MED: Microbiology Papers

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Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses
(PUBLIC LIBRARY SCIENCE, 2017-06-26) Daaboul, George G.; Freedman, David S.; Scherr, Steven M.; Carter, Erik; Rosca, Alexandru; Bernstein, David; Mire, Chad E.; Agans, Krystle N.; Hoenen, Thomas; Geisbert, Thomas W.; Unlu, M. Selim; Connor, John H.
Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm), while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm). Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.
Robust visualization and discrimination of nanoparticles by interferometric imaging
(2017-03) Trueb, Jacob; Avci, Oguzhan; Sevenler, Derin; Connor, John H.; Ünlü, M. Selim
Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.
Wolbachia wStri blocks Zika virus growth at two independent stages of viral replication
(AMER SOC MICROBIOLOGY, 2018-05-01) Schultz, M.J.; Tan, A.L.; Gray, C.N.; Isern, S.; Michael, S.F.; Frydman, Horacio M.; Connor, John H.
Mosquito-transmitted viruses are spread globally and present a great risk to human health. Among the many approaches investigated to limit the diseases caused by these viruses are attempts to make mosquitos resistant to virus infection. Coinfection of mosquitos with the bacterium Wolbachia pipientis from supergroup A is a recent strategy employed to reduce the capacity for major vectors in the Aedes mosquito genus to transmit viruses, including dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, a supergroup B Wolbachia wStri, isolated from Laodelphax striatellus, was shown to inhibit multiple lineages of ZIKV in Aedes albopictus cells. Here, we show that wStri blocks the growth of positive-sense RNA viruses DENV, CHIKV, ZIKV, and yellow fever virus by greater than 99.9%. wStri presence did not affect the growth of the negative-sense RNA viruses LaCrosse virus or vesicular stomatitis virus. Investigation of the stages of the ZIKV life cycle inhibited by wStri identified two distinct blocks in viral replication. We found a reduction of ZIKV entry into wStri-infected cells. This was partially rescued by the addition of a cholesterol-lipid supplement. Independent of entry, transfected viral genome was unable to replicate in Wolbachia-infected cells. RNA transfection and metabolic labeling studies suggested that this replication defect is at the level of RNA translation, where we saw a 66% reduction in mosquito protein synthesis in wStri-infected cells. This study’s findings increase the potential for application of wStri to block additional arboviruses and also identify specific blocks in viral infection caused by Wolbachia coinfection.
Reverse Genetic Characterization of the Natural Genomic Deletion in SARS-Coronavirus Strain Frankfurt-1 Open Reading Frame 7B Reveals an Attenuating Function of the 7B Protein in-Vitro and in-Vivo
(BioMed Central, 2009-8-24) Pfefferle, Susanne; Krähling, Verena; Ditt, Vanessa; Grywna, Klaus; Mühlberger, Elke; Drosten, Christian
During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells). In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic.
P05-10. Sequential Immunization with a Subtype B HIV-1 Envelope Quasispecies Elicits Broader Neutralization than Vaccination with a Single Envelope Clone
(BioMed Central, 2009-10-22) Malherbe, D. C.; Doria-Rose, N.; Misher, L.; Beckett, T.; Blay Puryear, Wendy; Barnett, S.; Srivastava, I.; Richardson, B.; Stamatatos, L.; Haigwood, N. L.
Cooperative Stimulation of Dendritic Cells by Cryptococcus neoformans Mannoproteins and CpG Oligodeoxynucleotides
(Public Library of Science, 2008-4-30) Dan, Jennifer M.; Wang, Jennifer P.; Lee, Chrono K.; Levitz, Stuart M.
While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4+ T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.
PPARγ and LXR Signaling Inhibit Dendritic Cell-Mediated HIV-1 Capture and trans-Infection
(Public Library of Science, 2010-7-1) Hanley, Timothy M.; Blay Puryear, Wendy; Gummuluru, Suryaram; Viglianti, Gregory A.
