MicrobiologyBytes

Marine natural products are a continued focus for drug discovery and have provided many important therapeutic agents. Lead compounds with biomedical potential have been isolated from marine invertebrates, bacteria, and fungi. Each year numerous compounds with an array of biological activities are reported, but to-date only 13 molecules have entered into the clinical pipeline. Four molecules have been approved for clinical use, one of which is approved only in the EU. The approved molecules include two nucleosides based on sponge-derived nucleosides, a cone snail peptide, and a metabolite isolated from a tunicate. Marine microbes have received growing attention as the sources for bioactive metabolites and have great potential to increase the number of marine natural products in clinical trials. The sustainable and economic supply of the active pharmaceutical ingredient is often easier to achieve for compounds produced through microbial fermentation approaches versus the cultivation of slower growing macroorganisms.

Marine natural products provide an excellent opportunity to study diverse and unique compounds not readily accessible from any other source leading to expansion of the pharmaceutical pipeline. Marine microbes can produce unique compounds covering new chemical space, and the utility of marine natural products is expanding beyond its original role in identification of new prototype drug leads into fields of study involving sustainable supplies of unique molecules using biosynthesis in conjunction with synthesis. Perhaps the greatest impact marine natural products has played is in revealing that unexplored and previously inaccessible chemical space can contribute to growth in the pharmaceutical pipeline. Improved methodologies in fermentation technologies, biosynthesis, and synthesis provide opportunities to both create and supply drug leads that would not be available by any single method independently. As a result pharmaceutical biotechnology in the future is certain to provide increasingly sophisticated molecular architecture assembled using biosynthesis and synthesis in concert.

The expanding role of marine microbes in pharmaceutical development. Curr Opin Biotechnol. Oct 16 2010
Marine microbes have received growing attention as sources of bioactive metabolites and offer a unique opportunity to both increase the number of marine natural products in clinical trials as well as expedite their development. This review focuses specifically on those molecules currently in the clinical pipeline that are established or highly likely to be produced by bacteria based on expanding circumstantial evidence. We also include an example of how compounds from harmful algal blooms may yield both tools for measuring environmental change as well as leads for pharmaceutical development. An example of the karlotoxin class of compounds isolated from the dinoflagellate Karlodinium veneficum reveals a significant environmental impact in the form of massive fish kills, but also provides opportunities to construct new molecules for the control of cancer and serum cholesterol assisted by tools associated with rational drug design.

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Enteroviruses are associated with a number of diverse syndromes such as myocarditis, febrile illness, and are the main causative agents of aseptic meningitis. No effective therapeutics exist to combat non-poliovirus enterovirus infections. A better understanding of the mechanisms by which these viruses infect host cells could lead to the design of effective therapeutic interventions. This study found that intracellular calcium stores in polarized endothelial monolayers are depleted upon exposure to coxsackievirus B (CVB) and that this release is mediated by viral attachment to its receptor decay-accelerating factor. It also discovered that the calcium release requires the activation of signaling molecules involved in calcium signaling such as Src tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3 on the ER membrane. A calcium-activated cystein protease, calpain-2, was activated and necessary for proper viral trafficking inside the cell. Interestingly, this signaling cascade was critical for CVB internalization into the endothelium, but was not involved in CVB entry into the epithelium. This is an important advance in our understanding of how enteroviruses hijack host endothelial cell signaling mechanisms in order to facilitate their entry and eventual spread.

Release of Intracellular Calcium Stores Facilitates Coxsackievirus Entry into Polarized Endothelial Cells. (2010) PLoS Pathog 6(10): e1001135. doi:10.1371/journal.ppat.1001135
Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.

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• Dynamics of picornavirus RNA replication
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Scientists are studying a retrovirus that has been dormant in chimpanzees and their ancestors for at least one million years. The virus, known as CERV2, is present in the genomes of chimpanzees, but not those of humans, suggesting that chimpanzees’ ancestors became infected after they diverged from human ancestors 5–6 million years ago. The virus was clearly replicating around the time that the first humans were trotting around Africa and beginning to think about colonizing the rest of the world. As a receptor, the ancient virus exploited a transport protein that normally transports copper into and out of cells.

