Leptospiral adhesins: from identification to future perspectives.

Leptospirosis is a significant zoonosis worldwide, with disease severity ranging from a mild non-specific illness to multi-organ dysfunction and hemorrhage. The disease is caused by pathogenic bacteria of the genus Leptospira, which are classified into pathogenic and saprophytic clades. Bacterial binding to host molecules and cells, coordinated by adhesin proteins, is an important step in pathogenesis. While many leptospiral adhesins have been identified, the vast majority have not been characterized in vivo. Herein, we present an overview of the current methodologies and successes in identifying adhesins in Leptospira, including known biological roles in vivo. We will also identify and discuss potential areas for future research.

P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi.

Innate immunity, the first line of defense against pathogens, relies on efficient elimination of invading agents by phagocytes. In the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through mimicry at the mammalian anti-phagocytic "don't eat me" signaling axis between CD47 (ligand) and SIRPα (receptor). We identified a protein, P66, on the surface of Borrelia burgdorferi that, like CD47, is necessary and sufficient to bind the macrophage receptor SIRPα. Expression of the gene encoding the protein is required for bacteria to bind SIRPα or a high-affinity CD47 reagent. Genetic deletion of p66 increases phagocytosis by macrophages. Blockade of P66 during infection promotes clearance of the bacteria. This study demonstrates that mimicry of the mammalian anti-phagocytic protein CD47 by B. burgdorferi inhibits macrophage-mediated bacterial clearance. Such a mechanism has broad implications for understanding of host-pathogen interactions and expands the function of the established innate immune checkpoint receptor SIRPα. Moreover, this report reveals P66 as a novel therapeutic target in the treatment of Lyme Disease.

Identification of amino acid domains of Borrelia burgdorferi P66 that are surface exposed and important for localization, oligomerization, and porin function of the protein.

P66, a bifunctional integral outer membrane protein, is necessary for Borrelia burgdorferi to establish initial infection and to disseminate in mice. The integrin binding function of P66 facilitates extravasation and dissemination, but the role of its porin function during murine infection has not been investigated. A limitation to studying P66 porin function during mammalian infection has been the lack of structural information for P66. In this study, we experimentally characterized specific domains of P66 with regard to structure and function. First, we aligned the amino acid sequences of P66 from Lyme disease-causing Borrelia and relapsing fever-causing Borrelia to identify conserved and unique domains between these disease-causing clades. Then, we examined whether specific domains of P66 are exposed on the surface of the bacteria by introducing c-Myc epitope tags into each domain of interest. The c-Myc epitope tag inserted C-terminally to E33 (highly conserved domain), to T187 (integrin binding region domain and a non-conserved domain), and to E334 (non-conserved domain) were all detected on the surface of Borrelia burgdorferi. The c-Myc epitope tag inserted C-terminally to E33 and D303 in conserved domains disrupted P66 oligomerization and porin function. In a murine model of infection, the E33 and D303 mutants exhibited decreased infectivity and dissemination. Taken together, these results suggest the importance of these conserved domains, and potentially P66 porin function, in vivo.

Heterologous production of the adhesin LIC13411 from pathogenic Leptospira facilitates binding of non-pathogenic Leptospira in vitro and in vivo.

Leptospirosis is an important cause of morbidity and mortality worldwide. Disease severity ranges from asymptomatic colonization to widespread hemorrhage and multiorgan dysfunction. The causative agents, Leptospira spp., are zoonotic Gram-negative spirochetes. One important step in pathogenesis is binding of bacterial adhesins to host components. Previously our laboratory identified two L. interrogans candidate adhesins, LIC11574 and LIC13411, that bind to VE-cadherin in vitro. In the current study, we demonstrate the ability of two strains of pathogenic L. interrogans to disrupt the localization of VE-cadherin, a protein important to maintaining inter-endothelial junctions. Purified MBP-LIC11574 and MBP-LIC13411 bind human dermal microvascular endothelial cells in a pattern reminiscent of VE-cadherin, but do not disrupt VE-cadherin localization. Genes encoding the candidate adhesins from pathogenic Leptospira were cloned in an overexpression vector and introduced into non-pathogenic L. biflexa, creating gain-of-function strains producing LIC11574 or LIC13411. Protein production and localization to the outer membrane were confirmed by Triton X-114 fractionation. Although these strains do not disrupt VE-cadherin localization, production of LIC13411 increases binding of non-pathogenic Leptospira to human endothelial cells and specifically to VE-cadherin. In a short-term murine model of infection, LIC13411 production led to increased burdens of the non-pathogen in the lung, liver, kidney, and bladder. These data confirm the role of LIC13411 as an adhesin in Leptospira spp. and implicate it in dissemination to multiple organs. Importantly, anti-adhesin therapy has been shown to have many benefits over classical antibiotics. Taken together, this work provides novel insight into the pathogenesis of Leptospira spp. and identifies LIC13411 as a potential prophylactic and therapeutic target.

