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1.  Identification of Cell-Binding Adhesins of Leptospira interrogans 
Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic Leptospira species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate L. interrogans sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its L. biflexa homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for L. interrogans. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in L. interrogans infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and vaccines.
Author Summary
Leptospirosis, caused by pathogenic species of the genus Leptospira, is an infectious disease that has emerged as a globally important health problem. Infection can either lead to mild illness or can progress to a severe disease form manifested by jaundice, kidney and liver dysfunction, and widespread blood vessel damage. It is thought that the ability of the bacteria to recognize and bind to human and animal cells is important for Leptospira spp. to cause the disease. Using phage display, we were able to identify bacterial proteins that mediate the binding of the bacteria to host cells. One of the identified proteins, LIC11574, attaches to different types of host cells, and to VE-cadherin, a cell surface protein previously identified as receptor for disease-causing L. interrogans. All bacterial proteins identified were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals. Our findings may be of value in leptospirosis control and prevention, with these bacterial surface proteins as new targets for serodiagnosis and vaccine development.
doi:10.1371/journal.pntd.0003215
PMCID: PMC4183468  PMID: 25275630
2.  Illuminating the roles of the Borrelia burgdorferi adhesins 
Trends in microbiology  2013;21(8):372-379.
The Lyme disease spirochetes, Borrelia burgdorferi (sensu lato), must cause persistent, disseminated infection to be maintained in the natural enzootic cycle. In human Lyme disease, spirochetes spread from the site of a tick bite to colonize multiple tissue sites, causing multisystem clinical manifestations. The Lyme spirochetes produce many adhesive surface proteins that collectively recognize diverse host substrates and cell types and are likely to promote dissemination and chronic infection in a variety of tissues. Recent application of state-of-the-art in vivo imaging technologies is illuminating mechanisms of interaction of B. burgdorferi with the host and the importance of multiple adhesins during mammalian infection.
doi:10.1016/j.tim.2013.06.005
PMCID: PMC3773214  PMID: 23876218
Lyme disease; Borrelia burgdorferi; adhesins; intravital imaging; dissemination; transmigration
3.  Leptospira interrogans Binds to Cadherins 
Leptospirosis, caused by pathogenic species of Leptospira, is the most widespread zoonosis and has emerged as a major public health problem worldwide. The adhesion of pathogenic Leptospira to host cells, and to extracellular matrix (ECM) components, is likely to be necessary for the ability of leptospires to penetrate, disseminate and persist in mammalian host tissues. Previous work demonstrated that pathogenic L. interrogans binds to host cells more efficiently than to ECM. Using two independent screening methods, mass spectrometry and protein arrays, members of the cadherin family were identified as potential L. interrogans receptors on mammalian host surfaces. We focused our investigation on vascular endothelial (VE)-cadherin, which is widely expressed on endothelia and is primarily responsible for endothelial cell-cell adhesion. Monolayers of EA.hy926 and HMEC-1 endothelial cells produce VE-cadherin, bind L. interrogans in vitro, and are disrupted upon incubation with the bacteria, which may reflect the endothelial damage seen in vivo. Dose-dependent and saturable binding of L. interrogans to the purified VE-cadherin receptor was demonstrated and pretreatment of purified receptor or endothelial cells with function-blocking antibody against VE-cadherin significantly inhibited bacterial attachment. The contribution of VE-cadherin to leptospiral adherence to host endothelial cell surfaces is biologically significant because VE-cadherin plays an important role in maintaining the barrier properties of the vasculature. Attachment of L. interrogans to the vasculature via VE-cadherin may result in vascular damage, facilitating the escape of the pathogen from the bloodstream into different tissues during disseminated infection, and may contribute to the hemorrhagic manifestations of leptospirosis. This work is first to describe a mammalian cell surface protein as a receptor for L. interrogans.
