PLoS Pathogens: New Articles

How Do Microbial Pathogens Make CENs?

2012-02-09T22:00:00Z

by Kaustuv Sanyal

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

2012-02-09T22:00:00Z

by Amanda R. Robinson, Swee Sen Kwek, Shannon C. Kenney

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells.

Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

2012-02-09T22:00:00Z

by Ling-An Kong, Jun Yang, Guo-Tian Li, Lin-Lu Qi, Yu-Jun Zhang, Chen-Fang Wang, Wen-Sheng Zhao, Jin-Rong Xu, You-Liang Peng

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.

The Arabidopsis Lectin Receptor Kinase LecRK-V.5 Represses Stomatal Immunity Induced by Pseudomonas syringae pv. tomato DC3000

2012-02-09T22:00:00Z

by Marie Desclos-Theveniau, Dominique Arnaud, Ting-Yu Huang, Grace Jui-Chih Lin, Wei-Yen Chen, Yi-Chia Lin, Laurent Zimmerli

Stomata play an important role in plant innate immunity by limiting pathogen entry into leaves but molecular mechanisms regulating stomatal closure upon pathogen perception are not well understood. Here we show that the Arabidopsis thaliana L-type lectin receptor kinase-V.5 (LecRK-V.5) negatively regulates stomatal immunity. Loss of LecRK-V.5 function increased resistance to surface inoculation with virulent bacteria Pseudomonas syringae pv tomato DC3000. Levels of resistance were not affected after infiltration-inoculation, suggesting that LecRK-V.5 functions at an early defense stage. By contrast, lines overexpressing LecRK-V.5 were more susceptible to Pst DC3000. Enhanced resistance in lecrk-V.5 mutants was correlated with constitutive stomatal closure, while increased susceptibility phenotypes in overexpression lines were associated with early stomatal reopening. Lines overexpressing LecRK-V.5 also demonstrated a defective stomatal closure after pathogen-associated molecular pattern (PAMP) treatments. LecRK-V.5 is rapidly expressed in stomatal guard cells after bacterial inoculation or treatment with the bacterial PAMP flagellin. In addition, lecrk-V.5 mutants guard cells exhibited constitutive accumulation of reactive oxygen species (ROS) and inhibition of ROS production opened stomata of lecrk-V.5. LecRK-V.5 is also shown to interfere with abscisic acid-mediated stomatal closure signaling upstream of ROS production. These results provide genetic evidences that LecRK-V.5 negatively regulates stomatal immunity upstream of ROS biosynthesis. Our data reveal that plants have evolved mechanisms to reverse bacteria-mediated stomatal closure to prevent long-term effect on CO₂ uptake and photosynthesis.

Specialized Peptidoglycan Hydrolases Sculpt the Intra-bacterial Niche of Predatory Bdellovibrio and Increase Population Fitness

2012-02-09T22:00:00Z

by Thomas R. Lerner, Andrew L. Lovering, Nhat Khai Bui, Kaoru Uchida, Shin-Ichi Aizawa, Waldemar Vollmer, R. Elizabeth Sockett

Bdellovibrio are predatory bacteria that have evolved to invade virtually all Gram-negative bacteria, including many prominent pathogens. Upon invasion, prey bacteria become rounded up into an osmotically stable niche for the Bdellovibrio, preventing further superinfection and allowing Bdellovibrio to replicate inside without competition, killing the prey bacterium and degrading its contents. Historically, prey rounding was hypothesized to be associated with peptidoglycan (PG) metabolism; we found two Bdellovibrio genes, bd0816 and bd3459, expressed at prey entry and encoding proteins with limited homologies to conventional dacB/PBP4 DD-endo/carboxypeptidases (responsible for peptidoglycan maintenance during growth and division). We tested possible links between Bd0816/3459 activity and predation. Bd3459, but not an active site serine mutant protein, bound β-lactam, exhibited DD-endo/carboxypeptidase activity against purified peptidoglycan and, importantly, rounded up E. coli cells upon periplasmic expression. A ΔBd0816 ΔBd3459 double mutant invaded prey more slowly than the wild type (with negligible prey cell rounding) and double invasions of single prey by more than one Bdellovibrio became more frequent. We solved the crystal structure of Bd3459 to 1.45 Å and this revealed predation-associated domain differences to conventional PBP4 housekeeping enzymes (loss of the regulatory domain III, alteration of domain II and a more exposed active site). The Bd3459 active site (and by similarity the Bd0816 active site) can thus accommodate and remodel the various bacterial PGs that Bdellovibrio may encounter across its diverse prey range, compared to the more closed active site that “regular” PBP4s have for self cell wall maintenance. Therefore, during evolution, Bdellovibrio peptidoglycan endopeptidases have adapted into secreted predation-specific proteins, preventing wasteful double invasion, and allowing activity upon the diverse prey peptidoglycan structures to sculpt the prey cell into a stable intracellular niche for replication.