Dendritic cells (DCs) contribute to human immunodeficiency virus type 1 (HIV-1) transmission and dissemination by capturing and transporting infectious virus from the mucosa to draining lymph nodes, and transferring these virus particles to CD4+ T cells with high efficiency. Toll-like receptor (TLR)-induced maturation of DCs enhances their ability to mediate trans-infection of T cells and their ability to migrate from the site of infection. Because TLR-induced maturation can be inhibited by nuclear receptor (NR) signaling, we hypothesized that ligand-activated NRs could repress DC-mediated HIV-1 transmission and dissemination. Here, we show that ligands for peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor (LXR) prevented proinflammatory cytokine production by DCs and inhibited DC migration in response to the chemokine CCL21 by preventing the TLR-induced upregulation of CCR7. Importantly, PPARγ and LXR signaling inhibited both immature and mature DC-mediated trans-infection by preventing the capture of HIV-1 by DCs independent of the viral envelope glycoprotein. PPARγ and LXR signaling induced cholesterol efflux from DCs and led to a decrease in DC-associated cholesterol, which has previously been shown to be required for DC capture of HIV-1. Finally, both cholesterol repletion and the targeted knockdown of the cholesterol transport protein ATP-binding cassette A1 (ABCA1) restored the ability of NR ligand treated cells to capture HIV-1 and transfer it to T cells. Our results suggest that PPARγ and LXR signaling up-regulate ABCA1-mediated cholesterol efflux from DCs and that this accounts for the decreased ability of DCs to capture HIV-1. The ability of NR ligands to repress DC mediated trans-infection, inflammation, and DC migration underscores their potential therapeutic value in inhibiting HIV-1 mucosal transmission. Author SummaryHeterosexual transmission is the primary mode of HIV transmission worldwide. In the absence of an effective vaccine, there is an increasing demand for the development of effective microbicides that block HIV sexual transmission. Dendritic cells (DCs) play a critical role in HIV transmission by efficiently binding virus particles, migrating to lymph nodes, and transmitting them to CD4+ T cells, a process called trans-infection. In addition, DCs secrete proinflammatory cytokines that create a favorable environment for virus replication. DC maturation by pathogen-encoded TLR ligands or proinflammatory cytokines dramatically increases their capacity to capture HIV, migrate to lymphoid tissue, and trans-infect T cells. Here, we report that signaling through the nuclear receptors PPARγ and LXR prevents DC maturation and proinflammatory cytokine production, as well as migration. In addition, PPARγ and LXR signaling prevents efficient DC capture and transfer of infectious HIV by increasing ABCA1-mediated cholesterol efflux. Our studies suggest that PPARγ and LXR may be targets for drugs that can inhibit specific aspects of HIV mucosal transmission, namely inflammation, migration, and virus capture and transfer. These findings provide a rationale for considering PPARγ and LXR agonists as potential combination therapies with conventional anti-viral microbicides that target other aspects of mucosal HIV transmission.
Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus
(Public Library of Science, 2010-1-15) Valmas, Charalampos; Grosch, Melanie N.; Schümann, Michael; Olejnik, Judith; Martinez, Osvaldo; Best, Sonja M.; Krähling, Verena; Basler, Christopher F.; Mühlberger, Elke
Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFN/α/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNa/α/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling. Author SummaryThe closely related members of the filovirus family, Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic disease in humans with high fatality rates. Infected individuals exhibit dysregulated immune responses which appear to result from several factors, including virus-mediated impairment of innate immune responses. Previous studies demonstrated that both MARV and EBOV block the type I interferon-induced Jak-STAT signaling pathway. For EBOV, the viral protein VP24 mediates the inhibitory effects by interfering with the nuclear translocation of activated STAT proteins. Here, we show that MARV uses a distinct mechanism to block IFN signaling pathways. Our data revealed that MARV blocks the phosphorylation of Janus kinases and their target STAT proteins in response to type I and type II interferon and interleukin 6. Surprisingly, the observed inhibition is not achieved by the MARV VP24 protein, but by the matrix protein VP40 which also mediates viral budding. Over-expression studies indicate that MARV VP40 globally antagonizes Jak1-dependent signaling. Further, we show that a MARV VP40 mutant defective for budding retains interferon antagonist function. Our results highlight a basic difference between EBOV and MARV, define a new function for MARV VP40 and reveal new targets for the development of anti-MARV therapies.