Author Summary
Leptospirosis is a globally widespread bacterial infection caused by pathogenic species of the genus Leptospira. The disease manifestations of leptospirosis range from mild, non-specific illness to a severe disease that includes multi-organ failure, widespread damage to blood vessels, and hemorrhage. Attachment to human or animal cells is likely to be important to the ability of the bacteria to spread and to cause disease. In this study, members of the cadherin family were identified as mammalian cell receptors that bind Leptospira. Cadherins are cell surface proteins that function to maintain cell-cell integrity by anchoring neighboring cells together. Disease-causing L. interrogans, but not the non-infectious L. biflexa, binds to cells that line blood vessels and VE-cadherin, the predominant cadherin found in this cell type. The binding of bacteria was reduced in the presence of antibodies against VE-cadherin, supporting the role of this protein in bacterial attachment. The attachment of L. interrogans to the inner lining of the vessels via VE-cadherin may result in damage, facilitating the escape of the pathogen from the bloodstream into different tissues, and may contribute to the hemorrhagic manifestations of leptospirosis. This work is first to identify a mammalian cell surface protein as a receptor for L. interrogans.
doi:10.1371/journal.pntd.0002672
PMCID: PMC3907533  PMID: 24498454
4.  The β3-Integrin Ligand of Borrelia burgdorferi is Critical for Infection of Mice but not Ticks 
Molecular microbiology  2012;85(6):1105-1118.
Summary
P66 is a Borrelia burgdorferi surface protein with β3 integrin binding and channel forming activities. In this study, the role of P66 in mammalian and tick infection was examined. B. burgdorferiΔp66 strains were not infectious in wild-type, TLR2−/− or MyD88−/− deficient mice. Strains with p66 restored to the chromosome restored near wild-type infectivity, while complementation with p66 on a shuttle vector did not restore infectivity. Δp66 mutants are cleared quickly from the site of inoculation, but analyses of cytokine expression and cellular infiltrates at the site of inoculation did not reveal a specific mechanism of clearance. The defect in these mutants cannot be attributed to nutrient limitation or an inability to adapt to the host environment in vivo as Δp66 bacteria were able to survive as well as wild-type in dialysis membrane chambers in the rat peritoneum. Δp66 bacteria were able to survive in ticks through the larva to nymph molt, but were non-infectious in mice when delivered by tick bite. Independent lines of evidence do not support any increased susceptibility of the Δp66 strains to factors in mammalian blood. This study is the first to define a B. burgdorferi adhesin as essential for mammalian, but not tick infection.
doi:10.1111/j.1365-2958.2012.08160.x
PMCID: PMC3438374  PMID: 22758390
5.  The Borrelia burgdorferi Integrin Ligand P66 Affects Gene Expression by Human Cells in Culture ▿  
Infection and Immunity  2011;79(8):3249-3261.
Borrelia burgdorferi, an agent of Lyme disease, establishes persistent infection in immunocompetent animals and humans. Although the infection in humans can be cleared by antibiotic therapy, persistence in reservoir animals is necessary for the maintenance of the bacterium in the natural reservoir host⇔tick vector infectious cycle. B. burgdorferi binds to β1- and β3-chain integrins, and the P66 outer membrane protein is responsible for at least some of the integrin binding activity of the spirochete. Because integrins are transmembrane, bidirectional signaling molecules, integrin binding may alter the nature of the host response to the bacteria. We used isogenic B. burgdorferi p66+ and Δp66 strains to analyze the responses of cultured human cells to P66-integrin interaction during infection. Microarray results suggest that the response differs according to the cell type, infection time, and experimental conditions. Clusters of genes in functionally related categories that showed significant changes included proteins involved in cell-extracellular matrix interactions, actin dynamics, stress response, and immune responses. Integrin binding by P66 may therefore help B. burgdorferi establish infection by facilitating tissue invasion and modulating the activation of the immune system to other components of the bacteria, e.g., lipoproteins. These results provide insight into how B. burgdorferi is able to establish infection in immunocompetent hosts.
doi:10.1128/IAI.05122-11
PMCID: PMC3147572  PMID: 21576330
6.  Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses 
Future microbiology  2010;5(9):1413-1425.
