance for surface antigen expression and evolution. Our data suggest that RNAi-mediated control of this genetic network by trans-acting RNAs allows rapid epigenetic adaptation by phenotypic variation in combination with long-term genetic adaptation by Darwinian evolution of antigen genes.

Proteomic analysis in Giardia duodenalis yields insights into strain virulence and antigenic variation.

Giardia duodenalis is a protozoan parasite of the small intestine in vertebrates, including humans. Assemblage A of G. duodenalis is one of the two discrete subtypes that infects humans, and is considered a zoonotic assemblage. Two G. duodenalis Assemblage A strains BRIS/95/HEPU/2041 and BRIS/83/HEPU/106, constituting virulent and control strains respectively, were analyzed in one of the first comparative shotgun proteomic studies performed in this parasite. Protein extracts were prepared using a multiplatform approach with both an in-gel and in-solution sample preparation to enable us to assess the complementarity for future Giardia proteomic studies. Protein analysis revealed that BRIS/95/HEPU/2041 possessed a wider and more varied repertoire of variant surface proteins (VSPs), which are hypothesized to be involved in host adaptation, immune evasion, and virulence. A total of 35 VSPs were identified, with three common to both strains, six unique to BRIS/82/HEPU/106, and twenty-six unique to BRIS/95/HEPU/2041. Additionally, up to 25.6% of all differentially expressed proteins in BRIS/95/HEPU/2041 belonged to the VSP family, a trend not seen in the control BRIS/83/HEPU/106. Greater antigen variation in BRIS/95/HEPU/2041 may explain aspects of virulence phenotypes in G. duodenalis, with a highly diverse population capable of evading host immune responses.

Histopathological findings, phenotyping of inflammatory cells, and expression of markers of nitritative injury in joint tissue samples from calves after vaccination and intraarticular challenge with Mycoplasma bovis strain 1067.

BackgroundThe pathogenesis of caseonecrotic lesions developing in lungs and joints of calves infected with Mycoplasma bovis is not clear and attempts to prevent M. bovis-induced disease by vaccines have been largely unsuccessful. In this investigation, joint samples from 4 calves, i.e. 2 vaccinated and 2 non-vaccinated, of a vaccination experiment with intraarticular challenge were examined. The aim was to characterize the histopathological findings, the phenotypes of inflammatory cells, the expression of class II major histocompatibility complex (MHC class II) molecules, and the expression of markers for nitritative stress, i.e. inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT), in synovial membrane samples from these calves. Furthermore, the samples were examined for M. bovis antigens including variable surface protein (Vsp) antigens and M. bovis organisms by cultivation techniques.ResultsThe inoculated joints of all 4 calves had caseonecrotic and inflammatory lesions. Necrotic foci were demarcated by phagocytic cells, i.e. macrophages and neutrophilic granulocytes, and by T and B lymphocytes. The presence of M. bovis antigens in necrotic tissue lesions was associated with expression of iNOS and NT by macrophages. Only single macrophages demarcating the necrotic foci were positive for MHC class II. Microbiological results revealed that M. bovis had spread to approximately 27% of the non-inoculated joints. Differences in extent or severity between the lesions in samples from vaccinated and non-vaccinated animals were not seen.ConclusionsThe results suggest that nitritative injury, as in pneumonic lung tissue of M. bovis-infected calves, is involved in the development of caseonecrotic joint lesions. Only single macrophages were positive for MHC class II indicating down-regulation of antigen-presenting mechanisms possibly caused by local production of iNOS and NO by infiltrating macrophages.

The Genome of Spironucleus salmonicida Highlights a Fish Pathogen Adapted to Fluctuating Environments

Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we identified energy-producing hydrogenosomes in S. salmonicida. Here we present a genome analysis of the fish parasite with a focus on the comparison to the more studied diplomonad Giardia intestinalis. We annotated 8067 protein coding genes in the ∼12.9 Mbp S. salmonicida genome. Unlike G. intestinalis, promoter-like motifs were found upstream of genes which are correlated with gene expression, suggesting a more elaborate transcriptional regulation. S. salmonicida can utilise more carbohydrates as energy sources, has an extended amino acid and sulfur metabolism, and more enzymes involved in scavenging of reactive oxygen species compared to G. intestinalis. Both genomes have large families of cysteine-rich membrane proteins. A cluster analysis indicated large divergence of these families in the two diplomonads. Nevertheless, one of S. salmonicida cysteine-rich proteins was localised to the plasma membrane similar to G. intestinalis variant-surface proteins. We identified S. salmonicida homologs to cyst wall proteins and showed that one of these is functional when expressed in Giardia. This suggests that the fish parasite is transmitted as a cyst between hosts. The extended metabolic repertoire and more extensive gene regulation compared to G. intestinalis suggest that the fish parasite is more adapted to cope with environmental fluctuations. Our genome analyses indicate that S. salmonicida is a well-adapted pathogen that can colonize different sites in the host.

