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1.  Epitope Mapping of Immunogenic and Adhesive Structures in Repetitive Domains of Mycoplasma bovis Variable Surface Lipoproteins 
Infection and Immunity  2000;68(2):680-687.
The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units RA1, RA2, RA4.1, RB2.1, RE1, and RF1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of M. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (RA1, RA2, RA4.1, RB2.1, RE1, and RF1) and in RF2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer RF1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.
PMCID: PMC97192  PMID: 10639433
2.  A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis. 
Infection and Immunity  1994;62(11):5075-5084.
A set of strain- and size-variant highly immunogenic membrane surface protein antigens of Mycoplasma bovis, which has been identified by a monoclonal antibody, is shown in this report to make up a family of antigenically and structurally related lipid-modified proteins, designated Vsps (variable surface proteins). By systematic analysis of several isogenic clonal lineages of the type strain PG45, three members of this family have been identified, VspA, VspB, and VspC, each of which was shown to undergo independent high-frequency changes in size as well as noncoordinate phase variation between ON and OFF expression states. The monoclonal antibody-defined epitope common to VspA, VspB, and VspC was accessible on the cell surface in most, but not all, of the clonal populations analyzed and was present on a C-terminal limit tryptic fragment of each Vsp variant that was released from the membrane surface. VspA and VspC were distinguished from VspB by their selective detection with colloidal gold and by their distinctive reaction with a polyclonal antibody against M. bovis D490. VspA, VspB, and VspC were further distinguishable from one another by their characteristic patterns of degradation at carboxypeptidase Y pause sites. While these Vsp-specific structural fingerprints with an irregular periodic spacing were constant for similarly sized variants of a defined Vsp product, they showed distinct differences among variants differing in size. This variability included gain or loss of individual bands within distinct subsets of bands, as well as shifts of the entire banding patterns up- or downwards, indicating that insertions or deletions underlying Vsp size variation can occur at various locations either within the C-terminal domain or within other regions of these proteins. This was similarly confirmed by comparative epitope mapping analysis of tryptic cleavage products generated from different Vsp size variants. The Vsp family of M. bovis described in this study represents a newly discovered system of surface antigenic variation in mycoplasmas displaying features which closely resemble but are also different from the characteristics reported for the Vlp (variable lipoprotein) system of M. hyorhinis. The isogenic lineages established here provide key populations for subsequent analysis of corresponding genes to further elucidate Vsp structure and variation, which may have important relevance for a better understanding of the pathogenicity of this agent.
PMCID: PMC303228  PMID: 7927790
3.  Juxtaposition of an Active Promoter to vsp Genes via Site-Specific DNA Inversions Generates Antigenic Variation in Mycoplasma bovis 
Journal of Bacteriology  2001;183(19):5698-5708.
Mycoplasma bovis, the most important etiological agent of bovine mycoplasmosis, undergoes extensive antigenic variation of major and highly immunogenic surface lipoprotein antigens (Vsps). A family of 13 related but divergent vsp genes, which occur as single chromosomal copies, was recently found in the chromosome of M. bovis. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal isolates exhibiting phase transitions of VspA or of VspC (i.e., ON→OFF→ON) has shown that DNA inversions occur during Vsp phase variation. The upstream region of each vsp gene contains two sequence cassettes. The first (cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhibits 98% homology among all vsp genes, while the second (cassette no. 2), upstream of cassette no. 1, ranges in size from 50 to 180 bp and is more divergent. Examination of the ends of the inverted fragments during VspA or VspC phase variation revealed that in both cases, a change in the organization of vsp upstream cassettes involving three vsp genes had occurred. Primer extension and Northern blot analysis have shown that a specific cassette no. 2, designated A2, is an active promoter and that juxtaposition of this regulatory element to a silent vsp gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that phase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present within the conserved cassette no. 1. A model for the control of Vsp phase variation is proposed.
PMCID: PMC95462  PMID: 11544233
4.  Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements. 
Journal of Bacteriology  1996;178(18):5395-5401.