Leptospirosis, the most widespread zoonosis in the world, is an emerging public health problem, particularly in large urban centers of developing countries. Several pathogenic species of the genus Leptospira can cause a wide range of clinical manifestations, from a mild, flu-like illness to a severe disease form characterized by multiorgan system complications leading to death. However, the mechanisms of pathogenesis of Leptospira are largely unknown. This article will address the animal models of acute and chronic leptospire infections, and the recent developments in the genetic manipulation of the bacteria, which facilitate the identification of virulence factors involved in pathogenesis and the assessment of their potential values in the control and prevention of leptospirosis.
doi:10.2217/fmb.10.102
PMCID: PMC3037011  PMID: 20860485
epidemiology; leptospirosis; pathogenesis; virulence
7.  Effect of Borrelia burgdorferi OspC at the Site of Inoculation in Mouse Skin▿  
Infection and Immunity  2010;78(11):4723-4733.
The Borrelia burgdorferi surface lipoprotein OspC is a critical virulence factor, but its precise role in the establishment of B. burgdorferi infection remains unclear. To determine whether OspC affects the host response at the site of inoculation of the bacterium, the recruitment of macrophages and neutrophils and the production of cytokines were examined at the site of infection by wild-type, ospC mutant, and complemented mutant B. burgdorferi strains. Of the 21 cytokines tested, monocyte chemoattractant protein 1 (MCP-1), keratinocyte-derived chemokine (KC, CXCL1), and vascular endothelial growth factor (VEGF) were found at increased levels at the site of inoculation of B. burgdorferi, and the levels varied with the production of OspC at one or more time points over the 1-week course of infection. The kinetics of expression and the dependence on OspC production by B. burgdorferi varied among the cytokines. The production of KC and MCP-1, and the appearance of monocytic infiltrates, correlated with the presence of the bacteria rather than with OspC specifically. In contrast, VEGF production was not correlated simply to the presence of the bacteria and is influenced by the presence of OspC. In in vitro assays, OspC and B. burgdorferi expressing OspC stimulated the growth of endothelial cells more than did the controls. These data suggest the possibility of a novel role for OspC in the life of B. burgdorferi at the interface of its mammalian and tick hosts.
doi:10.1128/IAI.00464-10
PMCID: PMC2976318  PMID: 20696825
8.  The Emergence of Severe Pulmonary Hemorrhagic Leptospirosis: Questions to Consider 
Since the 1980s, the incidence of severe pulmonary hemorrhage caused by Leptospira spp. infection has increased. The mild, non-specific symptoms or the more classical form of severe disease with hepatorenal manifestations, Weil’s syndrome, predominate world-wide. However, several regions of the world have seen increases in numbers of patients with pulmonary hemorrhage attributed to leptospirosis. The reasons behind the emergence of this syndrome, which carries a high mortality rate, are not known. Several avenues for future research may shed light on the mechanisms involved in development of pulmonary hemorrhage, and inform targeted therapeutics to improve outcomes. Possibilities to consider include: (1) emergence of new bacterial strains, (2) acquisition of virulence traits by strains in the endemic regions, (3) changes in underlying health of the affected human populations, and (4) increased recognition of the syndrome and better record keeping by the medical and veterinary communities. Determining the causes of emerging clinical manifestations presents challenges and opportunities for potentially life-saving research into the pathogenesis of a number of infectious diseases, including leptospirosis.
doi:10.3389/fcimb.2011.00024
PMCID: PMC3417368  PMID: 22919589
leptospirosis; Leptospira; pulmonary hemorrhage
9.  Responses of Human Endothelial Cells to Pathogenic and Non-Pathogenic Leptospira Species 
Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.
