M. Lamine Mbow

Borrelia burgdorferi Changes Its Surface Antigenic Expression in Response to Host Immune Responses

ABSTRACT The Lyme disease spirochete, Borrelia burgdorferi, causes persistent mammalian infection despite the development of vigorous immune responses against the pathogen. To examine spirochetal phenotypes that dominate in the hostile immune environment, the mRNA transcripts of four prototypic surface lipoproteins, decorin-binding protein A (DbpA), outer surface protein C (OspC), BBF01, and VlsE, were analyzed by quantitative reverse transcription-PCR under various immune conditions. We demonstrate that B. burgdorferi changes its surface antigenic expression in response to immune attack. dbpA expression was unchanged while the spirochetes decreased ospC expression by 446 times and increased BBF01 and vlsE expression up to 20 and 32 times, respectively, under the influence of immune pressure generated in immunocompetent mice during infection. This change in antigenic expression could be induced by passively immunizing infected severe combined immunodeficiency mice with specific Borrelia antisera or OspC antibody and appears to allow B. burgdorferi to resist immune attack.

Analysis of Borrelia burgdorferi gene expression during life cycle phases of the tick vector Ixodes scapularis

Borrelia burgdorferi exists in nature via an enzootic cycle whereby the organism must adapt to the diverse environmental conditions provided inside the arthropod transmission vector and the mammalian reservoir hosts. B. burgdorferi genes shown to be regulated by temperature, pH and/or cell density during the organisms growth in culture medium were assayed for expression during various stages of the tick feeding cycle by reverse transcription-polymerase chain reaction (RT-PCR). ospA, ospC, flaB, erpA/I/N, erpB/J/O, rev and mlpA, were transcriptionally active following the larval and nymphal stages of feeding as determined by qualitative RT-PCR. During tick resting periods between feedings, ospC, mlpA and rev transcription were undetectable, in contrast to ospA, flaB, erpA/I/N and erpB/J/O. bba64, a gene induced by environmental changes in culture and expressed during mammalian infection, was not detectable during any of the tick life cycle phases. Quantitative PCR to determine B. burgdorferi genome equivalents in these tick samples using DNA co-purified with the RNA allowed an estimation of gene expression relative to the numbers of B. burgdorferi present in the ticks. Although the spirochete totals varied widely between individual tick pools of fed, replete nymphs, the relative expression ratios between individual target genes following a nymphal feed were comparable. Similarly, borrelial gene transcription from the larval feeding and the nymphal feeding were observed and compared. These findings analogize B. burgdorferi gene expression observed by environmental stimuli in vitro with the transcriptional activity occurring during the organisms infectious cycle within the tick.

Effects of Single Nucleotide Polymorphisms on Toll-like Receptor 3 Activity and Expression in Cultured Cells

Recognition of double-stranded RNA by Toll-like receptor 3 (TLR3) will increase the production of cytokines and chemokines through transcriptional activation by the NF-κB protein. Over 136 single-nucleotide polymorphisms (SNPs) in TLR3 have been identified in the human population. Of these, four alter the sequence of the TLR3 protein. Molecular modeling suggests that two of the SNPs, N284I and L412F, could affect the packing of the leucine-rich repeating units in TLR3. Notably, L412F is reported to be present in 20% of the population and is higher in the asthmatic population. To examine whether the four SNPs affect TLR3 function, each were cloned and tested for their ability to activate the expression of TLR3-dependent reporter constructs. SNP N284I was nearly completely defective for activating reporter activity, and L412F was reduced in activity. These two SNPs did not obviously affect the level of TLR3 expression or their intracellular location in vesicles. However, N284I and L412F were underrepresented on the cell surface, as determined by flow cytometry analysis, and were not efficiently secreted into the culture medium when expressed as the soluble ectodomain. They were also reduced in their ability to act in a dominant negative fashion on the wild type TLR3 allele. These observations suggest that N284I and L412F affect the activities of TLR3 needed for proper signaling.

Structural and functional analyses of the human Toll-like receptor 3. Role of glycosylation.

Toll-like receptors (TLRs) play critical roles in bridging the innate and adaptive immune responses. The human TLR3 recognizes foreign-derived double-stranded RNA and endogenous necrotic cell RNA as ligands. Herein we characterized the contribution of glycosylation to TLR3 structure and function. Exogenous addition of purified extracellular domain of TLR3 (hTLR3 ECD) expressed in human embryonic kidney cells was found to inhibit TLR3-dependent signaling, thus providing a reagent for structural and functional characterization. Approximately 35% of the mass of the hTLR3 ECD was due to posttranslational modification, with N-linked glycosyl groups contributing substantially to the additional mass. Cells treated with tunicamycin, an inhibitor of glycosylation, prevented TLR3-induced NF-κB activation, confirming that N-linked glycosylation is required for bioactivity of this receptor. Further, mutations in two of these predicted glycosylation sites impaired TLR3 signaling without obviously affecting the expression of the protein. Single-particle structures reconstructed from electron microscopy images and two-dimensional crystallization revealed that hTLR3 ECD forms a horseshoe structure similar to the recently elucidated x-ray structure of the protein expressed in insect cells using baculovirus vectors (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585 and Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980). There are, however, notable differences between the human cell-derived and insect cell-derived structures, including features attributable to glycosylation.

Toll-Like Receptor 9 Can Be Expressed at the Cell Surface of Distinct Populations of Tonsils and Human Peripheral Blood Mononuclear Cells

ABSTRACT Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9). However, the nature and cellular localization of TLR9 in primary human cells remain controversial. Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface. The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed. Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9+ cells were confined to the major histocompatibility complex (MHC) class II+ CD19− populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II+ CD19+ cells. Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10%. In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression. Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization. Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9.