Mycoplasma bovis, an important pathogen of cattle, was recently shown to possess a family of phase- and size-variable membrane surface lipoprotein antigens (Vsps). These proteins spontaneously undergo noncoordinate phase variation between ON and OFF expression states, generating surface antigenic variation. In the present study, we show that the spontaneously high rate of Vsp phenotypic switching involves DNA rearrangements that occur at high frequency in the M. bovis chromosome. A 1.5-kb HindIII genomic fragment carrying the vspA gene from M. bovis PG45 was cloned and sequenced. The deduced VspA amino acid sequence revealed that 80% of the VspA molecule is composed of reiterated intragenic coding sequences, creating a periodic polypeptide structure. Four distinct internal regions of repetitive sequences in the form of in-tandem blocks extending from the N-terminal to the C-terminal portion of the Vsp product were identified. Southern blot analysis of phenotypically switched isogenic lineages representing ON or OFF phase states of Vsp products suggested that changes in the Vsp expression profile were associated with detectable changes at the DNA level. By using a synthetic oligonucleotide representing a sequence complementary to the repetitive vspA gene region as a probe, we could identify the vspA-bearing restriction fragment undergoing high-frequency reversible rearrangements during oscillating phase transition of vspA. The 1.5-kb HindIII fragment carrying the vspA gene (on state) rearranged and produced a 2.3-kb HindIII fragment (OFF state) and vice versa. Two newly discovered vsp genes (vspE and vspF) were localized on two HindIII fragments flanking the vsp gene upstream and downstream. Southern blot hybridization with vspE- and vspF-specific oligonucleotides as probes against genomic DNA of VspA phase variants showed that the organization and size of the fragments adjacent to the vspA gene remained unchanged during VspA ON-OFF switching. The mechanisms regulating the vsp genes are yet unknown; our findings suggest that a recombinative mechanism possibly involving DNA inversions, DNA insertion, or mobile genetic elements may play a role in generating the observed high-frequency DNA rearrangements.
PMCID: PMC178356  PMID: 8808927
5.  Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen 
Infection and Immunity  2001;69(6):3703-3712.
A family of 13 related but divergent vsp genes was recently found in the chromosome of the bovine pathogen Mycoplasma bovis. The vsp genomic locus was shown to undergo high-frequency rearrangements and to mediate phenotypic switching of variable lipoprotein antigens (Vsps) on the mycoplasma cell surface. Here we report that the vsp gene repertoire is subject to changes. Genetic analysis of M. bovis clonal isolates displaying distinct Vsp phenotypes showed that an intergenic recombination event between two closely related members of the vsp gene family, the formerly expressed vspA gene and the vspO gene, led to the formation of a new chimeric and functional vsp gene, vspC. The 5′ end of the recombination event was identified within the highly conserved vsp-upstream region, while the 3′ end was localized within the first repetitive domain (RA1) present in both vspA and vspO structural genes. As a result, the vspC gene is an embodiment of the following domains: an N-terminus-encoding region linked to the highly conserved vsp-upstream region provided by the vspO gene; and a C-terminus-encoding region and the more distal and divergent vsp-upstream region acquired from the vspA gene. The generation of chimeric genes encoding surface antigens may provide an important element of genetic variation and an additional source of antigenic diversification within the mycoplasma population.
PMCID: PMC98374  PMID: 11349034
6.  Characterization of VspB of Borrelia turicatae, a Major Outer Membrane Protein Expressed in Blood and Tissues of Mice 
Infection and Immunity  1999;67(9):4637-4645.
Serotypes A and B of the relapsing fever spirochete Borrelia turicatae produce different disease manifestations in infected mice. Whereas serotype B causes more severe arthritis and reaches higher densities in the blood of mice than serotype A, serotype A invades the central nervous system earlier than serotype B during infection. These differences between serotypes A and B in mice are associated with the expression of different surface proteins, VspA and VspB, respectively, in the culture medium. To determine whether these proteins, in particular, VspB, are also expressed in vivo, scid mice infected with B. turicatae were studied. The expression of VspB by spirochetes in the blood was demonstrated in Coomassie blue-stained polyacrylamide gels and Western blots with a specific monoclonal antibody. Indirect immunofluorescence and immunoperoxidase studies confirmed the expression of VspB in the blood and also demonstrated VspB expression in the joints and heart. The gene for VspB was next identified and cloned by using partial amino acid sequencing, reverse transcriptase PCR, and a specific monoclonal antibody. The vspB gene encodes a protein of 216 amino acids that is 68% identical to VspA of B. turicatae and 44 to 56% identical to representative Vsp and OspC lipoproteins of other Borrelia spp. The processed VspB protein was distinguished from 26 other Vsp and OspC proteins by a high predicted isoelectric point at 9.39. The promoter region for vspB was similar to the promoter region for the vsp33 gene of Borrelia hermsii and for the ospC gene of Borrelia burgdorferi, two genes known to be environmentally regulated. These studies established that the virulence-associated VspB protein is expressed by spirochetes in the mouse and that VspB is a novel member of the Vsp-OspC family of proteins.