Author Summary
Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but is seen occasionally in temperate regions as well. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Our analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa caused changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily maintaining cell layer integrity, while L. interrogans disrupted cell layers. In fact, L. interrogans caused significant disruption of endothelial cell layers, but this damage could be abrogated by the endothelial protective drug lisinopril. Our results suggest that L. interrogans binds to endothelial cells and disrupts endothelial barrier function, which may promote dissemination of the bacteria and contribute to severe disease manifestations. This disruption may be slowed by endothelial-protective drugs to decrease damage in leptospirosis.
doi:10.1371/journal.pntd.0000918
PMCID: PMC3001904  PMID: 21179504
10.  Leptospira interrogans Binds to Human Cell Surface Receptors Including Proteoglycans▿  
Infection and Immunity  2009;77(12):5528-5536.
Leptospirosis is a global public health problem, primarily in the tropical developing world. The pathogenic mechanisms of the causative agents, several members of the genus Leptospira, have been underinvestigated. The exception to this trend has been the demonstration of the binding of pathogenic leptospires to the extracellular matrix (ECM) and its components. In this work, interactions of Leptospira interrogans bacteria with mammalian cells, rather than the ECM, were examined. The bacteria bound more efficiently to the cells than to the ECM, and a portion of this cell-binding activity was attributable to attachment to glycosaminoglycan (GAG) chains of proteoglycans (PGs). Chondroitin sulfate B PGs appeared to be the primary targets of L. interrogans attachment, while heparan sulfate PGs were much less important. Inhibition of GAG/PG-mediated attachment resulted in partial inhibition of bacterial attachment, suggesting that additional receptors for L. interrogans await identification. GAG binding may participate in the pathogenesis of leptospirosis within the host animal. In addition, because GAGs are expressed on the luminal aspects of epithelial cells in the proximal tubules of the kidneys, this activity may play a role in targeting the bacteria to this critical site. Because GAGs are shed in the urine, GAG binding may also be important for transmission to new hosts through the environment.
doi:10.1128/IAI.00546-09
PMCID: PMC2786458  PMID: 19805539
11.  Interaction of Borrelia burgdorferi Hbb with the p66 promoter 
Nucleic Acids Research  2009;38(2):414-427.
Borrelia burgdorferi, an agent of Lyme disease, encodes the β3-chain integrin ligand P66. P66 is expressed by B. burgdorferi in the mammal, in laboratory media, and as the bacteria are acquired or transmitted by the tick, but is not expressed by the bacterium in unfed ticks. Attempts to reveal factors influencing expression revealed that P66 was expressed in all in vitro conditions investigated. Candidate regulators identified in a search of the B. burgdorferi genome for homologs to other bacterial transcription factors were cloned and introduced into E. coli carrying a p66 promoter-signal sequence-phoA (alkaline phosphatase, or AP) fusion. Three candidate transcription factors—two that decreased AP activity (Hbb and BB0527), and one that increased AP activity (BBA23)—were identified. BBA23 and BB0527 did not bind to the p66 promoter at physiologically relevant concentrations. In contrast, several promoter fragments, including p66, were bound by Hbb (BB0232), with slightly different affinities. Consistent with results from other laboratories, Hbb appears to recognize multiple DNA sequences. Changes in the expression of p66 and bb0232 in the tick at various points with respect to feeding on mice, along with the results of the reporter experiment in the surrogate host E. coli, are consistent with Hbb/BB0232 being involved in regulating p66 expression.
doi:10.1093/nar/gkp1027
PMCID: PMC2811001  PMID: 19910373
12.  High Prevalence of Spirochetosis in Cholera Patients, Bangladesh 
Emerging Infectious Diseases  2009;15(4):571-573.