Hematogenous dissemination of pathogenic and non-pathogenic Leptospira in a short-term murine model of infection.

Leptospirosis is an emerging zoonosis caused by pathogenic Leptospira spp. Because rodents are natural hosts of Leptospira, rodent models of pathogenesis have been limited, but are valuable to understand infection in reservoir animals even in the absence of disease. Mouse models of infection provide advantages due to genetic tractability, so developing murine models of Leptospira infection is crucial for further understanding the biology of this organism. Previously our laboratory developed a short-term murine model of Borrelia burgdorferi hematogenous dissemination to investigate the role of adhesion proteins on bacterial survival and dissemination within a host. Here we adapt this model to Leptospira. C3H/HeJ mice are anesthetized, inoculated intravenously, and then bacteria are allowed to circulate for up to twenty-four hours. Mice are euthanized, perfused with saline, and tissues are harvested for culture and DNA purification. Bacterial burdens are determined by quantitative PCR. Reproducible burdens of bacteria were found in tissues upon inoculation with pathogens and non-pathogens, demonstrating the utility of this model to probe different Leptospira species and strains. Pathogenic L. interrogans has a significantly higher burden in blood, liver, kidney, and bladder at one-hour post-inoculation when compared to non-pathogenic L. biflexa. Colonization of the kidney is essential to the life cycle of pathogenic Leptospira in nature. Measurable burdens of non-pathogenic L. biflexa were found in numerous organs and live leptospires were recovered from blood samples for at least three hours post-inoculation, contrary to the previous belief that non-pathogenic leptospires are rapidly cleared. This short-term murine model of Leptospira hematogenous dissemination will allow for the interrogation of virulence factors potentially important for tissue colonization and evasion of host defenses, and represents a novel animal model for investigating determinants of Leptospira infection.

VlsE, the nexus for antigenic variation of the Lyme disease spirochete, also mediates early bacterial attachment to the host microvasculature under shear force.

Hematogenous dissemination is a critical step in the evolution of local infection to systemic disease. The Lyme disease (LD) spirochete, which efficiently disseminates to multiple tissues, has provided a model for this process, in particular for the key early event of pathogen adhesion to the host vasculature. This occurs under shear force mediated by interactions between bacterial adhesins and mammalian cell-surface proteins or extracellular matrix (ECM). Using real-time intravital imaging of the Lyme spirochete in living mice, we previously identified BBK32 as the first LD spirochetal adhesin demonstrated to mediate early vascular adhesion in a living mouse; however, deletion of bbk32 resulted in loss of only about half of the early interactions, suggesting the existence of at least one other adhesin (adhesin-X) that promotes early vascular interactions. VlsE, a surface lipoprotein, was identified long ago by its capacity to undergo rapid antigenic variation, is upregulated in the mammalian host and required for persistent infection in immunocompetent mice. In immunodeficient mice, VlsE shares functional overlap with OspC, a multi-functional protein that displays dermatan sulfate-binding activity and is required for joint invasion and colonization. In this research, using biochemical and genetic approaches as well as intravital imaging, we have identified VlsE as adhesin-X; it is a dermatan sulfate (DS) adhesin that efficiently promotes transient adhesion to the microvasculature under shear force via its DS binding pocket. Intravenous inoculation of mice with a low-passage infectious B. burgdorferi strain lacking both bbk32 and vlsE almost completely eliminated transient microvascular interactions. Comparative analysis of binding parameters of VlsE, BBK32 and OspC provides a possible explanation why these three DS adhesins display different functionality in terms of their ability to promote early microvascular interactions.

