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Showing papers in "PLOS Pathogens in 2012"


Journal ArticleDOI
TL;DR: This review highlights immunological events that may accelerate the development of one of the two diseases in the presence of the co-infecting organism and describes gaps in knowledge and needs for future studies to develop preventive measures against theTwo diseases.
Abstract: Tuberculosis (TB) and HIV co-infections place an immense burden on health care systems and pose particular diagnostic and therapeutic challenges. Infection with HIV is the most powerful known risk factor predisposing for Mycobacterium tuberculosis infection and progression to active disease, which increases the risk of latent TB reactivation 20-fold. TB is also the most common cause of AIDS-related death. Thus, M. tuberculosis and HIV act in synergy, accelerating the decline of immunological functions and leading to subsequent death if untreated. The mechanisms behind the breakdown of the immune defense of the co-infected individual are not well known. The aim of this review is to highlight immunological events that may accelerate the development of one of the two diseases in the presence of the co-infecting organism. We also review possible animal models for studies of the interaction of the two pathogens, and describe gaps in knowledge and needs for future studies to develop preventive measures against the two diseases.

640 citations


Journal ArticleDOI
TL;DR: It is recommended that Salmonella classification by serotyping should be replaced by MLST or its equivalents as it confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings.
Abstract: Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.

552 citations


Journal ArticleDOI
TL;DR: A rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis is demonstrated.
Abstract: Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis.

536 citations


Journal ArticleDOI
TL;DR: The Dothideomycetes are one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts as mentioned in this paper.
Abstract: The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.

514 citations


Journal ArticleDOI
TL;DR: It is proposed that molecular epidemiology performed for surveillance and outbreak investigation and genotypic antimicrobial susceptibility testing for microbes that are difficult to grow represent the most immediate areas for application of WGS.
Abstract: Whole genome sequencing (WGS) promises to be transformative for the practice of clinical microbiology, and the rapidly falling cost and turnaround time mean that this will become a viable technology in diagnostic and reference laboratories in the near future. The objective of this article is to consider at a very practical level where, in the context of a modern diagnostic microbiology laboratory, WGS might be cost-effective compared to current alternatives. We propose that molecular epidemiology performed for surveillance and outbreak investigation and genotypic antimicrobial susceptibility testing for microbes that are difficult to grow represent the most immediate areas for application of WGS, and discuss the technical and infrastructure requirements for this to be implemented.

488 citations


Journal ArticleDOI
TL;DR: The Ross-Macdonald theory has since played a central role in development of research on mosquito-borne pathogen transmission and the development of strategies for mosquitoes-borne disease prevention.
Abstract: Ronald Ross and George Macdonald are credited with developing a mathematical model of mosquito-borne pathogen transmission. A systematic historical review suggests that several mathematicians and scientists contributed to development of the Ross-Macdonald model over a period of 70 years. Ross developed two different mathematical models, Macdonald a third, and various “Ross-Macdonald” mathematical models exist. Ross-Macdonald models are best defined by a consensus set of assumptions. The mathematical model is just one part of a theory for the dynamics and control of mosquito-transmitted pathogens that also includes epidemiological and entomological concepts and metrics for measuring transmission. All the basic elements of the theory had fallen into place by the end of the Global Malaria Eradication Programme (GMEP, 1955–1969) with the concept of vectorial capacity, methods for measuring key components of transmission by mosquitoes, and a quantitative theory of vector control. The Ross-Macdonald theory has since played a central role in development of research on mosquito-borne pathogen transmission and the development of strategies for mosquito-borne disease prevention.

481 citations


Journal ArticleDOI
TL;DR: A single-cell fluorescence resonance energy transfer (FRET)-based method is used to investigate the potential cytosolic access of M. tuberculosis and provides a novel mechanism how ESX-1 competent, virulent M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells.
Abstract: 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.