PMCID: PMC96788  PMID: 10456910
7.  A surface epitope undergoing high-frequency phase variation is shared by Mycoplasma gallisepticum and Mycoplasma bovis. 
Infection and Immunity  1994;62(11):4962-4968.
We have recently reported that three distinct size- and phase-variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis possess a common epitope recognized by monoclonal antibody 1E5. In the present study, we show that this epitope is also present on a size-variant protein (PvpA) of the avian pathogen Mycoplasma gallisepticum. Application of monoclonal antibody 1E5 in Western immunoblot analysis of Triton X-114 phase-fractionated proteins and in colony immunoblots, as well as in trypsin and carboxypeptidase digestion experiments, has demonstrated that (i) PvpA is an integral membrane protein with a free C terminus, (ii) the shared epitope is surface exposed, and (iii) PvpA is subjected to high-frequency phase variation in expression. By using serum antibodies from M. gallisepticum-infected chickens, we were able to demonstrate the immunogenic nature of PvpA and identify three additional highly immunogenic Triton X-114 phase proteins (p67, p72, and p75) also undergoing high-frequency phase variation spontaneously and independently. Metabolic labeling experiments with [14C]palmitate and [14C]oleate revealed that PvpA, in contrast to p67, p72, and p75, is not lipid modified. Southern blot hybridization with restriction fragments carrying the pvpA gene of M. gallisepticum or the vspA gene of M. bovis against digested genomic DNA of the two Mycoplasma species indicated the absence of genetic relatedness between the pvpA and vspA genes. The apparent complexity of the antigenic variation phenomenon in M. gallisepticum is discussed.
PMCID: PMC303213  PMID: 7523302
8.  The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features 
Journal of Bacteriology  1999;181(18):5734-5741.
Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogen Mycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395–5401, 1996). In the present study, 13 single-copy vsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of each vsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the various vsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5′ linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.
PMCID: PMC94094  PMID: 10482515
9.  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 
Mycoplasma 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.
Lung 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.
Suppurative 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.
The 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.
PMCID: PMC3287114  PMID: 22305416
Cattle, Mycoplasma bovis; pneumonia; immunoglobulins; CD4+ T cells; CD8+ cells; MHC class II
10.  Cross-Species Surface Display of Functional Spirochetal Lipoproteins by Recombinant Borrelia burgdorferi  
Infection and Immunity  2004;72(3):1463-1469.
Surface-exposed lipoproteins of relapsing fever (RF) and Lyme borreliosis Borrelia spirochetes mediate certain interactions of the bacteria with their arthropod and vertebrate hosts. RF spirochetes such as Borrelia hermsii serially evade the host's antibody response by multiphasic antigenic variation of Vsp and Vlp proteins. Furthermore, the expression of Vsp1 and Vsp2 by Borrelia turicatae is associated with neurotropism and higher blood densities, respectively. In contrast to RF Borrelia species, the Lyme borreliosis spirochete Borrelia burgdorferi is amenable to genetic manipulation. To facilitate structure-function analyses of RF surface lipoproteins, we used recombinant plasmids to introduce full-length vsp1 and vsp2 as well as two representative vlp genes into B. burgdorferi cells. Recombinant B. burgdorferi cells constitutively expressed the proteins under the control of the B. burgdorferi flaB promoter. Antibody and protease accessibility assays indicated proper surface exposure and folding. Expression of Vsp1 and Vsp2 conferred glycosaminoglycan binding to recombinant B. burgdorferi cells that was similar to that observed with purified recombinant proteins and B. turicatae expressing native Vsp. These data demonstrate that the lipoprotein modification and export mechanisms in the genus Borrelia are conserved. They also validate the use of recombinant B. burgdorferi in studies of surface lipoprotein structure-function and the biogenesis of spirochete membranes.
