Population genetics of genetically monomorphic bacterial pathogens
Simplifying molecular diagnostics for tropical diseases
Traditionally the world of molecular biology has been closed to diagnosing tropical diseases in the endemic areas they affect. However, there is now huge, global interest in developing the types of diagnostics that would be suitable for endemic settings, including rural health posts. Here, I will explore three examples, LAMP, GeneXpert and GeneDrive with reference to Tuberculosis, Malaria and Leishmaniasis. Is their performance good enough to implement? Where would these diagnostics fit best in a diagnostic algorithm and do they offer real advantages in terms of case detection?
Uncovering Past Human Infections – Sequencing Tuberculosis from Hungarian Mummies
During building works carried out in 1994 at the Dominican church in Vác, Hungary, a crypt was discovered which had been sealed since 1838. Inside workers found the remains of 263 individuals from prominent local families and clerics interred between the years 1731-1838; about two-thirds of the bodies were naturally-mummified. The contemporaneous church and civic archives have enabled the identification of some family groups including three members of Hausmann family (a mother and her two daughters) all of whom died between 1793 and 1797. The appearance of the remains as well as traditional ancient DNA analysis using PCR demonstrated that the three family members were each infected with a different strain of Mycobacterium tuberculosis (MTB). Using an open-ended metagenomic approach we found evidence of mixed infection in the elder daughter, Terézia Hausmann. From the patterns of deletions in the MTB metagenome we conclude that both strains fall within the cluster of the MTB Haarlem lineage and resemble a recent outbreak strain from German, 7199/99. This study demonstrates the power of metagenomics and wealth of information it can provide.
Dr Katherine Denby
Elucidating gene regulatory networks mediating plant defence
Transcription to Translation and some of the bits in-between
The central dogma of molecular biology states that DNA makes RNA makes Protein. However, what this doesn’t take into account are the molecular events that take place in order for a cell to function correctly – i.e. have all the components necessary for growth or survival present at exactly the right time. Technologies to monitor transcriptional events in great detail have given us a huge amount of useful information, allowing us to monitor regulons, understand what is required for a cell to grow or survive under different conditions and determine start and stop sites of transcription. Although this information is very informative, does it really explain the whole story? This presentation will describe three interesting regulatory concepts from the Gram-positive bacterium Bacillus subtilis that provide evidence it is not sufficient to take large “omics” studies at face value. 1. Observing a population at the single cell level - monitoring expression of the bacteriocin sublancin. Are all cells behaving the same? 2. Regulating transcription through translational control – Understanding how translation of YheJ controls the transcription of the bmrCD ABC transporter involved in drug efflux. 3. Post-transcriptional control - even though an mRNA is made how do we know that it is actually translated? Under specific conditions translation of particular mRNA’s can be manipulated through the use of sRNAs. How does an sRNA regulate a global transcriptional regulator at the posttranscriptional level and why does it use this mechanism?
Professor Gad Frankel
The interplay between the Escherichia coli type III secretion system effectors
Enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC) use a type III secretion system to deliver between 20 and 50 effectors that subvert cell signalling, including actin dynamics, apoptosis, inflammation and endosomal trafficking. While in the past we studied each of the effectors individually, we now know that their function is interdependent. I will give a few examples of how the effectors complement or antagonise each other, describe the mechanisms by which these pathogens subvert actin signalling and the role it plays in infection and colonisation, introduce a new category of invasive EPEC and describe a novel mechanism by which these pathogen protect cells from effector overload.
Cyanophage psbA – differential expression, intron splicing and an antisense RNA
The cyanophage S-PM2 carries the “photosynthesis” genes psbA and psbD, which encode the core photosystem II proteins PsbA and PsbD respectively. An unavoidable consequence of photosynthesis is damage to the PsbA protein, which can lead to photoinhibition. Cyanophage are thought to maintain photosynthesis by the expression of phage copies of psbA and psbD during the infection cycle, allowing PsbA to be turned over. The psbA gene of S-PM2 is unusual in that it contains both a self splicing group I intron and a non-coding RNA, CfrI, that is antisense to psbA. Under conditions of high-light compared to low-light there is differential rates of splicing of the psbA intron. This splicing is potentially controlled by the antisense RNA CfrI , which can bind to un-spliced psbA transcript and prevent intron splicing. Thus, providing a mechanism to for phage to respond to changes in environmental light conditions.
Adventures in translational genomics and metagenomics
Despite my training as medical microbiologist, during the nineties and noughties, my career was largely focussed on basic curiosity- and hypothesis-driven research on how bacterial cells function and how they cause disease. However, since 2010, my career has swerved back in the direction of translational research, riding the wave of opportunities that have arisen from high-throughout sequencing. Here, I will outline my adventures in translational genomics and metagenomics, including the genomic epidemiology of hospital pathogens such as Acinetobacter baumannii, the roller coaster of open-source genomics of the 2011 German outbreak of Shiga-toxigenic E. coli and our surprising recent successes with clinical metagenomics.
