MicrobiologyBytes

Herpes simplex virus (HSV) type-1 and type-2 have evolved numerous strategies to infect a wide range of hosts and cell types. The result is a very successful prevalence of the virus in the human population infecting 40-80% of people worldwide. HSV entry into host cell is a multistep process that involves the interaction of the viral glycoproteins with various cell surface receptors. Based on the cell type, HSV enter into host cell using different modes of entry. The combination of various receptors and entry modes has resulted in a virus that is capable of infecting virtually all cell types. Identifying the common rate limiting steps of the infection may help the development of antiviral agents that are capable of preventing the virus entry into host cell. This review describes the major features of HSV entry that have contributed to the wide susceptibility of cells to HSV infection.

Herpes simplex virus infects most cell types in vitro: clues to its succes. (2011) Virology Journal 8: 481 doi:10.1186/1743-422X-8-481

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Inflammatory responses generated during virus infections are critical for antiviral immune responses. The exact virus recognition elements that activate cells to induce pro-inflammatory signals are not completely characterized. Virus recognition elements such as dsRNA and 5′-triphosphate single-stranded RNA are recognized by several cellular pathways. The intracellular or extracellular interaction of cells with virus recognition elements results in activation of innate immune responses as indicated by expression of inflammatory cytokines and chemokines. In addition to the innate immune inflammation, virus recognition elements trigger establishment of an antiviral state, under which each cell resists virus infection. The resistance to virus infection is in part through inhibition of virus replication by perturbation of RNA and protein synthesis. Determining the exact mechanisms by which immune and antivirus responses are activated is essential for understanding virus pathogenesis.

RNA with high inosine content is not commonly found in normally growing eukaryotic cells but it is present during infections with DNA and RNA viruses such as polyomavirus, Rous-associated virus, vesicular stomatitis virus, measles virus, and respiratory syncytial virus. Extracellular Ino-RNA is generated during virus infections. Cell lysis occurs frequently during virus infections, which results in the release of cell content, including intracellular generated Ino-RNA, into the extracellular space. Extracellular dsRNA has been shown to be able to stimulate antiviral responses in neighboring, uninfected cells.

Using RSV infection as a model, this paper reports that the presence of inosines in ssRNA is a potent inducer of inflammatory cytokines and the antiviral state during virus infection and suggests that Ino-RNA, of virus or cellular origin, in the surrounding tissue after release from infected cells is a signal for the presence of virus infections.

Inosine-Containing RNA Is a Novel Innate Immune Recognition Element and Reduces RSV Infection. (2011) PLoS ONE 6(10): e26463. doi:10.1371/journal.pone.0026463
During viral infections, single- and double-stranded RNA (ssRNA and dsRNA) are recognized by the host and induce innate immune responses. The cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) activation in virally infected cells leads to presence of inosine-containing RNA (Ino-RNA). Here we report that ss-Ino-RNA is a novel viral recognition element. We synthesized unmodified ssRNA and ssRNA that had 6% to16% inosine residues. The results showed that in primary human cells, or in mice, 10% ss-Ino-RNA rapidly and potently induced a significant increase in inflammatory cytokines, such as interferon (IFN)-β (35 fold), tumor necrosis factor (TNF)-α (9.7 fold), and interleukin (IL)-6 (11.3 fold) (p

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As the incidence of type 1 diabetes in developed countries has been increasing at a rate far beyond the rate of population growth, environmental factors have been considered as likely candidates responsible for this change in disease incidence in recent decades. Of those factors, the gut microbiota have come under recent interest; supported in part by observations in both non-obese diabetic (NOD) mice and BioBreeding Diabetes Prone (BB-DP) rats where antibiotic use prevents the onset of diabetes.

