MicrobiologyBytes

The ciliated epithelium that lines the airways of the lung normally functions to transport hydrated mucus secretions out of the airways to maintain respiratory sterility. Cystic fibrosis (CF) lung disease results from reduced airway surface hydration leading to decreased mucus clearance that precipitates bacterial infection and progressive obstructive lung disease. CF is a genetic disease, and the mutant protein is a chloride ion channel (CFTR) that normally regulates ion and fluid transport on the airway surface. Restoration of corrected CFTR function to the airway epithelium of CF patients by delivering a new CFTR gene to airway epithelial cells has long been envisioned as a therapeutic strategy for CF lung disease. In 1989 scientists identified the gene mutation that causes cystic fibrosis (CF), which led to the hope that CF lung disease could be “cured” using gene therapy. The premise of gene therapy is that modified viruses or other gene-based systems could be used to deliver a corrected version of a gene into affected tissues. However, the projected cure has been hampered by the natural ability of the lung to limit the introduction of foreign genes into its cells.

Now scientists have found what may be the most efficient way to deliver a corrected gene to lung cells derived from CF patients, renewing hope that gene therapy for CF lung disease could be a successful future treatment. While cystic fibrosis is a multiple organ disease, it most devastatingly affects the lung. In people with CF the airways are clogged with mucus that is dehydrated and thicker than normal. The inability to clear mucus from the lung increases the susceptibility of CF patients to lung infections, which results in lung damage. Over the last two decades scientists have developed a variety of viral and non-viral vector systems suitable for delivering a corrected CF gene back into lung cells grown in the laboratory. Several of these vectors systems have been tested in human clinical trials. However, the efficiency of gene delivery achieved in the laboratory has not borne out in the clinical studies, suggesting that the cell models used in the laboratory do not represent the status of the cells in patients’ lungs. Scientists have since developed laboratory models of human lung cells derived from CF patients that recapitulate the architecture and function of the cells present in the human lung. Studies using such cell models have revealed that previously used vector systems cannot deliver the corrected CF gene to enough lung cells to be of clinical benefit to CF patients.

In this new study, scientists took a different approach and used parainfluenza virus, a virus known to infect human lung cells and to cause common colds. The researchers engineered this virus to contain the corrected CF gene and found that it could deliver this gene to 60-70% of lung cells although only 25% of cells needed to be targeted to restore normal function back to the tissue model. This study demonstrates efficient and efficacious CFTR delivery to CF ciliated airway epithelium and that CFTR delivered to approximately 25% of the surface epithelial cells restores normal levels of airway surface hydration and mucus transport. These studies serve as a benchmark for the efficiency of CFTR gene delivery to CF airways for future CF gene therapy studies in vivo.

This is the first demonstration in which medicine has been able to execute delivery in an efficient manner to a tissue that resembles what is present in the lung. When you consider that in past gene therapy clinical trials, the targeting efficiency has been somewhere around 0.1 percent of cells at best, you can see this is a giant leap forward. Now the researchers must work to ensure the safety of the delivery system. In a pleasant surprise, simply adding the CF gene to the virus significantly attenuated it, potentially reducing its ability to cause an inflammatory reaction. But the scientists may need to alter the virus further. Although they have not generated a vector that that can be used in patients right now, researchers are slowly but surely moving forward towards this goal. It is going to require a long term commitment from the CF gene therapy field that has achieved so much this far.

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium. PLoS Biol 7(7): e1000155 doi:10.1371/journal.pbio.1000155
Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to .60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl2 and Na+ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.

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Cells arrange their components – proteins, lipids, and nucleic acids – in organized and reproducible ways to optimize their activities and to improve cell efficiency and survival. Eukaryotic cells have a complex arrangement of subcellular structures such as membrane-bound organelles and cytoskeletal transport systems. However, subcellular organization is also important in prokaryotic cells, including rod-shaped bacteria such as E. coli, most of which lack such well-developed systems of organelles and motor proteins for transporting cellular cargoes. In fact, it has remained somewhat mysterious how bacteria are able to organize and spatially segregate their interiors.

