Untersuchungen zur Induktion von Filopodien durch das Amyloid Precursor Like Protein 1 (APLP1), ein Mitglied der APP-Genfamilie

Die Alzheimer Krankheit ist eine fortschreitendende Demenzerkrankung von der in Deutschland ca. 1,6 Millionen Menschen betroffen sind. Im Gehirn der Patienten finden sich sogenannte amyloide Plaques, deren Hauptbestandteil das Aβ-Protein ist. Dieses Peptid ist ein Spaltprodukt des APP-Proteins (engl. amyloid precursor protein). APP ist das namensgebende Mitglied der APP-Proteinfamilie zu der neben APP die beiden APP-Homologen APLP1 und APLP2 (engl. amyloid precursor like protein) gehören. Obwohl inzwischen über die pathologische Rolle dieser Proteinfamilie bei der Alzheimer Krankheit vieles bekannt ist, bleiben die physiologischen Funktionen dieser Proteine bisher größtenteils ungeklärt. Die vorliegende Arbeit beschreibt erstmals einen APLP1-spezifischen Effekt auf die Ausbildung von Filopodien. Sowohl das humane als auch das murine APLP1 induzierten nach transienter Überexpression die Bildung zahlreicher filopodialer Fortsätze auf der Membran von PC12-Zellen. Vergleichbare Resultate konnten mit beiden APLP1-Proteinen auch auf der Membran von embryonalen (E18.5), cortikalen Neuronen der Ratte gezeigt werden. Dass APLP1 einen derartigen Effekt auf Neuronen und PC12-Zellen zeigt, begründet die Annahme, dass APLP1 in vivo eine Funktion bei der Entwicklung und Differenzierung von Neuronen übernimmt. Anhand von Versuchen mit deletierten APLP1-Proteinen und APLP1/APLP2-Chimärproteinen konnte gezeigt werden, dass die von Exon 5 und Exon 6 codierten Bereiche des APLP1 für die Induktion der Filopodien essentiell sind. Unter Einbeziehung von in ihrer räumlichen Struktur bereits bekannten Domänen und aufgrund von Homologievergleichen der primären Aminosäuresequenz dieser Region mit entsprechenden Bereichen der APP- bzw. APLP2-Proteine wurde die wahrscheinliche Lage der Filopodien-induzierenden Domäne innerhalb des von Exon 6 codierten Bereiches diskutiert. Es konnte ferner gezeigt werden, dass die untersuchte Induktion von Filopodien durch die sogenannte α-Sekretierung moduliert werden kann. Unter den gewählten Versuchsbedingungen war nur membranständiges APLP1, nicht aber sekretiertes APLP1 in der Lage, Filopodien zu induzieren. Abschliessend wurden Ergebnisse gezeigt, die erste Einblicke in Signalkaskaden erlauben, die von APLP1 angesteuert werden und so die Enstehung der Filopodien auslösen. Bezüglich des primären Prozesses der Signalkaskade, der Bindung von APLP1 an einen bisher unbekannten Rezeptor, wurde die Möglichkeit diskutiert, ob APP oder APLP2 oder sogar APLP1 selbst als Rezeptor fungieren könnten. Die beobachteten Prozesse nach Überexpression von APLP1 entsprechen vermutlich einer physiologischen Funktion bei der Differenzierung von Neuronen, die mit der Interaktion einer extrazellulär gelegenen Domäne mit einem Rezeptor beginnt, die Aktivierung einer Signalkaskade zur Akrinreorganisation zu Folge hat und die Entstehung filopodialer Strukturen auslöst.

