PLoS Pathogens: New Articles

Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites

2010-02-05T08:00:00Z

Author Summary

Malaria remains a major public health problem in many parts of the tropical world. All the clinical symptoms of malaria are attributed to the blood stage of the parasite life cycle during which Plasmodium merozoites invade and multiply within host erythrocytes. Invasion by Plasmodium merozoites is a complex process that requires multiple molecular interactions between the invading parasite and target erythrocyte. Parasite proteins that mediate such interactions are localized in membrane bound internal organelles at the apical end of merozoites called micronemes and rhoptries. The timely secretion of microneme and rhoptry proteins to the merozoite surface to allow receptor binding is a crucial step in the invasion process. In this study, we demonstrate that exposure of Plasmodium falciparum merozoites to low potassium ion concentrations as found in blood plasma provides the natural signal that triggers a rise in intracellular calcium, which in turn triggers secretion of microneme proteins to the merozoite surface. Subsequently, binding of released microneme proteins with erythrocyte receptors provides the signal for release of rhoptry proteins. These studies open the path for analysis of signal transduction pathways involved in apical organelle secretion. A clear understanding of these pathways will enable development of inhibitors that block secretion of key parasite proteins required for receptor-binding. Such inhibitors will block erythrocyte invasion and inhibit parasite growth, providing promising leads for development of novel drugs against malaria.

Immunoelectron Microscopic Evidence for Tetherin/BST2 as the Physical Bridge between HIV-1 Virions and the Plasma Membrane

2010-02-05T08:00:00Z

Author Summary

Tetherin or BST2 is a cellular protein that was recently found to limit the ability of HIV to escape from cells. HIV counteracts this cellular restriction to its lifecycle by expressing the small viral accessory protein Vpu. Upon viral infection, cells expressing high levels of tetherin accumulate large clusters or strings of virions that remain attached to the plasma membrane by an unknown mechanism. The simplest explanation for this clustering is that tetherin itself physically attaches particles to the plasma membrane and to each other. In this article, we demonstrate that this is indeed the case. We found that particles released from cells by gentle protease treatment contain either cleaved or full-length tetherin. Using electron microscopy and immunogold staining, we show that tetherin is present as a physical link between viruses and the plasma membrane and sometimes between virus particles in large clusters or strings. Together this provides evidence that tetherin serves a direct, physical role in retaining particles on the surface of cells.

Fis Is Essential for Capsule Production in Pasteurella multocida and Regulates Expression of Other Important Virulence Factors

2010-02-05T08:00:00Z

Author Summary

Pasteurella multocida is an animal pathogen of worldwide economic significance. It causes fowl cholera in wild birds and poultry, hemorrhagic septicemia in ungulates, and atrophic rhinitis in swine. The major virulence factor in fowl cholera-causing isolates is the polysaccharide capsule, which is composed of hyaluronic acid. Although there have been reports of spontaneous capsule loss in some strains, to date there has been no systematic investigation into the molecular mechanisms of this phenomenon. In this study, we describe for the first time the underlying transcriptional mechanisms required for the expression of capsule in P. multocida, and identify a transcriptional regulator required for capsule production.

Structural and Biochemical Characterization of SrcA, a Multi-Cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

2010-02-05T08:00:00Z

Author Summary

Systemic typhoid fever caused by Salmonella enterica serovar Typhi leads to high mortality in the developing world and can be linked with chronic, persistent infections in survivors. To cause disease, Salmonella uses a specialized secretion device called a type III secretion system to disarm cells of the immune system and replicate within them. The assembly and function of this secretion system requires a set of chaperone proteins to direct the process, but the chaperone proteins themselves have remained elusive. Here, we found a new chaperone protein, called SrcA, which is required for proper function of the type III secretion system. Using a bacterial mutant lacking the srcA gene, we found that this chaperone was needed for Salmonella to compete against wild type cells during systemic disease because it controls secretion of at least 2 key proteins involved in immune escape and cell-to-cell transmission. This chaperone is present in all types of virulent Salmonella, but not in Salmonella that don't cause human infections, providing new insights into the pathogenic nature of this organism.

An Extensive Circuitry for Cell Wall Regulation in Candida albicans

2010-02-05T08:00:00Z

Author Summary

Candida albicans is the major fungal commensal and pathogen of humans. Like most microorganisms, C. albicans is surrounded and protected by a cell wall. This cell wall has two purposes: to act as a rigid “exoskeleton” to prevent cells from bursting, and to provide a surface where a cell can interact with the outside environment while protecting the cell itself from this environment. Maintenance of this structure has been well studied in the model fungus, Saccharomyces cerevisiae, but previous evidence suggested that C. albicans might have additional inputs to this process. By creating and testing a library of mutant strains for sensitivity to cell wall stress, we were able to identify a number of conserved genes with roles in this process not shared by their S. cerevisiae counterparts. Although some of these genes had previously been linked to cell wall integrity, it appears that they have increased impact on this process in C. albicans. For other genes, their role in cell wall integrity may represent a novel connection. Our findings provide insight into novel aspects of cell wall integrity in this pathogen and lay a foundation for its more detailed analysis.