PMCID: PMC356051  PMID: 14977951
11.  Isogenic Serotypes of Borrelia turicatae Show Different Localization in the Brain and Skin of Mice 
Infection and Immunity  2001;69(5):3389-3397.
Mice with severe combined immunodeficiency (scid mice) and infected with the relapsing fever agent Borrelia turicatae develop manifestations that resemble those of disseminated Lyme disease. We have characterized two isogenic serotypes, A and B, which differ in their variable small proteins (Vsps) and disease manifestations. Serotype A but not serotype B was cultured from the brain during early infection, and serotype B caused more severe arthritis, myocarditis, and vestibular dysfunction than serotype A. Here we compared the localization and number of spirochetes and the severity of inflammation in scid mice, using immunostained and hematoxylin-and-eosin-stained coronal sections of decalcified heads. Spirochetes in the brain localized predominantly to the leptomeninges, and those in peripheral tissues localized mainly to the extracellular matrix. There were significantly more serotype A than B spirochetes in the leptomeninges and more serotype B than A spirochetes in the skin. The first tissue where spirochetes were observed outside the vasculature was the dura mater. Inflammation was more severe in the skin than in the brain. VspA, VspB, and the periplasmic flagellin protein were expressed in all tissues examined. These findings indicate that isogenic but antigenically distinct Borrelia serotypes can have marked differences in their localization in tissues.
PMCID: PMC98298  PMID: 11292762
12.  Experimental Infections of Neonatal Mice with Cysts of Giardia lamblia Clone GS/M-83-H7 Are Associated with an Antigenic Reset of the Parasite  
Infection and Immunity  2004;72(8):4763-4771.
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.
PMCID: PMC470631  PMID: 15271938
13.  Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae, AvgC, with the Use of Synthetic Peptides  
Infection and Immunity  2002;70(1):171-176.
As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection.
PMCID: PMC127643  PMID: 11748179
14.  Multiplex Assay Detection of Immunoglobulin G Antibodies That Recognize Giardia intestinalis and Cryptosporidium parvum Antigens ▿  
Clinical and Vaccine Immunology : CVI  2010;17(11):1695-1707.
Giardiasis and cryptosporidiosis are common enteric parasitic diseases that have similar routes of transmission. In this work, we have identified epitopes within the Giardia variant-specific surface protein (VSP) sequences that are recognized by IgG antibodies from 13 of 14 (93%) sera from patients with stool-confirmed giardiasis. The conserved epitopes are shared among VSPs from both of the assemblages that commonly infect humans, and they are likely to be structural, as both sodium dodecyl sulfate treatment and dithiothreitol reduction decrease antibody recognition. In a multiplex bead assay (MBA), we used three VSP fragments from an assemblage A Giardia strain, three VSP fragments from assemblage B strains, and the α-1 giardin structural antigen to detect IgG antibodies to Giardia and used the recombinant 17- and 27-kDa antigens to simultaneously detect IgG antibodies to Cryptosporidium. The MBA differentiated between sera from Giardia and Cryptosporidium outbreaks and also identified a giardiasis outbreak that may have included cryptosporidiosis cases. Approximately 40% of cryptosporidiosis outbreak samples had high MBA responses for both the 27- and 17-kDa antigens, while <10% of nonoutbreak and giardiasis outbreak samples had high responses. At least 60% of giardiasis outbreak samples were positive for antibodies to multiple Giardia antigens, while ≤12% of nonoutbreak samples and samples from U.S. and British Columbia cryptosporidiosis outbreaks met our definition for Giardia seropositivity. A MBA using multiple parasite antigens may prove useful in the epidemiologic analysis of future waterborne or food-borne outbreaks of diarrheal disease.
PMCID: PMC2976096  PMID: 20876825
15.  A New Family of Giardial Cysteine-Rich Non-VSP Protein Genes and a Novel Cyst Protein 
PLoS ONE  2006;1(1):e44.
Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, “HCNCp” (High Cysteine Non-variant Cyst protein), that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs) that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many “CxxC” tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple “CxC” motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp C-terminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp) containing ≥20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents, vaccine candidates, and subjects for further research into Giardia biology.