Identification of avir-orthologous immune evasion gene family from primate malaria parasites

The immune evasion gene family of malaria parasites encodes variant surface proteins that are expressed at the surface of infected erythrocytes and help the parasite in evading the host immune response by means of antigenic variation. The identification of Plasmodium vivax vir orthologous immune evasion gene family from primate malaria parasites would provide new insight into the evolution of virulence and pathogenesis. Three vir subfamilies viz. vir-B, vir-D and vir-G were successfully PCR amplified from primate malaria parasites, cloned and sequenced. DNA sequence analysis confirmed orthologues of vir-D subfamily in Plasmodium cynomolgi, Plasmodium simium, Plasmodium simiovale and Plasmodium fieldi. The identified vir-D orthologues are 1-9 distinct members of the immune evasion gene family which have 68-83% sequence identity with vir-D and 71.2-98.5% sequence identity within the members identified from primate malaria parasites. The absence of other vir subfamilies among primate malaria parasites reflects the limitations in the experimental approach. This study clearly identified the presence of vir-D like sequences in four species of Plasmodium infecting primates that would be useful in understanding the evolution of virulence in malaria parasites.

Coexistence of sense and anti-sense mRNAs of variant surface protein in Giardia lamblia trophozoites

A strategy of the parasitic protozoan Giardia lamblia to evade attack from the host immune system is periodic changes of its surface antigen, a member of the variant surface protein (VSP) family. A post-transcriptional gene silencing mechanism has been proposed to explain the presence of only one among many possible VSPs at any time. To investigate this phenomenon further, we extracted total RNA from cultured trophozoites of the G. lamblia C2 isolate, and cDNA was reverse-transcribed from the RNA. Sense and anti-sense VSPs were amplified from the total cDNA using nested PCR with primers designed from the 3′-conserved region and the known 5′ or 3′ end of the cDNA library. Sequence analyses of the amplified products revealed more than 34 full-length antisense VSPs and a smear of sense VSPs. Sequence alignments and comparisons revealed that these VSPs contained variable N-termini and conserved C-termini, and could be classified into 5 clades based on the sizes and variations of the N-terminal sequence. All antisense VSPs existed in the sense forms, but no corresponding antisense VSP existed for sense RNA (snsRNA) 16. The coexistence of sense and antisense VSP mRNAs in cultured G. lamblia supports the post-transcriptional regulation of VSP expression. We propose that VSPs transcribed simultaneously in the sense and antisense forms form double-stranded RNAs (dsRNAs) which are degraded by the Dicer endonuclease, while a VSP without an antisense transcription (e.g., snsRNA16) will be expressed on the surface of Giardia. In addition, in the course of this investigation VSPs were identified that were previously not known. PCR-based amplification of specific sense and antisense VSP cDNAs can be used to identify the specific VSP on G. lamblia trophozoites, which is easier than using specific monoclonal antibody approaches.

Memory CD4 T cells in influenza.

Influenza A virus is a significant cause of morbidity and mortality worldwide, particularly among young children and the elderly. Current vaccines induce neutralizing antibody responses directed toward highly variable viral surface proteins, resulting in limited heterosubtypic protection to new viral serotypes. By contrast, memory CD4 T cells recognize conserved viral proteins and are cross-reactive to multiple influenza strains. In humans, virus-specific memory CD4 T cells were found to be the protective correlate in human influenza challenge studies, suggesting their key role in protective immunity. In mouse models, memory CD4 T cells can mediate protective responses to secondary influenza infection independent of B cells or CD8 T cells, and can influence innate immune responses. Importantly, a newly defined, tissue-resident CD4 memory population has been demonstrated to be retained in lung tissue and promote optimal protective responses to an influenza infection. Here, we review the current state of results regarding the generation of memory CD4 T cells following primary influenza infection, mechanisms for their enhanced efficacy in protection from secondary challenge including their phenotype, localization, and function in the context of both mouse models and human infection. We also discuss the generation of memory CD4 T cells in response to influenza vaccines and its future implications for vaccinology.