Comparative genomic characterization of Francisella tularensis strains belonging to low and high virulence subspecies
(Public Library of Science Pathogens, 2009-5-29) Champion, Mia D.; Zeng, Qiandong; Nix, Eli B.; Nano, Francis E.; Keim, Paul; Kodira, Chinnappa D.; Borowsky, Mark L.; Young, Sarah; Koehrsen, Michael; Engels, Reinhard; Pearson, Matthew; Howarth, Clint; Larson, Lisa; White, Jared; Alvarado, Lucia; Forsman, Mats; Bearden, Scott W.; Sjöstedt, Anders; Titball, Richard; Michell, Stephen L.; Birren, Bruce; Galagan, James
Tularemia is a geographically widespread, severely debilitating, and occasionally lethal disease in humans. It is caused by infection by a gram-negative bacterium, Francisella tularensis. In order to better understand its potency as an etiological agent as well as its potential as a biological weapon, we have completed draft assemblies and report the first complete genomic characterization of five strains belonging to the following different Francisella subspecies (subsp.): the F. tularensis subsp. tularensis FSC033, F. tularensis subsp. holarctica FSC257 and FSC022, and F. tularensis subsp. novicida GA99-3548 and GA99-3549 strains. Here, we report the sequencing of these strains and comparative genomic analysis with recently available public Francisella sequences, including the rare F. tularensis subsp. mediasiatica FSC147 strain isolate from the Central Asian Region. We report evidence for the occurrence of large-scale rearrangement events in strains of the holarctica subspecies, supporting previous proposals that further phylogenetic subdivisions of the Type B clade are likely. We also find a significant enrichment of disrupted or absent ORFs proximal to predicted breakpoints in the FSC022 strain, including a genetic component of the Type I restriction-modification defense system. Many of the pseudogenes identified are also disrupted in the closely related rarely human pathogenic F. tularensis subsp. mediasiatica FSC147 strain, including modulator of drug activity B (mdaB) (FTT0961), which encodes a known NADPH quinone reductase involved in oxidative stress resistance. We have also identified genes exhibiting sequence similarity to effectors of the Type III (T3SS) and components of the Type IV secretion systems (T4SS). One of the genes, msrA2 (FTT1797c), is disrupted in F. tularensis subsp. mediasiatica and has recently been shown to mediate bacterial pathogen survival in host organisms. Our findings suggest that in addition to the duplication of the Francisella Pathogenicity Island, and acquisition of individual loci, adaptation by gene loss in the more recently emerged tularensis, holarctica, and mediasiatica subspecies occurred and was distinct from evolutionary events that differentiated these subspecies, and the novicida subspecies, from a common ancestor. Our findings are applicable to future studies focused on variations in Francisella subspecies pathogenesis, and of broader interest to studies of genomic pathoadaptation in bacteria. Author SummaryTularemia is a zoonotic disease that is widely disseminated throughout the Northern Hemisphere and is caused by different strain types of bacteria belonging to the genus Francisella. In general, Francisella tularensis subspecies are able to infect a wide range of mammals including humans and are often transmitted via insect vectors such as ticks. Depending on the strain and route of infection the disease may be fatal in humans. In order to better understand F. tularensis as an etiological agent as well as its potential as a biological weapon, we have completed draft sequence assemblies of five globally diverse strains. We have performed a comparative analysis of these sequences with other available public Francisella sequences of strains of differing virulence. Our analysis suggests that genome rearrangements and gene loss in specific Francisella subspecies may underlie the evolution of niche adaptation and virulence of this pathogen.
Interpreting Expression Data with Metabolic Flux Models: Predicting Mycobacterium tuberculosis Mycolic Acid Production
(Public Library of Science, 2009-8-28) Colijn, Caroline; Brandes, Aaron; Zucker, Jeremy; Lun, Desmond S.; Weiner, Brian; Farhat, Maha R.; Cheng, Tan-Yun; Moody, D. Branch; Murray, Megan; Galagan, James E.