Reverse Genetics in Candida albicans Predicts ARF Cycling Is Essential for Drug Resistance and Virulence

2010-02-05T08:00:00Z

Author Summary

Candida albicans is a fungus that normally resides as part of the microflora in the human gut. Candida species can cause superficial infections like thrush in the healthy human population and life-threatening invasive infections in immunocompromised patients. Fungal infections are often treated with azole drugs, but due to the fungistatic nature of these agents, C. albicans can develop drug resistance, leading to therapy failure. To improve the action of azoles and convert them into fungicidal drugs, we first systematically analyzed the genetic requirements for tolerance to one such azole drug, fluconazole. We show, both genetically and pharmacologically, that components of the ARF cycling machinery are critical in mediating both azole and echinocandin tolerance in C. albicans as well as several other pathogenic Candida species and in the pathogenic mold Aspergillus fumigatus. We highlight the importance of ARF cycling in drug resistance by showing that genetic compromise of ARF functions overrides common drug resistance mechanisms in clinical samples and other key regulators of azole/echinocandin tolerance. We validated the therapeutic potential of ARF cycling in two mouse models and provide evidence that drug treatment is more efficacious when ARF activities are genetically compromised. Our study demonstrates a new mechanism involved in two important aspects of the biology of human fungal pathogens and provides a potential route for improved antifungal therapies.

TgMORN1 Is a Key Organizer for the Basal Complex of Toxoplasma gondii

2010-02-05T08:00:00Z

Author Summary

The disease toxoplasmosis is the result of uncontrolled growth and proliferation of the intracellular parasite Toxoplasma gondii, which is pathogenic for most warm-blooded animals. If growth of the parasite is blocked, then it does not cause disease, even though it may persist in the host as a chronic infection. Proper assembly of the cytoskeleton of T. gondii is known to be essential for its growth, and consequently required for virulence. In this study, we investigated the function of a novel cytoskeletal protein, TgMORN1, in T. gondii. TgMORN1 is a major component of the basal complex, a novel cytoskeletal assembly located at the posterior end of the parasite. We found that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end and is important for ensuring successful separation of daughters at late stage of parasite replication. In addition, infection with parasites deficient in TgMORN1 not only failed to kill mice but also provided protective immunity against a lethal challenge infection, indicating the importance of TgMORN1 in T. gondii growth both in vitro and in vivo.

Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice

2010-02-05T08:00:00Z

Author Summary

When infectious agents enter our body, neutrophils are the first cells recruited to the scene. In addition to their capacity to kill microbes, neutrophils secrete molecules that attract other cells also involved in immune defense, such as dendritic cells (DCs). Here, we investigate the secretion of DC-attracting chemokines by neutrophils after inoculation of mice with Leishmania major, a protozoan parasite that can cause long-lasting, skin ulcers in man. Following parasite inoculation, most inbred strains of mice (e.g.C57BL/6) develop self-resolving lesions, while in a few strains (e.g. BALB/c) lesions fail to heal. We report here that in “healer” C57BL/6 mice, higher numbers of DCs were attracted at the site of infection than in “non-healer” BALB/c mice. DC recruitment correlated with secretion by neutrophils of the chemokine CCL3, as indeed a markedly decreased DC recruitment was observed in C57BL/6 mice depleted of neutrophils or deprived of the capacity to produce CCL3. DC recruitment was restored upon transfer of normal neutrophils to CCL3 deficient mice. Our results reveal a crucial role for neutrophil-secreted CCL3 in early recruitment of DCs in L. major-infected “healer” mice, and suggest that the type of chemokine secreted by neutrophils will have consequences in the launching of pathogen-specific immune response.

Exacerbated Innate Host Response to SARS-CoV in Aged Non-Human Primates

2010-02-05T08:00:00Z

Author Summary

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection causes acute lung injury that may develop into the life-threatening acute respiratory distress syndrome (ARDS) in mostly elderly individuals. Although SARS-CoV infection can be fatal, most patients recover, suggesting that protective host responses are operational to combat the viral infection. Therefore, we used age as predisposing factor to obtain insight into the pathogenesis of SARS-CoV. In this study, we show that SARS-CoV-infected aged macaques developed significantly more pathology than young adult animals, which could not be contributed to differences in viral replication. Using comparative microarray analyses, it was shown that although the nature of the host response to SARS-CoV infection was similar in aged and young adult macaques, the severity was significantly different, with aged macaques displaying an increase in differential expression of genes associated with inflammation. Interestingly, type I IFN-β mRNA levels correlated negatively with gross pathology. Therapeutic treatment of aged macaques with type I IFN reduced pathology without affecting virus replication. However, pro-inflammatory gene expression was significantly diminished. Thus, modulation of the host response by type I IFNs provides a promising outlook for novel intervention strategies.

IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

2010-02-05T08:00:00Z

Author Summary

West Nile virus (WNV) is a mosquito-transmitted RNA virus that has emerged in the Western hemisphere and is now the leading cause of arboviral encephalitis in the United States. However, the virus/host interface that controls WNV pathogenesis is not well understood. Previous studies have established that the innate immune response and interferon (IFN) defenses are essential for controlling virus replication and dissemination. In this study, we assessed the importance of the RIG-I like receptor (RLR) signaling pathway in WNV pathogenesis through analysis of mice lacking IPS-1, the central adaptor molecule of RLR signaling. Our studies revealed that IPS-1 is essential for protection against WNV infection and that it regulates processes that control virus replication and triggering of innate immune defenses. We found that IPS-1 plays an important role in establishing adaptive immunity through an innate/adaptive interface that elicits effective antibody responses and controls the expansion of regulatory T cells. Thus, RLRs are essential for pathogen recognition of WNV infection and their signaling programs help orchestrate immune response maturation, regulation of inflammation, and immune homeostasis that define the outcome of WNV infection.

PLoS Pathogens Issue Image | Vol. 6(1) January 2010

2010-01-29T08:00:00Z

Listeria monocytogenes infection of the human placenta.

L. monocytogenes causes abortions and preterm labor. Robbins et al. (see doi:10.1371/journal.ppat.1000732) used first trimester human placental organ cultures to quantitatively analyze how L. monocytogenes breaches the maternal-fetal barrier. The syncytiotrophoblast (red), which constitutes most of the placental surface and is bathed in maternal blood in vivo, is highly resistant to L. monocytogenes infection. Instead, extravillous cytotrophoblasts, which anchor the placenta in the uterus, serve as the primary portal of entry for L. monocytogenes (green). Nuclei are blue.

Image Credit: Jennifer R. Robbins and Anna I. Bakardjiev, University of California, San Francisco

The Deadly Chytrid Fungus: A Story of an Emerging Pathogen

2010-01-29T08:00:00Z

Killing a Killer: What Next for Smallpox?

2010-01-29T08:00:00Z

CD8+ T Cell Control of HIV—A Known Unknown

2010-01-29T08:00:00Z

Social Motility in African Trypanosomes

2010-01-29T08:00:00Z

Author Summary

African trypanosomes, e.g. Trypanosoma brucei, and related kinetoplastid parasites cause morbidity and mortality in several million people worldwide. Trypanosomes are protists and are thus generally considered to behave as single-celled microorganisms. In other microorganisms, social interactions among individuals lead to development of multicellular communities with specialized and advantageous capabilities versus single cells. The concept of bacteria acting as groups of cells communicating and cooperating with one another has had a major impact on our understanding of bacterial physiology and pathogenesis, but this paradigm has not been applied to parasitic protozoa. Here we report that T. brucei is capable of social behavior when exposed to semisolid surfaces. This behavior, termed social motility, is characterized by the assembly of parasites into multicellular communities with emergent properties that are not evident in single cells. Parasites within communities exhibit polarized movements and cooperate to coordinate their movements in response to an external stimulus. Social motility offers many potential advantages, such as facilitating colonization and navigation through host tissues. The identification of social behavior in T. brucei reveals a novel and unexpected aspect of parasite biology and provides new concepts for considering host-parasite interactions.

Nucleoporin 153 Arrests the Nuclear Import of Hepatitis B Virus Capsids in the Nuclear Basket

2010-01-29T08:00:00Z

Author Summary

Viral capsids facilitate protection of the enclosed viral genome and participate in the intracellular transport of the genome. At the site of replication capsids have to release the genome. The particular factors triggering genome liberation are not well understood. Like other karyophilic cargos, hepatitis B virus (HBV) capsids are transported through the nuclear pore using nuclear transport receptors of the importin ß superfamily. Unlike physiological cargos, HBV capsids become arrested within the nuclear basket, which is a filamentous structure on the nuclear side of the nuclear pore. Asking which interaction causes this unique strategy, we found that the capsids bind to a protein of the basket periphery, nucleoporin 153 (Nup153). The findings were confirmed in situ using digitonin-permeabilized cells that support physiological genome delivery into the nucleus. We observed that HBV capsids bound to Nup153 irrespective of the maturation of the encapsidated genome. But while capsids with an immature genome remained in arrested state, capsids with a mature genome disassembled and released their DNA.

Upregulation of xCT by KSHV-Encoded microRNAs Facilitates KSHV Dissemination and Persistence in an Environment of Oxidative Stress

2010-01-29T08:00:00Z

Author Summary

Herpesviruses are the most common etiologic agents of cancer in patients with suppressed immune function, and the Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the most common causes of cancer in this setting. KSHV infection of new cell targets is critical for tumor progression, and a better understanding of how viral receptors on the surface of cells are regulated in the tumor microenvironment may lead to new therapies. KSHV encodes unique RNAs called microRNAs (KSHV miRNAs) that regulate a variety of cell functions. In this study, we show that KSHV miRNAs increase the susceptibility of cells to KSHV infection and protect infected cells from death induced by cancer-promoting reactive nitrogen species (RNS). They accomplish this in large part by increasing cell surface expression of a transport protein subunit called xCT. We also show that KSHV miRNAs increase secretion of RNS by infected cells, and that blocking RNS secretion reduces the ability of KSHV to infect cells. Therefore, by regulating xCT and RNS, we find KSHV is able to “fine-tune” cell function in order to maintain a stable population of infected cells which secrete cancer-promoting factors in the local environment. This work has important implications for developing new therapies to target xCT and reduce survival of KSHV-infected tumor cells.