Identification of a receptor for an extinct virus. PNAS USA October 25, 2010 doi: 10.1073/pnas.101234410
The resurrection of endogenous retroviruses from inactive molecular fossils has allowed the investigation of interactions between extinct pathogens and their hosts that occurred millions of years ago. Two such paleoviruses, chimpanzee endogenous retrovirus-1 and -2 (CERV1 and CERV2), are relatives of modern MLVs and are found in the genomes of a variety of Old World primates, but are absent from the human genome. No extant CERV1 and -2 proviruses are known to encode functional proteins. To investigate the host range restriction of these viruses, we attempted to reconstruct functional envelopes by generating consensus genes and proteins. CERV1 and -2 enveloped MLV particles infected cell lines from a range of mammalian species. Using CERV2 Env-pseudotyped MLV reporters, we identified copper transport protein 1 (CTR1) as a receptor that was presumably used by CERV2 during its ancient exogenous replication in primates. Expression of human CTR1 was sufficient to confer CERV2 permissiveness on otherwise resistant hamster cells, and CTR1 knockdown or CuCl2 treatment specifically inhibited CERV2 infection of human cells. Mutations in highly conserved CTR1 residues that have rendered hamster cells resistant to CERV2 include a unique deletion in a copper-binding motif. These CERV2 receptor-inactivating mutations in hamster CTR1 are accompanied by apparently compensating changes, including an increased number of extracellular copper-coordinating residues, and this may represent an evolutionary barrier to the acquisition of CERV2 resistance in primates.

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Wolbachia pipientis is an intracellular maternally-inherited bacterial symbiont of invertebrates that is very common in insects, including a number of mosquito species. It can manipulate host reproduction in several ways, including cytoplasmic incompatibility, whereby certain crosses are rendered effectively sterile. Females that are uninfected produce infertile eggs when they mate with males that carry Wolbachia, while there is a “rescue” effect in Wolbachia-infected embryos such that infected females can reproduce successfully with any males. Therefore uninfected females suffer a frequency-dependent reproductive disadvantage. Wolbachia is able to rapidly invade populations using this powerful mechanism

Malaria is one of the world’s most devastating diseases, particularly in Africa, and new control strategies are desperately needed. Here we show that the presence of Wolbachia bacteria inhibits the development of a malaria parasite in the most important Anopheles mosquito species of Africa. In addition it shows that the presence of Wolbachia results in the switching on of immune genes that are known to affect development of many species of malaria parasite. When added to the lifespan-shortening effects of this particular strain of Wolbachia, and the general ability of Wolbachia to spread through insect populations, this study provides a stimulus for the development of Wolbachia-based malaria control methods. It also provides new insights into the wide range of effects of Wolbachia in insects.

Wolbachia Stimulates Immune Gene Expression and Inhibits Plasmodium Development in Anopheles gambiae. (2010) PLoS Pathog 6(10): e1001143. doi:10.1371/journal.ppat.1001143
The over-replicating wMelPop strain of the endosymbiont Wolbachia pipientis has recently been shown to be capable of inducing immune upregulation and inhibition of pathogen transmission in Aedes aegypti mosquitoes. In order to examine whether comparable effects would be seen in the malaria vector Anopheles gambiae, transient somatic infections of wMelPop were created by intrathoracic inoculation. Upregulation of six selected immune genes was observed compared to controls, at least two of which (LRIM1 and TEP1) influence the development of malaria parasites. A stably infected An. gambiae cell line also showed increased expression of malaria-related immune genes. Highly significant reductions in Plasmodium infection intensity were observed in the wMelPop-infected cohort, and using gene knockdown, evidence for the role of TEP1 in this phenotype was obtained. Comparing the levels of upregulation in somatic and stably inherited wMelPop infections in Ae. aegypti revealed that levels of upregulation were lower in the somatic infections than in the stably transinfected line; inhibition of development of Brugia filarial nematodes was nevertheless observed in the somatic wMelPop infected females. Thus we consider that the effects observed in An. gambiae are also likely to be more pronounced if stably inherited wMelPop transinfections can be created, and that somatic infections of Wolbachia provide a useful model for examining effects on pathogen development or dissemination. The data are discussed with respect to the comparative effects on malaria vectorial capacity of life shortening and direct inhibition of Plasmodium development that can be produced by Wolbachia.