Vaccination induced antibodies to recombinant avian influenza A virus M2 protein or synthetic M2e peptide do not bind to the M2 protein on the virus or virus infected cells

BackgroundInfluenza viruses are characterized by their highly variable surface proteins HA and NA. The third surface protein M2 is a nearly invariant protein in all Influenza A strains. Despite extensive studies in other animal models, this study is the first to describe the use of recombinant M2 protein and a peptide coding for the extracellular part of the M2 protein (M2e) to vaccinate poultry.MethodsFour groups of layer chickens received a prime-boost vaccination with recombinant M2 protein, M2e, a tetrameric construct from M2e peptide bound to streptavidin and a control tetrameric construct formulated with Stimune adjuvant.ResultsWe determined the M2-specific antibody (Ab) responses in the serum before vaccination, three weeks after vaccination and two weeks after booster, at days 21, 42 and 56 of age. The group vaccinated with the M2 protein in combination with Stimune adjuvant showed a significant Ab response to the complete M2 protein as compared to the other groups. In addition an increased Ab response to M2e peptide was found in the group vaccinated with the M2e tetrameric construct. None of the vaccinated animals showed seroconversion to AI in a commercial ELISA. Finally no Ab’s were found that bound to M2 expressed on in vitro AI infected MDCK cells.ConclusionAlthough Ab’s are formed against the M2 protein and to Streptavidin bound M2e peptide in a tetrameric conformation these Ab’s do not recognize of M2 on the virus or on infected cells.

An ER‐directed transcriptional response to unfolded protein stress in the absence of conserved sensor‐transducer proteins in Giardia lamblia

The protozoan Giardia lamblia has a minimized organelle repertoire, and most strikingly lacks a classical stacked Golgi apparatus. Nevertheless, Giardia trophozoites constitutively secrete variant surface proteins, and dramatically increase the volume of protein secretion during differentiation to cysts. Eukaryotic cells have evolved an elaborate system for quality control (QC) of protein folding and capacity in the endoplasmic reticulum (ER). Upon ER-overload, an unfolded protein response (UPR) is triggered on transcriptional/translational level aiming at alleviating ER stress. In Giardia, a minimized secretory machinery and absence of glycan-dependent QC suggests that a genetically conserved UPR (or functional equivalent) to cope with insults to the secretory system has been eliminated. We tested this hypothesis of UPR elimination by profiling the transcriptional response during induced ER-folding stress. We show that on the contrary, ER-folding stress triggers a stressor-specific, ER-directed response with upregulation of only ~ 30 genes, with different kinetics and scope compared with the UPR of other eukaryotes. Computational genomics revealed conserved cis-acting motifs in upstream regions of responder genes capable of stressor-specific gene regulation in transfected cells. Interestingly, the sensors/transducers of folding stress, well conserved in model eukaryotes, are absent in Giardia suggesting the presence of a novel version of this essential eukaryotic function.

Nuclear Pores and Perinuclear Expression Sites ofvarand Ribosomal DNA Genes Correspond to Physically Distinct Regions in Plasmodium falciparum

The human malaria parasite Plasmodium falciparum modifies the erythrocyte it infects by exporting variant proteins to the host cell surface. The var gene family that codes for a large, variant adhesive surface protein called P. falciparum erythrocyte membrane protein 1 (PfEMP1) plays a particular role in this process, which is linked to pathogenesis and immune evasion. A single member of this gene family is highly transcribed while the other 59 members remain silenced. Importantly, var gene transcription occurs at a spatially restricted, but yet undefined, perinuclear site that is distinct from repressed var gene clusters. To advance our understanding of monoallelic expression, we investigated whether nuclear pores associate with the var gene expression site. To this end, we studied the nuclear pore organization during the asexual blood stage using a specific antibody directed against a subunit of the nuclear pore, P. falciparum Nup116 (PfNup116). Ring and schizont stage parasites showed highly polarized nuclear pore foci, whereas in trophozoite stage nuclear pores redistributed over the entire nuclear surface. Colocalization studies of var transcripts and anti-PfNup116 antibodies showed clear dissociation between nuclear pores and the var gene expression site in ring stage. Similar results were obtained for another differentially transcribed perinuclear gene family, the ribosomal DNA units. Furthermore, we show that in the poised state, the var gene locus is not physically linked to nuclear pores. Our results indicate that P. falciparum does form compartments of high transcriptional activity at the nuclear periphery which are, unlike the case in yeast, devoid of nuclear pores.