Lyme Disease Pathogenesis.

Lyme disease Borrelia are obligately parasitic, tick- transmitted, invasive, persistent bacterial pathogens that cause disease in humans and non-reservoir vertebrates primarily through the induction of inflammation. During transmission from the infected tick, the bacteria undergo significant changes in gene expression, resulting in adaptation to the mammalian environment. The organisms multiply and spread locally and induce inflammatory responses that, in humans, result in clinical signs and symptoms. Borrelia virulence involves a multiplicity of mechanisms for dissemination and colonization of multiple tissues and evasion of host immune responses. Most of the tissue damage, which is seen in non-reservoir hosts, appears to result from host inflammatory reactions, despite the low numbers of bacteria in affected sites. This host response to the Lyme disease Borrelia can cause neurologic, cardiovascular, arthritic, and dermatologic manifestations during the disseminated and persistent stages of infection. The mechanisms by which a paucity of organisms (in comparison to many other infectious diseases) can cause varied and in some cases profound inflammation and symptoms remains mysterious but are the subjects of diverse ongoing investigations. In this review, we provide an overview of virulence mechanisms and determinants for which roles have been demonstrated in vivo, primarily in mouse models of infection.

BBB07 contributes to, but is not essential for, Borrelia burgdorferi infection in mice.

Borrelia burgdorferi, a causative agent of Lyme disease, encodes a protein BBB07 on the genomic plasmid cp26. BBB07 was identified as a candidate integrin ligand based on the presence of an RGD tripeptide motif, which is present in a number of mammalian ligands for ß1 and ß3 integrins . Previous work demonstrated that BBB07 in recombinant form binds to ß1 integrins and induces inflammatory responses in synovial cells in culture. Several transposon mutants in bbb07 were attenuated in an in vivo screen of the transposon library in mice. We therefore tested individual transposon mutant clones in single-strain infections in mice and found that they were attenuated in terms of ID50 but did not have significantly reduced tissue burdens in mice. Based on data presented here we conclude that BBB07 is not essential for, but does contribute to, B. burgdorferi infectivity in mice.

Method to Overcome Inefficiencies in Site-Directed Mutagenesis of A/T-Rich DNA.

Site-directed mutagenesis (SDM) is an invaluable technique that enables the manipulation of DNA and therefore the primary structure and function of any encoded gene products. Commercial protocols for SDM have been optimized for Escherichia coli and mean A/T content but may hinder generation of desired products using other templates. Mutagenesis of A/T-rich DNA is often hindered by low oligodeoxynucleotide (oligo)-annealing temperatures, requiring oligos longer than manufacturer protocol recommendations. However, longer oligos can result in primer dimer formation and decreased SDM efficiencies. Commercially available kits proved inefficient at generating AT-rich mutants. We sought to generate a modified protocol that generated SDM products detectable using gel electrophoresis and that did not require an apparent limit on oligo length.

Strain-specific joint invasion and colonization by Lyme disease spirochetes is promoted by outer surface protein C.

Lyme disease, caused by Borrelia burgdorferi, B. afzelii and B. garinii, is a chronic, multi-systemic infection and the spectrum of tissues affected can vary with the Lyme disease strain. For example, whereas B. garinii infection is associated with neurologic manifestations, B. burgdorferi infection is associated with arthritis. The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner. Murine infection by isogenic B. burgdorferi strains differing only in their ospC coding region revealed that two OspC variants capable of binding dermatan sulfate promoted colonization of all tissues tested, including joints. However, an isogenic strain producing OspC from B. garinii strain PBr, which binds fibronectin but not dermatan sulfate, colonized the skin, heart and bladder, but not joints. Moreover, a strain producing an OspC altered to recognize neither fibronectin nor dermatan sulfate displayed dramatically reduced levels of tissue colonization that were indistinguishable from a strain entirely deficient in OspC. Finally, intravital microscopy revealed that this OspC mutant, in contrast to a strain producing wild type OspC, was defective in promoting joint invasion by B. burgdorferi in living mice. We conclude that OspC functions as an ECM-binding adhesin that is required for joint invasion, and that variation in OspC sequence contributes to strain-specific differences in tissue tropism displayed among Lyme disease spirochetes.