Metabolism is central to cell physiology, and metabolic disturbances play a role in numerous disease states. Despite its importance, the ability to study metabolism at a global scale using genomic technologies is limited. In principle, complete genome sequences describe the range of metabolic reactions that are possible for an organism, but cannot quantitatively describe the behaviour of these reactions. We present a novel method for modeling metabolic states using whole cell measurements of gene expression. Our method, which we call E-Flux (as a combination of flux and expression), extends the technique of Flux Balance Analysis by modeling maximum flux constraints as a function of measured gene expression. In contrast to previous methods for metabolically interpreting gene expression data, E-Flux utilizes a model of the underlying metabolic network to directly predict changes in metabolic flux capacity. We applied E-Flux to Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB). Key components of mycobacterial cell walls are mycolic acids which are targets for several first-line TB drugs. We used E-Flux to predict the impact of 75 different drugs, drug combinations, and nutrient conditions on mycolic acid biosynthesis capacity in M. tuberculosis, using a public compendium of over 400 expression arrays. We tested our method using a model of mycolic acid biosynthesis as well as on a genome-scale model of M. tuberculosis metabolism. Our method correctly predicts seven of the eight known fatty acid inhibitors in this compendium and makes accurate predictions regarding the specificity of these compounds for fatty acid biosynthesis. Our method also predicts a number of additional potential modulators of TB mycolic acid biosynthesis. E-Flux thus provides a promising new approach for algorithmically predicting metabolic state from gene expression data. Author Summary The ability of cells to survive and grow depends on their ability to metabolize nutrients and create products vital for cell function. This is done through a complex network of reactions controlled by many genes. Changes in cellular metabolism play a role in a wide variety of diseases. However, despite the availability of genome sequences and of genome-scale expression data, which give information about which genes are present and how active they are, our ability to use these data to understand changes in cellular metabolism has been limited. We present a new approach to this problem, linking gene expression data with models of cellular metabolism. We apply the method to predict the effects of drugs and agents on Mycobacterium tuberculosis (M. tb). Virulence, growth in human hosts, and drug resistance are all related to changes in M. tb's metabolism. We predict the effects of a variety of conditions on the production of mycolic acids, essential cell wall components. Our method successfully identifies seven of the eight known mycolic acid inhibitors in a compendium of 235 conditions, and identifies the top anti-TB drugs in this dataset. We anticipate that the method will have a range of applications in metabolic engineering, the characterization of disease states, and drug discovery.
Vesicular Stomatitis Virus-Based Ebola Vaccine Is Well-Tolerated and Protects Immunocompromised Nonhuman Primates
(Public Library of Science Pathogens, 2008-11-28) Geisbert, Thomas W.; Daddario-DiCaprio, Kathleen M.; Lewis, Mark G.; Geisbert, Joan B.; Grolla, Allen; Leung, Anders; Paragas, Jason; Matthias, Lennox; Smith, Mark A.; Jones, Steven M.; Hensley, Lisa E.; Feldmann, Heinz; Jahrling, Peter B.
Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVΔG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVΔG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV. Author SummaryEbola virus is among the most lethal microbes known to man, with case fatality rates often exceeding 80%. Since its discovery in 1976, outbreaks have been sporadic and geographically restricted, primarily to areas of Central Africa. However, concern about the natural or unnatural introduction of Ebola outside of the endemic areas has dramatically increased both research interest and public awareness. A number of candidate vaccines have been developed to combat Ebola virus, and these vaccines have shown varying degrees of success in nonhuman primate models. Safety is a significant concern for any vaccine and in particular for vaccines that replicate in the host. Here, we evaluated the safety of our replication-competent vesicular stomatitus virus (VSV)-based Ebola vaccine in SHIV-infected rhesus monkeys. We found that the vaccine caused no evidence of overt illness in any of these immunocompromised animals. We also demonstrated that this vaccine partially protected the SHIV-infected monkeys against a lethal Ebola challenge and that there appears to be an association with levels of CD4+ lymphocytes and survival. Our study suggests that the VSV-based Ebola vaccine will be safe in immunocompromised populations and supports further study and development of this promising vaccine platform for its use in humans.