PMCID: PMC1762436  PMID: 17183673
16.  vsp Gene Expression by Giardia lamblia Clone GS/M-83-H7 during Antigenic Variation In Vivo and In Vitro 
Infection and Immunity  2001;69(9):5278-5285.
Giardia lamblia infections are associated with antigenic variation of the parasite, which is generated by a continuous change of the variant-specific surface proteins (VSPs). Many investigations on the process of antigenic variation were based on the use of G. lamblia clone GS/M-83-H7, which expresses VSP H7 as its major surface antigen. In the present study, mice were infected with the aforementioned clonal line to investigate vsp gene expression during the complex process of antigenic variation of the parasite. Trophozoites collected from the intestines of individual animals at different time points postinfection (p.i.) were analyzed directly for their vsp gene expression patterns, i.e., without cultivating the recovered parasites in vitro. Because few trophozoites were recovered at late time points p.i., a combined 5′ rapid amplification of cDNA ends–reverse transcription-PCR approach was utilized. This allowed detection and subsequent sequence analysis of vsp gene transcripts upon generation of amplified cDNA analogues. The same PCR approach was applied for analysis of vsp gene expression in variants obtained after negative selection of axenic GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. In an overall view of the entire panel of in vivo- and in vitro-derived parasite populations, expression of 29 different vsp gene sequences could be demonstrated. In vivo antigenic variation of G. lamblia clone GS/M-83-H7 was shown to be a continuous process involving the consecutive appearance of relatively distinct sets of vsp transcripts. During the 42-day infection period investigated, this process activated at least 22 different vsp genes. Comparative molecular analyses of the amino acid level demonstrated that all cDNA segments identified encode structural elements typical of the terminal segment of Giardia VSP. The similarity of most of the GS/M-83-H7 VSP sequences identified in the present study supports previous suggestions that vsp gene diversification in G. lamblia is the result of ancestral gene duplication, mutation, and/or recombination events.
PMCID: PMC98636  PMID: 11500396
17.  Crystal Structure of Neurotropism-Associated Variable Surface Protein 1 (Vsp1) of Borrelia turicatae†  
Journal of Bacteriology  2006;188(12):4522-4530.
Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to allow bacterial persistence in spite of an immune response. Two isogenic serotypes of Borrelia turicatae strain Oz1 differ in their Vsp sequences and in disease manifestations in infected mice: Vsp1 is associated with the selection of a neurological niche, while Vsp2 is associated with blood and skin infection. We report here crystal structures of the Vsp1 dimer at 2.7 and 2.2 Å. The structures confirm that relapsing fever Vsp proteins share a common helical fold with OspCs of Lyme disease-causing Borrelia. The fold features an inner stem formed by highly conserved N and C termini and an outer “dome” formed by the variable central residues. Both Vsp1 and OspC structures possess small water-filled cavities, or pockets, that are lined largely by variable residues and are thus highly variable in shape. These features appear to signify tolerance of the Vsp-OspC fold for imperfect packing of residues at its antigenic surface. Structural comparison of Vsp1 with a homology model for Vsp2 suggests that observed differences in disease manifestation may arise in part from distinct differences in electrostatic surface properties; additional predicted positively charged surface patches on Vsp2 compared to Vsp1 may be sufficient to explain the relative propensity of Vsp2 to bind to acidic glycosaminoglycans.
