Biology subjects

Osburne, M. S.

Publications and source records attributed to Osburne, M. S..

3 recordsLinked to original sources

bioRxiv2021

Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

Spirochetal pathogens such as the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, encode an abundance of lipoproteins; however, due in part to their evolutionary distance from more well-studied bacteria such as Proteobacteria and Firmicutes, very few spirochetal lipoproteins have assigned functions. Indeed, B. burgdorferi devotes almost 8% of its genome to lipoprotein genes and interacts with its environment primarily through the production of at least eighty surface-exposed lipoproteins throughout its tick vector-vertebrate host lifecycle (57). Several B. burgdorferi lipoproteins have been shown to serve diverse roles, such as cellular adherence or immune evasion, but the functions for most B. burgdorferi surface lipoproteins remain unknown. In this study, we developed a B. burgdorferi lipoproteome screening platform utilizing intact spirochetes that enables the identification of previously unrecognized host interactions. As spirochetal survival in the bloodstream is essential for dissemination, we targeted our screen to C1, the first component of the classical (antibody-mediated) complement pathway. We identified two high-affinity C1 interactions by the paralogous lipoproteins, ErpB and ErpQ. Using biochemical, microbiological, and biophysical approaches, we demonstrated that ErpB and ErpQ inhibit the activated forms of the C1 proteases, C1r and C1s, and represent a new mechanistic class of C1 inhibitors that protect the spirochete from antibody-mediated complement killing by allosteric regulation. In addition to identifying a novel mode of complement inhibition, our study establishes a lipoproteome screening methodology as a discovery platform for identifying direct host-pathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent. Significance StatementSpirochetal pathogens encode an abundance of lipoproteins that can provide a critical interface with the host environment. For example, Borrelia burgdorferi, the model species for spirochetal biology, must survive an enzootic life cycle defined by fluctuations between vector (tick) and vertebrate host. While B. burgdorferi expresses over eighty surface lipoproteins-- many of which likely contribute to host survival--the B. burgdorferi lipoproteome is poorly characterized. Here, we generated a platform to rapidly identify targets of B. burgdorferi surface lipoproteins and identified two orthologs that allosterically inhibit complement C1 subcomponents, conferring resistance to classical complement killing. This work expands our understanding of complement evasion mechanisms and points towards a discovery approach for identifying host-pathogen interactions that are central to spirochete pathogenesis.

microbiology↗

bioRxiv2019

Toward a genetic system in the marine cyanobacterium Prochlorococcus

As the smallest and most abundant primary producer in the oceans, the cyanobacterium Prochlorococcus is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically.\n\nMultiple attempts have been made to develop an efficient genetic transformation method for Prochlorococcus over the years; all have been unsuccessful to date, despite some success with their close relative, Synechococcus. To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into Prochlorococcus cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.

microbiology↗

bioRxiv2018

Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection

Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice.\n\nSome commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacterrodentium lysogenized with the Stx2-encoding phage {Phi}stx2dact. This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that {Phi}stx2dact infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium ({Phi}stx2dact) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium ({Phi}stx2dact) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not.\n\nAuthor summaryEnterohemorrhagic Escherichia coli (EHEC), a food-borne pathogen that produces Shiga toxin, is associated with serious disease outbreaks worldwide, including over 390 food poisoning outbreaks in the U.S. in the last two decades. Humans acquire EHEC by ingesting contaminated food or water, or through contact with animals or their environment. Infection and toxin production may result in localized hemorrhagic colitis, but may progress to life-threatening systemic hemolytic uremic syndrome (HUS), the leading cause of kidney failure in children. Treatment for EHEC or HUS remains elusive, as antibiotics have been shown to exacerbate disease.\n\nShiga toxin genes reside on a dormant bacterial virus present in the EHEC genome, but are expressed when the virus is induced to leave its dormant state and begin to replicate. Extensive virus replication has been thought necessary to produce sufficient toxin to cause disease.\n\nUsing viral and bacterial mutants in our EHEC disease mouse model, we showed that whereas an inducing signal needed to begin viral replication was essential for lethal disease, virus production was not: sufficient Shiga toxin was produced to cause lethal mouse disease, even without viral replication. Future analyses of EHEC-infected human samples will determine whether this same phenomenon applies, potentially directing intervention strategies.

microbiology↗