Zoomorphism and the danger of analogy

Myxobacteria are soil-dwelling microbes that live by killing and consuming other members of the soil microbial community. They aren’t fussy, eating all varieties of bacteria and also fungi, making them of considerable interest as potential sources of new antimicrobials.
Myxobacterial feeding has been described as employing a ‘wolf-pack’ strategy, as it seems to be more efficient when performed by larger by numbers of cells. Myxobacterial cells secrete toxic molecules of death (Figure 1) into the extracellular milieu, killing surrounding prey cells, and releasing nutrients which can then be taken up for biomass assimilation.
Wolf-pack predation conjures up images of sophisticated pack behaviour, with several organisms orchestrating their attacks. And so it seems to be with myxobacterial predation. There are reports of myxobacteria luring prey towards themselves, probably through the secretion of prey food (perhaps more akin to a fishing trip than a pack hunt). We recently found that the signals prey send to each other (acyl-homoserine lactones – AHLs) can be eavesdropped on by myxobacteria, who then up-regulate their predatory behaviour in response (Lloyd and Whitworth, 2017).

Figure 1. Myxobacteria (yellow rods) secrete toxic molecules of death (black circles) which kill neighbouring prey cells. Nutrients released from prey are taken up by the myxobacteria, allowing them to reproduce.

In order to identify the genes involved in co-ordinating the response to prey, we undertook a transcriptomics experiment, assessing gene expression of predator and prey during predation (Livingstone et al., 2018). Myxobacteria have large genomes (with ~7,000 genes) and shifting them between nutrient-rich and nutrient-poor substantially changes the expression of around 1500 genes. When myxobacteria are exposed to prey, only 3 genes are switched on (and their role seems to deal with the indirect osmotic consequence of prey presence). The transcriptional response of myxobacteria to prey is a resounding ‘who cares?’.
Behavioural changes in bacteria can be modulated by post-translational mechanisms without recourse to switching genes on or off, but it is hard to imagine a process that involves the secretion of ~100 proteins to be unregulated transcriptionally. To explain this, we invoked a model of constitutive feeding, with regulated nutrient assimilation triggered by the presence of decaying prey (adding dead prey to myxobacteria causes them to up-regulate a much more respectable 124 genes). This was certainly not wolf-pack behaviour and we were reminded more of spiders – creating external secretions (a web) which captures/kills prey, while signalling to the predator that prey is present.
So are myxobacteria wolves or spiders? Obviously they are neither. Nor are they fishermen. We often feel the need to anthropomorphise, and when doing so we often unconsciously taint our analogies with extraneous associations. Zoomorphising is no better.

Livingstone, P.G., Millard, A.D., Swain, M.T. and Whitworth, D.E. (2018) Transcriptional changes when Myxococcus xanthus preys on Escherichia coli suggest myxobacterial predators are constitutively toxic but regulate their feeding. Microbial Genomics. 2018: 5.
Lloyd, D.G. and Whitworth, D.E. (2017) The myxobacterium Myxococcus xanthus can sense and respond to the quorum signals secreted by potential prey organisms. Frontiers in Microbiology. 8: 439.

Finding Novel Antimicrobial Peptides with AMPLY

Rise of the superbugs
The rise of antibiotic resistant strains of microbes (bacteria, parasites, viruses and fungi) is probably the leading threat facing humankind. Increasingly desperate warnings about the real-world implications of the increasing resistance are now front page news. The problem is a multifaceted one. Antimicrobial resistance is not just about the loss of human life, but inextricably intertwined with increased patient morbidity and massive economic consequences for global healthcare systems. There are two possible solutions, either a socio-political/behavioural change or a technical/scientific response. Humankind has shown itself remarkably intransigent when faced with doom laden prophecies that require behavioural modification to circumvent (see also Climate Change), therefore it is probably prudent to assume that a managed technical response may be our best hope. But new antibiotics are unlikely to arise spontaneously. Mokyr highlights one of the issues with relying on the existing pharmaceutical industry to address the problem: “…few economies have ever left [decisions like these] entirely to the decentralized decision-making processes of competitive firms. The market test by itself is not always enough” (Mokyr, 1998).