The Type III Secretion Effector NleE Inhibits NF-κB Activation

2010-01-29T08:00:00Z

Author Summary

The innate immune system senses intruding pathogens and in response, mounts an inflammatory reaction. Essential for this response is the activation of the transcription factor NF-κB, which mediates reprogramming of gene expression in the host. The bacteria Escherichia coli is usually a non-pathogenic resident of our intestinal track. Some E. coli strains, however, cause disease or food poisoning; one of these pathogenic strains is enteropathogenic E. coli (EPEC). This pathogen employs a syringe-like organelle, termed type three secretion system (TTSS), to inject into the intestinal host cell a battery of toxic proteins termed effectors. We found that two of the effectors that EPEC injects into the host cell upon infection block the activation of NF-κB and thus interfere with the host immune response. These findings elucidate the intricate cross-talk between the host immune system and the pathogen.

Parasite-Derived Plasma Microparticles Contribute Significantly to Malaria Infection-Induced Inflammation through Potent Macrophage Stimulation

2010-01-29T08:00:00Z

Author Summary

Although parasite materials are responsible for the activation of the immune system during malaria infection, exactly how the immune response is initiated during infection is extremely unclear. In this study we demonstrate that sub micron particles (microparticles) are produced by malaria infected red blood cells during malaria infection, and we show that these microparticles can promote strong inflammatory responses by activating macrophages. We show that infected red blood cell-derived microparticles are produced in higher numbers as infection progresses, and that the host's own pro-inflammatory immune response is not required for the generation of these microparticles. We have also examined the receptors and signalling pathways required for macrophage activation by microparticles, and we show that the pathway of microparticle-induced activation is distinct from other previously reported pathways. In summary, we have defined a novel pathway of immune response activation during malaria infection, which may be important for promoting parasite control and/or causing pathology.

Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

2010-01-29T08:00:00Z

Author Summary

Influenza A viruses generally infect individuals of all ages and cause severe respiratory disease in very young children and elderly people (>65 years). However, the 2009 pandemic H1N1 virus infection is predominantly seen in children and adults (<35 years of age), but rarely in people older than 65 years of age. Recent serological studies indicate that older people carry antibodies that recognize the 2009 H1N1 virus. This suggests that they may have been exposed to or vaccinated with an influenza virus similar to 2009 H1N1 virus. In this study, we wanted to identify the older H1N1 virus(es) that may confer protection to the elderly population. Using 11 different inactivated influenza A viruses that have circulated between 1918 to 2007, we immunized mice and challenged them with a lethal dose of the 2009 novel H1N1 virus. We find that mice vaccinated with human H1N1 viruses that circulated in 1918 and in 1943 were protected from the 2009 H1N1 virus. Also, the 1976 swine origin H1N1 virus, against which nearly 40 million people were immunized in 1976 in the United States, protects mice from death by the 2009 H1N1 virus. This indicates that people carrying antibodies against H1N1 viruses that circulated between 1918–1943 and to the 1976 swine origin H1N1 virus are likely to be protected against the 2009 pandemic H1N1. Importantly, our data underscores the significance of vaccinating people under 35 year of age, since the majority of them do not have protective antibodies against the 2009 H1N1, and provide a possible mechanism by which pandemic viruses could arise from antigenically frozen influenza viruses harbored in the swine population.

CD8+ Lymphocytes Control Viral Replication in SIVmac239-Infected Rhesus Macaques without Decreasing the Lifespan of Productively Infected Cells

2010-01-29T08:00:00Z

Author Summary

Despite overwhelming evidence that CD8+ T cells are important in controlling virus replication during HIV and simian immunodeficiency virus (SIV) infections, the mechanisms responsible for this antiviral effect in vivo remain poorly understood. This lack of knowledge represents a key obstacle to our efforts to develop a CD8+ T cell-based AIDS vaccine. In this study, we implemented a new experimental system in which we determined the lifespan of productively SIV-infected cells in vivo, either in the presence or absence of CD8+ lymphocytes. The lifespan of productively infected cells was calculated based on the slope of the decline of SIV plasma viremia after initiation of ART using a widely accepted mathematical model. Using this novel approach, we determined that CD8+ lymphocytes control virus replication without noticeably decreasing the lifespan of productively infected cells, thus suggesting that the major mechanism of antiviral activity by CD8+ lymphocytes during pathogenic SIV infection may not be direct cell killing of productively SIV-infected cells.