The E. coli chemotaxis network, a system important for the bacterial response to environmental cues, is one of the best-understood biological signal transduction pathways and serves as a useful model for studying bacterial spatial organization because its components display a nonrandom, periodic distribution in mature cells. Chemotaxis receptors aggregate and cluster into large sensory complexes that localize to the poles of bacteria. To understand how these clusters form and what controls their size and density, a recent study used ultrahigh-resolution light microscopy, called photoactivated localization microscopy, to visualize individual chemoreceptors in single E. coli cells. From these high-resolution images, the authors were able to determine that receptors are not actively distributed or attached to specific locations in cells. Instead, it appears that random receptor diffusion and receptor–receptor interactions are sufficient to generate the observed complex, ordered pattern. This simple mechanism, termed stochastic self-assembly, may prove to be widespread in both prokaryotic and eukaryotic cells.

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Self-Organization of the Escherichia coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy. 2009 PLoS Biol 7(6): e1000137 doi:10.1371/journal.pbio.1000137
The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

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Rabies causes an estimated 55,000 human deaths globally each year, 23,750 of which occur in Africa. Moreover, 11 million people undergo rabies postexposure prophylaxis (PEP) worldwide each year. Rabies is a zoonotic disease with dogs remaining the principal host in Asia, parts of America, and large parts of Africa, and rabid dogs are the cause of most human rabies cases. Between 30% to 60% of the victims of dog bites are children under the age of 15. Inappropriate dog vaccination programs, limited access to vaccination, and postexposure treatment of individuals that have been exposed to rabid dogs are major problems in developing countries.

Rabies virus (RV), a negative-stranded RNA virus of the rhabdoviridae family, has a relatively simple, modular genome that encodes 5 structural proteins: a RNA-dependent RNA polymerase (L), a nucleoprotein (N), a phosphorylated protein (P), a matrix protein (M), and an external surface glycoprotein (G). The N, P, and L together with the genomic RNA form the ribonucleoprotein complex (RNP). The main feature of rabies virus is neuroinvasiveness, which refers to its unique ability to invade the CNS from peripheral sites. Virus uptake, axonal transport, trans-synaptic spread, and the rate of virus replication are key factors that determine the neuroinvasiveness of RV.

The regulation of virus replication also appears to be one of the important mechanisms contributing to RV pathogenesis. Pathogenic RV strains replicate at a lower rate than attenuated strains, which helps preserve the structure of neurons that is used by the viruses to reach the CNS. In addition, the lower expression levels of virus antigens, in particular the RV G, which is the major viral antigen responsible for the induction of protective immunity, hinders early detection by the host immune system. In contrast to wildlife RVs, most attenuated RV strains replicate very quickly and express large amounts of G, thereby inducing strong adaptive immune responses that result in virus clearance. These properties provide the basis for the use of attenuated RV strains for the pre- and PEP of rabies. A live-attenuated RV vaccine is likely to provide effective immunization with a single dose, which has practical, cost, and logistical advantages over conventional multi-dose vaccines with respect to the worldwide eradication of dog rabies. In addition, because live-attenuated RV vaccines are capable of inducing immune responses that can clear virulent RVs from the CNS, there is the possibility that such vaccines could serve as the foundation for the treatment of early stage human rabies.

Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus. PNAS USA 2009 106(27): 11300-5
Rabies remains an important public health problem with more than 95% of all human rabies cases caused by exposure to rabid dogs in areas where effective, inexpensive vaccines are unavailable. Because of their ability to induce strong innate and adaptive immune responses capable of clearing the infection from the CNS after a single immunization, live-attenuated rabies virus (RV) vaccines could be particularly useful not only for the global eradication of canine rabies but also for late-stage rabies postexposure prophylaxis of humans. To overcome concerns regarding the safety of live-attenuated RV vaccines, we developed the highly attenuated triple RV G variant, SPBAANGAS-GAS-GAS. In contrast to most attenuated recombinant RVs generated thus far, SPBAANGAS-GAS-GAS is completely nonpathogenic after intracranial infection of mice that are either developmentally immunocompromised (e.g., 5-day-old mice) or have inherited deficits in immune function (e.g., antibody production or type I IFN signaling), as well as normal adult animals. In addition, SPBAANGAS-GAS-GAS induces immune mechanisms capable of containing a CNS infection with pathogenic RV, thereby preventing lethal rabies encephalopathy. The lack of pathogenicity together with excellent immunogenicity and the capacity to deliver immune effectors to CNS tissues makes SPBAANGAS-GAS-GAS a promising vaccine candidate for both the preexposure and postexposure prophylaxis of rabies.

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This summer on MicrobiologyBytes we’ll be revisiting a few old favourites – some of the most popular posts on this site. Today’s post is:

The results of a new study have provided fresh insights into influenza pandemics by raising the possibility that all three pandemic influenza strains of the 20th century may have been generated through a series of multiple reassortment events and emerged over a period of years before pandemic recognition. The results indicate that each of these strains was produced by reassortment between the previously circulating human virus and at least one virus of animal origin. The novel gene segments for the H2N2/1957 and H3N2/1968 pandemics seem to have originated from avian hosts, but the zoonotic sources of the introduced viral gene segments for the 1918 pandemic remain ambiguous. However, evidence suggests that, over a number of years, avian gene virus segments have entered mammalian populations where the viruses may have undergone reassortment with the prevailing human virus. Given the frequent interspecies transmission of influenza viruses between swine and humans, it is most likely that such reassortment events occurred in pigs before pandemic emergence.

This work suggests that in the 1918 and 1957 pandemics, novel NA and internal genes may have been introduced into the prevailing human virus strains before the acquisition of the novel pandemic HA. Frequent detection of seasonal human influenza strains in pigs indicates that pandemic precursor viruses probably have circulated in either swine or human populations. The precursors to the H2N2 and H3N2 pandemics have not been detected, probably because they originated in Asia where little or no surveillance was conducted at that time.

If future pandemics arise in this manner, this interval may provide the best opportunity for health authorities to intervene to mitigate the effects of a pandemic or even to abort its emergence. However, the findings argue the need for highthroughput characterization of all 8 gene segments of human virus isolates, even those that have unremarkable HA antigens, particularly of human viruses isolated in hotspots for zoonotic infections with avian influenza viruses. At present, global influenza surveillance in humans focuses attention primarily on hemagglutinin. Although this focus will continue to be required for strain selection for seasonal influenza vaccines, our findings argue that this surveillance will not suffice for early warning of an incipient pandemic.

Dating the emergence of pandemic influenza viruses. PNAS USA July 13, 2009. doi: 10.1073/pnas.0904991106
Pandemic influenza viruses cause significant mortality in humans. In the 20th century, 3 influenza viruses caused major pandemics: the 1918 H1N1 virus, the 1957 H2N2 virus, and the 1968 H3N2 virus. These pandemics were initiated by the introduction and successful adaptation of a novel hemagglutinin subtype to humans from an animal source, resulting in antigenic shift. Despite global concern regarding a new pandemic influenza, the emergence pathway of pandemic strains remains unknown. Here we estimated the evolutionary history and inferred date of introduction to humans of each of the genes for all 20th century pandemic influenza strains. Our results indicate that genetic components of the 1918 H1N1 pandemic virus circulated in mammalian hosts, i.e., swine and humans, as early as 1911 and was not likely to be a recently introduced avian virus. Phylogenetic relationships suggest that the A/Brevig Mission/1/1918 virus (BM/1918) was generated by reassortment between mammalian viruses and a previously circulating human strain, either in swine or, possibly, in humans. Furthermore, seasonal and classic swine H1N1 viruses were not derived directly from BM/1918, but their precursors co-circulated during the pandemic. Mean estimates of the time of most recent common ancestor also suggest that the H2N2 and H3N2 pandemic strains may have been generated through reassortment events in unknown mammalian hosts and involved multiple avian viruses preceding pandemic recognition. The possible generation of pandemic strains through a series of reassortment events in mammals over a period of years before pandemic recognition suggests that appropriate surveillance strategies for detection of precursor viruses may abort future pandemics.