The microbes that accompany the etiologic agent of cholera, Vibrio cholerae, are only now being defined. In this study, spirochetes from the genus Brachyspira were identified at high titers in more than one third of cholera patients in Bangladesh. Spirochetosis should now be tracked in the setting of cholera outbreaks.
doi:10.3201/eid1504.081214
PMCID: PMC2671413  PMID: 19331734
Spirochetosis; spirochete; cholera; Vibrio cholerae; Brachyspira; coinfection; secretory diarrhea; Bangladesh; dispatch
13.  Borrelia burgdorferi Adhesins Identified Using In Vivo Phage Display 
Molecular microbiology  2007;66(1):262-276.
Summary
Borrelia burgdorferi, the agent of Lyme disease, disseminates from the site of deposition by Ixodes ticks to cause systemic infection. Dissemination occurs through the circulation and through tissue matrices, but the B. burgdorferi molecules that mediate interactions with the endothelium in vivo have not yet been identified. In vivo selection of filamentous phage expressing B. burgdorferi protein fragments on the phage surface identified several new candidate adhesins, and verified the activity of one adhesin that had been previously characterized in vitro. P66, a B. burgdorferi ligand for β3-chain integrins, OspC, a protein that is essential for the establishment of infection in mammals, and Vls, a protein that undergoes antigenic variation in the mammal, were all selected for binding to the murine endothelium in vivo. Additional B. burgdorferi proteins for which no functions have been identified, including all four members of the OspF family and BmpD, were identified as candidate adhesins. The use of in vivo phage display is one approach to the identification of adhesins in pathogenic bacteria that are not easily grown in the laboratory, or for which genetic manipulations are not straightforward.
doi:10.1111/j.1365-2958.2007.05924.x
PMCID: PMC2651023  PMID: 17784908
14.  Borrelia burgdorferi BBB07 Interaction With Integrin α3β1 Stimulates Production of Pro-Inflammatory Mediators in Primary Human Chondrocytes 
Cellular microbiology  2007;10(2):320-331.
Summary
Borrelia burgdorferi, the causative agent of Lyme disease, activates multiple signaling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin α3β1on human chondrocytes activates signaling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin α3β1and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin-binding motif, the RGD (Arg-Gly-Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin α3β1 and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro-inflammatory mediators in primary human chondrocyte cells by interaction with integrin α3β1. This interaction is specific, as P66, another integrin ligand of B. burgdorferi, does not activate signaling through α3β1. In summary, we have identified a B. burgdorferi protein, BBB07 that interacts with integrin α3β1 and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells.
doi:10.1111/j.1462-5822.2007.01043.x
PMCID: PMC2586958  PMID: 17822440
15.  Regulators of Expression of the Oligopeptide Permease A Proteins of Borrelia burgdorferi▿  
Journal of Bacteriology  2007;189(7):2653-2659.
Borrelia burgdorferi undergoes an infectious cycle that requires adaptation to different hosts and marked differences in environment. B. burgdorferi copes with its different environments by regulating the expression of proteins required for survival in specific settings. The B. burgdorferi oligopeptide permease (Opp) is one of only a few transporters encoded by the B. burgdorferi genome. Opp proteins in other bacteria serve multiple environmental adaptation functions. B. burgdorferi appears to broaden the usage of this transporter by utilizing five different substrate binding proteins (OppA proteins) that interact with the integral membrane components of the transporter. Expression of the OppA proteins is individually regulated and may play different roles in adaptation to host environments. Very little is known about the mechanisms used by B. burgdorferi to regulate the expression of different OppA proteins. Here we show that the alternative sigma factors, RpoS and RpoN, regulate the expression of oppA5 but not that of other oppA genes. Using a reporter assay with Escherichia coli and gel shift binding assays, we also show that the B. burgdorferi BosR/Fur homologue interacts with the oppA4 promoter and that another candidate transcription factor, EbfC, interacts with the oppA5 promoter. Binding to the promoters was confirmed by gel shift assays. Expression of BosR/Fur in its different hosts does appear to parallel the expression of oppA4. A better understanding of the factors involved in gene regulation in B. burgdorferi will help to identify coregulated proteins that may cooperate to allow the organism to survive in a specific environment.
doi:10.1128/JB.01760-06
PMCID: PMC1855802  PMID: 17237172
16.  The emergence of Lyme disease 
Journal of Clinical Investigation  2004;113(8):1093-1101.