PMCID: PMC1482977  PMID: 16740958
18.  Antigenic variation in Giardia lamblia and the host's immune response. 
Giardia lamblia, a protozoan parasite of the small intestine of humans and other animals, undergoes surface antigenic variation. The antigens involved belong to a family of variant-specific surface proteins (VSPs), which are unique, cysteine-rich zinc finger proteins. The patterns of infection in humans and animals fail to show the expected cyclical waves of increasing and decreasing numbers of parasites expressing unique VSPs. Nevertheless, changes in VSP expression occur within the population in vivo owing to selection of VSPs by both immune and non-immune mechanisms. After inoculation of a single G. lamblia clone (able to persist in the absence of immune pressure) expressing one VSP (> or = 90%) into mice or humans, the original VSP continues to be expressed until 2 weeks post inoculation (p.i.), when many other VSPs gradually replace it. Selection by immune-mediated processes is suggested because switching occurs at the same time that humoral responses are first detected. In most mouse strains, switching also occurs at about two weeks. Almost all trophozoites are eliminated at three weeks (p.i.), but a barely detectable infection persists over months. In neonatal mice, apparent self-cure is delayed until the sixth or seventh week. Antigenic switching does not occur in adult or neonatal severe combined immunodeficiency disease (SCID) mice, but does occur in neonatal nude mice, thus implicating B-cell-mediated mechanisms in immune switching. Not all VSPs are expressed to the same degree in vivo. Some VSPs appear to be preferentially selected whereas others are eliminated on a non-immune basis. In infections in which immunity does not play a role, such as in SCID mice, and during the first week of infection in immunocompetent mice or gerbils, persisting VSPs are preferentially expressed and maintained whereas non-persisting VSPs are replaced within the first week of infection. The purpose of antigenic variation may be presentation of a wide assortment of VSPs to hosts, increasing the chance of a successful initial infection or reinfection. Immune selection of variants comes into play following biological selection.
PMCID: PMC1692022  PMID: 9355129
19.  Experimental Verification of the Identity of Variant-Specific Surface Proteins in Giardia lamblia Trophozoites 
mBio  2013;4(3):e00321-13.
The cell membrane of a Giardia lamblia trophozoite is covered with a single species of variant-specific surface protein (VSP) that is replaced by another VSP every 6 to 13 generations of cell growth, possibly for an evasion of host immunity. Experimentally, only six VSP species have been verified to localize to the cell membrane thus far. By assuming that VSP contains multiple CXXC motifs, 219 vsp genes were annotated in GiardiaDB of the WB isolate. By further assuming that VSP possesses both CXXC motifs and a CRGKA tail at the C terminus, Adam et al. (BMC Genomics 11:424, 2010) identified a total of 303 potential vsp genes in Giardia WB. The discrepancies between these two assumed VSP identities have caused some confusion. Here, we used experimental approaches to further verify what is required of the structures of a VSP to localize to the surface of cell membrane. The data led to the following conclusions. (i) The C-terminal CRGKA sequence is not essential for localizing VSPs to the cell membrane. (ii) A “motif 1” of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic “motif 2” of 38 residues at the C terminus are both essential and sufficient for localizing the protein to the cell membrane. (ii) An N-terminal sequence upstream from motif 1 is not required for targeting VSPs to the cell membrane. By these criteria, we are able to identify 73 open reading frames as the putative vsp genes in Giardia.
The intestinal pathogen Giardia lamblia expresses only one variant-specific surface protein (VSP) on the cell membrane surface at a given time, but it changes spontaneously every 6 to 13 generations of growth, presumably for evading the host immunity. Only 6 VSPs have been empirically shown to localize to the cell membrane surface thus far. Here, we used mutations of VSPs and methods of identifying their locations in Giardia cells and found that a “motif 1” of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic “motif 2” of 38 residues at the C terminus are the only essential and sufficient structural requirements for localizing a protein to the cell membrane. By these criteria, 73 genes are identified in the Giardia WB strain genome database as the putative repertoire of VSPs.
PMCID: PMC3656445  PMID: 23695837
20.  Serological analysis of antigenic heterogeneity of Giardia lamblia variant surface proteins. 
Infection and Immunity  1996;64(4):1385-1390.
Neonatal ZU.ICR mice were infected with trophozoites of Giardia lamblia clone GS/M-83-H7 expressing the variant surface protein (VSP) H7 and were subsequently investigated for their serum antibody response directed against VSPH7. Recombinant polypeptides, representing overlapping segments of VSPH7, and native Giardia proteins were used as antigenic reagents to examine the antigenic substructure of VSPH7 and the extent of antigenic variation in vivo. VSPH7 proved to be the predominant antigen of the parasite with respect to serum antibody reactivity. The data indicated that VSPH7 basically consists of two antigenically distinct parts: (i) a unique, variant-specific 314-amino-acid N-terminal region which elicits a low antibody response preferentially detectable during the early phase of the infection and (ii) a 171-amino-acid C-terminal region which elicits a high antibody response during the later phase or after resolution of the infection. The epitopes of the C-terminal region appear to be shared by other, as yet uncharacterized, variant antigens. The highly conserved 34-amino-acid stretch at the extreme C terminus of VSPH7 exhibited no immunoreactivity to any of the sera tested. Further investigations indicated that antigenic variation of the intestinal parasite population was associated with a diversification into at least six to nine new antigen types. These variant antigens were extremely heterogeneous in size ranging from approximately 50 to 115 kDa. None of these proteins shared antigenic epitopes with the 314-amino-acid N-terminal portion, but several of them cross-reacted with antibodies specific to the 171-amino-acid C-terminal portion of the VSP from the original inoculum.