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This summer on MicrobiologyBytes we’ll be revisiting a few old favourites – some of the most popular posts on this site. Today’s post is:

In the early 1980s, Kary Mullis developed the polymerase chain reaction, an elegant way to make copies of a DNA strand using the enzyme polymerase and some basic DNA “building blocks.” Mullis shared the 1993 Nobel Prize in Chemistry for developing this technique. Most recently, he’s been taking a look at immunity; a recent patent from his company Altermune describes the redirection of an existing immune response to a new pathogen. Video:

“In my day” i.e. when I started my PhD back in 197<cough>, the first few weeks were spent joining the Enzyme Club. This encompassed all the biomedical researchers at the University of Leicester. Each new student would prepare a batch enzyme for recombinant DNA work. In my case, I made Hsu I (an isoschizomer of Hae III but allegedly easier to prepare). Since it was years ago, I can’t remember how many litres of the organism I grew up, but I remember very clearly doing the first assay on two litres of crude extract, and figuring out I was holding £40 million pounds worth of enzyme at the then current market prices. The first affinity column cut it down to £15 million, and a quick gel filtration to couple of millions pounds worth – still pretty good for two weeks work, especially when you remember that two million pounds was enough to buy you a house back in the 1970s!

Why did the Enzyme Club exist? Because these reagents were scarce in the 1970s, and rationed both by price and availability. Only a few years before, the only way to get hold of any of these enzymes was to make your own. This type of open science made sense. Why did the Enzyme Club cease to exist? Gradually, it became clear that the batch of enzyme I made wasn’t very good. It had a persistent exonuclease activity which meant it was fine for restriction analysis but rubbish for cloning, and it went off very quickly in storage, so that after three months there wasn’t much activity left. And although I’ve always been a rubbish protein chemist, that was a pretty common experience. Gradually, the companies dropped their prices and improved both the quality and availability of commercial enzymes. The day came when the Enzyme Club didn’t make sense any more, and it quietly died. It’s probably still moldering in the back of a coldroom over in the MSB.

So boys and girls, this is a story of the economics of open science, which made sense in response to scarce resources. When the availability of enzymes was limiting, this open approach made sense. When time became limiting, we all retreated back into our laboratories and got on with whatever we needed to do to get a PhD. The moral of this story is that open science pops up it’s head when times are hard and resources are scarce, but retreats quickly as the balance changes.

Regulation of gene expression at the level of mRNA translation is important for the control of numerous biological processes including cell growth, differentiation, development and the response to environmental stress. Unlike prokaryotes, the vast majority of eukaryotic mRNAs are unable to recognize ribosomes directly and rely instead on an intricate set of translation initiation factors that assemble a specialized multisubunit complex onto the mRNA 5′ terminus to recruit the 40S ribosome subunit. The responsiveness of individual constituents of this complex to a wide spectrum of cellular signals allows the translational machinery to respond rapidly to diverse physiological effectors. The 4E-BP translational repressor family, for example, sequesters eIF4E and prevents binding to eIF4G, limiting ribosome recruitment. Similarly, the ERK and p38-responsive eIF4G-associated kinase Mnk1 modulates eIF4E phosphorylation, which in specific instances has been associated with increased translation rates. Thus, regulated translation initiation factor complex assembly and modification is poised to potentiate important developmental decisions by controlling global and specific mRNA translation.