Since its identification nearly 30 years ago, Lyme disease has continued to spread, and there have been increasing numbers of cases in the northeastern and north central US. The Lyme disease agent, Borrelia burgdorferi, causes infection by migration through tissues, adhesion to host cells, and evasion of immune clearance. Both innate and adaptive immune responses, especially macrophage- and antibody-mediated killing, are required for optimal control of the infection and spirochetal eradication. Ecological conditions favorable to the disease, and the challenge of prevention, predict that Lyme disease will be a continuing public health concern.
doi:10.1172/JCI200421681
PMCID: PMC385417  PMID: 15085185
17.  Characterization of Pseudomonas aeruginosa Exoenzyme S as a Bifunctional Enzyme in J774A.1 Macrophages  
Infection and Immunity  2003;71(9):5296-5305.
Pseudomonas aeruginosa exoenzyme S (ExoS) is a type III secretion (TTS) effector, which includes both a GTPase-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteins and an ADP-ribosyltransferase (ADPRT) activity that targets LMWG proteins in the Ras, Rab, and Rho families. The coordinate function of both activities of ExoS in J774A.1 macrophages was assessed by using P. aeruginosa strains expressing and translocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity. Distinct and coordinated functions were identified for both domains. The GAP activity was required for the antiphagocytic effect of ExoS and was linked to interference of lamellopodium and membrane ruffle formation. Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and was linked to effects on filopodium formation. The cellular mechanism of ExoS GAP activity included an inactivation of Rac1 function, as determined in p21-activated kinase 1-glutathione S-transferase (GST) pull-down assays. The ADPRT activity of ExoS targeted Ras and RalA but not Rab or Rho proteins, and Ral binding protein 1-GST pull-down assays identified an effect of ExoS ADPRT activity on RalA activation. The results from these studies confirm the bifunctional nature of ExoS activity within macrophages when translocated by TTS.
doi:10.1128/IAI.71.9.5296-5305.2003
PMCID: PMC187317  PMID: 12933877
18.  Regulation of Expression of the Borrelia burgdorferi β3-Chain Integrin Ligand, P66, in Ticks and in Culture  
Infection and Immunity  2003;71(2):1001-1007.
Borrelia burgdorferi is maintained in an infection cycle between mammalian and arthropod hosts. Appropriate gene expression by B. burgdorferi at different stages of this cycle is probably essential for transmission and establishment of infection. The B. burgdorferi β3 integrin ligand P66 is expressed by the bacteria in mammals, laboratory culture, and engorged but not unfed ticks. No in vitro culture conditions in which P66 expression reflected that in the unfed tick were found, suggesting that there are aspects of B. burgdorferi-tick interaction that remain unexplored.
doi:10.1128/IAI.71.2.1001-1007.2003
PMCID: PMC145366  PMID: 12540584
19.  Delineation of Borrelia burgdorferi p66 Sequences Required for Integrin αIIbβ3 Recognition 
Infection and Immunity  2001;69(5):3455-3459.
The outer membrane protein p66 of the Lyme disease agent, Borrelia burgdorferi, has been identified as a candidate ligand for β3-chain integrins. To identify portions of p66 required for integrin recognition, fusions of maltose-binding protein to fragments of p66 were tested for binding to integrin αIIbβ3, and synthetic peptides derived from the p66 amino acid sequence were tested for the ability to inhibit B. burgdorferi attachment to the same integrin. The data identify two noncontiguous segments of p66 that are important for αIIbβ3 recognition, suggesting that, as is true for other integrin ligands, the tertiary structure of p66 is important for receptor recognition.
doi:10.1128/IAI.69.5.3455-3459.2001
PMCID: PMC98311  PMID: 11292775
20.  Recognition of Multiple Antibody Epitopes throughout Borrelia burgdorferi p66, a Candidate Adhesin, in Patients with Early or Late Manifestations of Lyme Disease 
Infection and Immunity  2001;69(3):1953-1956.