In Vivo CD8+ T-Cell Suppression of SIV Viremia Is Not Mediated by CTL Clearance of Productively Infected Cells

2010-01-29T08:00:00Z

Author Summary

The recognition and elimination of infected host cells by CD8+ T-lymphocytes is held to be a key component of the immune response against viral pathogens. However, this basic tenet of viral immunology may not hold true for HIV and the related SIV. In the current work, we eliminated CD8+ T-cells by treating simian immunodeficiency virus (SIV) infected macaques with a CD8-depleting monoclonal antibody then treated the animals with antiretroviral drugs and measured virus levels. Viral levels fell just as fast for the animals with or without CD8+ T-cells, implying that survival of infected cells producing SIV was not impacted by the presence or absence of CD8+ T-cells. Virus obtained after CD8+ T-cell depletion showed changes in the types of sequences in a viral protein (Nef) that is expressed early after infection of a cell but not in a viral protein (Gag) that is expressed later. These findings suggest CD8+ T-cells have a limited ability to kill cells already expressing SIV but instead may be restricted to non-killing mechanisms or to targeting cells during earlier stages of infection before virus production begins. Understanding and overcoming the factors that prevent CD8+ T-cells from effectively eliminating infected cells producing virus could advance HIV vaccine efforts.

Persistent ER Stress Induces the Spliced Leader RNA Silencing Pathway (SLS), Leading to Programmed Cell Death in Trypanosoma brucei

2010-01-22T08:00:00Z

Author Summary

Trypanosomes are the causative agent of major parasitic diseases such as African sleeping sickness, leishmaniasis and Chagas' disease that affect millions of people mostly in developing countries. These organisms diverged very early from the eukaryotic linage and possess unique molecular mechanisms such as trans-splicing and RNA editing. Trypanosomes lack polymerase II promoters that govern the transcription of protein coding genes. Eukaryotes respond to unfolding of proteins in the endoplasmic reticulum (ER) by a distinct transcriptional programming known as the unfolded protein response (UPR). In this study, we demonstrate that despite the lack of transcriptional regulation, procyclic trypanosomes change their transcriptome as a response to ER stress by differential mRNA stabilization. Prolonged ER stress induces a unique process, the spliced leader RNA silencing (SLS), that shuts off the trans-splicing and the production of all mRNAs. SLS is induced both by prolonged ER stress and by knock-down of factors involved in ER translocation in both life stages of the parasite. SLS induces programmed cell death (PCD) evident by the hallmark of apoptosis in metazoa (DNA fragmentation, membrane flipping and ultrastructural changes). We propose that SLS serves as a unique death pathway replacing the conventional caspase-mediated PCD observed in higher eukaryotes.

Placental Syncytiotrophoblast Constitutes a Major Barrier to Vertical Transmission of Listeria monocytogenes

2010-01-22T08:00:00Z

Author Summary

Placental infections can lead to severe pregnancy complications as well as infection of the fetus and newborn with significant morbidity and mortality. Pathogens that are able to cross the maternal-fetal barrier typically have life cycles inside host cells. Among these is the facultative intracellular bacterial pathogen Listeria monocytogenes, which is highly amenable to experimental analysis. Our study is the first to use early gestation primary human placental organ cultures to identify the mechanisms by which L. monocytogenes breaches the human maternal-fetal barrier. We found that the placenta has evolved multiple mechanisms to resist infection. The main portal of entry into the placenta was a small subpopulation of fetally derived trophoblast cells (extravillous cytotrophoblasts), which anchor the placenta in the decidua, the lining of the pregnant uterus. These cells could be infected via two mechanisms: direct invasion of extracellular bacteria and cell-to-cell spread. The extravillous cytotrophoblasts are not readily accessible from the maternal blood stream. This is a significant finding because it provides a novel explanation why almost all placental pathogens have intracellular life cycles: they may need maternal cells to reach the decidua and infect the placenta.

Polyoma Virus-Induced Osteosarcomas in Inbred Strains of Mice: Host Determinants of Metastasis

2010-01-22T08:00:00Z

Author Summary

The oncogenic mouse polyoma virus and its mutants have previously been used to investigate viral determinants of tumor induction using a standard inbred mouse strain as a common host. Here we use wild type virus to investigate the role of the host genetic background, focusing on two host strains that differ with respect to bone tumor metastasis. Comparing osteosarcoma cell lines from these mice, we have identified a molecular pathway that underlies invasive behavior in vitro and correlates with metastasis in vivo. The pathway involves secretion of the metalloproteinase MMP-2 under partial control of NFAT as a transcriptional regulator. This virus-host system reflects an important feature of human osteosarcoma with respect to pulmonary metastasis. Based on naturally occurring differences among inbred mice, the model differs from genetically engineered models targeting p53 or pRb as known risk factors in the human disease. Here, metastatic osteosarcomas retain functional p53. As noted by others, the frequency of p53 loss in patients with localized versus metastatic disease is the same, suggesting that events beyond p53 loss are important in metastasis. While the downstream effectors of metastasis in the genetically engineered models remain unknown, evidence presented here implicates upregulation of an NFAT → MMP-2 pathway in the development of metastatic osteosarcoma.

