Deletion of the Major Facilitator Superfamily Transporter fptB Alters Host Cell Interactions and Attenuates Virulence of Type A Francisella tularensis.

Francisella tularensis is a Gram-negative, facultative, intracellular coccobacillus that can infect a wide variety of hosts. In humans, F. tularensis causes the zoonosis tularemia following insect bites, ingestion, inhalation, and the handling of infected animals. The fact that a very small inoculum delivered by the aerosol route can cause severe disease, coupled with the possibility of its use as an aerosolized bioweapon, has led to the classification of Francisella tularensis as a category A select agent and has renewed interest in the formulation of a vaccine. To this end, we engineered a type A strain SchuS4 derivative containing a targeted deletion of the major facilitator superfamily (MFS) transporter fptB Based on the attenuating capacity of this deletion in the F. tularensis LVS background, we hypothesized that the deletion of this transporter would alter the intracellular replication and cytokine induction of the type A strain and attenuate virulence in the stringent C57BL/6J mouse model. Here we demonstrate that the deletion of fptB significantly alters the intracellular life cycle of F. tularensis, attenuating intracellular replication in both cell line-derived and primary macrophages and inducing a novel cytosolic escape delay. Additionally, we observed prominent differences in the in vitro cytokine profiles in human macrophage-like cells. The mutant was highly attenuated in the C57BL/6J mouse model and provided partial protection against virulent type A F. tularensis challenge. These results indicate a fundamental necessity for this nutrient transporter in the timely progression of F. tularensis through its replication cycle and in pathogenesis.

Global Network Analysis of Neisseria gonorrhoeae Identifies Coordination between Pathways, Processes, and Regulators Expressed during Human Infection.

Neisseria gonorrhoeae is a Gram-negative diplococcus that is responsible for the sexually transmitted infection gonorrhea, a high-morbidity disease in the United States and worldwide. Over the past several years, N. gonorrhoeae strains resistant to antibiotics used to treat this infection have begun to emerge across the globe. Thus, new treatment strategies are needed to combat this organism. Here, we utilized N. gonorrhoeae transcriptomic data sets, including those obtained from natural infection of the human genital tract, to infer the first global gene coexpression network of this pathogen. Interrogation of this network revealed genes central to the network that are likely critical for gonococcal growth, metabolism, and virulence, including genes encoding hypothetical proteins expressed during mucosal infection. In addition, network analysis revealed overlap in the response of N. gonorrhoeae to incubation with neutrophils and exposure to hydrogen peroxide stress in vitro Network analysis also identified new targets of the gonococcal global regulatory protein Fur, while examination of the network neighborhood of genes allowed us to assign additional putative categories to several proteins. Collectively, the characterization of the first gene coexpression network for N. gonorrhoeae described here has revealed new regulatory pathways and new categories for proteins and has shown how processes important to gonococcal infection in both men and women are linked. This information fills a critical gap in our understanding of virulence strategies of this obligate human pathogen and will aid in the development of new treatment strategies for gonorrhea.IMPORTANCE Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, a disease with high morbidity worldwide with an estimated 87 million cases annually. Current therapeutic and pharmacologic approaches to treat gonorrhea have been compromised by increased antibiotic resistance worldwide, including to the most recent FDA-approved antibiotic. New treatment strategies are urgently needed to combat this organism. In this study, we used network analysis to interrogate and define the coordination of pathways and processes in N. gonorrhoeae An analysis of the gonococcal network was also used to assign categories to genes and to expand our understanding of regulatory strategies. Network analysis provides important insights into pathogenic mechanisms of this organism that will guide the design of new strategies for disease treatment.

Engaging Ly-6A/Sca-1 triggers lipid raft-dependent and -independent responses in CD4+ T-cell lines.

INTRODUCTION: The lymphocyte antigen 6 (Ly-6) supergene family encodes proteins of 12-14 kda in molecular mass that are either secreted or anchored to the plasma membrane through a glycosyl-phosphatidylinisotol (GPI) lipid anchor at the carboxy-terminus. The lipidated GPI-anchor allows localization of Ly-6 proteins to the 10-100 nm cholesterol-rich nano-domains on the membrane, also known as lipid rafts. Ly-6A/Sca-1, a member of Ly-6 gene family is known to transduce signals despite the absence of transmembrane and cytoplasmic domains. It is hypothesized that the localization of Ly-6A/Sca-1 with in lipid rafts allows this protein to transduce signals to the cell interior. METHODS AND RESULTS: In this study, we found that cross-linking mouse Ly-6A/Sca-1 protein with a monoclonal antibody results in functionally distinct responses that occur simultaneously. Ly-6A/Sca-1 triggered a cell stimulatory response as gauged by cytokine production with a concurrent inhibitory response as indicated by growth inhibition and apoptosis. While production of interleukin 2 (IL-2) cytokine by CD4+ T cell line in response to cross-linking Ly-6A/Sca-1 was dependent on the integrity of lipid rafts, the observed cell death occurred independently of it. Growth inhibited CD4+ T cells showed up-regulated expression of the inhibitory cell cycle protein p27kip but not of p53. In addition, Ly-6A/Sca-1 induced translocation of cytochrome C to the cytoplasm along with activated caspase 3 and caspase 9, thereby suggesting an intrinsic apoptotic cell death mechanism. CONCLUSIONS: We conclude that opposing responses with differential dependence on the integrity of lipid rafts are triggered by engaging Ly-6A/Sca-1 protein on the membrane of transformed CD4+ T cells.