Characterising the Mucosal and Systemic Immune Responses to Experimental Human Hookworm Infection

2012-02-09T22:00:00Z

by Soraya Gaze, Henry J. McSorley, James Daveson, Di Jones, Jeffrey M. Bethony, Luciana M. Oliveira, Richard Speare, James S. McCarthy, Christian R. Engwerda, John Croese, Alex Loukas

The mucosal cytokine response of healthy humans to parasitic helminths has never been reported. We investigated the systemic and mucosal cytokine responses to hookworm infection in experimentally infected, previously hookworm naive individuals from non-endemic areas. We collected both peripheral blood and duodenal biopsies to assess the systemic immune response, as well as the response at the site of adult worm establishment. Our results show that experimental hookworm infection leads to a strong systemic and mucosal Th2 (IL-4, IL-5, IL-9 and IL-13) and regulatory (IL-10 and TGF-β) response, with some evidence of a Th1 (IFN-γ and IL-2) response. Despite upregulation after patency of both IL-15 and ALDH1A2, a known Th17-inducing combination in inflammatory diseases, we saw no evidence of a Th17 (IL-17) response. Moreover, we observed strong suppression of mucosal IL-23 and upregulation of IL-22 during established hookworm infection, suggesting a potential mechanism by which Th17 responses are suppressed, and highlighting the potential that hookworms and their secreted proteins offer as therapeutics for human inflammatory diseases.

Essential Roles for Soluble Virion-Associated Heparan Sulfonated Proteoglycans and Growth Factors in Human Papillomavirus Infections

2012-02-09T22:00:00Z

by Zurab Surviladze, Agnieszka Dziduszko, Michelle A. Ozbun

A subset of human papillomavirus (HPV) infections is causally related to the development of human epithelial tumors and cancers. Like a number of pathogens, HPV entry into target cells is initiated by first binding to heparan sulfonated proteoglycan (HSPG) cell surface attachment factors. The virus must then move to distinct secondary receptors, which are responsible for particle internalization. Despite intensive investigation, the mechanism of HPV movement to and the nature of the secondary receptors have been unclear. We report that HPV16 particles are not liberated from bound HSPG attachment factors by dissociation, but rather are released by a process previously unreported for pathogen-host cell interactions. Virus particles reside in infectious soluble high molecular weight complexes with HSPG, including syndecan-1 and bioactive compounds, like growth factors. Matrix mellatoproteinase inhibitors that block HSPG and virus release from cells interfere with virus infection. Employing a co-culture assay, we demonstrate HPV associated with soluble HSPG-growth factor complexes can infect cells lacking HSPG. Interaction of HPV-HSPG-growth factor complexes with growth factor receptors leads to rapid activation of signaling pathways important for infection, whereas a variety of growth factor receptor inhibitors impede virus-induced signaling and infection. Depletion of syndecan-1 or epidermal growth factor and removal of serum factors reduce infection, while replenishment of growth factors restores infection. Our findings support an infection model whereby HPV usurps normal host mechanisms for presenting growth factors to cells via soluble HSPG complexes as a novel method for interacting with entry receptors independent of direct virus-cell receptor interactions.

Degradation of Cellular miR-27 by a Novel, Highly Abundant Viral Transcript Is Important for Efficient Virus Replication In Vivo

2012-02-09T22:00:00Z

by Lisa Marcinowski, Mélanie Tanguy, Astrid Krmpotic, Bernd Rädle, Vanda J. Lisnić, Lee Tuddenham, Béatrice Chane-Woon-Ming, Zsolt Ruzsics, Florian Erhard, Corinna Benkartek, Marina Babic, Ralf Zimmer, Joanne Trgovcich, Ulrich H. Koszinowski, Stipan Jonjic, Sébastien Pfeffer, Lars Dölken

Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the ∼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3′-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3′-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3′-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo.