PMCID: PMC173930  PMID: 8606105
21.  Developmental changes in abundance of the VSPβ protein following nuclear transformation of maize with the Soybean vspβ cDNA 
BMC Plant Biology  2005;5:3.
Developing monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the vspA and B genes encode subunits of a dimeric vegetative storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize (Zea Mays L. Hi-II hybrid) lines were created expressing soybean vspB from a maize ubiquitin Ubi-1 promoter.
From 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced vspB transcripts and VSPβ polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPβ accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R0), but to only 0.03% in R1 plants. During seed-filling (silage-stage) in R1 plants, the VSPβ protein was no longer detected in leaves and stems despite continued presence of the vspB RNA. The RNA transcripts for this peptide either became less efficiently translated, or the VSPβ protein became unstable during seed-fill.
Developmental differences in the accumulation of soybean VSPβ when transgenically expressed in maize show that despite no changes in the vspB transcript level, VSPβ protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.
PMCID: PMC554770  PMID: 15743517
22.  The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence 
PLoS Pathogens  2014;10(2):e1003791.
microRNAs (miRNA) have been detected in the deeply branched protist, Giardia lamblia, and shown to repress expression of the family of variant-specific surface proteins (VSPs), only one of which is expressed in Giardia trophozoite at a given time. Three next-generation sequencing libraries of Giardia Argonaute-associated small RNAs were constructed and analyzed. Analysis of the libraries identified a total of 99 new putative miRNAs with a size primarily in the 26 nt range similar to the size previously predicted by the Giardia Dicer crystal structure and identified by our own studies. Bioinformatic analysis identified multiple putative miRNA target sites in the mRNAs of all 73 VSPs. The effect of miRNA target sites within a defined 3′-region were tested on two vsp mRNAs. All the miRNAs showed partial repression of the corresponding vsp expression and were additive when the targeting sites were separately located. But the combined repression still falls short of 100%. Two other relatively short vsp mRNAs with 15 and 11 putative miRNA target sites identified throughout their ORFs were tested with their corresponding miRNAs. The results indicate that; (1) near 100% repression of vsp mRNA expression can be achieved through the combined action of multiple miRNAs on target sites located throughout the ORF; (2) the miRNA machinery could be instrumental in repressing the expression of vsp genes in Giardia; (3) this is the first time that all the miRNA target sites in the entire ORF of a mRNA have been tested and shown to be functional.
Author Summary
Giardia lamblia is a protozoan parasite causing the diarrheal disease giardiasis. Variant-specific surface proteins (VSP) in Giardia are likely involved in its evasion of host immune response. Their expression is regulated by microRNAs (miRNA). To determine the full complement of miRNAs in Giardia, three cDNA libraries of Giardia Argonaute associated small RNAs were constructed and analyzed to identify a total of 105 miRNAs. Bioinformatic target identification showed that 102 of the 105 miRNAs find their putative target sites in vsp mRNAs. When only the target sites within the 3′ region,100 nts upstream of the stop codon, were tested against their corresponding miRNAs, however, only partial repression of VSP expression was observed. When all the miRNA target sites in the open reading frames of vsp mRNAs were examined, however, they all turned out to be functional. A saturation of them with the corresponding miRNAs resulted in a full repression of VSP expression, suggesting that this is the mechanism of miRNA repression of VSP expression in Giardia. The ability of miRNAs to regulate target sites throughout the entire open reading frame also provides the first indication that all the miRNA target sites in an mRNA are functional.
PMCID: PMC3937270  PMID: 24586143
23.  Molecular Epidemiological Analysis of Mycoplasma bovis Isolates from the United Kingdom Shows Two Genetically Distinct Clusters 
Journal of Clinical Microbiology  2004;42(10):4556-4565.