Antibody responses to p66, a candidate integrin ligand of Borrelia burgdorferi, were studied in 79 patients with early or late manifestations of Lyme disease. The central portion of p66 was previously shown to contain all of the information required for specific recognition of β3-chain integrins, but work by others had suggested that the C-terminal portion of the protein contains a single surface-exposed, immunodominant loop. In examining antibody responses to full-length p66 and to three overlapping fragments of the protein, we found that the majority of Lyme disease patients had immunoglobulin M (IgM) and/or IgG responses to p66 and that, particularly early in the disease, epitopes throughout p66 were recognized. Among patients with later manifestations of the illness, antibody responses to the C-terminal portion of the protein were more prominent. These results demonstrate that Lyme disease patient sera recognize epitopes throughout p66.
doi:10.1128/IAI.69.3.1953-1956.2001
PMCID: PMC98111  PMID: 11179382
21.  Macrophages and Epithelial Cells Respond Differently to the Pseudomonas aeruginosa Type III Secretion System 
Infection and Immunity  1999;67(6):3151-3154.
The multiple effects of Pseudomonas aeruginosa type III secretion have largely been attributed to variations in cytotoxin expression between strains. Here we show that the target cell type is also important. While lung epithelial cells showed significant changes in morphology but not viability when infected with P. aeruginosa, macrophages were efficiently killed by P. aeruginosa. Both responses were dependent on the type III secretion system.
PMCID: PMC96636  PMID: 10338535
22.  Integrins αvβ3 and α5β1 Mediate Attachment of Lyme Disease Spirochetes to Human Cells 
Infection and Immunity  1998;66(5):1946-1952.
Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin αIIbβ3 was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins αvβ3 and α5β1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified αvβ3 and α5β1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified αvβ3 and α5β1 was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to αvβ3 was also shown by using a transfected cell line that expresses this receptor but not αIIbβ3. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both α5β1 and αvβ3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.
PMCID: PMC108148  PMID: 9573074
23.  Different Classes of Proteoglycans Contribute to the Attachment of Borrelia burgdorferi to Cultured Endothelial and Brain Cells 
Infection and Immunity  1998;66(3):994-999.
The Lyme disease spirochete, Borrelia burgdorferi, infects multiple tissues, such as the heart, joint, skin, and nervous system and has been shown to recognize heparan sulfate and dermatan sulfate proteoglycans. In this study, we examined the contribution of different classes of proteoglycans to the attachment of the infectious B. burgdorferi strain N40 to several immortalized cell lines and primary cultured cells, including endothelial cells and brain cells. Bacterial attachment was inhibited by exogenous proteoglycans or by treatment of host cells with inhibitors of proteoglycan synthesis or sulfation, indicating that proteoglycans play a critical role in bacterial binding to diverse cell types. Binding to primary bovine capillary endothelial cells or a human endothelial cell line was also inhibited by digestion with heparinase or heparitinase but not with chondroitinase ABC. In contrast, binding to glial cell-enriched brain cell cultures or to a neuronal cell line was inhibited by all three lyases. Binding of strain N40 to immobilized heparin could be completely inhibited by dermatan sulfate, and conversely, binding to dermatan sulfate could be completely blocked by heparin. As measured by 50% inhibitory dose, heparin was a better inhibitor of binding than dermatan sulfate, regardless of whether the substrate was heparin or dermatan sulfate. These results are consistent with the hypotheses that the species of proteoglycans recognized by B. burgdorferi vary with cell type and that bacterial recognition of different proteoglycans is mediated by the same bacterial molecule(s).
PMCID: PMC108007  PMID: 9488387

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