The TPR Domain in the Host Cyp40-like Cyclophilin Binds to the Viral Replication Protein and Inhibits the Assembly of the Tombusviral Replicase

2012-02-09T22:00:00Z

by Jing-Yi Lin, Venugopal Mendu, Judit Pogany, Jun Qin, Peter D. Nagy

Replication of plus-stranded RNA viruses is greatly affected by numerous host-coded proteins acting either as susceptibility or resistance factors. Previous genome-wide screens and global proteomics approaches with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of cyclophilins, which are a large family of host prolyl isomerases, in TBSV replication. In this paper, we identified those members of the large cyclophilin family that interacted with the viral replication proteins and inhibited TBSV replication. Further characterization of the most effective cyclophilin, the Cyp40-like Cpr7p, revealed that it strongly inhibits many steps during TBSV replication in a cell-free replication assay. These steps include viral RNA recruitment inhibited via binding of Cpr7p to the RNA-binding region of the viral replication protein; the assembly of the viral replicase complex and viral RNA synthesis. Since the TPR (tetratricopeptide repeats) domain, but not the catalytic domain of Cpr7p is needed for the inhibitory effect on TBSV replication, it seems that the chaperone activity of Cpr7p provides the negative regulatory function. We also show that three Cyp40-like proteins from plants can inhibit TBSV replication in vitro and Cpr7p is also effective against Nodamura virus, an insect pathogen. Overall, the current work revealed a role for Cyp40-like proteins and their TPR domains as regulators of RNA virus replication.

Structural and Functional Insights into the Pilotin-Secretin Complex of the Type II Secretion System

2012-02-09T22:00:00Z

by Shuang Gu, Saima Rehman, Xiaohui Wang, Vladimir E. Shevchik, Richard W. Pickersgill

Gram-negative bacteria secrete virulence factors and assemble fibre structures on their cell surface using specialized secretion systems. Three of these, T2SS, T3SS and T4PS, are characterized by large outer membrane channels formed by proteins called secretins. Usually, a cognate lipoprotein pilot is essential for the assembly of the secretin in the outer membrane. The structures of the pilotins of the T3SS and T4PS have been described. However in the T2SS, the molecular mechanism of this process is poorly understood and its structural basis is unknown. Here we report the crystal structure of the pilotin of the T2SS that comprises an arrangement of four α-helices profoundly different from previously solved pilotins from the T3SS and T4P and known four α-helix bundles. The architecture can be described as the insertion of one α-helical hairpin into a second open α-helical hairpin with bent final helix. NMR, CD and fluorescence spectroscopy show that the pilotin binds tightly to 18 residues close to the C-terminus of the secretin. These residues, unstructured before binding to the pilotin, become helical on binding. Data collected from crystals of the complex suggests how the secretin peptide binds to the pilotin and further experiments confirm the importance of these C-terminal residues in vivo.

Genome-wide Transcriptional Profiling of Appressorium Development by the Rice Blast Fungus Magnaporthe oryzae

2012-02-09T22:00:00Z

by Darren M. Soanes, Apratim Chakrabarti, Konrad H. Paszkiewicz, Angus L. Dawe, Nicholas J. Talbot

The rice blast fungus Magnaporthe oryzae is one of the most significant pathogens affecting global food security. To cause rice blast disease the fungus elaborates a specialised infection structure called an appressorium. Here, we report genome wide transcriptional profile analysis of appressorium development using next generation sequencing (NGS). We performed both RNA-Seq and High-Throughput SuperSAGE analysis to compare the utility of these procedures for identifying differential gene expression in M. oryzae. We then analysed global patterns of gene expression during appressorium development. We show evidence for large-scale gene expression changes, highlighting the role of autophagy, lipid metabolism and melanin biosynthesis in appressorium differentiation. We reveal the role of the Pmk1 MAP kinase as a key global regulator of appressorium-associated gene expression. We also provide evidence for differential expression of transporter-encoding gene families and specific high level expression of genes involved in quinate uptake and utilization, consistent with pathogen-mediated perturbation of host metabolism during plant infection. When considered together, these data provide a comprehensive high-resolution analysis of gene expression changes associated with cellular differentiation that will provide a key resource for understanding the biology of rice blast disease.