472 citations


Journal ArticleDOI
TL;DR: The morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those from the closely related flavivirus.
Abstract: All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

456 citations


Journal ArticleDOI
TL;DR: There is good reason for optimism in the field of TB vaccines that it will be possible to develop better vaccines than BCG, which is still the only vaccine available against TB.
Abstract: In this review we discuss recent progress in the development, testing, and clinical evaluation of new vaccines against tuberculosis (TB). Over the last 20 years, tremendous progress has been made in TB vaccine research and development: from a pipeline virtually empty of new TB candidate vaccines in the early 1990s, to an era in which a dozen novel TB vaccine candidates have been and are being evaluated in human clinical trials. In addition, innovative approaches are being pursued to further improve existing vaccines, as well as discover new ones. Thus, there is good reason for optimism in the field of TB vaccines that it will be possible to develop better vaccines than BCG, which is still the only vaccine available against TB.

425 citations


Journal ArticleDOI
TL;DR: Evidence is presented that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections, and the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status.
Abstract: The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.

413 citations


Journal ArticleDOI
TL;DR: The mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface are explored, illustrating the critical interaction between matrix architecture and pH heterogeneity in the 3D environment.
Abstract: Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.

Journal ArticleDOI
TL;DR: It is shown that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition ofDENV infection and spread in mice.
Abstract: Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.

Journal ArticleDOI
TL;DR: An integrated analysis of the population and molecular changes associated with the collapse of honeybees infested by the parasitic mite Varroa destructor shows that this parasite can de-stabilise the within-host dynamics of Deformed wing virus (DWV), transforming a cryptic and vertically transmitted virus into a rapidly replicating killer, which attains lethal levels late in the season.
Abstract: The health of the honeybee and, indirectly, global crop production are threatened by several biotic and abiotic factors, which play a poorly defined role in the induction of widespread colony losses. Recent descriptive studies suggest that colony losses are often related to the interaction between pathogens and other stress factors, including parasites. Through an integrated analysis of the population and molecular changes associated with the collapse of honeybee colonies infested by the parasitic mite Varroa destructor, we show that this parasite can de-stabilise the within-host dynamics of Deformed wing virus (DWV), transforming a cryptic and vertically transmitted virus into a rapidly replicating killer, which attains lethal levels late in the season. The de-stabilisation of DWV infection is associated with an immunosuppression syndrome, characterized by a strong down-regulation of the transcription factor NF-κB. The centrality of NF-κB in host responses to a range of environmental challenges suggests that this transcription factor can act as a common currency underlying colony collapse that may be triggered by different causes. Our results offer an integrated account for the multifactorial origin of honeybee losses and a new framework for assessing, and possibly mitigating, the impact of environmental challenges on honeybee health.

Journal ArticleDOI
TL;DR: It is shown that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance, illustrating how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.
Abstract: Pest and pathogen losses jeopardise global food security and ever since the 19 th century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

Journal ArticleDOI
TL;DR: It is concluded that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation.
Abstract: Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

Journal ArticleDOI
TL;DR: New in vivo models that recapitulate persistent infections associated with viral pathogens are required and new ways of interacting with the host miRNA machinery including noncanonical miRNA biogenesis and new mechanisms of posttranscriptional cis gene regulation are revealed.
Abstract: MicroRNAs (miRNAs) are small RNAs that play important roles in the regulation of gene expression. First described as posttranscriptional gene regulators in eukaryotic hosts, virus-encoded miRNAs were later uncovered. It is now apparent that diverse virus families, most with DNA genomes, but at least some with RNA genomes, encode miRNAs. While deciphering the func- tions of viral miRNAs has lagged behind their discovery, recent functional studies are bringing into focus these roles. Some of the best characterized viral miRNA functions include subtle roles in prolonging the longevity of infected cells, evading the immune response, and regulating the switch to lytic infection. Notably, all of these functions are particularly important during persis- tent infections. Furthermore, an emerging view of viral miRNAs suggests two distinct groups exist. In the first group, viral miRNAs mimic host miRNAs and take advantage of conserved networks of host miRNA target sites. In the larger second group, viral miRNAs do not share common target sites conserved for host miRNAs, and it remains unclear what fraction of these targeted transcripts are beneficial to the virus. Recent insights from multiple virus families have revealed new ways of interacting with the host miRNA machinery including noncanonical miRNA biogenesis and new mechanisms of posttranscriptional cis gene regulation. Exciting challeng- es await the field, including determining the most relevant miRNA targets and parlaying our current understanding of viral miRNAs into new therapeutic strategies. To accomplish these goals and to better grasp miRNA function, new in vivo models that recapitulate persistent infections associated with viral pathogens are required.