RAB3GAP1 und RAB3GAP2 (RAB3 GTPase-activating Protein 1/2) beeinflussen die Autophagie in C. elegans und humanen Zellen

Die Proteinhomöostase wird in der Zelle von drei Stoffwechselwegen reguliert: den molekularen Chaperonen, dem Ubiquitin-Proteasom-System und dem autophagosomalen Abbauweg. Die (Makro)Autophagie verpackt und transportiert zytosolische Komponenten in Autophagosomen zu den Lysosomen, wo sie abgebaut werden. Eine Störung dieses Abbauwegs wirkt auf die Proteostase.rnIn dieser Dissertation wurde C. elegans als Modellorganismus zur Erforschung von Proteinstabilität genutzt. In einer RNAi-vermittelten Proteostase-Analyse von Chromosom I und ausgewählter zusätzlicher Gene wurde ein Wurmstamm, der ein Luc::GFP-Konstrukt im Muskel exprimiert, genutzt. Dieses Reporterprotein aggregiert unter Hitzestressbedingungen und diese Aggregation kann durch Modulatoren der Proteostase beeinflusst werden. Dabei wurden mögliche neue Faktoren der Proteinhomöostase entdeckt. Durch weitere Experimente bei denen die Aggregation von PolyQ35::YFP im AM140-System, der Paralyse-Phänotyp und die Akkumulation Thioflavin S-gefärbter Aggregate von Aβ42 im CL2006-Wurmstamm und die Effekte auf die Autophagie mittels eines GFP::LGG1-Konstrukt analysiert wurden, konnten rbg-1 und rbg-2 als neue Modulatoren der Proteinhomöostase, insbesondere der Autophagie, identifiziert werden.rnIm Säuger bilden beide Orthologe dieser Gene, RAB3GAP1 und RAB3GAP2 den heterodimeren RAB3GAP-Komplex, der bisher nur bekannt war für die Stimulation der Umwandlung der GTP-gebundenen aktiven Form zur GDP-gebundenen inaktiven Form der RAB GTPase RAB3. In Immunoblot-Analysen und mikroskopischen Darstellungen im Säugersystem konnte gezeigt werden, dass die Effekte auf die Proteostase über den autophagosomalen Abbauweg wirken. RAB3GAP1/2 wirken als positive Stimulatoren, wenn die Lipidierung von LC3-I und der autophagische Flux von LC3-II und p62/SQSTM1 betrachtet werden. Diese Effekte werden aber nicht über die RAB GTPase RAB3 vermittelt. Die Proteine FEZ1 und FEZ2 haben einen antagonistischen Effekt auf die Autophagie und wenn alle vier Komponenten RAB3GAP1, RAB3GAP2, FEZ1 und FEZ2 zusammen herunter- oder hochreguliert werden, heben sich diese Effekte auf. In Co-Immunopräzipitationen und proteomischen Analysen konnte keine direkte Interaktion zwischen dem RAB3GAP-Komplex und FEZ1/2 oder zu anderen Autophagie-Genen nachgewiesen werden.rnHier konnte der RAB3GAP-Komplex funktionell mit Proteostase und Autophagie in C. elegans und Säugerzellen assoziiert werden. Dieser Komplex zeigt Einflüsse auf die autophagosomale Biogenese indem sie die Proteostase und die Bildung von (prä)autophagosomalen Strukturen in C. elegans und die Lipidierung von LC3 und damit den autophagischen Flux der Autophagiesubstrate LC3-II und p62/SQSTM1 in Säugerzellen beeinflusst. Darüber hinaus wirkt RAB3GAP der komplexen Autophagie-Unterdrückung durch FEZ1 und FEZ2 entgegen. Somit konnte gezeigt werden, dass RAB3GAP als neuartiger Faktor auf die autophagosomale Biogenese und somit auf die Proteostase wirkt.rn

The Ras inhibitors caveolin-1 and docking protein 1 activate peroxisome proliferator-activated receptor ? through spatial relocalization at helix 7 of its ligand-binding domain

Peroxisome proliferator-activated receptor ? (PPAR?) is a transcription factor that promotes differentiation and cell survival in the stomach. PPAR? upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPAR? is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPAR? signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPAR? and enhanced nuclear translocation and ligand-independent transcription of PPAR? target genes. In contrast, Cav1 overexpression sequestered PPAR? in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPAR?'s ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPAR? and to inhibit cell proliferation. Ligand-activated PPAR? also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPAR? regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPAR? to ligands, limiting proliferation of gastric epithelial cells.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Elevated systemic monocyte chemoattractrant protein-1 in hepatic steatosis without significant hepatic inflammation

