Philip W Fowler

University of Oxford | OX · Nuffield Department of Clinical Medicine

Viral sequencing has been critical in the COVID-19 pandemic response, but sequencing and bioinformatics capacity remain inconsistent. To examine the utility of a cloud-based sequencing analysis platform for SARS-CoV-2 sequencing, we conducted a cross-sectional study incorporating seven countries in July 2022. Sites submitted sequential SARS-CoV-2 s...

Objectives Fluoroquinolone resistance poses a threat to the successful treatment of tuberculosis. WGS, and the subsequent detection of catalogued resistance-associated mutations, offers an attractive solution to fluoroquinolone susceptibility testing but sensitivities are often less than 90%. We hypothesize that this is partly because the bioinform...

Background: Bedaquiline is a core drug for the treatment of multidrug-resistant tuberculosis; however, the understanding of resistance mechanisms is poor, which is hampering rapid molecular diagnostics. Some bedaquiline-resistant mutants are also cross-resistant to clofazimine. To decipher bedaquiline and clofazimine resistance determinants, we co...

Synopsis Fluoroquinolone resistance poses a threat to the successful treatment of tuberculosis. Whole genome sequencing (WGS), and the subsequent detection of catalogued resistance-associated mutations, offers an attractive solution to fluoroquinolone susceptibility testing. However, the bioinformatic pipelines used often mask the recognition of mi...

Drug resistant Mycobacterium tuberculosis, which mostly results from single nucleotide polymorphisms in antibiotic target genes, poses a major threat to tuberculosis treatment outcomes. Relative binding free energy (RBFE) calculations can rapidly predict the effects of mutations, but this approach has not been tested on large, complex proteins. We...

Background: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a serious health problem in India, also contributing to one-fourth of the global MDR tuberculosis (TB) burden. About 36% of the MDR MTBC strains are reported fluoroquinolone (FQ) resistant leading to high pre-extensively drug-resistant (pre-XDR) and XDR-TB...

The emergence of drug-resistant tuberculosis is a major global public health concern that threatens the ability to control the disease. Whole-genome sequencing as a tool to rapidly diagnose resistant infections can transform patient treatment and clinical practice. While resistance mechanisms are well understood for some drugs, there are likely man...

The Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) presents here a data compendium of 12,289 Mycobacterium tuberculosis global clinical isolates, all of which have undergone whole-genome sequencing and have had their minimum inhibitory concentrations to 13 antitubercular drugs measured in a single assay....

Tuberculosis is a respiratory disease that is treatable with antibiotics. An increasing prevalence of resistance means that to ensure a good treatment outcome it is desirable to test the susceptibility of each infection to different antibiotics. Conventionally this is done by culturing a clinical sample and then exposing aliquots to a panel of anti...

Viral sequence data from clinical samples frequently contain contaminating human reads, which must be removed prior to sharing for legal and ethical reasons. To enable host read removal for SARS-CoV-2 sequencing data on low-specification laptops, we developed ReadItAndKeep, a fast lightweight tool for Illumina and nanopore data that only keeps read...

Background: Molecular diagnostics are considered the most promising route to achieving rapid, universal drug susceptibility testing for Mycobacterium tuberculosiscomplex (MTBC). We aimed to generate a WHO endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: A c...

Background Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Method...

Viral sequence data from clinical samples frequently contain human contamination, which must be removed prior to sharing for legal and ethical reasons. To enable host read removal for SARS-CoV-2 sequencing data on low-specification laptops, we developed ReadItAndKeep, a fast lightweight tool for Illumina and nanopore data that only keeps reads matc...

Genome sequencing is pivotal to SARS-CoV-2 surveillance, elucidating the emergence and global dissemination of acquired genetic mutations. Amplicon sequencing has proven very effective for sequencing SARS-CoV-2, but prevalent mutations disrupting primer binding sites have necessitated the revision of sequencing protocols in order to maintain perfor...

Several bioinformatics genotyping algorithms are now commonly used to characterise antimicrobial resistance (AMR) gene profiles in whole genome sequencing (WGS) data, with a view to understanding AMR epidemiology and developing resistance prediction workflows using WGS in clinical settings. Accurately evaluating AMR in Enterobacterales, particularl...

Tuberculosis is a respiratory disease that is treatable with antibiotics. An increasing prevalence of resistance means that to ensure a good treatment outcome it is desirable to test the susceptibility of each infection to different antibiotics. Conventionally this is done by culturing a clinical sample and then exposing aliquots to a panel of anti...

