scholarly journals Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure

2021 ◽  
Vol 15 (9) ◽  
pp. e0009753
Author(s):  
Michelle J. Lin ◽  
Austin M. Haynes ◽  
Amin Addetia ◽  
Nicole A. P. Lieberman ◽  
Quynh Phung ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Antigenic Variation ◽  
Treponema Pallidum ◽  
Sequence Diversity ◽  
Immune Selection ◽  
Syphilis Infection ◽  
Variable Regions ◽  
Immune Pressure

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.

scholarly journals Longitudinal accumulation of in vivo and in vitro-grown Treponema pallidum subsp. pallidum TprK variants in the presence and absence of immune pressure

2021 ◽  
Author(s):  
Michelle J Lin ◽  
Austin Haynes ◽  
Amin Addetia ◽  
Nicole A.P. Lieberman ◽  
Quynh Phung ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Antigenic Variation ◽  
Treponema Pallidum ◽  
Sequence Diversity ◽  
Immune Selection ◽  
Variable Regions ◽  
Immune Pressure ◽  
Presence And Absence

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.

scholarly journals TprK Sequence Diversity Accumulates during Infection of Rabbits with Treponema pallidum subsp. pallidum Nichols Strain

2006 ◽  
Vol 74 (3) ◽  
pp. 1896-1906 ◽  
Author(s):  
Rebecca E. LaFond ◽  
Arturo Centurion-Lara ◽  
Charmie Godornes ◽  
Wesley C. Van Voorhis ◽  
Sheila A. Lukehart
Keyword(s):  
Antigenic Variation ◽  
Treponema Pallidum ◽  
Adaptive Immune Response ◽  
Serial Passage ◽  
Sequence Diversity ◽  
Adaptive Immune ◽  
Immune Pressure ◽  
V Region ◽  
Slow Passage

ABSTRACT The tprK gene in Treponema pallidum undergoes antigenic variation. In all T. pallidum isolates examined to date, except the Nichols type strain, heterogeneous tprK sequences have been identified. This heterogeneity is localized to seven variable (V) regions, and tprK sequence diversity accumulates with serial passage in naïve rabbits. The T. pallidum Nichols genome described a single tprK sequence, and after decades of independent passage, only minor tprK sequence diversity is seen among the Nichols strains from different laboratories. We hypothesized that T. pallidum Nichols is capable of only limited tprK diversification. To address this hypothesis, we passaged the T. pallidum Nichols strain in naïve rabbits at the peak of infection (rapid passage) or after the adaptive immune response had cleared most organisms in vivo (slow passage). After 22 rapid passages (9- to 10-day intervals), no tprK V region sequence changes were observed. In contrast, after two slow passages (30- to 35-day intervals), three V regions had sequences that were completely different from that of the original inoculum. New sequences were observed in all seven V regions by the fifth slow passage. In contrast to the rapid-passaged Nichols strain, rapid-passaged Chicago C, a clonal strain isolated from the highly diverse parent Chicago strain, developed significant tprK diversification. These findings suggest that tprK variation can occur, but at a lower rate, in Nichols and that immune pressure may be required for accumulation of bacteria with diverse tprK sequences. Adaptation to growth in rabbits may explain the limited repertoire of V region sequences seen in the Nichols strain.

scholarly journals Sequence Diversity of Treponema pallidum subsp. pallidum tprK in Human Syphilis Lesions and Rabbit-Propagated Isolates

2003 ◽  
Vol 185 (21) ◽  
pp. 6262-6268 ◽  
Author(s):  
Rebecca E. LaFond ◽  
Arturo Centurion-Lara ◽  
Charmie Godornes ◽  
Anne M. Rompalo ◽  
Wesley C. Van Voorhis ◽  
...  
Keyword(s):  
Clustering Analysis ◽  
Type Strain ◽  
Signal Sequence ◽  
Treponema Pallidum ◽  
Sequence Diversity ◽  
Variable Regions ◽  
Member Gene ◽  
Mixed Populations ◽  
Heterogeneous Clustering ◽  
Different Levels