Viruses provide attractive models to study simple developmental decisions. In prokaryotes, much has been learned using bacteriophage λ to investigate how the lysis-lysogeny decision is made. In eukaryotes, latent herpesviruses exist in one of two developmental states within their hosts and must resolve an analogous question of whether to remain latent or initiate productive viral growth. Different herpesviruses permanently colonize distinct specialized host cell-types. Those that establish residency in dividing cell populations, exemplified by members of the γ-herpesvirus subfamily that includes Kaposi’s sarcoma associated herpesvirus (KSHV/HHV8), express a limited subset of viral genes that stimulate cell proliferation, allow for viral minichromosome replication and segregation, and evade antiviral defenses. In response to poorly understood environmental cues, these viruses can switch to a developmental program that results in productive replication. This alternate pathway involves activating a temporally coordinated cascade of viral lytic gene expression, which in turn results in massive viral DNA amplification, progeny virus production, and ultimately host cell destruction. To effectively switch its gene expression program, all herpesviruses produce a new population of viral mRNAs transcribed by the cellular RNA polymerase II, which are mostly capped and polyadenylated like their host counterparts, and these must successfully engage and reprogram the host cell translational apparatus. This is a critical component of the developmental switch because viruses are absolutely dependent upon the translational machinery resident in their hosts. Manipulating host translation initiation factors to ensure that nascent viral mRNAs successfully recruit ribosomes will therefore determine the overall level and efficacy with which the newly transcribed developmental instructions are executed. While we have a general understanding of how the viral transcriptome is altered for many viruses, a role for translational control in the developmental switch from a latent to a productively replicating state has not been described.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an important human pathogen and, like all herpesviruses, establishes a state of permanent residency in the infected host called latency. Major sites of KSHV latency are cells of the immune system and cells lining blood vessels. In individuals with weakened immunity, inappropriate growth of these cells driven by the resident virus can give rise to primary effusion lymphoma and Kaposi’s sarcoma, respectively. These life-threatening cancers are most common in patients with HIV/AIDS and have become a major source of mortality in parts of sub-Saharan Africa. Under appropriate stimuli, herpesviruses change their relationship with the host cell and begin to manufacture proteins required to assemble new infectious virus particles that can be released and spread. To achieve this, the virus hijacks key processes within the cell and conscripts them into producing viral proteins. A recent study describes for the first time how KSHV carefully manipulates the host protein synthesis machinery during the switch from latency to this specialized infectious virus production mode. The results show that although overall protein synthesis is diminished, key components of the host’s protein manufacturing machinery are actually stimulated, presumably to accelerate virus protein production.

Activation of Host Translational Control Pathways by a Viral Developmental Switch. PLoS Pathog 5(3): e1000334. doi:10.1371/journal.ppat.1000334
In response to numerous signals, latent herpesvirus genomes abruptly switch their developmental program, aborting stable host–cell colonization in favor of productive viral replication that ultimately destroys the cell. To achieve a rapid gene expression transition, newly minted capped, polyadenylated viral mRNAs must engage and reprogram the cellular translational apparatus. While transcriptional responses of viral genomes undergoing lytic reactivation have been amply documented, roles for cellular translational control pathways in enabling the latent-lytic switch have not been described. Using PEL-derived B-cells naturally infected with KSHV as a model, we define efficient reactivation conditions and demonstrate that reactivation substantially changes the protein synthesis profile. New polypeptide synthesis correlates with 4E-BP1 translational repressor inactivation, nuclear PABP accumulation, eIF4F assembly, and phosphorylation of the cap-binding protein eIF4E by Mnk1. Significantly, inhibiting Mnk1 reduces accumulation of the critical viral transactivator RTA through a post-transcriptional mechanism, limiting downstream lytic protein production, and impairs reactivation efficiency. Thus, herpesvirus reactivation from latency activates the host cap-dependent translation machinery, illustrating the importance of translational regulation in implementing new developmental instructions that drastically alter cell fate.

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