Discovery of AMPs

Figure 1: A cationic, helical AMP (Taliecin-1)

The discovery of AMPs dates back to 1939, when Dubos extracted an antimicrobial agent from a soil Bacillus strain. The designation of AMPs has been extended to encompass a general view of them as a group of anionic antimicrobial proteins/peptides; host defence peptides; cationic amphipathic peptides and cationic AMPs. In contrast to acquired immune mechanisms these endogenous peptides provide a fast and effective means of defence against pathogens as part of the innate immune response. Antimicrobial peptides are evolutionary ancient weapons and their ubiquity throughout the animal and plant kingdoms supports the hypothesis that they have played a key role in the successful evolution of complex multi-cellular organisms. Such is their diversity they can be found in locations as disparate as the skin secretions of a frog to the defensive arsenal of a protozoa.

Dolby Bioinformatics

Figure 2: The Dolby certification logo (dolby.com)

One specific feature of AMPs that makes them difficult to find is that they’re small (often less than 20 amino acids in length – which is comparatively tiny compared to typical proteins). In a typical ‘omic dataset containing, potentially millions and millions of datapoints, isolating interesting AMPs for synthesis and testing is a challenging test. For inspiration we can look to the music industry. In the mid-20th century recordings were made on magnetic tape and engineers wrestled with an ever present low level of hissing noise in the background that threatened to drown out the music. Various ingenious solutions were deigned to mitigate the persistent hiss from forms of “low-noise” tape which recorded more signal; running the tape at a higher speed, or using dynamic pre-emphasis during recording and a form of dynamic de-emphasis during playback. This latter approach became the backbone of the Dolby noise reduction system, which became all pervasive in home audio equipment from the late 60s onwards. The audio engineer’s struggle to maximise signal-to-noise is the same core problem that faces computational biologists and the ongoing analysis of ‘omic “big data” in the search for tiny novel AMPs. There is music there, but at the moment the hiss is tremendous.

The detection of AMPs in metagenomic data is a tantalising low-hanging fruit for computational biologists, however. Post-computational wet-lab work is relatively cheap with spot synthesis of peptides up to around 25aas long possible from a wide array of third party companies with prices from as low as £2.50 per amino acid. A well organised screening program can screen in excess of 100 peptides a day, per person, against a model bacterial organism to test for activity. As a potential workflow the rapid assessment of multiple ‘omic datasets; identification of homologues of pattern matched AMPs; rapid synthesis and screening and a rush to publication would appear to provide a grant-friendly drug-discovery goldmine! But to tap this rich vein, improving the hit rate of putative AMPs from ‘omic data needs to be streamlined and improved.

The AMPLY Pipeline
Finding small sequences (you’re interested in) that often look a lot like other small sequences (you’re not interested in) in datafiles that can contain potentially gigabytes of data is a trickier task than it first appears. Annotation in metagenomics is an art and the determination of what’s real and what’s not often relies purely on defining mutually agreed thresholds. However, as the length of the aligned data being identified starts to shorten, a lot of the assumptions on PercentageID, BitScore and E-Value thresholds begins to fall away. It’s here we return to the Dolby signal-to-noise analogy – the “music” of the AMPs in metagenomic datasets are often drowned out by the sheer volume of background noise and to find them we need to adopt a novel strategy of aggressive emphasis.

Designed by Ben Thomas at Aberystwyth University in the CreeveyLab (http://www.creeveylab.org/) and funded by Life Science Wales (https://www.lifescienceshubwales.com/), AMPLY (http://amply.info) is a pipeline designed to plug this gap between the ‘omic data and lab work. AMPLY is designed to provide a basis to sift-out AMPs suitable as synthesis candidates and provide potential regions for crude synthesis by adopting a hyper-wide “balance of evidence” approach. AMPLY passes over data with a series of detection methods, then wrapping the summative results of both them and presenting the final results into a final tableau (known as the “bitpad”) where each potential AMP can be evaluated on the strength of a series of hundreds of datapoints, rather than just a couple of numeric values.

Figure 3: The AMPLY workflow

To date, AMPLY has been used to find, characterise and synthesise over 800 potentially novel AMPs which have been lab screened in partnership with Tika Diagnostics (http://tikadiagnostics.com/) at St. George’s Hospital in London. Among the AMPs discovered by AMPLY many are highly active against MRSA (a key superbug) and offer encouraging potential treatment avenues for future development. While there is still much work to be done, results so far have been extremely promising: AMPLY has been used to find bioactive AMPs in datasets as diverse as the skin of Peruvian poison dart frogs to the testicles of a Salamander so the only limitation in the AMPLY pipeline is the diversity of the stream of ‘omic data provided to it….