ABSTRACT The tprK gene of Treponema pallidum subsp. pallidum, the causative agent of venereal syphilis, belongs to a 12-member gene family and encodes a protein with a predicted cleavable signal sequence and predicted transmembrane domains. Except for the Nichols type strain, all rabbit-propagated isolates of T. pallidum examined thus far are comprised of mixed populations of organisms with heterogeneous tprK sequences. We show that tprK sequences in treponemes obtained directly from syphilis patients are also heterogeneous. Clustering analysis demonstrates that primary chancre tprK sequences are more likely to cluster within a sample than among samples and that tighter clustering is seen within chancre samples than within rabbit-propagated isolates. Closer analysis of tprK sequences from a rabbit-propagated isolate reveals that individual variable regions have different levels of diversity, suggesting that variable regions may have different intrinsic rates of sequence change or may be under different levels of selection. Most variable regions show increased sequence diversity upon passage. We speculate that the diversification of tprK during infection allows organisms to evade the host immune response, contributing to reinfection and persistent infection.

scholarly journals Antigenic Variation inTreponema pallidum: TprK Sequence Diversity Accumulates in Response to Immune Pressure during Experimental Syphilis

2010 ◽  
Vol 184 (7) ◽  
pp. 3822-3829 ◽  
Author(s):  
Lorenzo Giacani ◽  
Barbara J. Molini ◽  
Eric Y. Kim ◽  
B. Charmie Godornes ◽  
B. Troy Leader ◽  
...  
Keyword(s):  
Antigenic Variation ◽  
Sequence Diversity ◽  
Immune Pressure

scholarly journals Anaplasma phagocytophilum MSP2(P44)-18 Predominates and Is Modified into Multiple Isoforms in Human Myeloid Cells

2008 ◽  
Vol 76 (5) ◽  
pp. 2090-2098 ◽  
Author(s):  
Madhubanti Sarkar ◽  
Matthew J. Troese ◽  
Sarah A. Kearns ◽  
Tian Yang ◽  
Dexter V. Reneer ◽  
...  
Keyword(s):  
Anaplasma Phagocytophilum ◽  
Structural Integrity ◽  
Antigenic Variation ◽  
Surface Protein ◽  
Etiologic Agent ◽  
In Vitro Cultivation ◽  
Immune Pressure ◽  
Molecular Approaches ◽  
Dna And Rna

ABSTRACT Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis. MSP2(P44), the bacterium's major surface protein, is encoded by a paralogous gene family and has been implicated in a variety of pathobiological processes, including antigenic variation, host adaptation, adhesion, porin activity, and structural integrity. The consensus among several studies performed at the DNA and RNA levels is that a heterogeneous mix of a limited number of msp2(p44) transcripts is expressed by A. phagocytophilum during in vitro cultivation. Such analyses have yet to be extended to the protein level. In this study, we used proteomic and molecular approaches to determine that MSP2(P44)-18 is the predominant if not the only paralog expressed and is modified into multiple 42- to 44-kDa isoforms by A. phagocytophilum strain HGE1 during infection of HL-60 cells. The msp2(p44) expression profile was homogeneous for msp2(p44)-18. Thus, MSP2(P44)-18 may have a fitness advantage in HL-60 cell culture in the absence of selective immune pressure. Several novel 22- to 27-kDa MSP2 isoforms lacking most of the N-terminal conserved region were also identified. A. phagocytophilum MSP2(P44) orthologs expressed by other pathogens in the family Anaplasmataceae are glycosylated. Gas chromatography revealed that recombinant MSP2(P44)-18 is modified by glucose, galactose, xylose, mannose, and trace amounts of other glycosyl residues. These data are the first to confirm differential modification of any A. phagocytophilum MSP2(P44) paralog and the first to provide evidence for expression of truncated versions of such proteins.

scholarly journals New Tools for Syphilis Research

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Sheila A. Lukehart
Keyword(s):  
Protein Function ◽  
Antigenic Variation ◽  
Treponema Pallidum ◽  
Content Type ◽  
Genetic Tools ◽  
Rapid Isolation

ABSTRACTSyphilis research has been severely limited by the necessity to propagateTreponema pallidumin vivoin rabbits. After decades of erroneous or irreproducible reports of cultivation ofT. pallidum, the recent very convincing report of its successful long-termin vitropropagation opens numerous opportunities for development of genetic tools for studying pathogenesis and protein function, antigenic variation, and surface exposure of antigens. The possibility of more rapid isolation of new strains will expand our knowledge of this organism beyond the century-old Nichols strain.