To explore specific differences in the microbial communities responsible for T1D modulation, a metagenomic analysis of bacteria from susceptible rodents was performed. This revealed bacteria whose members were either positively or negatively correlated with diabetes. Lactobacillus and Bifidobacterium were more abundant in BB-DR (Diabetes Resistant) rats while Bacteroides and Clostridium were more abundant in BB-DP (Diabetes Prone) rats. Both Lactobacillus and Bifidobacterium are well known to have members with probiotic characteristics. These data suggest a model for the role of bacteria in a healthy gut. The total number of lactic acid producing and butyrate producing bacteria is higher in controls than in diabetic animals. This suggest that microbial-induced butyrate production, and subsequent mucin synthesis, with a corresponding enhancement of tight junctions may contribute to the development of autoimmunity for type 1 diabetes in humans.

Gut Microbiome Metagenomics Analysis Suggests a Functional Model for the Development of Autoimmunity for Type 1 Diabetes. (2011) PLoS ONE 6(10): e25792. doi:10.1371/journal.pone.0025792
Recent studies have suggested a bacterial role in the development of autoimmune disorders including type 1 diabetes (T1D). Over 30 billion nucleotide bases of Illumina shotgun metagenomic data were analyzed from stool samples collected from four pairs of matched T1D case-control subjects collected at the time of the development of T1D associated autoimmunity (i.e., autoantibodies). From these, approximately one million open reading frames were predicted and compared to the SEED protein database. Of the 3,849 functions identified in these samples, 144 and 797 were statistically more prevalent in cases and controls, respectively. Genes involved in carbohydrate metabolism, adhesions, motility, phages, prophages, sulfur metabolism, and stress responses were more abundant in cases while genes with roles in DNA and protein metabolism, aerobic respiration, and amino acid synthesis were more common in controls. These data suggest that increased adhesion and flagella synthesis in autoimmune subjects may be involved in triggering a T1D associated autoimmune response. Extensive differences in metabolic potential indicate that autoimmune subjects have a functionally aberrant microbiome. Mining 16S rRNA data from these datasets showed a higher proportion of butyrate-producing and mucin-degrading bacteria in controls compared to cases, while those bacteria that produce short chain fatty acids other than butyrate were higher in cases. Thus, a key rate-limiting step in butyrate synthesis is more abundant in controls. These data suggest that a consortium of lactate- and butyrate-producing bacteria in a healthy gut induce a sufficient amount of mucin synthesis to maintain gut integrity. In contrast, non-butyrate-producing lactate-utilizing bacteria prevent optimal mucin synthesis, as identified in autoimmune subjects.

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Filoviruses cause lethal hemorrhagic fever in humans and nonhuman primates. The family Filoviridae includes two genera: Marburgvirus (MARV) and Ebolavirus (EBOVs). MARV was discovered in 1967 in Marburg, Germany during an outbreak in laboratory staff exposed to tissues from monkeys imported from Uganda. The Zaire virus was discovered in 1976 in Yambuku, Zaire during a 312-person outbreak associated with 90% mortality. With the exception of Reston Ebolavirus that appears to be pathogenic in nonhuman primates but not in humans and is endemic in the Philippines, all known filoviruses are pathogenic in primates including humans and are endemic in Africa. Bats are implicated as reservoirs and vectors for transmission of filoviruses in Africa. Ebolavirus sequences have been found in various bats. Bats naturally or experimentally infected with Ebolaviruses are healthy and shed virus in their feces for up to 3 weeks.

In 2002, colonies of Schreiber’s bats (Miniopterus schreibersii), sustained massive die-offs in caves in France, Spain and Portugal. This paper report the first discovery of an ebolavirus-like filovirus in bats from Europe.

Discovery of an Ebolavirus-Like Filovirus in Europe. (2011) PLoS Pathog 7(10): e1002304. doi:10.1371/journal.ppat.1002304
Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain.

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This review gives an overview of the current state-of-the-art of bacteriophage therapy and discusses the challenges to be overcome before phage therapy can become a part of Western medicine.