Melanoma Differentiation-Associated Gene 5 (MDA5) Is Involved in the Innate Immune Response to Paramyxoviridae Infection In Vivo

2010-01-22T08:00:00Z

Author Summary

The innate immune system possesses an array of sensory molecules which are purposed in detecting viral nucleic acids. Our understanding of how these molecular sensors detect viral nucleic acids continues to evolve. Herein, we demonstrate that MDA5, a member of the RIG-I-like receptor family, is involved in the detection of paramyxovirus infection in vivo. Specifically, MDA5 appears to trigger antiviral cytokines that inhibit paramyxovirus replication. In this regard, mice that are deficient in MDA5 are unable to express sustained levels of these cytokines and thus succumb to extensive viral propagation and disease. Our findings are largely discordant from previous in vitro studies using cultured cells, where it has been shown that RIG-I and not MDA5 is involved in the innate response to negative sense RNA viruses. Thus, our data provides strong evidence of MDA5-based detection of negative sense RNA viruses, and furthermore underscore the importance of organism-based analysis of the innate system.

Evolutionary Trajectories of Beta-Lactamase CTX-M-1 Cluster Enzymes: Predicting Antibiotic Resistance

2010-01-22T08:00:00Z

Author Summary

Antimicrobial resistance in bacterial organisms is a worldwide problem widely discussed from clinical, economical and social points of view. The number of new resistance mechanisms and microorganisms resistant to new drugs is increasing all over the world. The development and spread of antibiotic resistance in bacterial communities represents an excellent model for testing the predictive potential of evolutionary principles at short time-scales. A number of studies have tried to predict the selection of resistant variants when a new drug is commercialized. However, in many cases there is no correlation between in vitro predictions and in-field observations. For this reason, it can be suspected that the ability to predict the emergence of new resistant variants might be incomplete unless we know the evolutionary forces acting on the genetic diversification processes. Using the CTX-M β-lactamases as a model, a combination of molecular phylogenetic approaches and experimental site-specific mutagenesis has allowed us to establish evolutionary trajectories. We have demonstrated that two synthetic antibiotics, cefotaxime and ceftazidime, were the selective forces driving the diversification of CTX-M enzymes, but only when both antibiotics were simultaneously present in the environment. We also predict that, if the current selective landscape is not modified, variants carrying the mutation D240G will be more prevalent and diverse in the future.

Protease-Sensitive Synthetic Prions

2010-01-22T08:00:00Z

Author Summary

Prions are infectious proteins that cause heritable, sporadic, and transmissible diseases in humans and other mammals. These infectious proteins arise when the normal form of the prion protein (PrP) adopts a self-perpetuating conformation. This disease-causing PrP form is frequently distinguished from normal PrP by its resistance to digestion by proteases although considerable evidence shows that protease-sensitive prions occur naturally in humans and sheep. Here we describe the generation of novel protease-sensitive synthetic prions. After producing recombinant PrP of the wild-type mouse sequence in Escherichia coli, we polymerized the protein into an amyloid fiber conformation. Mice inoculated with these amyloid fibers developed extensive neurodegeneration characteristic of prion disease, but did not generate protease-resistant PrP. Prions from sick animals were transmitted to healthy animals, which likewise developed neurodegeneration but not protease-resistant prions. These novel synthetic prions demonstrate that truncated wild-type PrP can undergo a conformational change that becomes infectious yet the protein remains protease sensitive.

Histone Deacetylases Play a Major Role in the Transcriptional Regulation of the Plasmodium falciparum Life Cycle

2010-01-22T08:00:00Z

Author Summary

Plasmodium falciparum, a parasitic protozoan, causes the most lethal form of human malaria, killing more than 2 million people per year. It has a complex life cycle that involves distinct morphological stages accompanied by stage specific gene expression in both the mosquito and human hosts. The lack of a vaccine for malaria and widespread resistance highlights the urgency for new anti-malarial drugs that act on different parasite targets. We show that inhibition of histone deacetylase activities results in activation and repression of transcriptionally regulated genes in multiple stages of the P. falciparum asexual life cycle. We also show that inhibition disrupts the steady-state level of histone acetylation and methylation across the P. falciparum genome. Our data strongly implies that in P. falciparum, inhibition of histone deacetylase activity leads to a dramatic increase in global acetylation of histones and subsequently disruption of stage specific gene expression. This process then leads to a collapse of the transcriptional cascade of P. falciparum. Therefore, the essential role of histone deacetylases in Plasmodium parasites suggests their high potential as molecular targets for malaria intervention strategies.

TRIM5α Modulates Immunodeficiency Virus Control in Rhesus Monkeys

2010-01-22T08:00:00Z

Author Summary

The cytoplasmic TRIM5α restricts the replication of a broad range of retroviruses in a species-specific manner. In the present study we show that TRIM5α is more than a species barrier for retroviruses. We show that naturally occurring B30.2(SPRY) polymorphisms affect retrovirus infection. These observations demonstrate the importance of SIV/B30.2(SPRY) interactions in vivo. These findings are the first demonstration of the importance of such a pathogen/host protein interaction in vivo. Importantly, the striking variability in the clinical course of HIV-infected individuals has long puzzled the biomedical community. A large number of investigators have devoted considerable effort to determine what genetically determined factors might contribute to the containment of HIV replication, reasoning that an understanding of the determinants of effective control of HIV spread will provide important targets for both drug and vaccine development. Our demonstration in the present study that B30.2(SPRY) polymorphisms have a dramatic effect on the clinical outcome of an AIDS virus infection highlight the extraordinary importance of TRIM5α on the control of an AIDS virus infection.