Pathogenic antiphospholipid antibody: an antigen-selected needle in a haystack

Blood ◽  
2004 ◽  
Vol 104 (6) ◽  
pp. 1711-1715 ◽  
Author(s):  
Patricia Lieby ◽  
Vincent Poindron ◽  
Stamatiki Roussi ◽  
Cyril Klein ◽  
Anne-Marie Knapp ◽  
...  
Keyword(s):  
Antiphospholipid Antibodies ◽  
Germ Line ◽  
Annexin A5 ◽  
Antiphospholipid Antibody ◽  
Variable Regions ◽  
Anionic Phospholipids ◽  
Pregnant Mice

Abstract Antiphospholipid antibodies represent a heterogeneous group of autoantibodies directed against anionic phospholipids (PLs) usually linked to protein cofactors. Their presence during the antiphospholipid syndrome is associated with risks of thrombosis and fetal losses. Among 5 randomly selected monoclonal antiphospholipid antibodies, all originating from a single patient suffering from this autoimmune disease, only 1 induced fetal losses when passively injected into pregnant mice. Its antiphospholipid activity was dependent on annexin A5, and its variable regions contained mainly 3 replacement mutations. To clarify the role of these mutations in the pathogenicity of the antibody, they were in vitro reverted to the germ line configuration. The resulting “germ line” antibody reacted with multiple self-antigens and only partially lost its reactivity against PLs, but it was no more dependent on annexin A5 and, more importantly, was no more pathogenic. This study illustrates that the in vivo antigen-driven maturation process of natural autoreactive B cells can be responsible for pathogenicity. (Blood. 2004;104:1711-1715)

scholarly journals Surface alterations of erythrocytes in Plasmodium falciparum malaria. Antigenic variation, antigenic diversity, and the role of the spleen.

1983 ◽  
Vol 157 (4) ◽  
pp. 1137-1148 ◽  
Author(s):  
M Hommel ◽  
P H David ◽  
L D Oligino
Keyword(s):  
Plasmodium Falciparum ◽  
Developmental Stages ◽  
Antigenic Variation ◽  
Plasmodium Falciparum Malaria ◽  
Immune Serum ◽  
Antigenic Determinants ◽  
Original Population ◽  
Antigenic Diversity ◽  
Immune Pressure

The surface of erythrocytes infected with late developmental stages of Plasmodium falciparum is profoundly altered and new antigenic determinants can be detected by surface immunofluorescence using immune squirrel monkey serum. The expression of these parasite-specific antigenic determinants on the surface of the host erythrocyte can be modulated by the presence or absence of the spleen and by immune pressure. An antigenic switch occurred when a cloned population of the Ugandan Palo Alto strain of P. falciparum was transferred from a splenectomized into an intact monkey and this switch was reversible. In another strain (Indochina-1), we showed that the parasites isolated during secondary and recrudescent peaks expressed erythrocyte-associated surface antigens different from the parasites isolated during the primary infection; six variant antigenic types distinct from the original population were isolated in this way. The passive transfer of immune serum can induce antigenic variation and this can occur in a cloned parasite. The various mechanisms of antigenic variation in P. falciparum are discussed in the context of strain-specific diversity and the role of antigenic diversity in acquired immunity.

scholarly journals Effects of vlsE Complementation on the Infectivity of Borrelia burgdorferi Lacking the Linear Plasmid lp28-1

2004 ◽  
Vol 72 (11) ◽  
pp. 6577-6585 ◽  
Author(s):  
Matthew B. Lawrenz ◽  
R. Mark Wooten ◽  
Steven J. Norris
Keyword(s):  
Borrelia Burgdorferi ◽  
Gene Conversion ◽  
Sequence Variation ◽  
Antigenic Variation ◽  
Shuttle Vector ◽  
Immunoblot Analysis ◽  
Linear Plasmid ◽  
Shuttle Plasmid ◽  
Immunocompetent Mice

