Gram-positive bacteria cause the majority of skin and soft tissue infections (SSTIs), resulting in the most common reason for clinic visits in the United States. Recently, it was discovered that Gram-positive pathogens use a unique heme biosynthesis pathway, which implicates this pathway as a target for development of antibacterial therapies. We report here the identification of a small-molecule activator of coproporphyrinogen oxidase (CgoX) from Gram-positive bacteria, an enzyme essential for heme biosynthesis. Activation of CgoX induces accumulation of coproporphyrin III and leads to photosensitization of Gram-positive pathogens. In combination with light, CgoX activation reduces bacterial burden in murine models of SSTI. Thus, small-molecule activation of CgoX represents an effective strategy for the development of light-based antimicrobial therapies.

Staphylococcus aureus is a pathogen that causes significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for multiple cellular processes. Although required for pathogenesis, excess heme is toxic. S. aureus employs a two-component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for the pathogenesis of numerous Gram-positive organisms, yet the mechanism of heme sensing remains unknown. Compound '3981 was identified in a high-throughput screen as an activator of staphylococcal HssRS that triggers HssRS independently of heme accumulation. '3981 is toxic to S. aureus; however, derivatives of '3981 were synthesized that lack toxicity while retaining HssRS activation, enabling the interrogation of the heme stress response without confounding toxic effects of the parent molecule. Using '3981 derivatives as probes of the heme stress response, numerous genes required for '3981-induced activation of HssRS were uncovered. Specifically, multiple genes involved in the production of nitric oxide were identified, including the gene encoding bacterial nitric oxide synthase (bNOS). bNOS protects S. aureus from oxidative stress imposed by heme. Taken together, this work identifies bNOS as crucial for the S. aureus heme stress response, providing evidence that nitric oxide synthesis and heme sensing are intertwined.

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.

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.

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.