Surface exposure and protease insensitivity of Borrelia burgdorferi Erp (OspEF-related) lipoproteins

Borrelia burgdorferi can encode numerous lipoproteins of the Erp family. Although initially described as outer surface proteins, the technique used in that earlier study has since been demonstrated to disrupt bacterial membranes and allow labelling of subsurface proteins. Data are now presented from additional analyses indicating that Erp proteins are indeed surface exposed in the outer membrane. Surface localization of these infection-associated proteins indicates the potential for interactions of Erp proteins with vertebrate tissues. Some Erp proteins were resistant to in situ digestion by certain proteases, suggesting that those proteins fold in manners which hide protease cleavage sites, or that they interact with other protective membrane components. Additionally, cultivation of B. burgdorferi in the presence of antibodies directed against Erp proteins inhibited bacterial growth.

Immunization with Leishmania major Exogenous Antigens Protects Susceptible BALB/c Mice against Challenge Infection with L. major

ABSTRACT The potential of Leishmania major culture-derived soluble exogenous antigens (SEAgs) to induce a protective response in susceptible BALB/c mice challenged with L. major promastigotes was investigated. Groups of BALB/c mice were immunized with L. major SEAgs alone, L. major SEAgs coadministered with either alum (aluminum hydroxide gel) or recombinant murine interleukin-12 (rmIL-12), L. major SEAgs coadministered with both alum and rmIL-12, and L. major SEAgs coadministered with Montanide ISA 720. Importantly and surprisingly, the greatest and most consistent protection against challenge with L. major was seen in mice immunized with L. major SEAgs alone, in the absence of any adjuvant. Mice immunized with L. major SEAgs had significantly smaller lesions that at times contained more than 100-fold fewer parasites. When lymphoid cells from L. major SEAg-immunized mice were stimulated with leishmanial antigen in vitro, they proliferated and secreted a mixed profile of type 1 and type 2 cytokines. Finally, analyses with Western blot analyses and antibodies against three surface-expressed and secreted molecules of L. major (lipophosphoglycan, gp46/M2/PSA-2, and gp63) revealed that two of these molecules are present in L. major SEAgs, lipophosphoglycan and the molecules that associate with it and gp46/M2/PSA-2.

Indomethacin treatment slows disease progression and enhances a Th1 response in susceptible BALB/c mice infected with Leishmania major.

Prostaglandins of the E series inhibit the development of Th1 responses. When infected with Leishmania major, BALB/c mice fail to develop a Th1 response, but instead mount a Th2 response and die of the disease. Therefore, we treated L. major‐infected BALB/c mice with indomethacin, which inhibits prostaglandin production. Indomethacin lessened disease severity (parasite burden and pathology), and promoted a Th1 response, but the mice still succumbed to infection. The explanation for these observations may be two‐fold: (1) the beneficial effects of indomethacin were predominantly observed later in infection (beyond two weeks), a time at which indomethacin was unable to sufficiently block the development of a Th2 response; (2) indomethacin was unable to induce a Th1 response in BALB/c mice that was of the same magnitude as the Th1 response observed in C57BL/6 mice infected with L. major.

Leishmania major induces differential expression of costimulatory molecules on mouse epidermal cells

Levels of expression of costimulatory molecules have been proposed to influence the outcome of antigen‐specific T cell priming. We found that Leishmania major selectively modulated the expression of costimulatory molecules on various populations of epidermal cells. B7.2 expression was down‐regulated on Thy1.2+ epidermal cells (keratinocytes) from disease‐resistant C3H mice, but not from disease‐susceptible BALB/c mice. In addition, epidermal cells from BALB/c mice showed a down‐regulation of B7.1 expression on NLDC 145+ Langerhans cells. In vitroT cell priming experiments, using syngeneic epidermal cells as antigen‐presenting cells (APC), showed that the production of IFN‐γ was inhibited when either B7.1 or B7.2 signaling pathways wereblocked. Blockade of B7.2, but not B7.1, significantly inhibited the ability of epidermal cells to induce IL‐4 production from CD4+ T cells. In addition, C3H CD4+ T cells, which were unable to secrete detectable levels of IL‐4 in cultures with syngeneic APC, were now able to secrete IL‐4 following presentation of L. major antigens by congenic BALB/K epidermal cells. Conversely, C3H epidermal cells supported the priming of BALB/K CD4+ T cells for IL‐4 production in vitro. Thus, the differential expression of B7 molecules on epidermal cells may notrepresent the sole factor governing the polarization of L. major‐specific CD4+ T cells in vitro.

Borrelia burgdorferi-specific monoclonal antibodies derived from mice primed with Lyme disease spirochete-infected Ixodes scapularis ticks.

We have generated a panel of IgG monoclonal antibodies (MAbs) directed against Borrelia burgdorferi strain B31 antigens, using a method whereby mice were primed with organisms naturally inoculated by Ixodes scapularis nymphal ticks. Western blot analysis showed that these MAbs recognized several B. burgdorferi B31 antigens, including the complement inhibitor factor H-binding proteins ErpA/I/N and ErpC. Two other MAbs were specific for the RevA protein, and have enabled characterization of that previously unknown protein. The data presented here suggest that the production of MAbs from animals infected by tick-bite is a potentially useful tool for the identification of novel proteins synthesized by B. burgdorferi during mammalian infection.