Substance P Causes Seizures in Neurocysticercosis

2012-02-09T22:00:00Z

by Prema Robinson, Armandina Garza, Joel Weinstock, Jose A. Serpa, Jerry Clay Goodman, Kristian T. Eckols, Bahrom Firozgary, David J. Tweardy

Neurocysticercosis (NCC), a helminth infection of the brain, is a major cause of seizures. The mediators responsible for seizures in NCC are unknown, and their management remains controversial. Substance P (SP) is a neuropeptide produced by neurons, endothelial cells and immunocytes. The current studies examined the hypothesis that SP mediates seizures in NCC. We demonstrated by immunostaining that 5 of 5 brain biopsies from NCC patients contained substance P (SP)-positive (+) cells adjacent to but not distant from degenerating worms; no SP+ cells were detected in uninfected brains. In a rodent model of NCC, seizures were induced after intrahippocampal injection of SP alone or after injection of extracts of cysticercosis granuloma obtained from infected wild type (WT), but not from infected SP precursor-deficient mice. Seizure activity correlated with SP levels within WT granuloma extracts and was prevented by intrahippocampal pre-injection of SP receptor antagonist. Furthermore, extracts of granulomas from WT mice caused seizures when injected into the hippocampus of WT mice, but not when injected into SP receptor (NK1R) deficient mice. These findings indicate that SP causes seizures in NCC, and, suggests that seizures in NCC in humans may be prevented and/or treated with SP-receptor antagonists.

Viral Mediated Redirection of NEMO/IKKγ to Autophagosomes Curtails the Inflammatory Cascade

2012-02-02T22:00:00Z

by Patricia M. Fliss, Tali Pechenick Jowers, Melanie M. Brinkmann, Barbara Holstermann, Claudia Mack, Paul Dickinson, Heinrich Hohenberg, Peter Ghazal, Wolfram Brune

The early host response to viral infections involves transient activation of pattern recognition receptors leading to an induction of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Subsequent activation of cytokine receptors in an autocrine and paracrine manner results in an inflammatory cascade. The precise mechanisms by which viruses avert an inflammatory cascade are incompletely understood. Nuclear factor (NF)-κB is a central regulator of the inflammatory signaling cascade that is controlled by inhibitor of NF-κB (IκB) proteins and the IκB kinase (IKK) complex. In this study we show that murine cytomegalovirus inhibits the inflammatory cascade by blocking Toll-like receptor (TLR) and IL-1 receptor-dependent NF-κB activation. Inhibition occurs through an interaction of the viral M45 protein with the NF-κB essential modulator (NEMO), the regulatory subunit of the IKK complex. M45 induces proteasome-independent degradation of NEMO by targeting NEMO to autophagosomes for subsequent degradation in lysosomes. We propose that the selective and irreversible degradation of a central regulatory protein by autophagy represents a new viral strategy to dampen the inflammatory response.

Enhancement of Chemokine Function as an Immunomodulatory Strategy Employed by Human Herpesviruses

2012-02-02T22:00:00Z

by Abel Viejo-Borbolla, Nadia Martinez-Martín, Hendrik J. Nel, Patricia Rueda, Rocío Martín, Soledad Blanco, Fernando Arenzana-Seisdedos, Marcus Thelen, Padraic G. Fallon, Antonio Alcamí

Herpes simplex virus (HSV) types 1 and 2 are highly prevalent human neurotropic pathogens that cause a variety of diseases, including lethal encephalitis. The relationship between HSV and the host immune system is one of the main determinants of the infection outcome. Chemokines play relevant roles in antiviral response and immunopathology, but the modulation of chemokine function by HSV is not well understood. We have addressed the modulation of chemokine function mediated by HSV. By using surface plasmon resonance and crosslinking assays we show that secreted glycoprotein G (SgG) from both HSV-1 and HSV-2 binds chemokines with high affinity. Chemokine binding activity was also observed in the supernatant of HSV-2 infected cells and in the plasma membrane of cells infected with HSV-1 wild type but not with a gG deficient HSV-1 mutant. Cell-binding and competition experiments indicate that the interaction takes place through the glycosaminoglycan-binding domain of the chemokine. The functional relevance of the interaction was determined both in vitro, by performing transwell assays, time-lapse microscopy, and signal transduction experiments; and in vivo, using the air pouch model of inflammation. Interestingly, and in contrast to what has been observed for previously described viral chemokine binding proteins, HSV SgGs do not inhibit chemokine function. On the contrary, HSV SgGs enhance chemotaxis both in vitro and in vivo through increasing directionality, potency and receptor signaling. This is the first report, to our knowledge, of a viral chemokine binding protein from a human pathogen that increases chemokine function and points towards a previously undescribed strategy of immune modulation mediated by viruses.