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• Wolbachia
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Staphylococcus epidermidis frequently causes chronic infections, indicating pronounced capacity to evade host defenses. However, S. epidermidis is in general much less aggressive than its close relative, S. aureus. This paper looks at the molecular reasons for that discrepancy by showing that S. epidermidis immune evasion mechanisms are limited to those involving molecules that protect against or eliminate antimicrobial agents secreted by white blood cells, while immune evasion mechanisms of virulent S. aureus include the production of destructive toxins. This is especially noteworthy, because we demonstrate here for the first time that S. epidermidis has the capacity to produce a toxin with great potential to destroy white blood cells, but keeps its production at a very limited level. This study shows that two closely related human pathogens have adapted specific molecular mechanisms to evade host defenses, reflecting the unique approach used by each to cause human disease.

Staphylococcus epidermidis Strategies to Avoid Killing by Human Neutrophils. (2010) PLoS Pathog 6(10): e1001133. doi:10.1371/journal.ppat.1001133
Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus.

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I spent 10 years working on Human T Lymphotropic Virus (HTLV), so it’s dear to me, although it’s fallen out of fashion recently, which is why it’s good to see this excellent review paper from my former UCLA colleague Pat Green:

Human T Lymphotropic Virus Type 1 (HTLV-1): Molecular Biology and Oncogenesis. (2010) Viruses 2(9): 2037-2077. doi:10.3390/v2092037
Human T lymphotropic viruses (HTLVs) are complex deltaretroviruses that do not contain a proto-oncogene in their genome, yet are capable of transforming primary T lymphocytes both in vitro and in vivo. There are four known strains of HTLV including HTLV type 1 (HTLV-1), HTLV-2, HTLV-3 and HTLV-4. HTLV-1 is primarily associated with adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-2 is rarely pathogenic and is sporadically associated with neurological disorders. There have been no diseases associated with HTLV-3 or HTLV-4 to date. Due to the difference in the disease manifestation between HTLV-1 and HTLV-2, a clear understanding of their individual pathobiologies and the role of various viral proteins in transformation should provide insights into better prognosis and prevention strategies. In this review, we aim to summarize the data accumulated so far in the transformation and pathogenesis of HTLV-1, focusing on the viral Tax and HBZ and citing appropriate comparisons to HTLV-2.

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Many organisms are able to adapt their development to the severity of their environment based on specific cues, and we have identified such a phenomenon, termed phenotypic plasticity, in the filarial parasite Litomosoides sigmodontis. Filarial nematodes infect about 200 million people worldwide, and much effort is going into finding a vaccine that would complement current drug treatments. Although anti-filarial immunity can be achieved, we show, in accord with evolutionary theory, that when these parasites infect a new host, they are able to adjust their development and reproduction to the presence of immune cells specialized in anti-helminth attack. These developmental schedules are determined within hours and impact their lifelong reproductive strategy; when immune attack is strong, and thus mortality is likely to be high, they produce offspring earlier and in greater numbers. Because current experimental vaccines rely on the very immune elements to which these nematodes adjust their development, their phenotypic plasticity could mitigate the expected reduction of disease burden in vaccinated populations.

