MicrobiologyBytes

Bacteriophage lysis is a precisely scheduled process controlled by proteins of the holin family. Holins are a diverse class of small phage-encoded membrane proteins. The best studied holin is S105, a 105-residue polypeptide with three transmembrane domains (TMDs) encoded by the S gene of phage λ. Throughout the period of late gene expression and particle assembly, S105 accumulates in the cytoplasmic membrane of Escherichia coli without any effect on its integrity. Suddenly, at a programmed time, S105 triggers to form a lesion, or hole, in the membrane; this allows the λ-endolysin, R, to escape from the cytoplasm and attack the cell wall. In phages of Gram-negative hosts, there is a third step to complete the lysis pathway involving a protein or protein complex, the spanin, which connects the cytoplasmic and outer membranes. In λ, the spanin complex consists of the cytoplasmic membrane protein, Rz, and the outer membrane lipoprotein, Rz1. This complex is thought to act by disrupting the outer membrane, possibly by fusion with the inner membrane. The large holes observed can be viewed as supporting the notion that at the time of lethal triggering, the S105 holin exists in such large aggregates, leading to one or a small number of holes rather than many smaller holes distributed throughout the membrane.

Micron-scale holes terminate the phage infection cycle. PNAS USA January 11 2010. doi: 10.1073/pnas.0914030107
Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the phage infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or “holes.” In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.

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• How can lytic viruses be evolutionarily competitive?

Invasive fungal infections are an increasing threat to human health. In the developed world, these infections predominantly occur in increasingly aggressive immunosuppressive therapies. The overall mortality for invasive diseases caused by Candida spp. and Aspergillus spp. is 30–50%, despite the advent of new diagnostic and therapeutic strategies. In the developing world, there are 1 million cases of cryptococcal disease per year, resulting in 675,000 deaths. Allergic fungal syndromes are increasingly recognised. Continued efforts are required to improve the often suboptimal therapeutic outcomes associated with fungal infections.

The high degree of phylogenetic relatedness between fungi and humans means that there are relatively few differential targets to be exploited for antifungal drug development. Fungi are involved in an interminable struggle for survival with each other and with other microbes. They produce a vast array of extracellular enzymes and secondary metabolites to counteract and digest the external world. Many antimicrobial agents have been isolated from fungi themselves. The best example is penicillin, which was isolated from Penicillium notatum (now Penicillium chrysogenum) by Fleming, and later purified for medical use by Florey and Chain. Similarly, the echinocandins, a novel class of antifungal compounds now in widespread clinical use, are semisynthetic derivatives of fungal-derived cyclic hexapeptides. Currently, five classes of antifungal agents are used orally or intravenously for the treatment of fungal infections in humans: polyenes, pyrimidine analogues, allylamines, azoles and the echinocandins. Each antifungal compound has advantages and limitations related to its spectrum of activity, route of administration, drug interactions and toxicity profile.

This review describes the role and limitations of these agents for the treatment of the most common and medically important syndromes, and provides a perspective on the current status of drug therapy for invasive fungal diseases, together with priorities for the future development of novel compounds. Key opportunities for new drugs include production of orally bioavailable agents for the treatment of invasive aspergillosis, invasive candidiasis, cryptococcal meningitis and mucosal and urinary Candida infections. Orally bioavailable agents for the treatment of chronic pulmonary and allergic aspergillosis are also required, as well as new potent drugs against a range of medically important moulds. Antifungal resistance is a problem in certain contexts, but is generally less of a problem than bacterial infections. Earlier and more complete mycological diagnosis and improvements in underlying risk estimation will improve outcomes.

Therapy for fungal diseases: opportunities and priorities. Trends Microbiol. March 5, 2010

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• All About Fungi: Part 1
• All About Fungi: Infection

Before World War II, diphtheria was a major killer disease of childhood. In this article in Microbiology Today (pdf) Philip Mortimer looks at why Britain did not start its national efforts to vaccinate the population against this bacterial infection sooner and the lessons that have been learnt since:

Delay and indifference characterized the interwar period of British public health provision, at least in respect of diphtheria prophylaxis. The two decades witnessed a gradual decline in numbers of diphtheria cases and deaths, mainly attributable to the increasingly effective use of diphtheria antitoxin; but the UK signally failed to take advantage of the opportunity that, from the mid-1920s, diphtheria toxoid (i.e. vaccine) offered to eliminate the disease. The failure was, at its root, an administrative rather than a scientific or professional one, and the emergency measures of 1940 that repaired the lapse in effect acknowledged that.