The next generation of bacteriophage therapy Curr Opin Microbiol. (2011)14(5): 524-531
Bacteriophage therapy for bacterial infections is a concept with an extensive but controversial history. There has been a recent resurgence of interest into bacteriophages owing to the increasing incidence of antibiotic resistance and virulent bacterial pathogens. Despite these efforts, bacteriophage therapy remains an underutilized option in Western medicine due to challenges such as regulation, limited host range, bacterial resistance to phages, manufacturing, side effects of bacterial lysis, and delivery. Recent advances in biotechnology, bacterial diagnostics, macromolecule delivery, and synthetic biology may help to overcome these technical hurdles. These research efforts must be coupled with practical and rigorous approaches at academic, commercial, and regulatory levels in order to successfully advance bacteriophage therapy into clinical settings.

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Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification.

A new paper describes a genome-wide screen of human cells to identify host factors required for Ebola virus entry. Cells defective for the homotypic fusion and vacuole protein-sorting (HOPS) complex or cholesterol transporter protein Niemann–Pick C1 (NPC1) are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. Membrane fusion mediated by filovirus glycoproteins and virus escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. These findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

Ebola virus entry requires the cholesterol transporter Niemann–Pick C1. (2011) Nature 477: 7364 doi:10.1038/nature10348
Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann–Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

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Cellular movement is key to life and in the case of intracellular parasites, provides a vital mechanism to gain access to the protected niche they require. The parasite Toxoplasma gondii is a model for a group of parasites called apicomplexans, which move by an actin-dependent process referred to as gliding motility. This form of motility is distinct from that used by ciliated or flagellated cells, and from the crawling behavior of amoeba and many mammalian cells.

A new paper demonstrates that the normally highly conserved protein actin is divergent in these parasites and that it displays unusual kinetic properties that result in formation of short unstable filaments, in contrast to the highly stable nature of mammalian actin. The findings reveal that the short dynamic nature of parasite actins is due to a small number of amino acid differences that affect stability of the filament. These properties are essential to normal parasite motility since reversion of these residues to match those seen in mammalian cells was detrimental to gliding movement. The dependence of parasites on rapid turnover of highly unstable actins renders them extremely sensitive to toxins that stabilize actin filaments, thus providing a potential target for development of specific intervention.

Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites. (2011) PLoS Pathog 7(10): e1002280. doi:10.1371/journal.ppat.1002280
Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

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Researchers have discovered a toxin – SElX – released by methicillin-resistant Staphylococcus aureus (MRSA) which leads the body’s immune system to go into overdrive and damage healthy cells. SElX is made by 95 per cent of S. aureus strains, making it a potential drug target to fight this hospital superbug. SElX belongs to a family of toxins known as superantigens that can invoke an extreme immune response. When it is released it triggers an over multiplication of immune cells, which can lead to high fever, toxic shock and potentially fatal lung infections looked at a strain of MRSA known as USA300 that can cause severe infections in otherwise healthy individuals. If we can find ways to target this toxin, we may be able to stop it from triggering an over-reaction of the body’s immune system and prevent severe infections.

A Novel Core Genome-Encoded Superantigen Contributes to Lethality of Community-Associated MRSA Necrotizing Pneumonia. (2011) PLoS Pathog 7(10): e1002271. doi:10.1371/journal.ppat.1002271

Other research has linked a naturally occurring mutation in the bacterium Clostridium difficile to severe and debilitating diarrhoea in hospital patients undergoing antibiotic therapy. These antibiotics destroy the “good” bacteria in the gut, which allows this “bad” bacterium to colonise the colon, where it causes bowel infections that are difficult to treat. The mutation wipes out an inbuilt disease regulator, called anti-sigma factor TcdC, producing hypervirulent strains of C. difficile that are resistant to antibiotics and which have been found to circulate in Canada, the US, UK, Europe and Australia. The results suggest that bacterial strains carrying this mutation have the potential to produce more of the harmful toxins that cause disease in susceptible individuals – commonly patients aged 65 years or over. As we now have a better understanding of these strains, we can design new strategies to prevent, control and treat these infections.

The Anti-Sigma Factor TcdC Modulates Hypervirulence in an Epidemic BI/NAP1/027 Clinical Isolate of Clostridium difficile. (2011) PLoS Pathog 7(10): e1002317. doi:10.1371/journal.ppat.1002317

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