A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

2012-02-02T22:00:00Z

by Amy Huei-Yi Lee, Brenden Hurley, Corinna Felsensteiner, Carmen Yea, Wenzislava Ckurshumova, Verena Bartetzko, Pauline W. Wang, Van Quach, Jennifer D. Lewis, Yulu C. Liu, Frederik Börnke, Stephane Angers, Andrew Wilde, David S. Guttman, Darrell Desveaux

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.

The Role of Animal Models for Research on Severe Malaria

2012-02-02T22:00:00Z

by Alister G. Craig, Georges E. Grau, Chris Janse, James W. Kazura, Dan Milner, John W. Barnwell, Gareth Turner, Jean Langhorne, on behalf of the participants of the Hinxton Retreat meeting on “Animal Models for Research on Severe Malaria”

Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death

2012-02-02T22:00:00Z

by Roxane Simeone, Alexandre Bobard, Juliane Lippmann, Wilbert Bitter, Laleh Majlessi, Roland Brosch, Jost Enninga

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective.

Absence of HIV-1 Evolution in the Gut-Associated Lymphoid Tissue from Patients on Combination Antiviral Therapy Initiated during Primary Infection

2012-02-02T22:00:00Z

by Teresa H. Evering, Saurabh Mehandru, Paul Racz, Klara Tenner-Racz, Michael A. Poles, Amir Figueroa, Hiroshi Mohri, Martin Markowitz

Mucosal mononuclear (MMC) CCR5+CD4+ T cells of the gastrointestinal (GI) tract are selectively infected and depleted during acute HIV-1 infection. Despite early initiation of combination antiretroviral therapy (cART), gut-associated lymphoid tissue (GALT) CD4+ T cell depletion and activation persist in the majority of HIV-1 positive individuals studied. This may result from ongoing HIV-1 replication and T-cell activation despite effective cART. We hypothesized that ongoing viral replication in the GI tract during cART would result in measurable viral evolution, with divergent populations emerging over time. Subjects treated during early HIV-1 infection underwent phlebotomy and flexible sigmoidoscopy with biopsies prior to and 15–24 months post initiation of cART. At the 2^nd biopsy, three GALT phenotypes were noted, characterized by high, intermediate and low levels of immune activation. A representative case from each phenotype was analyzed. Each subject had plasma HIV-1 RNA levels <50 copies/ml at 2^nd GI biopsy and CD4+ T cell reconstitution in the peripheral blood. Single genome amplification of full-length HIV-1 envelope was performed for each subject pre- and post-initiation of cART in GALT and PBMC. A total of 280 confirmed single genome sequences (SGS) were analyzed for experimental cases. For each subject, maximum likelihood phylogenetic trees derived from molecular sequence data showed no evidence of evolved forms in the GALT over the study period. During treatment, HIV-1 envelope diversity in GALT-derived SGS did not increase and post-treatment GALT-derived SGS showed no substantial genetic divergence from pre-treatment sequences within transmitted groups. Similar results were obtained from PBMC-derived SGS. Our results reveal that initiation of cART during acute/early HIV-1 infection can result in the interruption of measurable viral evolution in the GALT, suggesting the absence of de-novo rounds of HIV-1 replication in this compartment during suppressive cART.

Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

2012-02-02T22:00:00Z

by Susana Gardete, Choonkeun Kim, Boris M. Hartmann, Michael Mwangi, Christelle M. Roux, Paul M. Dunman, Henry F. Chambers, Alexander Tomasz

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system.

Biochemical Properties of Highly Neuroinvasive Prion Strains

2012-02-02T22:00:00Z

by Cyrus Bett, Shivanjali Joshi-Barr, Melanie Lucero, Margarita Trejo, Pawel Liberski, Jeffery W. Kelly, Eliezer Masliah, Christina J. Sigurdson

Infectious prions propagate from peripheral entry sites into the central nervous system (CNS), where they cause progressive neurodegeneration that ultimately leads to death. Yet the pathogenesis of prion disease can vary dramatically depending on the strain, or conformational variant of the aberrantly folded and aggregated protein, PrP^Sc. Although most prion strains invade the CNS, some prion strains cannot gain entry and do not cause clinical signs of disease. The conformational basis for this remarkable variation in the pathogenesis among strains is unclear. Using mouse-adapted prion strains, here we show that highly neuroinvasive prion strains primarily form diffuse aggregates in brain and are noncongophilic, conformationally unstable in denaturing conditions, and lead to rapidly lethal disease. These neuroinvasive strains efficiently generate PrP^Sc over short incubation periods. In contrast, the weakly neuroinvasive prion strains form large fibrillary plaques and are stable, congophilic, and inefficiently generate PrP^Sc over long incubation periods. Overall, these results indicate that the most neuroinvasive prion strains are also the least stable, and support the concept that the efficient replication and unstable nature of the most rapidly converting prions may be a feature linked to their efficient spread into the CNS.