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and the metabolic syndrome. It encompasses a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine monocyte chemoattractant protein 1 (MCP-1) plays an important role in the progression of hepatic inflammation and fibrosis, and both increased hepatic expression and circulating serum levels have been described in NASH. Here, we aimed to investigate MCP-1 expression in simple hepatic steatosis. Upon feeding a high-fat diet mice developed hepatic steatosis in the absence of significant hepatic inflammation, but elevated hepatic MCP-1 expression compared to control mice fed a standard chow. Interestingly, high-fat diet fed mice had significantly higher MCP-1 serum levels, and MCP-1 mRNA expression was significantly increased in visceral adipose tissue. Furthermore, MCP-1 serum levels were also elevated in patients with ultrasound-diagnosed NAFLD and correlated with the body-mass index and fasting glucose. In conclusion, our data indicate both the liver and adipose tissue as cellular sources of elevated circulating MCP-1 levels already in the early phase of hepatic steatosis. Since MCP-1 derived from visceral adipose tissue reaches the liver via portal circulation at high concentrations it may significantly contribute to the progression of simple steatosis to NASH.

Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism

Background Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages. Results We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers. Conclusions Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.

Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.

Tocopherol-associated protein-1 accelerates apoptosis induced by alpha-tocopheryl succinate in mesothelioma cells

Alpha-tocopheryl succinate (alpha-TOS), a redox-silent analogue of vitamin E, induces apoptosis in multiple cell lines in a selective manner, by activating the intrinsic pathway. Since it is a highly hydrophobic compound, it may require a carrier protein for its trafficking to intracellular targets like mitochondria. We studied the role of the ubiquitous tocopherol-associated protein-1 (TAP1 or sec14-like 2) in apoptosis induction by alpha-TOS in malignant mesothelioma (MM) cells. Over-expression of TAP1 in MM cells sensitised them to apoptosis by low doses of alpha-TOS which were sub-apoptotic for the parental cells. Apoptosis induced in TAP1-over-expressing cells was mitochondria- and caspase-dependent, as suggested by dissipation of mitochondrial trans-membrane potential and inhibition by zVAD-fmk, respectively. Binding assays showed affinity of alpha-TOS for TAP1. Finally, TAP1 over-expressing cells accumulated alpha-TOS at higher levels compared to their normal counterparts. We suggest that TAP1 may act as an intracellular shuttle for alpha-TOS, promoting apoptosis initiated by this vitamin E analogue, as shown here for MM cells.

Regulation of endothelial cell cytoskeletal reorganization by a secreted frizzled-related protein-1 and frizzled 4- and frizzled 7-dependent pathway: role in neovessel formation

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3beta. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3 beta and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3beta and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.

Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale

Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

Reduction in rat phosphatidylethanolamine binding protein-1 (PEBP1) after chronic corticosterone treatment may be paralleled by cognitive impairment: a first study

Chronic stress is associated with hippocampal atrophy and cognitive dysfunction. This study investigates how long-lasting administration of corticosterone as a mimic of experimentally induced stress affects psychometric performance and the expression of the phosphatidylethanolamine binding protein (PEBP1) in the adult hippocampus of one-year-old male rats. Psychometric investigations were conducted in rats before and after corticosterone treatment using a holeboard test system. Rats were randomly attributed to 2 groups (n = 7) for daily subcutaneous injection of either 26.8 mg/kg body weight corticosterone or sesame oil (vehicle control). Treatment was continued for 60 days, followed by cognitive retesting in the holeboard system. For protein analysis, the hippocampal proteome was separated by 2D electrophoresis (2DE) followed by image processing, statistical analysis, protein identification via peptide mass fingerprinting and gel matching and subsequent functional network mapping and molecular pathway analysis. Differential expression of PEBP1 was additionally quantified by Western blot analysis. Results show that chronic corticosterone significantly decreased rat hippocampal PEBP1 expression and induced a working and reference memory dysfunction. From this, we derive the preliminary hypothesis that PEBP1 may be a novel molecular mediator influencing cognitive integrity during chronic corticosterone exposure in rat hippocampus.

Cathepsin D specifically cleaves the chemokines macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, and SLC that are expressed in human breast cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation.

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.