Journal ArticleDOI
TL;DR: It is revealed that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment, and myc-regulated host mi RNAs from the miR-17/92 cluster can regulate latent viral gene expression.
Abstract: 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.

Journal ArticleDOI
TL;DR: The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges.
Abstract: Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ~2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (~six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a basis for developing Marburg hemorrhagic fever risk reduction strategies.


Journal ArticleDOI
TL;DR: Hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death, revealing new levels of functional complexity for this fungal organ.
Abstract: Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death.

Journal ArticleDOI
TL;DR: It is concluded that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity, which represents a novel strategy for establishing a biotrophic interaction.
Abstract: The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1) as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H2O2 strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction.

Journal ArticleDOI
TL;DR: It is suggested that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication.
Abstract: The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma. We also present evidence that suggests virus-derived piRNA-like small RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication.

Journal ArticleDOI
TL;DR: Dating estimates, together with striking phylogenetic and quantitative human-bacterial similarities show that H. pylori is approximately as old as are anatomically modern humans, and suggest that it may have been acquired via a single host jump from an unknown, non-human host.
Abstract: When modern humans left Africa ca. 60,000 years ago (60 kya), they were already infected with Helicobacter pylori, and these bacteria have subsequently diversified in parallel with their human hosts. But how long were humans infected by H. pylori prior to the out-of-Africa event? Did this co-evolution predate the emergence of modern humans, spanning the species divide? To answer these questions, we investigated the diversity of H. pylori in Africa, where both humans and H. pylori originated. Three distinct H. pylori populations are native to Africa: hpNEAfrica in Afro-Asiatic and Nilo-Saharan speakers, hpAfrica1 in Niger-Congo speakers and hpAfrica2 in South Africa. Rather than representing a sustained co-evolution over millions of years, we find that the coalescent for all H. pylori plus its closest relative H. acinonychis dates to 88–116 kya. At that time the phylogeny split into two primary super-lineages, one of which is associated with the former hunter-gatherers in southern Africa known as the San. H. acinonychis, which infects large felines, resulted from a later host jump from the San, 43–56 kya. These dating estimates, together with striking phylogenetic and quantitative human-bacterial similarities show that H. pylori is approximately as old as are anatomically modern humans. They also suggest that H. pylori may have been acquired via a single host jump from an unknown, non-human host. We also find evidence for a second Out of Africa migration in the last 52,000 years, because hpEurope is a hybrid population between hpAsia2 and hpNEAfrica, the latter of which arose in northeast Africa 36–52 kya, after the Out of Africa migrations around 60 kya.

Journal ArticleDOI
TL;DR: The discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity is reported, and data is presented showing that soluble PSM peptides disperse biofilms while polymerized peptides do not.
Abstract: Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms.

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TL;DR: Virulence factors, most notably, type III secretion systems, Vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and various factors essential for the intracellular life cycle of S. enterica have been characterized.
Abstract: Salmonella enterica infections result in diverse clinical manifestations. Typhoid fever, caused by S. enterica serovar Typhi (S. Typhi) and S. Paratyphi A, is a bacteremic illness but whose clinical features differ from other Gram-negative bacteremias. Non-typhoidal Salmonella (NTS) serovars cause self-limiting diarrhea with occasional secondary bacteremia. Primary NTS bacteremia can occur in the immunocompromised host and infants in sub-Saharan Africa. Recent studies on host–pathogen interactions in Salmonellosis using genome sequencing, murine models, and patient studies have provided new insights. The full genome sequences of numerous S. enterica serovars have been determined. The S. Typhi genome, compared to that of S. Typhimurium, harbors many inactivated or disrupted genes. This can partly explain the different immune responses both serovars induce upon entering their host. Similar genome degradation is also observed in the ST313 S. Typhimurium strain implicated in invasive infection in sub-Saharan Africa. Virulence factors, most notably, type III secretion systems, Vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and various factors essential for the intracellular life cycle of S. enterica have been characterized. Genes for these factors are commonly carried on Salmonella Pathogenicity Islands (SPIs). Plasmids also carry putative virulence-associated genes as well as those responsible for antimicrobial resistance. The interaction of Salmonella pathogen-associated molecular patterns (PAMPs) with Toll-like receptors (TLRs) and NOD-like receptors (NLRs) leads to inflammasome formation, activation, and recruitment of neutrophils and macrophages and the production of pro-inflammatory cytokines, most notably interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and interferon-gamma (IFN)-γ. The gut microbiome may be an important modulator of this immune response. S. Typhimurium usually causes a local intestinal immune response, whereas S. Typhi, by preventing neutrophil attraction resulting from activation of TLRs, evades the local response and causes systemic infection. Potential new therapeutic strategies may lead from an increased understanding of infection pathogenesis.