Direct inhibition of T-cell responses by the cryptococcus capsular polysaccharide glucuronoxylomannan
(Public Library of Science Pathogens, 2006-11-10) Yauch, Lauren E.; Lam, Jennifer S.; Levitz, Stuart M.
The major virulence factor of the pathogenic fungi Cryptococcus neoformans and C. gattii is the capsule. Glucuronoxylomannan (GXM), the major component of the capsule, is a high-molecular-weight polysaccharide that is shed during cryptococcosis and can persist in patients after successful antifungal therapy. Due to the importance of T cells in the anticryptococcal response, we studied the effect of GXM on the ability of dendritic cells (DCs) to initiate a T-cell response. GXM inhibited the activation of cryptococcal mannoprotein-specific hybridoma T cells and the proliferation of OVA-specific OT-II T cells when murine bone marrow-derived DCs were used as antigen-presenting cells. Inhibition of OT-II T-cell proliferation was observed when either OVA protein or OVA323-339 peptide was used as antigen, indicating GXM did not merely prevent antigen uptake or processing. We found that DCs internalize GXM progressively over time; however, the suppressive effect did not require DCs, as GXM directly inhibited T-cell proliferation induced by anti-CD3 antibody, concanavalin A, or phorbol-12-myristate-13-acetate/ionomycin. Analysis of T-cell viability revealed that the reduced proliferation in the presence of GXM was not the result of increased cell death. GXM isolated from each of the four major cryptococcal serotypes inhibited the proliferation of human peripheral blood mononuclear cells stimulated with tetanus toxoid. Thus, we have defined a new mechanism by which GXM can impart virulence: direct inhibition of T-cell proliferation. In patients with cryptococcosis, this could impair optimal cell-mediated immune responses, thereby contributing to the persistence of cryptococcal infections. SynopsisInfections due to the pathogenic yeast Cryptococcus are a significant cause of morbidity and mortality in persons with impaired T-cell functions, particularly those with AIDS. The major virulence factor of Cryptococcus is its capsule, which is composed primarily of the polysaccharide glucuronoxylomannan (GXM). The capsule not only surrounds the organism but also is shed during cryptococcosis. GXM is taken up by macrophages in vitro and in vivo; however, little is known about the interaction between GXM and dendritic cells, which are the most potent cells capable of activating T cells. Because of the importance of T cells in the anticryptococcal response, the authors investigated the effect of GXM on the ability of dendritic cells to initiate a T-cell response. They found the polysaccharide was internalized by dendritic cells and inhibited antigen-specific T-cell responses. Furthermore, GXM had a direct, inhibitory effect on T-cell proliferation, independent of the effect on dendritic cells. These findings may help explain the persistence of cryptococcal infections and suggest that GXM could be therapeutic in situations where suppression of T-cell responses is desired.
Development of an Acute and Highly Pathogenic Nonhuman Primate Model of Nipah Virus Infection
(Public Library of Science, 2010-5-18) Geisbert, Thomas W.; Daddario-DiCaprio, Kathleen M.; Hickey, Andrew C.; Smith, Mark A.; Chan, Yee-Peng; Wang, Lin-Fa; Mattapallil, Joseph J.; Geisbert, Joan B.; Bossart, Katharine N.; Broder, Christopher C.
