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.

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.

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.

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 β1 and β3 integrins . Previous work demonstrated that BBB07 in recombinant form binds to β1 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.

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.