Recent discoveries and advancements in research on the Lyme disease spirochete Borrelia burgdorferi.

This review highlights some of the highest-profile developments and advancements in the research on Borrelia burgdorferi, the Lyme disease spirochete, that have emerged in the last two years. Particular emphasis is placed on the controversy surrounding genus nomenclature, antigenic variation at the vlsE locus, genes involved in infectivity and virulence, membrane characteristics of B. burgdorferi, and developments in experimental approaches.

An extracellular Leptospira interrogans leucine-rich repeat protein binds human E- and VE-cadherins.

Pathogenic Leptospira bacteria are the causative agents of leptospirosis, a zoonotic disease affecting animals and humans worldwide. These pathogenic species have the ability to rapidly cross host tissue barriers by a yet unknown mechanism. A comparative analysis of pathogens and saprophytes revealed a higher abundance of genes encoding proteins with leucine-rich repeat (LRR) domains in the genomes of pathogens. In other bacterial pathogens, proteins with LRR domains have been shown to be involved in mediating host cell attachment and invasion. One protein from the pathogenic species Leptospira interrogans, LIC10831, has been previously analysed via X-ray crystallography, with findings suggesting it may be an important bacterial adhesin. Herein we show that LIC10831 elicits an antibody response in infected animals, is actively secreted by the bacterium, and binds human E- and VE-cadherins. These results provide biochemical and cellular evidences of LRR protein-mediated host-pathogen interactions and identify a new multireceptor binding protein from this infectious Leptospira species.

Characterization of Stress and Innate Immunity Resistance of Wild-Type and Δp66 Borrelia burgdorferi.

Borrelia burgdorferi is a causative agent of Lyme disease, the most common arthropod-borne disease in the United States. B. burgdorferi evades host immune defenses to establish a persistent, disseminated infection. Previous work showed that P66-deficient B. burgdorferi (Δp66) is cleared quickly after inoculation in mice. We demonstrate that the Δp66 strain is rapidly cleared from the skin inoculation site prior to dissemination. The rapid clearance of Δp66 bacteria is not due to inherent defects in multiple properties that might affect infectivity: bacterial outer membrane integrity, motility, chemotactic response, or nutrient acquisition. This led us to the hypothesis that P66 has a role in mouse cathelicidin-related antimicrobial peptide (mCRAMP; a major skin antimicrobial peptide) and/or neutrophil evasion. Neither wild-type (WT) nor Δp66 B. burgdorferi was susceptible to mCRAMP. To examine the role of neutrophil evasion, we administered neutrophil-depleting antibody anti-Ly6G (1A8) to C3H/HeN mice and subsequently monitored the course of B. burgdorferi infection. Δp66 mutants were unable to establish infection in neutrophil-depleted mice, suggesting that the important role of P66 during early infection is through another mechanism. Neutrophil depletion did not affect WT B. burgdorferi bacterial burdens in the skin (inoculation site), ear, heart, or tibiotarsal joint at early time points postinoculation. This was unexpected given that prior in vitro studies demonstrated neutrophils phagocytose and kill B. burgdorferi These data, together with our previous work, suggest that despite the in vitro ability of host innate defenses to kill B. burgdorferi, individual innate immune mechanisms have limited contributions to controlling early B. burgdorferi infection in the laboratory model used.

Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival.

Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced.

Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells.

Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways including endothelial barrier damage and inflammation, potentially leading to vascular hyperpermeability and severe illness in vivo. This work provides new insights into the pathophysiological mechanisms of Leptospira infection.

Multifunctional and Redundant Roles of Borrelia burgdorferi Outer Surface Proteins in Tissue Adhesion, Colonization, and Complement Evasion.