Nipah virus (NiV) is an enigmatic emerging pathogen that causes severe and often fatal neurologic and/or respiratory disease in both animals and humans. Amongst people, case fatality rates range between 40 and 75 percent and there are no vaccines or treatments approved for human use. Guinea pigs, hamsters, cats, ferrets, pigs and most recently squirrel monkeys (New World monkey) have been evaluated as animal models of human NiV infection, and with the exception of the ferret, no model recapitulates all aspects of NiV-mediated disease seen in humans. To identify a more viable nonhuman primate (NHP) model, we examined the pathogenesis of NiV in African green monkeys (AGM). Exposure of eight monkeys to NiV produced a severe systemic infection in all eight animals with seven of the animals succumbing to infection. Viral RNA was detected in the plasma of challenged animals and occurred in two of three subjects as a peak between days 7 and 21, providing the first clear demonstration of plasma-associated viremia in NiV experimentally infected animals and suggested a progressive infection that seeded multiple organs simultaneously from the initial site of virus replication. Unlike the cat, hamster and squirrel monkey models of NiV infection, severe respiratory pathology, neurological disease and generalized vasculitis all manifested in NiV-infected AGMs, providing an accurate reflection of what is observed in NiV-infected humans. Our findings demonstrate the first consistent and highly pathogenic NHP model of NiV infection, providing a new and critical platform in the evaluation and licensure of either passive and active immunization or therapeutic strategies for human use.
Effect of Differential N-linked and O-linked Mannosylation on Recognition of Fungal Antigens by Dendritic Cells
(Public Library of Science, 2007-10-10) Lam, Jennifer S.; Huang, Haibin; Levitz, Stuart M.
BACKGROUND. An experimental approach for improving vaccine efficacy involves targeting antigens to mannose receptors (MRs) on dendritic cells (DCs) and other professional antigen presenting cells. Previously, we demonstrated that mannosylated Pichia pastoris-derived recombinant proteins exhibited increased immunogenicity compared to proteins lacking mannosylation. In order to gain insight into the mechanisms responsible for this observation, the present study examined the cellular uptake of the mannosylated and deglycosylated recombinant proteins. METHODOLOGY/PRINCIPAL FINDINGS. Utilizing transfected cell lines, roles for the macrophage mannose receptor (MMR, CD206) and DC-SIGN (CD209) in the recognition of the mannosylated, but not deglycosylated, antigens were demonstrated. The uptake of mannosylated antigens into murine bone marrow-derived DCs (BMDCs) was inhibited by yeast mannans (YMs), suggesting a mannose-specific C-type lectin receptor-dependent process, while the uptake of deglycosylated antigens remained unaffected. In particular, antigens with both N-linked and extensive O-linked mannosylation showed the highest binding and uptake by BMDCs. Finally, confocal microscopy studies revealed that both mannosylated and deglycosylated P. pastoris-derived recombinant proteins localized in MHC class II+ compartments within BMDCs. CONCLUSIONS/SIGNIFICANCE. Taken together with our previous results, these data suggest that increased uptake by mannose-specific C-type lectin receptors is the major mechanism responsible for the enhanced antigenicity seen with mannosylated proteins. These findings have important implications for vaccine design and contribute to our understanding of how glycosylation affects the immune response to eukaryotic pathogens.
Toll-Like Receptors and Innate Immune Responses in Systemic Lupus Erythematosus
(BioMed Central, 2007-11-29) Lafyatis, Robert; Marshak-Rothstein, Ann
A series of discoveries over the past several years has provided a new paradigm for understanding autoimmunity in systemic lupus erythematosus. The discoveries of pattern recognition receptors and of how these receptors can be recruited into autoimmune responses underpin this paradigm. The implications of these observations continue to unfold with ongoing investigation into the range and specificity of pattern recognition receptors, into how immune complexes containing nucleic acids trigger these receptors, into how endogenous macromolecular 'danger signals' stimulate innate immune responses, and into the effect of pattern recognition receptor activation on various cell types in initiating and perpetuating autoimmunity. The development of clinical trials using therapeutic agents that target components of the innate immune system suggests that these advances may soon culminate in new medications for treating patients with systemic lupus erythematosus.
A Blind Deconvolution Approach to High-Resolution Mapping of Transcription Factor Binding Sites from ChIP-Seq Data
(BioMed Central, 2009-12-22) Lun, Desmond S.; Sherrid, Ashley; Weiner, Brian; Sherman, David R.; Galagan, James E.