A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

2010-01-15T08:00:00Z

Author Summary

Toxoplasma gondii and related parasites within the Phylum Apicomplexa are collectively responsible for a great deal of human disease and death worldwide. The ability of apicomplexan parasites to invade cells of their hosts, disseminate through tissues and cause disease depends critically on parasite motility. Motility is driven by a complex of proteins that is well conserved within the phylum; however, very little is known about how the unconventional myosin motor protein at the heart of this motility machinery is regulated. T. gondii serves as a powerful model system for studying apicomplexan motile mechanisms. We show here that a recently identified pharmacological inhibitor of T. gondii motility induces a posttranslational modification of TgMLC1, a protein that binds to the myosin motor protein, TgMyoA. The compound-induced modification of TgMLC1 is associated with a decrease in TgMyoA mechanical activity. These data provide the first glimpse into how TgMyoA is regulated and how a change in the activity of the T. gondii myosin motor complex can affect the motility and infectivity of this important human pathogen.

Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

2010-01-15T08:00:00Z

Author Summary

The closely related members of the filovirus family, Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic disease in humans with high fatality rates. Infected individuals exhibit dysregulated immune responses which appear to result from several factors, including virus-mediated impairment of innate immune responses. Previous studies demonstrated that both MARV and EBOV block the type I interferon-induced Jak-STAT signaling pathway. For EBOV, the viral protein VP24 mediates the inhibitory effects by interfering with the nuclear translocation of activated STAT proteins. Here, we show that MARV uses a distinct mechanism to block IFN signaling pathways. Our data revealed that MARV blocks the phosphorylation of Janus kinases and their target STAT proteins in response to type I and type II interferon and interleukin 6. Surprisingly, the observed inhibition is not achieved by the MARV VP24 protein, but by the matrix protein VP40 which also mediates viral budding. Over-expression studies indicate that MARV VP40 globally antagonizes Jak1-dependent signaling. Further, we show that a MARV VP40 mutant defective for budding retains interferon antagonist function. Our results highlight a basic difference between EBOV and MARV, define a new function for MARV VP40 and reveal new targets for the development of anti-MARV therapies.

Type I Interferon Induction Is Detrimental during Infection with the Whipple's Disease Bacterium, Tropheryma whipplei

2010-01-15T08:00:00Z

Author Summary

Innate immune cells are sentinels allowing the host to sense invading pathogens. Among them, macrophages are highly microbicidal and are able to kill microorganisms. However, several pathogens have evolved strategies to hijack macrophage responses in order to survive or replicate. Tropheryma whipplei is the agent of Whipple's disease, a systemic disease that associates arthropathy, weight loss and gastrointestinal symptoms. It has been known for several years that this bacterium has a tropism for macrophages, in which it replicates. In this study, we have shown that T. whipplei induces host cell apoptosis and a surprising macrophage activation, characterized by anti-inflammatory molecules and type I interferon (IFN) signaling, which is generally associated to viral infections. We demonstrate that this type I IFN response is critical for bacterial pathogenicity, as it is required for bacterial replication and provides the first step of the apoptotic program of infected macrophages. By identifying these signaling events induced in macrophage by T. whipplei, we can now better understand the molecular basis of the pathophysiology of Whipple's disease, of interest for clinical and therapeutic ends.

Structure of the HCMV UL16-MICB Complex Elucidates Select Binding of a Viral Immunoevasin to Diverse NKG2D Ligands

2010-01-15T08:00:00Z

Author Summary

Cytotoxic lymphocytes such as natural killer (NK) cells or CD8 T cells have the ability to detect and destroy cells infected by viruses. They therefore are tools on which the human immune system critically depends in order to control viral infections. To avoid discovery by cytotoxic lymphocytes and to allow for longtime persistence in the human host, the human cytomegalovirus (HCMV) has developed a multitude of immune evasive strategies that are mediated by so-called immunoevasins. We present here a structure-function analysis of one of the best-known HCMV immunevasins, UL16, and its interaction with a cellular ligand for NK cells, MICB. The normal function of MICB is to activate NK cells by engaging the most well-known NK receptor, NKG2D. Our results provide molecular evidence for the strategy used by UL16 to disable NK cell activation. In a rare example of structural mimicry that has likely arisen through convergent evolution, UL16 mimics a central binding motif of the structurally unrelated NKG2D protein. This allows UL16 to engage and disable several diverse NKG2D ligands, while others have apparently evolved to escape recognition by UL16 through alteration of key residues at strategic locations.