Viral Cyclins Mediate Separate Phases of Infection by Integrating Functions of Distinct Mammalian Cyclins

2012-02-02T22:00:00Z

by Katherine S. Lee, Andrea L. Suarez, David J. Claypool, Taylor K. Armstrong, Erin M. Buckingham, Linda F. van Dyk

Gammaherpesvirus cyclins have expanded biochemical features relative to mammalian cyclins, and promote infection and pathogenesis including acute lung infection, viral persistence, and reactivation from latency. To define the essential features of the viral cyclin, we generated a panel of knock-in viruses expressing various viral or mammalian cyclins from the murine gammaherpesvirus 68 cyclin locus. Viral cyclins of both gammaherpesvirus 68 and Kaposi's sarcoma-associated herpesvirus supported all cyclin-dependent stages of infection, indicating functional conservation. Although mammalian cyclins could not restore lung replication, they did promote viral persistence and reactivation. Strikingly, distinct and non-overlapping mammalian cyclins complemented persistence (cyclin A, E) or reactivation from latency (cyclin D3). Based on these data, unique biochemical features of viral cyclins (e.g. enhanced kinase activation) are not essential to mediate specific processes during infection. What is essential for, and unique to, the viral cyclins is the integration of the activities of several different mammalian cyclins, which allows viral cyclins to mediate multiple, discrete stages of infection. These studies also demonstrated that closely related stages of infection, that are cyclin-dependent, are in fact genetically distinct, and thus predict that cyclin requirements may be used to tailor potential therapies for virus-associated diseases.

Selecting an Invertebrate Model Host for the Study of Fungal Pathogenesis

2012-02-02T22:00:00Z

by Athanasios Desalermos, Beth Burgwyn Fuchs, Eleftherios Mylonakis

Discrete Cyclic di-GMP-Dependent Control of Bacterial Predation versus Axenic Growth in Bdellovibrio bacteriovorus

2012-02-02T22:00:00Z

by Laura Hobley, Rowena K. Y. Fung, Carey Lambert, Maximilian A. T. S. Harris, Jayesh M. Dabhi, Simon S. King, Sarah M. Basford, Kaoru Uchida, Robert Till, Rashidah Ahmad, Shin-Ichi Aizawa, Mark Gomelsky, R. Elizabeth Sockett

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways.

Msb2 Shedding Protects Candida albicans against Antimicrobial Peptides

2012-02-02T22:00:00Z

by Eva Szafranski-Schneider, Marc Swidergall, Fabien Cottier, Denis Tielker, Elvira Román, Jesus Pla, Joachim F. Ernst

Msb2 is a sensor protein in the plasma membrane of fungi. In the human fungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinase pathway to maintain cell wall integrity and allow filamentous growth. Msb2 doubly epitope-tagged in its large extracellular and small cytoplasmic domain was efficiently cleaved during liquid and surface growth and the extracellular domain was almost quantitatively released into the growth medium. Msb2 cleavage was independent of proteases Sap9, Sap10 and Kex2. Secreted Msb2 was highly O-glycosylated by protein mannosyltransferases including Pmt1 resulting in an apparent molecular mass of >400 kDa. Deletion analyses revealed that the transmembrane region is required for Msb2 function, while the large N-terminal and the small cytoplasmic region function to downregulate Msb2 signaling or, respectively, allow its induction by tunicamycin. Purified extracellular Msb2 domain protected fungal and bacterial cells effectively from antimicrobial peptides (AMPs) histatin-5 and LL-37. AMP inactivation was not due to degradation but depended on the quantity and length of the Msb2 glycofragment. C. albicans msb2 mutants were supersensitive to LL-37 but not histatin-5, suggesting that secreted rather than cell-associated Msb2 determines AMP protection. Thus, in addition to its sensor function Msb2 has a second activity because shedding of its glycofragment generates AMP quorum resistance.