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• Vaccination is up to 99% effective
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Leishmania donovani is a protozoan parasite that causes severe disease in humans with associated pathology in the spleen and liver. In experimental models of L. donovani infection, the hepatic response to infection is characterised by the presence of a focal mononuclear cell-rich inflammatory response (a granuloma) surrounding cells infected with intracellular amastigotes. Granulomas provide focus to the ensuing immune response, helping to contain parasite dissemination and providing the major effector site responsible for parasites elimination from the liver. Although granulomas are believed to form around infected resident liver macrophages (Kupffer cells), the role of these cells in intra-granuloma antigen presentation is currently unknown. Researchers used sophisticated microscopy to identify how killer T lymphocytes behaved when they enter sites of inflammation caused by L. donovani, and which infected cells they were able to recognise.

Leishmaniasis is a globally important but neglected disease, affecting approximately two million people every year. For most people, infection results in a slow-to-heal skin ulcer. In others, however, the parasite targets the liver, spleen and bone marrow, leading to over 70,000 deaths annually. The Leishmania parasite is eventually contained by a characteristic type of inflammatory response that forms cellular structures called granulomas. Little is known about the inner workings of these granulomas, in spite of their occurrence in other human diseases, from tuberculosis to rheumatoid arthritis. The scientists used an advanced laser-based microscopy technique, called “2-photon imaging”, to view the inner workings of the granuloma in mice infected with Leishmania. This enabled them to study how killer lymphocytes, such as those that can be induced by vaccination, are able to enter into the granulomas, penetrate deep into the core of the structure and seek out specific types of parasite-infected cells. Although this technique can not be used currently for the study of inflammatory disease in humans, the insights provided into the biology of granulomas and the hidden world of inflammation should help to improve vaccines and drugs, and allow researchers to now construct in silico models for this type of inflammatory process. These data have important implications for the understanding of how granulomas function to limit infection and may have important implications for the development of vaccines to Leishmania.

Dynamic Imaging of Experimental Leishmania donovani-Induced Hepatic Granulomas Detects Kupffer Cell-Restricted Antigen Presentation to Antigen-Specific CD8+ T Cells. PLoS Pathog 6(3): e1000805. doi:10.1371/journal.ppat.1000805
Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intravital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

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• Novel application for an old drug: tamoxifen in the treatment of Leishmania infection
• Foamy macrophages allow TB agent to survive in infected individuals

Tuesday 30 March 2010

Follow this event on Twitter: #sgmed10

When asked what they are looking for in a microbiology graduate, employers often stipulate a well-rounded individual who is self-motivated, can solve problems and interacts productively with fellow scientists. This symposium, organised jointly by the SGM and the HE Academy UK Centre for Bioscience, will give you the opportunity to discuss innovative approaches to delivering this paragon. The first half will look at how we can foster the key skills of creativity, problem-solving and enquiry-based learning in the laboratory and field. This will include a keynote presentation about an exciting ‘Phage-Hunting’ project from the University of Pittsburgh, which allows students from a range of educational backgrounds to engage in authentic scientific enquiry. We will then explore the impact of social networking tools, both on our students’ learning and on our own teaching practice. The symposium should be of interest to anyone involved in teaching microbiologists who wants to learn how to exploit the power of emerging approaches and technologies. If you think Twitter is just for chatting to celebrities, it’s time to take a fresh look!

Promoting key skills in microbiology teaching:
0830 Carol Wakeford, University of Manchester: A culture of creativity: techniques to foster new ideas.
0930 Tina Overton, Higher Education Academy, Hull Problem solving.
1100 Gus Cameron, eBioLabs, University of Bristol: Laboratory skills.
1400 Graham Hatfull, University of Pittsburgh: Where there’s smoke there’s PHIRE: authentic research projects for novice researchers.

Using Web2.0 technologies for teaching microbiology: information overload or filter failure?
1500 Cameron Neylon, STFC Rutherford Appleton Laboratory, Didcot: Information overload or filter failure?
1530 Vincent Racaniello, Columbia University Medical Center, New York: Social media in microbiology education and research.
1630 Kevin Emamy / Jason Hoyt, CiteULike, London / Mendeley, London: Automated discovery of scientific literature.