Comparative phylogenetics of the ICEHin1056 family reveals deep evolutionary relationships of mobile genetic elements
Integrating and conjugating elements (ICEs) are self-transmissible mobile genetic elements. ICEs are composed of modules of conserved genes, with accessory genes at hotspots. Antibiotic resistance genes are often encoded on ICEs, leading to rapid intra and inter-specific spread of resistance. Our aim was to study ICEs with homology to ICEHin1056 in Haemophilus influenzae using the large number of whole genome sequences now available. We identified over a hundred whole or partial sequences in the ICEHin1056 family in a-, b- and g- proteobacteria. This is the largest comparative phylogenetic study of ICEs performed to date and demonstrates extensive lateral gene transfer across the whole phylum. The three core ICE modules encode: replication, type IV secretion and excision/integration. The conservation of synteny implies a powerful selective advantage of the ICE. GC content of the core modules mirrors that of the host chromosome, suggesting co-existence deep in evolutionary history. Absence of core genes or modules represents “lifestyle” adaptations of the mobile genetic element. Absence of an integrase and presence of a replicative DNA helicase are markers of a “plasmid lifestyle”. A variety of accessory genes are found at hotspots; they confer survival advantage in the ecological niche of the organism, which ranges from eukaryotic pathogens to extreme environments.
Mechanisms of host cell manipulation by bacterial pathogens
Bacteria are adept at manipulating the host environment to their own advantage through interactions with host factors. Surface-associated and secreted proteins are key to such interactions and are critical in mediating bacterial virulence. Gram positive pathogens like Staphylococcus aureus have evolved sophisticated pathways including specialized secretion systems to export proteins required for virulence. Although we know much about extracellular virulence factors and their role in staphylococcal pathogenesis, little is known about staphylococcal factors modulating intracellular infection. To understand how S. aureus invades the host cell, we have performed preliminary invasion screens to identify cell surface proteins important for entry into epithelial cells. Characterization of new invasins will provide a better understanding of invasion mechanisms employed by S. aureus. In order to investigate proteins modulating bacterial survival within cells, we have examined the role of the staphylococcal type VII secretion system, Ess, during intracellular infection. Ess encodes for substrates EsxA and EsxB, which are virulence factors and potential vaccine candidates. We find that staphylococcal Esx proteins modulate host cell survival by interfering with cell death pathways. Molecular studies on the interactions of the Esx systems with cellular and immune pathways will help comprehend the role of Esx proteins during persistent staphylococcal infection.
A systems biology level analysis of human host adaptation of the nematode symbiont Photorhabdus asymbiotica
We have used powerful post genomic techniques to understand how the nematode symbiont Photorhabdus asymbiotica can infect both insects and humans. Comparative genomics, functional genomics, RNAseq digital transcriptomics, proteomics and pheno-array studies have provided detailed insights into how P. asymbiotica achieves this duel pathogenic state. In addition to allowing us to construct a systems level model of gene expression in different hosts, it has provided an excellent discovery platform for bioactive proteins and small drug like secondary metabolites. Such bioactive molecules include insecticides, antimicrobials and immune modulatory molecules. Studying the expression of these has allowed us to begin to unravel how this pathogen can combat the innate immune systems of both insects and people. Furthermore our network model analysis suggests that temperature adaptation and changes in metabolic activity are crucail to this host shift from insect to man.
Professor Elizabeth Wellington
Mycobacterial pathogens in the environment: indicators of infection and potential reservoirs of disease
Tuberculosis (TB) is one of the most widespread infectious diseases and a major cause of death for adults worldwide. Although much attention has focused on treatment and prevention of human tuberculosis, human tuberculosis from animal origin (zoonotic tuberculosis due to Mycobacterium bovis) is an important and re-emerging public health concern in developing countries. Within the UK, despite a current (cattle) and past (badger) test and elimination policy, there has been an average 18% increase in the annual number of new confirmed herd breakdowns since the mid-1980s with an estimated cost of approximately £500 million during the past decade. In the developing world where there are little or no animal control measures in place, the impact is much wider with the economy, ecosystems and human health affected. The WHO has recently designated bovine tuberculosis as a neglected zoonosis, with particular reference to the developed world. M. bovis has been shown to persist in the environment for a substantial period of time, several months to years, raising questions about the role of environmental reservoirs in the chronic persistence of bTB in some cattle herds and wildlife populations. bTB is prevalent in cattle in the United Kingdom, Republic of Ireland, North America, Africa, New Zealand and Australia where a variety of wildlife reservoirs have been identified. In the United Kingdom, European badgers (Meles meles) are implicated in transmitting M. Bovis to cattle and vice versa. We have developed a number of methods for environmental detection of M. bovis and other members of the MTBC, we hypothesise that faecal shedding is a proxy for respiratory shedding and correlates with disease severity and so is a valuable indicator of the presence of infectious hosts and can be used to measure infection risk to other animals. We have data from studies being conducted in Republic of Ireland, UK and Africa to test this hypothesis and investigate impacts of control measures. In addition a wide range of mycobacteria including slow growing species can be detected in soil and water, the prevalence of which may have implications for human health. A study in Ethiopia provided an opportunity to investigate the diversity of mycobacteria in relation to biotic and abiotic environmental variables and disease prevalence. Results provided evidence of distinct patterns in prevalence which may have implications for disease and the exposure to certain prevalent species may reduce BCG efficacy.