CSdeconv is a novel method for determining the location of transcription factor binding from ChIP-seq data that discriminates closely-spaced sites. We present CSDeconv, a computational method that determines locations of transcription factor binding from ChIP-seq data. CSDeconv differs from prior methods in that it uses a blind deconvolution approach that allows closely-spaced binding sites to be called accurately. We apply CSDeconv to novel ChIP-seq data for DosR binding in Mycobacterium tuberculosis and to existing data for GABP in humans and show that it can discriminate binding sites separated by as few as 40 bp.
Accumulation of Marginal Zone B Cells and Accelerated Loss of Follicular Dendritic Cells in NF-κB p50-Deficient Mice
(BioMed Central, 2005-4-18) Ferguson, Andrew R.; Corley, Ronald B.
BACKGROUND: Marginal zone (MZ) B cells play important roles in the early phases of humoral immune responses. In addition to possessing an inherent capacity to rapidly differentiate into antibody secreting cells, MZ B cells also help to regulate the fate of both T-independent and T-dependent blood-borne antigens in the spleen. For T-dependent antigens, MZ B cells bind IgM-antigen complexes in a complement-dependent manner. Once MZ B cells bind IgM-containing immune complexes (IgM-IC), they transport them into B cell follicles for deposition onto follicular dendritic cells (FDCs), an important component of secreted IgM's ability to enhance adaptive immune responses. To further define the requirement for MZ B cells in IgM-IC deposition, mice deficient in the NF-κB protein p50, which have been reported to lack MZ B cells, were analyzed for their ability to trap IgM-IC onto FDCs. RESULTS: Mice (2 months of age) deficient in p50 (p50-/-) had small numbers of MZ B cells, as determined by cell surface phenotype and localization in the splenic MZ. These cells bound high levels of IgM-IC both in vivo and in vitro. Subsequent to the binding of IgM-IC by the MZ B cells in p50-/- mice, small amounts of IgM-IC were found localized on FDCs, suggesting that the MZ B cells retained their ability to transport these complexes into splenic follicles. Strikingly, MZ B cells accumulated with age in p50-/- mice. By 6 months of age, p50-/- mice contained normal numbers of these cells as defined by CD21/CD23 profile and high level expression of CD1d, CD9, and IgM, and by their positioning around the marginal sinus. However, FDCs from these older p50-/- mice exhibited a reduced capacity to trap IgM-IC and retain complement components. CONCLUSION: These results demonstrate that while the p50 component of the NF-κB transcription complex plays an important role in the early development of MZ B cells, MZ B cells can develop and accumulate in mice lacking this protein. These results highlight the interface between genetic deficiencies and age, and suggest that different transcription factors may play distinct roles in the development and maintenance of cell populations at different ages.
Framework for Leadership and Training of Biosafety Level 4 Laboratory Workers
(Centers for Disease Control and Prevention, 2011-12-29) Le Duc, James W.; Anderson, Kevin; Bloom, Marshall E.; Estep, James E.; Feldmann, Heinz; Geisbert, Joan B.; Geisbert, Thomas W.; Hensley, Lisa; Holbrook, Michael; Jahrling, Peter B.; Ksiazek, Thomas G.; Korch, George; Patterson, Jean; Skvorak, John P.; Weingartl, Hana
One-sentence summary for table of contents: Training should include theoretical consideration of biocontainment principles, practical hands-on training, and mentored on-the-job experience. Construction of several new Biosafety Level 4 (BSL-4) laboratories and expansion of existing operations have created an increased international demand for well-trained staff and facility leaders. Directors of most North American BSL-4 laboratories met and agreed upon a framework for leadership and training of biocontainment research and operations staff. They agreed on essential preparation and training that includes theoretical consideration of biocontainment principles, practical hands-on training, and mentored on-the-job experiences relevant to positional responsibilities as essential preparation before a person's independent access to a BSL-4 facility. They also agreed that the BSL-4 laboratory director is the key person most responsible for ensuring that staff members are appropriately prepared for BSL-4 operations. Although standardized certification of training does not formally exist, the directors agreed that facility-specific, time-limited documentation to recognize specific skills and experiences of trained persons is needed.

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