Immune Subversion and Quorum-Sensing Shape the Variation in Infectious Dose among Bacterial Pathogens

2012-02-02T22:00:00Z

by João Alves Gama, Sophie S. Abby, Sara Vieira-Silva, Francisco Dionisio, Eduardo P. C. Rocha

Many studies have been devoted to understand the mechanisms used by pathogenic bacteria to exploit human hosts. These mechanisms are very diverse in the detail, but share commonalities whose quantification should enlighten the evolution of virulence from both a molecular and an ecological perspective. We mined the literature for experimental data on infectious dose of bacterial pathogens in humans (ID50) and also for traits with which ID50 might be associated. These compilations were checked and complemented with genome analyses. We observed that ID50 varies in a continuous way by over 10 orders of magnitude. Low ID50 values are very strongly associated with the capacity of the bacteria to kill professional phagocytes or to survive in the intracellular milieu of these cells. Inversely, high ID50 values are associated with motile and fast-growing bacteria that use quorum-sensing based regulation of virulence factors expression. Infectious dose is not associated with genome size and shows insignificant phylogenetic inertia, in line with frequent virulence shifts associated with the horizontal gene transfer of a small number of virulence factors. Contrary to previous proposals, infectious dose shows little dependence on contact-dependent secretion systems and on the natural route of exposure. When all variables are combined, immune subversion and quorum-sensing are sufficient to explain two thirds of the variance in infectious dose. Our results show the key role of immune subversion in effective human infection by small bacterial populations. They also suggest that cooperative processes might be important for successful infection by bacteria with high ID50. Our results suggest that trade-offs between selection for population growth-related traits and selection for the ability to subvert the immune system shape bacterial infectiousness. Understanding these trade-offs provides guidelines to study the evolution of virulence and in particular the micro-evolutionary paths of emerging pathogens.

The CTLA-4 and PD-1/PD-L1 Inhibitory Pathways Independently Regulate Host Resistance to Plasmodium-induced Acute Immune Pathology

2012-02-02T22:00:00Z

by Julius Clemence R. Hafalla, Carla Claser, Kevin N. Couper, Georges Emile Grau, Laurent Renia, J. Brian de Souza, Eleanor M. Riley

The balance between pro-inflammatory and regulatory immune responses in determining optimal T cell activation is vital for the successful resolution of microbial infections. This balance is maintained in part by the negative regulators of T cell activation, CTLA-4 and PD-1/PD-L, which dampen effector responses during chronic infections. However, their role in acute infections, such as malaria, remains less clear. In this study, we determined the contribution of CTLA-4 and PD-1/PD-L to the regulation of T cell responses during Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM) in susceptible (C57BL/6) and resistant (BALB/c) mice. We found that the expression of CTLA-4 and PD-1 on T cells correlates with the extent of pro-inflammatory responses induced during PbA infection, being higher in C57BL/6 than in BALB/c mice. Thus, ECM develops despite high levels of expression of these inhibitory receptors. However, antibody-mediated blockade of either the CTLA-4 or PD-1/PD-L1, but not the PD-1/PD-L2, pathways during PbA-infection in ECM-resistant BALB/c mice resulted in higher levels of T cell activation, enhanced IFN-γ production, increased intravascular arrest of both parasitised erythrocytes and CD8⁺ T cells to the brain, and augmented incidence of ECM. Thus, in ECM-resistant BALB/c mice, CTLA-4 and PD-1/PD-L1 represent essential, independent and non-redundant pathways for maintaining T cell homeostasis during a virulent malaria infection. Moreover, neutralisation of IFN-γ or depletion of CD8⁺ T cells during PbA infection was shown to reverse the pathologic effects of regulatory pathway blockade, highlighting that the aetiology of ECM in the BALB/c mice is similar to that in C57BL/6 mice. In summary, our results underscore the differential and complex regulation that governs immune responses to malaria parasites.

