Developing a system of mutagenesis in Francisella tularensis LVS

Francisella tularensis is a gram-negative facultative intracellular coccobacillus that primarily infects macrophages. The causative agent of tularemia, this bacterium is considered among the most infectious organisms known, requiring fewer than ten organisms to cause disease. Although ubiquitous in nature, transmission to humans is rare but can occur via insect bites, direct contact with infected animals, ingestion of contaminated water, or through the inhalation of aerosols. There are several species of Francisella, however the majority of infections are caused by F. tularensis. Species of F. tularensis are further classified into two groups according to pathogenesis, the Type A highly virulent strain and the Type B, less pathogenic strains. Type A pathogens cause a variety of clinical manifestations including several glandular infections and the most life threatening, pneumonic tularemia. Given the many routes of transmission, low infectious dose and severity of the illness, F. tularensis has become a concern for potential development of the bacteria into a bioweapon and has been classified as a Category A pathogen by the Centers for Disease Control and Prevention (CDC). Previous studies have attempted to investigate the pathogenicity of F. tularensis using a variety of genetic manipulation techniques. However due to the unique challenges of applying current genetic techniques in F. tularensis, few genes important for Francisella virulence have been identified. This study aims to develop a random transposon mutagenesis library and primary screening assay to rapidly identify virulence factors associated with intra-macrophage survival. A potential library was generated using plasmid pFT-mariner, a Francisella mutagenesis vector constructed for this study. This plasmid utilizes a eukaryotic mariner himar-1 transposase and transposon cassette. An arabinose inducible promoter that regulates transposase activity, controls transposition of the kanamycin flanked transposon cassette. The pFT-mariner plasmid was introduced into F. tularensis live vaccine strain (LVS) through conjugation and resulted in several potential library founder clones. Founder clones were screened by polymerase chain reaction (PCR) and found to contain pFT-mariner components in several generations of passed bacteria. Select clones were incubated with arabinose to induce transposon integration into the genome. A counter-selection method was used to eliminate the pFT-mariner plasmid. DNA from potential library clones was screened by PCR to detect the integration of the transposon and to verify the loss of the remaining plasmid. Following confirmation of transposition, several methods were used to try to determine the site of insertion. To screen for pathogenicity, any identified mutants would be applied to a macrophage infection assay and compared to a F. tularensis LVS infection. This study generated multiple potential library founder clones and developed a rapid screening assay for intra-macrophage survival of F. tularensis LVS. However in our investigation we encountered several difficulties; while we were able to detect transposon integration immediately following transposase induction, these failed to be identified again in subsequent investigation. Ultimately, similar to previously reported mutagenesis attempts our potential library of transposon mutants was determined to be unstable. Thus, future transposon mutagenesis efforts should focus on verifying stability of the vector and transposon

Glycosaminoglycan Binding Facilitates Entry of a Bacterial Pathogen into Central Nervous Systems

Certain microbes invade brain microvascular endothelial cells (BMECs) to breach the blood-brain barrier (BBB) and establish central nervous system (CNS) infection. Here we use the leading meningitis pathogen group B Streptococcus (GBS) together with insect and mammalian infection models to probe a potential role of glycosaminoglycan (GAG) interactions in the pathogenesis of CNS entry. Site-directed mutagenesis of a GAG-binding domain of the surface GBS alpha C protein impeded GBS penetration of the Drosophila BBB in vivo and diminished GBS adherence to and invasion of human BMECs in vitro. Conversely, genetic impairment of GAG expression in flies or mice reduced GBS dissemination into the brain. These complementary approaches identify a role for bacterial-GAG interactions in the pathogenesis of CNS infection. Our results also highlight how the simpler yet genetically conserved Drosophila GAG pathways can provide a model organism to screen candidate molecules that can interrupt pathogen-GAG interactions for future therapeutic applications

Host and Pathogen Glycosaminoglycan-Binding Proteins Modulate Antimicrobial Peptide Responses in Drosophila melanogaster▿ †

During group B streptococcal infection, the alpha C protein (ACP) on the bacterial surface binds to host cell surface heparan sulfate proteoglycans (HSPGs) and facilitates entry of bacteria into human epithelial cells. Previous studies in a Drosophila melanogaster model showed that binding of ACP to the sulfated polysaccharide chains (glycosaminoglycans) of HSPGs promotes host death and is associated with higher bacterial burdens. We hypothesized that ACP-glycosaminoglycan binding might determine infection outcome by altering host responses to infection, such as expression of antimicrobial peptides. As glycosaminoglycans/HSPGs also interact with a number of endogenous secreted signaling molecules in Drosophila, we examined the effects of host and pathogen glycosaminoglycan/HSPG-binding structures in host survival of infection and antimicrobial peptide expression. Strikingly, host survival after infection with wild-type streptococci was enhanced among flies overexpressing the endogenous glycosaminoglycan/HSPG-binding morphogen Decapentaplegic—a transforming growth factor β-like Drosophila homolog of mammalian bone morphogenetic proteins—but not by flies overexpressing a mutant, non-glycosaminoglycan-binding Decapentaplegic, or the other endogenous glycosaminoglycan/HSPG-binding morphogens, Hedgehog and Wingless. While ACP-glycosaminoglycan binding was associated with enhanced transcription of peptidoglycan recognition proteins and antimicrobial peptides, Decapentaplegic overexpression suppressed transcription of these genes during streptococcal infection. Further, the glycosaminoglycan-binding domain of ACP competed with Decapentaplegic for binding to the soluble glycosaminoglycan heparin in an in vitro assay. These data suggest that, in addition to promoting bacterial entry into host cells, ACP competes with Decapentaplegic for binding to glycosaminoglycans/HSPGs during infection and that these bacterial and endogenous glycosaminoglycan-binding structures determine host survival and regulate antimicrobial peptide transcription

Biomarkers of Nutrition for Development (BOND): Vitamin B-12 Review

This report on vitamin B-12 (B12) is part of the Biomarkers of Nutrition for Development (BOND) Project, which provides state-of-the art information and advice on the selection, use, and interpretation of biomarkers of nutrient exposure, status, and function. As with the other 5 reports in this series, which focused on iodine, folate, zinc, iron, and vitamin A, this B12 report was developed with the assistance of an expert panel (BOND B12 EP) and other experts who provided information during a consultation. The experts reviewed the existing literature in depth in order to consolidate existing relevant information on the biology of B12, including known and possible effects of insufficiency, and available and potential biomarkers of status. Unlike the situation for the other 5 nutrients reviewed during the BOND project, there has been relatively little previous attention paid to B12 status and its biomarkers, so this report is a landmark in terms of the consolidation and interpretation of the available information on B12 nutrition. Historically, most focus has been on diagnosis and treatment of clinical symptoms of B12 deficiency, which result primarily from pernicious anemia or strict vegetarianism. More recently, we have become aware of the high prevalence of B12 insufficiency in populations consuming low amounts of animal-source foods, which can be detected with ≥1 serum biomarker but presents the new challenge of identifying functional consequences that may require public health interventions