Filarial Parasites Develop Faster and Reproduce Earlier in Response to Host Immune Effectors That Determine Filarial Life Expectancy. (2010) PLoS Biol 8(10): e1000525. doi:10.1371/journal.pbio.1000525
Humans and other mammals mount vigorous immune assaults against helminth parasites, yet there are intriguing reports that the immune response can enhance rather than impair parasite development. It has been hypothesized that helminths, like many free-living organisms, should optimize their development and reproduction in response to cues predicting future life expectancy. However, immune-dependant development by helminth parasites has so far eluded such evolutionary explanation. By manipulating various arms of the immune response of experimental hosts, we show that filarial nematodes, the parasites responsible for debilitating diseases in humans like river blindness and elephantiasis, accelerate their development in response to the IL-5 driven eosinophilia they encounter when infecting a host. Consequently they produce microfilariae, their transmission stages, earlier and in greater numbers. Eosinophilia is a primary host determinant of filarial life expectancy, operating both at larval and at late adult stages in anatomically and temporally separate locations, and is implicated in vaccine-mediated protection. Filarial nematodes are therefore able to adjust their reproductive schedules in response to an environmental predictor of their probability of survival, as proposed by evolutionary theory, thereby mitigating the effects of the immune attack to which helminths are most susceptible. Enhancing protective immunity against filarial nematodes, for example through vaccination, may be less effective at reducing transmission than would be expected and may, at worst, lead to increased transmission and, hence, pathology.

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• Elimination of elephantiasis

Viruses are obligate intracellular parasites which have evolved as natural, biological delivery vehicles. This makes them an attractive choice of vector for various clinical gene therapy applications. Human adenoviruses (Ad) are currently the most widely used viral vectors for gene therapy for several reasons; their basic biology has been studied extensively, the viral genome can accommodate large heterologous transgene insertions, they readily infect quiescent and dividing cells, they can be amplified to high titers and they have previously been shown to be relatively safe for use in humans. The family Adenoviridae consists of five genera, including genus Mastadenovirus and genus Aviadenovirus, which infect mammals and birds respectively. The Adenoviridae are non-enveloped, icosahedral virions which contain a linear, monopartite, double-stranded DNA genome approximately 36 kb in size. As of now, there are at least 55 different human adenoviruses which can be distinguished on the basis of their serological cross-reactivity, hemagglutinating properties or according to their phylogenetic sequence similarity. The adenovirus vector most commonly used for clinical trials and experimental gene therapy applications is species C adenovirus, HAdV-C5 (referred to as Ad5 in this review).

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors. Viruses 2010, 2(10), 2290-2355 doi:10.3390/v2102290
Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated “stealth” vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications.

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• Adenovirus vectors – new genes, new vaccines

From the first molecules of oxygen produced by marine cyanobacteria ~3.5 billion years ago to the methanogens luxuriating in the warm, carbon-rich swamps of the Carboniferous period, microbial processes have long been key drivers of, and responders to, climate change. It is widely accepted that microorganisms have played a key part in determining the atmospheric concentrations of greenhouse gases, including carbon dioxide (CO₂), methane (CH₄) and nitrous oxide (N₂O) (which have the greatest impact on radiative forcing), throughout much of Earth’s history. What is more open to debate is the part that they will play in the coming decades and centuries, the climate feedbacks that will be important, and how humankind might harness microbial processes to manage climate change. The feedback responses of microorganisms to climate change in terms of greenhouse gas flux may either amplify (positive feedback) or reduce (negative feedback) the rate of climate change. With the twenty-first century projected to experience some of the most rapid climatic changes in our planet’s history, and with biogenic fluxes of the main anthropogenic greenhouse gases being tied integrally to microorganisms, improving our understanding of microbial processes has never been so important.

Microorganisms and climate change: terrestrial feedbacks and mitigation options. (2010) Nature Reviews Microbiology 8, 779 doi:10.1038/nrmicro2439
Microbial processes have a central role in the global fluxes of the key biogenic greenhouse gases (carbon dioxide, methane and nitrous oxide) and are likely to respond rapidly to climate change. Whether changes in microbial processes lead to a net positive or negative feedback for greenhouse gas emissions is unclear. To improve the prediction of climate models, it is important to understand the mechanisms by which microorganisms regulate terrestrial greenhouse gas flux. This involves consideration of the complex interactions that occur between microorganisms and other biotic and abiotic factors. The potential to mitigate climate change by reducing greenhouse gas emissions through managing terrestrial microbial processes is a tantalizing prospect for the future.

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