Identification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus

2012-01-26T22:00:00Z

by Teresa A. Figueiredo, Rita G. Sobral, Ana Madalena Ludovice, João Manuel Feio de Almeida, Nhat K. Bui, Waldemar Vollmer, Hermínia de Lencastre, Alexander Tomasz

The glutamic acid residues of the peptidoglycan of Staphylococcus aureus and many other bacteria become amidated by an as yet unknown mechanism. In this communication we describe the identification, in the genome of S. aureus strain COL, of two co-transcribed genes, murT and gatD, which are responsible for peptidoglycan amidation. MurT and GatD have sequence similarity to substrate-binding domains in Mur ligases (MurT) and to the catalytic domain in CobB/CobQ-like glutamine amidotransferases (GatD). The amidation of glutamate residues in the stem peptide of S. aureus peptidoglycan takes place in a later step than the cytoplasmic phase – presumably the lipid phase - of the biosynthesis of the S. aureus cell wall precursor. Inhibition of amidation caused reduced growth rate, reduced resistance to beta-lactam antibiotics and increased sensitivity to lysozyme which inhibited culture growth and caused degradation of the peptidoglycan.

The Viral and Cellular MicroRNA Targetome in Lymphoblastoid Cell Lines

2012-01-26T22:00:00Z

by Rebecca L. Skalsky, David L. Corcoran, Eva Gottwein, Christopher L. Frank, Dong Kang, Markus Hafner, Jeffrey D. Nusbaum, Regina Feederle, Henri-Jacques Delecluse, Micah A. Luftig, Thomas Tuschl, Uwe Ohler, Bryan R. Cullen

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3′UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3′UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.

The Murine Coronavirus Hemagglutinin-esterase Receptor-binding Site: A Major Shift in Ligand Specificity through Modest Changes in Architecture

2012-01-26T22:00:00Z

by Martijn A. Langereis, Qinghong Zeng, Balthasar Heesters, Eric G. Huizinga, Raoul J. de Groot

The hemagglutinin-esterases (HEs), envelope glycoproteins of corona-, toro- and orthomyxoviruses, mediate reversible virion attachment to O-acetylated sialic acids (O-Ac-Sias). They do so through concerted action of distinct receptor-binding (“lectin”) and receptor-destroying sialate O-acetylesterase (”esterase”) domains. Most HEs target 9-O-acetylated Sias. In one lineage of murine coronaviruses, however, HE esterase substrate and lectin ligand specificity changed dramatically as these viruses evolved to use 4-O-acetylated Sias instead. Here we present the crystal structure of the lectin domain of mouse hepatitis virus (MHV) strain S HE, resolved both in its native state and in complex with a receptor analogue. The data show that the shift from 9-O- to 4-O-Ac-Sia receptor usage primarily entailed a change in ligand binding topology and, surprisingly, only modest changes in receptor-binding site architecture. Our findings illustrate the ease with which viruses can change receptor-binding specificity with potential consequences for host-, organ and/or cell tropism, and for pathogenesis.

IL-10R Blockade during Chronic Schistosomiasis Mansoni Results in the Loss of B Cells from the Liver and the Development of Severe Pulmonary Disease

2012-01-26T22:00:00Z

by Keke C. Fairfax, Eyal Amiel, Irah L. King, Tori C. Freitas, Markus Mohrs, Edward J. Pearce

In schistosomiasis patients, parasite eggs trapped in hepatic sinusoids become foci for CD4⁺ T cell-orchestrated granulomatous cellular infiltrates. Since the immune response is unable to clear the infection, the liver is subjected to ongoing cycles of focal inflammation and healing that lead to vascular obstruction and tissue fibrosis. This is mitigated by regulatory mechanisms that develop over time and which minimize the inflammatory response to newly deposited eggs. Exploring changes in the hepatic inflammatory infiltrate over time in infected mice, we found an accumulation of schistosome egg antigen-specific IgG1-secreting plasma cells during chronic infection. This population was significantly diminished by blockade of the receptor for IL-10, a cytokine implicated in plasma cell development. Strikingly, IL-10R blockade precipitated the development of portal hypertension and the accumulation of parasite eggs in the lungs and heart. This did not reflect more aggressive Th2 cell responsiveness, increased hepatic fibrosis, or the emergence of Th1 or Th17 responses. Rather, a role for antibody in the prevention of severe disease was suggested by the finding that pulmonary involvement was also apparent in mice unable to secrete class switched antibody. A major effect of anti-IL-10R treatment was the loss of a myeloid population that stained positively for surface IgG1, and which exhibited characteristics of regulatory/anti-inflammatory macrophages. This finding suggests that antibody may promote protective effects within the liver through local interactions with macrophages. In summary, our data describe a role for IL-10-dependent B cell responses in the regulation of tissue damage during a chronic helminth infection.