More information

Cystic fibrosis is a widespread genetic disease that leads to progressive disability and early death. The principal cause of mortality and morbidity in CF patients is a progressive deterioration of the respiratory system caused by a chronic infection of the patients’ lungs, mainly by the opportunistic bacterial pathogen Pseudomonas aeruginosa. CF lung infections can be treated with antibiotics, however full clearance is not possible due to the protective environment of the CF lung and the adaptation of infective species to a persistent lifestyle. This presents serious challenges for the long-term chemotherapy of CF patients.

New research reveals that Small Colony Variants (SCVs) of P. aeruginosa are be a hallmark of chronic infection in cystic fibrosis (CF) patients. These new results suggest that SCV-mediated persistence might be a good target for antimicrobial chemotherapy. Adaptive P. aeruginosa morphotypes include SCVs, slow-growing and strongly adherent variants that frequently arise in chronic lung infections. Because the appearance of SCVs correlates with poor lung function and antibiotic resistance, they have long been suspected of mediating the P. aeruginosa persistence phenotype in CF infections. In this study, the researchers characterized a signaling system in P. aeruginosa called YfiBNR, mutations in which lead to the generation of SCV variants. Activation of YfiBNR resulted in increased levels of the signaling molecule c-di-GMP, which in turn triggered massive production of exopolysaccharides and drastically reduced growth rates, two hallmarks of SCV behavior. YfiN-mediated SCVs were shown to be highly resistant to macrophage phagocytosis, suggesting a role for the SCV phenotype in immune system evasion. Consistent with this, activation of YfiN significantly increased the persistence of P. aeruginosa in long-term infections in a mouse model, establishing a firm causal link between SCV and persistence in chronic P. aeruginosa infections. The authors conclude that c-di-GMP has long been a key suspect in chronic behavior of bacterial pathogens. The finding that the c-di-GMP-mediated SCV phenotype confers a persistent advantage in mice provides the first direct evidence in favor of such a model. This study opens up new avenues to specifically counteract persistent infections in CF.

YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa. PLoS Pathog 6(3): e1000804. doi:10.1371/journal.ppat.1000804
During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCVrelated loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to subinhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

Related:

• Can the Common Cold Cure Cystic Fibrosis?
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Aedes aegypti is the urban vector of dengue viruses worldwide. While climate influences the geographical distribution of this mosquito species, other factors also determine the suitability of the physical environment for this mosquito. Importantly, the close association of Ae. aegypti with humans and the domestic environment allows this species to persist in regions that may otherwise be unsuitable based on climatic factors alone. This review highlights the need to incorporate the impact of the urban environment in attempts to model the potential distribution of Ae. aegypti and briefly discuss the potential for future technology to aid management and control of this widespread vector species.

The dengue vector Aedes aegypti: What comes next. Microbes Infect. Jan 20 2010. doi:10.1016/j.micinf.2009.12.011

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• Population movement a critical factor in dengue virus spread
• Dengue Virus
• Rethinking dengue hemorrhagic fever
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In the era of microbial genomics, much effort has gone into understanding the functions of individual genes. Complex metabolic events, however, rely on the interaction of multiple genes that play multiple roles and are best understood using whole-system modelling. In this article in Microbiology Today (pdf) Johnjoe McFadden explains how integrated systems methods are ideal for studying microbes and postulates that his approach could help us take on tuberculosis, one of the biggest global killers:

Microbial genome sequencing projects have released a flood of genes for most medically and industrially important microbes, including the TB bacillus. Much effort in microbial genetics is directed towards identifying the functions of these genes, particularly “orphan genes” that lack a functional homologue in other organisms…

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• Why is TB more common in men than in women?
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There is strong evidence to suggest that climate change has, and will continue to affect the occurrence, distribution and prevalence of livestock diseases in Great Britain (GB). This paper reviews how climate change could affect livestock diseases in GB. Factors influenced by climate change and that could affect livestock diseases include the molecular biology of the pathogen itself; vectors (if any); farming practice and land use; zoological and environmental factors; and the establishment of new microenvironments and microclimates. The interaction of these factors is an important consideration in forecasting how livestock diseases may be affected. Risk assessments should focus on looking for combinations of factors that may be directly affected by climate change, or that may be indirectly affected through changes in human activity, such as land use (e.g. deforestation), transport and movement of animals, intensity of livestock farming and habitat change. A risk assessment framework is proposed, based on modules that accommodate these factors. This framework could be used to screen for the emergence of unexpected disease events.

The effect of climate change on the occurrence and prevalence of livestock diseases in Great Britain: a review. J Appl Microbiol. (2009) 106(5): 1409-1423

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• Bluetongue virus
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