The Genotyping Potential of the Mycoplasma synoviae vlhA Gene

Mycoplasma synoviae (MS) continues to cause significant losses to poultry producers, and studying the epizootiology of infection is an important component of MS control. The partial variable lipoprotein hemagglutinin A (vlhA) gene is the only genomic target identified so far for MS sequence typing. The vlhA gene codes for two variable cell surface proteins, lipoprotein and hemagglutinin, and the proposed mechanism for the variation is gene conversion between a single expressed gene and an array of pseudogenes. The upstream portion of the vlhA gene is present in the genome in a single copy (not present in the pseudogenes), and it is the only part of the gene that can be used for targeted sequence typing. However, the 3' end of the vlhA single copy" as well as this region's discriminatory potential for genotyping purposes has not been established. The purpose of this study was to identify the exact limit and the genotyping potential of the vlhA single copy region. New PCR assays were developed to amplify the entire conserved region and part of the variable region of the vlhA gene. Amplification and sequencing were performed on a variety of MS samples and on in vitro sequential generations of a standard MS strain. Sequence analyses determined the site and composition of the most proximal sequence variation that could be attributed to a gene conversion event, and they predicted the end point of the vlhA single copy region. The results indicated that a currently available "revised Hammond" PCR spans the whole single copy of the vlhA gene and exploits the full genotyping potential of this MS genomic target. In addition, this study allows interesting insight into the gene conversion mechanism of MS and offers the opportunity for further investigation this mechanism in mycoplasmas.

Differential dissolved protein expression throughout the life cycle of Giardia lamblia

Giardia lamblia (G. lamblia) has a simple life cycle that alternates between a cyst and a trophozoite, and this parasite is an important human and animal pathogen. To increase our understanding of the molecular basis of the G. lamblia encystment, we have analyzed the soluble proteins expressed by trophozoites and cysts extracted from feces by quantitative proteomic analysis. A total of 63 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and were categorized as cytoskeletal proteins, a cell-cycle-specific kinase, metabolic enzymes and stress resistance proteins. Importantly, we demonstrated that the expression of seven proteins differed significantly between trophozoites and cysts. In cysts, the expression of three proteins (one variable surface protein (VSP), ornithine carbamoyltransferase (OTC), β-tubulin) increased, whereas the expression of four proteins (14-3-3 protein, α-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein disulfide isomerase 2 (PDI-2)) decreased significantly when compared with the levels of these proteins in trophozoites. The mRNA expression patterns of four of these proteins (OTC, α-tubulin, GAPDH, VSP) were similar to the expression levels of the proteins. These seven proteins appear to play an important role in the completion of the life cycle of G. lamblia.

The profile of snoRNA-derived microRNAs that regulate expression of variant surface proteins in Giardia lamblia.

In the current investigation, we analysed all the known small nucleolar RNAs (snoRNAs) in the deeply branching protozoan parasite Giardia lamblia for potential microRNAs (miRNAs) that might be derived from them. Two putative miRNAs have since been identified by Northern blot, primer extension, 3' RACE and co-immunoprecipitation with Giardia Argonaute (GlAgo), and designated miR6 and miR10. Giardia Dicer (GlDcr) is capable of processing the snoRNAs into the corresponding miRNAs in vitro. Potential miR6 and miR10 binding sites in Giardia genome were predicted bio-informatically. A miR6 binding site was found at the 3' untranslated regions (UTR) of 44 variant surface protein (vsp) genes, whereas a miR10 binding site was identified at the 3' end of 159 vsp open-reading frames. Thirty-three of these vsp genes turned out to contain binding sites for both miR6 and miR10. A reporter mRNA tagged with the 3' end of vsp1267, which contains the target sites for both miRNAs, was translationally repressed by both miRNAs in Giardia. Episomal expression of an N-terminal c-myc tagged VSP1267 was found significantly repressed by introducing either miR6 or miR10 into the cells and the repressive effects were additive. When the 2'-O-methyl antisense oligos (ASOs) of either miR6 or miR10 was introduced, however, there was an enhancement of tagged VSP1267 expression suggesting an inhibition of the repressive effects of endogenous miR6 or miR10 by the ASOs. Of the total 220 vsp genes in Giardia, we have now found 178 of them carrying putative binding sites for all the miRNAs that have been currently identified, suggesting that miRNAs are likely the regulators of VSP expression in Giardia.