B Cell Activation by Outer Membrane Vesicles—A Novel Virulence Mechanism

2010-01-15T08:00:00Z

Author Summary

Outer membrane vesicles secreted by pathogenic bacteria are recognized as a long-distance delivery system which transports diverse virulence factors, and allows pathogens to interact with the host, and hence the possibility to modify the immune response without close contact. Our study shows that Moraxella catarrhalis outer membrane vesicles that also exist in patients can activate human B cells isolated from pharyngeal lymphoid tissue. These findings have significant implications for understanding Moraxella pathogenesis since palatine tonsils are a potential tissue reservoir. Vesicles secreted by Moraxella bind to tonsillar B cells through the superantigen Moraxella IgD-binding protein designated MID. The interaction between MID and the B cell receptor induces Ca²⁺ mobilization and receptor clustering in lipid raft motifs followed by internalization of vesicles. Mainly Toll-like receptor 9, a pathogen recognition receptor of the innate immune system, participates in the signaling induced by vesicles through sensing of DNA associated with vesicles. The vesicle-dependent B cell activation induces up-regulation of surface activation markers in addition to IL-6 and IgM secretion. Vesicle secretion provides Moraxella with a sophisticated mechanism to modify the host immune response, avoiding contact between bacteria and the host.

Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis

2010-01-15T08:00:00Z

Author Summary

While both viral and bacterial pathogens have been shown to undergo genetic changes over the course of a chronic infection, this phenomenon has not been studied during an acute infection and as such is not well understood. Here, we examined within-host evolution of the pathogen Burkholderia pseudomallei during acute infection. B. pseudomallei causes the disease melioidosis, a significant cause of morbidity and mortality in many tropical regions of the world. We obtained multiple B. pseudomallei colonies from several tissue sites of four patients presenting with acute melioidosis in order to characterize how this bacterium evolves within the human host over a short period of time. By monitoring changes in rapidly evolving genetic regions, we found high levels of diversity of B. pseudomallei populations within a single patient, and even within a single body site. Comparison of these within-host mutation rates with in vitro mutation data enabled us to identify the most likely spatial migration of within-host populations and correlate these findings with clinical data to determine, in most cases, the origin of infection. Our study provides new insights into the evolution of bacterial pathogens during an acute infection, and lays the foundation for similar studies in other infectious agents.

β-Neurexin Is a Ligand for the Staphylococcus aureus MSCRAMM SdrC

2010-01-15T08:00:00Z

Author Summary

Staphylococcus aureus is an opportunistic pathogen, distinguished by its potential to cause serious and life-threatening infections in animals and humans. The ability of this bacterium to adhere to host tissues is considered an early, essential event in the disease process and contributes to the success of the organism as a pathogen. Adherence to host tissues is mediated by a subfamily of cell-wall anchored proteins named MSCRAMMs (microbial surface component recognizing adhesive matrix molecules). Work in our laboratory suggested that many of these proteins share a common ligand binding mechanism targeting linear amino acid sequences. Based on these observations, we hypothesized that screening a phage display library of random peptides may identify receptors for MSCRAMMs. Using this method, we demonstrate that the putative MSCRAMM SdrC recognizes a sequence in the neuronal protein β-neurexin. Furthermore, we show that intact β-neurexin 1 is a functional ligand for the S. aureus MSCRAMM SdrC. Successful implementation of this approach may open avenues for the identification of additional host ligands and the design of anti-staphylococcal therapeutics able to inhibit these interactions.

Two Plant Viral Suppressors of Silencing Require the Ethylene-Inducible Host Transcription Factor RAV2 to Block RNA Silencing

2010-01-15T08:00:00Z

Author Summary

RNA silencing is an important antiviral defense in plants, and many plant viruses encode proteins that block RNA silencing. However, the mechanism of action of the viral suppressors is complex, and little is known about the role of host plant proteins in the process. Here we report the first example of a host protein that plays a required role in viral suppression of silencing—a transcription factor called RAV2 that is required for suppression of silencing by two different and unrelated viral proteins. Analysis of plant gene expression patterns shows that RAV2 is required for induction of many genes involved in other stress and defense pathways, including genes implicated as plant suppressors of silencing. Overall, the results suggest that RAV2 is an important factor in viral suppression of silencing and that the role of RAV2 is to divert host defenses toward responses that interfere with antiviral silencing.

The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner

2010-01-15T08:00:00Z

Author Summary

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide. The virus specifically targets subpopulations of macrophages, central players in the immune system, and can persist in animals for extended periods of time due to a hampered immunity. At present, no vaccines are available that are both safe and effective and it is clear that a more rational vaccine design is needed to solve this problem. Therefore, advancing our fundamental understanding of PRRSV biology is crucial. The macrophage-specific lectin sialoadhesin is a crucial viral receptor on macrophages and although its role in PRRSV infection is well documented, its viral counterparts have remained unknown. Using a soluble form of sialoadhesin, we identified the M/GP₅ glycoprotein complex of PRRSV as the ligand for sialoadhesin and found this ligand-receptor interaction to be critically dependent on the lectin activity of sialoadhesin and on sialic acids on the GP₅ glycoprotein. These data represent a major breakthrough in the understanding of the role of PRRSV proteins in viral entry and pave the way for the development of a new generation of PRRSV vaccines capable of inducing an immunity that specifically blocks the interaction between viral M/GP₅ and sialoadhesin.