Borrelia burgdorferi is the causative agent of Lyme disease in the U.S., with at least 25,000 cases reported to the CDC each year. B. burgdorferi is thought to enter and exit the bloodstream to achieve rapid dissemination to distal tissue sites during infection. Travel through the bloodstream requires evasion of immune surveillance and pathogen clearance in the host, a process at which B. burgdorferi is adept. B. burgdorferi encodes greater than 19 adhesive outer surface proteins many of which have been found to bind to host cells or components of the extracellular matrix. Several others bind to host complement regulatory factors, in vitro. Production of many of these adhesive proteins is tightly regulated by environmental cues, and some have been shown to aid in vascular interactions and tissue colonization, as well as survival in the blood, in vivo. Recent work has described multifaceted and redundant roles of B. burgdorferi outer surface proteins in complement component interactions and tissue targeted adhesion and colonization, distinct from their previously identified in vitro binding capabilities. Recent insights into the multifunctional roles of previously well-characterized outer surface proteins such as BBK32, DbpA, CspA, and OspC have changed the way we think about the surface proteome of these organisms during the tick-mammal life cycle. With the combination of new and old in vivo models and in vitro techniques, the field has identified distinct ligand binding domains on BBK32 and DbpA that afford tissue colonization or blood survival to B. burgdorferi. In this review, we describe the multifunctional and redundant roles of many adhesive outer surface proteins of B. burgdorferi in tissue adhesion, colonization, and bloodstream survival that, together, promote the survival of Borrelia spp. throughout maintenance in their multi-host lifestyle.

Live Attenuated Borrelia burgdorferi Targeted Mutants in an Infectious Strain Background Protect Mice from Challenge Infection.

Borrelia burgdorferi, B. garinii, and B. afzelii are all agents of Lyme disease in different geographic locations. If left untreated, Lyme disease can cause significant and long-term morbidity, which may continue after appropriate antibiotic therapy has been administered and live bacteria are no longer detectable. The increasing incidence and geographic spread of Lyme disease are renewing interest in the vaccination of at-risk populations. We took the approach of vaccinating mice with two targeted mutant strains of B. burgdorferi that, unlike the parental strain, are avirulent in mice. Mice vaccinated with both strains were protected against a challenge with the parental strain and a heterologous B. burgdorferi strain by either needle inoculation or tick bite. In ticks, the homologous strain was eliminated but the heterologous strain was not, suggesting that the vaccines generated a response to antigens that are produced by the bacteria both early in mammalian infection and in the tick. Partial protection against B. garinii infection was also conferred. Protection was antibody mediated, and reactivity to a variety of proteins was observed. These experiments suggest that live attenuated B. burgdorferi strains may be informative regarding the identification of protective antigens produced by the bacteria and recognized by the mouse immune system in vivo Further work may illuminate new candidates that are effective and safe for the development of Lyme disease vaccines.

Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein.

Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.

A short-term Borrelia burgdorferi infection model identifies tissue tropisms and bloodstream survival conferred by adhesion proteins.

Borrelia burgdorferi, the causative agent of Lyme disease in the United States, is able to persist in the joint, heart, skin, and central nervous system for the lifetime of its mammalian host. Borrelia species achieve dissemination to distal sites in part by entry into and travel within the bloodstream. Much work has been performed in vitro describing the roles of many B. burgdorferi outer surface proteins in adhesion to host cell surface proteins and extracellular matrix components, although the biological relevance of these interactions is only beginning to be explored in vivo. A need exists in the field for an in vivo model to define the biological roles of B. burgdorferi adhesins in tissue-specific vascular interactions. We have developed an in vivo model of vascular interaction of B. burgdorferi in which the bacteria are injected intravenously and allowed to circulate for 1 h. This model has shown that the fibronectin binding protein BB0347 has a tropism for joint tissue. We also have shown an importance of the integrin binding protein, P66, in binding to vasculature of the ear and heart. This model also revealed unexpected roles for Borrelia adhesins BBK32 and OspC in bacterial burdens in the bloodstream. The intravenous inoculation model of short-term infection provides new insights into critical B. burgdorferi interactions with the host required for initial survival and tissue colonization.