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TL;DR: It is demonstrated that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells in the large intestine through the TLR2/MyD88 pathway.
Abstract: Specific intestinal microbiota has been shown to induce Foxp3+ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103+ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103+ DCs from Il10−/−, Tlr2−/−, and Myd88−/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103+ DCs failed to induce IL-10 production from co-cultured Il27ra−/− T cells. B. breve treatment of Tlr2−/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103+ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naive CD4+ T cells from wild-type mice, but not Il10−/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.

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TL;DR: A meeting to discuss the relative merits of a range of animal models and their overlap with the complex clinical syndromes of human disease brought the two research communities closer together to identify further work to provide information needed to validate the model and revitalise the development of other animal models displaying features of human pathology.
Abstract: In light of the recent controversies over the role of animal models for research into the development of new treatments for severe malaria, particularly cerebral disease, a group of scientists came together to discuss the relative merits of a range of animal models and their overlap with the complex clinical syndromes of human disease. While it was not possible to fully resolve differences over the utility of the Plasmodium berghei ANKA model of experimental cerebral malaria, the meeting did bring the two research communities closer together to identify further work to provide information needed to validate the model and revitalise the development of other animal models displaying features of human pathology. The driving force behind this was the desire to ensure better translation of experimental findings into effective treatments for severe malaria.

Journal ArticleDOI
TL;DR: Results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.
Abstract: Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.

Journal ArticleDOI
TL;DR: It is shown that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis and a new, physiologically relevant protein substrate of LT is identified.
Abstract: NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.

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TL;DR: The results reinforce the idea that the fundamental mechanism involved in viral interference in Drosophila and Aedes is not dependent on the up-regulation of the immune effectors examined, although it cannot be excluded that immune priming in the heterologous mosquito host might enhance the virus interference trait.
Abstract: The non-virulent Wolbachia strain wMel and the life-shortening strain wMelPop-CLA, both originally from Drosophila melanogaster, have been stably introduced into the mosquito vector of dengue fever, Aedes aegypti. Each of these Wolbachia strains interferes with viral pathogenicity and/or dissemination in both their natural Drosophila host and in their new mosquito host, and it has been suggested that this virus interference may be due to host immune priming by Wolbachia. In order to identify aspects of the mosquito immune response that might underpin virus interference, we used whole-genome microarrays to analyse the transcriptional response of A. aegypti to the wMel and wMelPop-CLA Wolbachia strains. While wMel affected the transcription of far fewer host genes than wMelPop-CLA, both strains activated the expression of some immune genes including anti-microbial peptides, Toll pathway genes and genes involved in melanization. Because the induction of these immune genes might be associated with the very recent introduction of Wolbachia into the mosquito, we also examined the same Wolbachia strains in their original host D. melanogaster. First we demonstrated that when dengue viruses were injected into D. melanogaster, virus accumulation was significantly reduced in the presence of Wolbachia, just as in A. aegypti. Second, when we carried out transcriptional analyses of the same immune genes up-regulated in the new heterologous mosquito host in response to Wolbachia we found no over-expression of these genes in D. melanogaster, infected with either wMel or wMelPop. These results reinforce the idea that the fundamental mechanism involved in viral interference in Drosophila and Aedes is not dependent on the up-regulation of the immune effectors examined, although it cannot be excluded that immune priming in the heterologous mosquito host might enhance the virus interference trait.