Mycoplasma bovis is an important veterinary pathogen causing pneumonia, arthritis, and mastitis in infected cattle. We investigated the genetic diversity of 53 isolates collected in the United Kingdom between 1996 and 2002 with pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD) analysis. In addition, the influence of variable surface protein (Vsp) profiles on the profiles generated with molecular typing techniques was studied. Both AFLP and RAPD separated the isolates into two distinct groups, but PFGE showed less congruence with the other techniques. There was no clear relationship between the geographic origin or year of isolation of the isolates and the profiles produced. No correlation between Vsp profiles and any of the molecular typing techniques was observed. We propose that RAPD and AFLP provide valuable tools for molecular typing of M. bovis.
PMCID: PMC522341  PMID: 15472309
24.  Structure-Function Investigation of Vsp Serotypes of the Spirochete Borrelia hermsii 
PLoS ONE  2009;4(10):e7597.
Relapsing fever (RF) spirochetes are notable for multiphasic antigenic variation of polymorphic outer membrane lipoproteins, a phenomenon responsible for immune evasion. An additional role in tissue localization is suggested by the finding that isogenic serotypes 1 (Bt1) and 2 (Bt2) of the RF spirochete Borrelia turicatae, which differ only in the Vsp they express, exhibit marked differences in clinical disease severity and tissue localization during infection.
Methodology/Principal Findings
Here we used known vsp DNA sequences encoding for B. turicatae and Borrelia hermsii Vsp proteins with variable regions and then studied whether there are differences in disease expression and tissue localization of their corresponding serotypes during mouse infection. For sequence and structural comparisons we focused exclusively on amino acid residues predicted to project away from the spirochetes surface, referred to as the Vsp dome. Disease severity and tissue localization were studied during persistent infection with individual or mixed serotypes in SCID mice. The results showed that all Vsp domes clustered into 3 main trunks, with the domes for B. turicatae Vsp1 (BtVsp1) and BtVsp2 clustering into separate ones. B. hermsii serotypes whose Vsp domes clustered with the BtVsp1 dome were less virulent but localized to the brain more. The BtVsp2 dome was the oddball among all and Bt2 was the only serotype that caused severe arthritis.
These findings indicate that there is significant variability in Vsp dome structure, disease severity, and tissue localization among serotypes of B. hermsii.
PMCID: PMC2766631  PMID: 19888463
25.  Variable Lipoprotein Genes of Mycoplasma agalactiae Are Activated In Vivo by Promoter Addition via Site-Specific DNA Inversions  
Infection and Immunity  2003;71(7):3821-3830.
Mycoplasma agalactiae, the etiological agent of contagious agalactia of small ruminants, has a family of related genes (avg genes) which encode surface lipoprotein antigens that undergo phase variation. A series of 13 M. agalactiae clonal isolates, obtained from one chronically infected animal over a period of 7 months, were found to undergo major rearrangement events within the avg genomic locus. We show that these rearrangements regulate the phase-variable expression of individual avg genes. Northern blot analysis and reverse transcription-PCR showed that only one avg gene is transcribed, while the other avg genes are transcriptionally silent. Sequence analysis and primer extension experiments with two M. agalactiae clonal isolates showed that a specific 182-bp avg 5′ upstream region (avg-B2) that is present as a single chromosomal copy serves as an active promoter and exhibits a high level of homology with the vsp promoter of the bovine pathogen Mycoplasma bovis. PCR analysis showed that each avg gene is associated with the avg-B2 promoter in a subpopulation of cells that is present in each subclone. Multiple sequence-specific sites for DNA recombination (vis-like), which are presumably recognized by site-specific recombinase, were identified within the conserved avg 5′ upstream regions of all avg genes and were found to be identical to the recombination sites of the M. bovis vsp locus. In addition, a gene encoding a member of the integrase family of tyrosine site-specific recombinases was identified adjacent to the variable avg locus. The molecular genetic basis for avg phase-variable expression appears to be mediated by site-specific DNA inversions occurring in vivo that allow activation of a silent avg gene by promoter addition. A model for the control of avg genes is proposed.
PMCID: PMC162021  PMID: 12819065

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