ABSTRACT The loss of linear plasmid lp28-1, which contains the vls antigenic variation locus, is associated with reduced infectivity of Borrelia burgdorferi in immunocompetent mice. The recombinant shuttle vector pBBE22, which includes the virulence determinant BBE22 from lp25 and restores infectivity to readily transformable B. burgdorferi lacking lp25 and lp56, was used to determine the effect of trans expression of vlsE on virulence. Spirochetes lacking lp28-1 were complemented with the plasmid pBBE22:vlsE, containing both BBE22 and vlsE. VlsE protein produced by this construct was expressed and surface accessible in in vitro-cultured B. burgdorferi, as determined by surface proteolysis and immunoblot analysis. Clones lacking lp25 but containing lp28-1 and either pBBE22 or pBBE22:vlsE were reisolated consistently from immunocompetent mice 8 weeks after infection. In contrast, a clone lacking both lp25 and lp28-1 and complemented with pBBE22:vlsE was isolated from only a single tissue of one of six C3H/HeN mice 8 weeks postinfection. These results indicate that either an intact vls antigenic variation locus or another determinant on lp28-1 is required to restore complete infectivity. In addition, an isogenic clone that retained lp28-1 was complemented with the vlsE shuttle plasmid and was examined for vlsE sequence variation and infectivity. Sequence variation was not observed for the shuttle plasmid, indicating that the cis arrangement of vlsE and the vls silent cassettes in lp28-1 facilitate vlsE gene conversion. Lack of vlsE sequence variation on the shuttle plasmid thus did not result in clearance of the trans-complemented strain in immunocompetent mice under the conditions tested.

DC-SIGN Binds Preferentially Highly Glycosylated IgM to Trigger Classical BCR Signaling in Follicular Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2968-2968 ◽  
Author(s):  
Rada Amin ◽  
Frédéric Mourcin ◽  
Fabrice Uhel ◽  
Marion Guirriec ◽  
Thierry Lamy ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Genetic Alterations ◽  
Variable Regions ◽  
Bcr Signaling

Abstract BCR is supposed to deliver key stimulatory signals to malignant B cells in various mature hematological malignancies. In follicular lymphoma (FL), unlike in aggressive lymphomas, very few genetic alterations in BCR signaling pathway have been described, and BCR activation has been reported to rely on the dynamic interaction of mannosylated Ig with C-type lectins, such as Mannose receptor (MR) and DC-SIGN. In fact, the variable regions of FL Ig genes are characterized by the specific accumulation of N-glycan motifs introduced by the somatic hypermutation process and positively selected during lymphomagenesis. BCR activation is thus induced by an antigen-independent functional bridge between FL B cells and lectin-expressing non-malignant cells within tumor cell niche. In agreement, FL cells are well known to be strongly dependent on their microenvironment, including in particular stromal cells, CD4pos T cells, and macrophages that collectively support malignant cell growth and suppress antitumor immune response. In particular, a high number of infiltrating tumor associated macrophages (TAM) is associated with poor prognosis in patients treated by conventional therapy. Since DC-SIGN and MR are expressed by myeloid cells, we explored the possibility that they could trigger FL BCR activation in vitro and in situ. We first demonstrated that primary FL B cells of IgM isotype exhibited a higher response to anti-Ig triggering than normal germinal center B cells, as highlighted by phosphorylation of CD79a, Syk, BLNK, and Erk. Interestingly, IgG-expressing FL B cells showed reduced BCR activation compared to IgM-expressing malignant B cells, an observation that shed new light on the potential role of allelic paradox in FL, where malignant B cells essentially express membrane IgM despite active class-switch recombination on the translocated allele. Furthermore, only IgM-expressing FL cells were able to bind recombinant DC-SIGN with variable affinity, correlated to the level of glycosylation of Ig. Crosslinking of FL IgM by DC-SIGN induces an immune complex where CD19 was detected and led to a sustained phosphorylation of Syk, Akt, and Erk, whereas crosslinking by anti-IgM antibodies triggered a quick and transient activation. Co-culture of FL cells expressing highly glycosylated IgM with DC-SIGNhi M2 macrophages, unlike with DC-SIGNlo M1 macrophages, led to relocalization of DC-SIGN at the M2/B-cell interface and activation of Syk, Akt, and Erk in malignant B cells. Interestingly, M2-dependent activation of Erk was abrogated by BCR inhibitors such as imatinib. Finally, IL-4, which is overexpressed by infiltrating CD4pos follicular helper T cells in FL, upregulated DC-SIGN expression on macrophages, thus favoring such BCR-dependent crosstalk. Overall, our results support the hypothesis that glycosylated BCR contributes to DC-SIGN-dependent activation in FL and reveal the role of IgM isotype in this process. Such study paves the way for a better understanding of TAM/B cell crosstalk, which could constitute an important therapeutic target in this still fatal malignancy. Disclosures No relevant conflicts of interest to declare.

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