So, if you’re reading this blog and have interesting data and would like to be part of the drive to find new antimicrobials then get in touch for potential collaborations. We are always interested.

Contact Ben Thomas at bet16@aber.ac.uk, or via Twitter @flwrs4algrnon

Mokyr, Joel. “The political economy of technological change.” Technological revolutions in Europe (1998): 39-64.

Successful PhD Defence on High Through-put Screening of Schistosoma

Successful PhD student with bottle of wine in labOn January 16, 2018, IBERS-funded PhD student Kezia Whatley successfully defended her thesis entitled: “Synergistic application of high-throughput screening (HTS) and high content imaging (HCI) technologies with in silico drug repositioning techniques to identify new chemotherapeutic targets in Schistosoma mansoni”. Supervised by Prof. Karl Hoffmann, Kezia helped set up a HTS platform based on a sister screening platform at the London School of Hygiene and Tropical Medicine as part of her PhD. This HTS platform, named Roboworm, enables the screening of compound collections against the larval stage of S. mansoni. This parasite is one of several Schistosoma sp., responsible to for causing the neglected tropical disease schistosomiasis which affects >218 million people globally. Additionally to this, Kezia also set up a simple and cost efficient cytotoxicity assay against Human Caucasian Hepatocyte Carcinoma (HepG2) cells, to enable an initial determination of compound toxicity. The skill sets obtained during her PhD have enabled Kezia to continue working in Prof. Karl Hoffmann’s research group on two research projects. Initially, Kezia worked collaboratively with industrial and academic partners to screen new compound libraries against S. mansoni. This project, funded by the Life Sciences National Research Network Wales, facilitated the screening and publication of several compounds synthesised by PhD students in IBERS and collaborations between IBERS and other academic institutions. Kezia is currently working on a Welsh Government funded Life Sciences Bridging Fund project to develop Roboworm for screening other parasitic worm species such as Fasciola hepatica and Haemonchus contortus. Both of these parasites are responsible for causing high economic loss in the farming industry (~£110 million per annum in the UK alone). These translational projects and collaborations have identified that there is a demand for a screening service against both veterinary and biomedically relevant parasites. It is hoped that continued development of the Roboworm platform will enable medicinal chemists to screen previously untested compounds against these parasites, which will help identify new antiparasitic compounds urgently needed for combating human and animal pathogens.

By Ifat Parveen

Potential class of HIV-1 integrase inhibitors

Cameron Garty is currently completing an MPhil in natural product drug discovery and medicinal chemistry in Dr Shah’s research group.

To date, seventy-eight million people in the world, have become infected with HIV and over thirty-five million deaths have resulted from HIV/AIDs and related diseases. Currently, there are between thirty-six million people living with HIV, of which two million are under the age of fifteen. The number of people with HIV receiving treatment in resource-poor countries has dramatically increased over the past decade. However, variations of HIV that develop with current medicines have led to drug-resistant strains; the search for successful therapies has not been more imperative. Understanding the function of the CD4 cells has made it possible for scientists to design antiretroviral drugs that inhibit the production of HIV by halting the process at the different stages of the life cycle. These include entry inhibitors, fusion inhibitors, reverse transcriptase inhibitors; nucleotide inhibitors, non-nucleotide inhibitors, integrase inhibitors and protease inhibitors. Currently, treatment does not cure HIV. The antiretroviral drug (ARV) therapy struggles with the issues of patient obedience, side effects, the huge cost and evolving drug resistances. More drugs against HIV targets are critical in preventing the HIV epidemic and the long term efficacy of ART. Lithospermic acid, isolated from red sage (Salvia miltiorrhiza), has been shown to inhibit HIV-1 integrase with a reported IC50 value of 0.48 M (Abd-Elazem et al., 2002). Further studies have shown it to strongly suppress HIV-1 infection in model organisms (H9 cells) with a reported IC50 value of 2 μM (Abd-Elazem et al., 2002). Currently, lithospermic acid is undergoing clinical trials as an anti-HIV drug. The overall aim of the project is to identify novel clinically active anti-HIV drugs. The objectives of the study are to synthesise a portfolio of compounds structurally similar to lithospermic acid and test against HIV-1 integrase.