Representational difference analysis identifies specific genes in the interaction of Giardia duodenalis with the murine intestinal epithelial cell line, IEC-6

Giardia duodenalis is a re-emerging protozoan parasite that causes diarrhoea in humans, significantly affecting the health of many people globally. To date, little is known about the genetic events underpinning the establishment of infection in host cells; however, the parasite’s ventral disc, proteases and variable surface proteins (VSPs) are recognised as important pathogenic factors. In this study, representational difference analysis (RDA) was used to identify differentially expressed genes in four different Giardia isolates (WB, P-1, NF and GS/M) during the first 2h of in vitro interaction with the rat intestinal epithelial cell line, IEC-6. RDA showed that more than 40 genes were differentially expressed in each of the four Giardia isolates upon IEC-6 cells infection. Most of the up-regulated genes were common to the four isolates except for those encoding proteins possibly involved in immune evasion such as VSPs, high cysteine membrane proteins (HCMp), hypothetical proteins, and oxygen defence proteins (e.g., thioredoxin, peroxiredoxin 1). Differences in the expressed VSPs and HCMp may account for the variation in symptoms during giardiasis. Interestingly, the NF isolate solely expressed genes involved in encystation during interaction with IEC-6 (e.g., glucosamine 6-phosphate isomerase, dynamin, acid sphingomyelinase-like phosphodiesterase) suggesting that encystation signals could be different for this isolate. Common to the four isolates, transcripts for genes involved in glycolysis (e.g., glucose-6-phosphate dehydrogenase, fructose bisphosphate aldolase, enolase), attachment (γ and α1 giardins) and cysteine proteases were frequently detected. Genes involved in transcription, translation, signalling and cell cycle control were also up-regulated. This study shows that the RDA technique has selectively isolated genes involved in host–parasite interactions and complements previous microarray data. Some of the detected genes are also discussed as potential virulence factors and treatment targets in giardiasis.

Molecular Structure of Isolated MvspI, a Variable Surface Protein of the Fish Pathogen Mycoplasma mobile

Mycoplasma mobile is a parasitic bacterium that causes necrosis in the gills of freshwater fishes. This study examines the molecular structure of its variable surface protein, MvspI, whose open reading frame encodes 2,002 amino acids. MvspI was isolated from mycoplasma cells by a biochemical procedure to 92% homogeneity. Gel filtration and analytical ultracentrifugation suggested that this protein is a cylinder-shaped monomer with axes of 66 and 2.7 nm. Rotary shadowing transmission electron microscopy of MvspI showed that the molecule is composed of two rods 30 and 45 nm long; the latter rod occasionally features a bulge. Immuno-electron microscopy and epitope mapping showed that the bulge end of the molecular image corresponds to the C terminus of the amino acid sequence. Partial digestion by various proteases suggested that the N-terminal part, comprised of 697 amino acids, is flexible. Analysis of the predicted amino acid sequence showed that the molecule features a lipoprotein and 16 repeats of about 90 residues; 15 positions exist between residues 88 and 1479, and the other position is between residues 1725 and 1807. The amino acid sequence of MvspI was mapped onto a molecular image obtained by electron microscopy. The present study is the first to elucidate the molecular shape of a variable surface protein of mycoplasma.

“Mycoplasmal Antigen Modulation,” a Novel Surface Variation Suggested for a Lipoprotein Specifically Localized on Mycoplasma mobile

Mycoplasma mobile, a pathogen of freshwater fish, glides easily across surfaces, colonizes on the fish gill, and causes necrosis. The cell surface is differentiated into three parts: the head, neck, and body. Mobile variable surface proteins (Mvsps) localizing at each of these parts may be involved in surface variation including phase variation and antigenic variation, although no proof exists. In this study, we examined this possibility by focusing on MvspI, the largest Mvsp. Immunofluorescence microscopy showed that MvspI is expressed on the surfaces of all cells. When anti-MvspI antibody was added at concentrations over 0.8 nM, MvspI was observed to decrease over time. After 72 h of cultivation with the antibody, the fluorescence intensity and amount of MvspI decreased up to 13 and 39%, respectively, compared to those of cells grown without antibody. These changes were reversed by the removal of the antibody. Such effects were not observed when another antibody targeting other Mvsps was used, suggesting that the decrease is specific to the relationship between MvspI and the antibody. Cell growth was also inhibited by the antibody, but the decrease in MvspI could not be explained by the selective growth of MvspI-negative variants or by the inhibition of growth with other conditions. The decrease in MvspI caused by the antibody binding may suggest a novel type of surface variation, designated here as "mycoplasmal antigen modulation."

Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: Characterization of lung pathology, persistence of variable surface protein antigens and local immune response

BackgroundMycoplasma bovis is associated with pneumonia in calves characterized by the development of chronic caseonecrotic lesions with the agent persisting within the lesion. The purposes of this study were to characterize the morphology of lung lesions, examine the presence of M. bovis variable surface protein (Vsp) antigens and study the local immune responses in calves after infection with M. bovis strain 1067.MethodsLung tissue samples from eight calves euthanased three weeks after experimental infection with M. bovis were examined by bacteriology and pathology. Lung lesions were evaluated by immunohistochemical (IHC) staining for wide spectrum cytokeratin and for M. bovis Vsp antigens and pMB67 antigen. IHC identification and quantitative evaluation of CD4+ and CD8+ T lymphocytes and immunoglobulin (IgG1, IgG2, IgM, IgA)-containing plasma cells was performed. Additionally, expression of major histocompatibility complex class II (MHC class II) was studied by IHC.ResultsSuppurative pneumonic lesions were found in all calves. In two calves with caseonecrotic pneumonia, necrotic foci were surrounded by epithelial cells resembling bronchial or bronchiolar epithelium. In all calves, M. bovis Vsp antigens were constantly present in the cytoplasm of macrophages and were also present extracellularly at the periphery of necrotic foci. There was a considerable increase in numbers of IgG1- and IgG2-positive plasma cells among which IgG1-containing plasma cells clearly predominated. Statistical evaluation of the numbers of CD4+ and CD8+ T cells, however, did not reveal statistically significant differences between inoculated and control calves. In M. bovis infected calves, hyperplasia of bronchus-associated lymphoid tissue (BALT) was characterized by strong MHC class II expression of lymphoid cells, but only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II.ConclusionsThe results from this study show that infection of calves with M. bovis results in various lung lesions including caseonecrotic pneumonia originating from bronchioli and bronchi. There is long-term persistence of M. bovis as demonstrated by bacteriology and immunohistochemistry for M. bovis antigens, i.e. Vsp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from local downregulation of antigen presenting mechanisms and an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins.

Regulation of a Myb Transcription Factor by Cyclin-dependent Kinase 2 in Giardia lamblia

The protozoan Giardia lamblia parasitizes the human small intestine to cause diseases. It undergoes differentiation into infectious cysts by responding to intestinal stimulation. How the activated signal transduction pathways relate to encystation stimulation remain largely unknown. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately up-regulated by a Myb2 transcription factor. Because cell differentiation is linked to cell cycle regulation, we tried to understand the role of cell cycle regulators, cyclin-dependent kinases (Cdks), in encystation. We found that the recombinant Myb2 was phosphorylated by Cdk-associated complexes and the levels of phosphorylation increased significantly during encystation. We have identified a putative cdk gene (cdk2) by searching the Giardia genome database. Cdk2 was found to localize in the cytoplasm with higher expression during encystation. Interestingly, overexpression of Cdk2 resulted in a significant increase of the levels of cwp gene expression and cyst formation. In addition, the Cdk2-associated complexes can phosphorylate Myb2 and the levels of phosphorylation increased significantly during encystation. Mutations of important catalytic residues of Cdk2 resulted in a significant decrease of kinase activity and ability of inducing cyst formation. Addition of a Cdk inhibitor, purvalanol A, significantly decreased the Cdk2 kinase activity and the levels of cwp gene expression and cyst formation. Our results suggest that the Cdk2 pathway may be involved in phosphorylation of Myb2, leading to activation of the Myb2 function and up-regulation of cwp genes during encystation. The results provide insights into the use of Cdk inhibitory drugs in disruption of Giardia differentiation into cysts.