Tuberculosis (TB) is the leading cause of death from a single infectious agent and in 2019 an estimated 10 million people worldwide contracted the disease. Although treatments for TB exist, continual emergence of drug-resistant variants necessitates urgent development of novel antituberculars. An important new target is the lipid transporter MmpL3,...

Bedaquiline (BDQ) and clofazimine (CFZ) are core drugs for treatment of multidrug resistant tuberculosis (MDR-TB), however, our understanding of the resistance mechanisms for these drugs is sparse which is hampering rapid molecular diagnostics. To address this, we employed a unique approach using experimental evolution, protein modelling, genome se...

We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a UK teaching hospital using naso-/oro-pharyngeal PCR testing and immunoassays for IgG antibodies. 1128/10,034(11.2%) staff had evidence of Covid-19 at some time. Using questionnaire data provided on potential risk-factors, staff with a confirmed household...

We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a UK teaching hospital using naso-/oro-pharyngeal PCR testing and immunoassays for IgG antibodies. 1128/10,034 (11.2%) staff had evidence of Covid-19 at some time. Using questionnaire data provided on potential risk-factors, staff with a confirmed household...

We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a UK teaching hospital using naso-/oro-pharyngeal PCR testing and immunoassays for IgG antibodies. 1128/10,034 (11.2%) staff had evidence of Covid-19 at some time. Using questionnaire data provided on potential risk-factors, staff with a confirmed household...

Background Personal protective equipment (PPE) and social distancing are key measures designed to mitigate the risk of occupational SARS-CoV-2 infection in hospitals. Why healthcare workers nevertheless remain at increased risk is uncertain. Methods We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a large...

Synopsis Background Pyrazinamide is one of four first-line antibiotics used to treat tuberculosis, however antibiotic susceptibility testing for pyrazinamide is challenging. Resistance to pyrazinamide is primarily driven by genetic variation in pncA, an enzyme that converts pyrazinamide into its active form. Methods We curated a dataset of 664 no...

Background Resistance to co-amoxiclav in Escherichia coli is rising globally, yet susceptibility testing remains challenging as different methods yield different results. Predicting co-amoxiclav susceptibility via whole-genome sequencing (WGS) may be more reliable. Methods 976 isolates from 968 E. coli bloodstream infection cases occurring in Oxfor...

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Al...

UKMYC5 is a 96-well microtitre plate designed by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to enable the measurement of minimum inhibitory concentrations (MICs) of 14 different anti-TB compounds for >30,000 clinical tuberculosis isolates. Unlike the MYCOTB plate, on which UKMYC5 is based, the pl...

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis , they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection A...

UKMYC5 is a 96-well microtitre plate designed by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to enable the measurement of minimum inhibitory concentrations (MICs) of 14 different anti-TB compounds for >30,000 clinical tuberculosis isolates. Unlike the MYCOTB plate, on which UKMYC5 is based, the pl...

The rise of antibiotic resistance threatens modern medicine; to combat it new diagnostic methods are required. Sequencing the whole genome of a pathogen offers the potential to accurately determine which antibiotics will be effective to treat a patient. A key limitation of this approach is that it cannot classify rare or previously unseen mutations...

Cell membranes are crowded and complex environments. To investigate the effect of protein-lipid interactions on dynamic organization in mammalian cell membranes, we have performed coarse-grained molecular dynamics simulations containing >100 copies of an inwardly rectifying potassium (Kir) channel which forms specific interactions with the regulato...

The first transmembrane (TM1) helix in the red cell anion exchanger (AE1, Band 3, SLC4A1) acts as an internal signal anchor that binds the signal recognition particle and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) membrane where it moves from the translocon laterally into the lipid bilayer. The sequence N-terminal to TM...

The ease with which a cell membrane can bend and deform is important for a wide range of biological functions. Peripheral proteins that induce curvature in membranes (e.g. BAR domains) have been studied for a number of years. Little is known, however, about the effect of integral membrane proteins on the stiffness of a membrane (characterised by th...

Characterizing the nanoscale dynamic organization within lipid bilayer membranes is central to our understanding of cell membranes at a molecular level. We investigate phase separation and communication across leaflets in ternary lipid bilayers, including saturated lipids with between 12 and 20 carbons per tail. Coarse-grained molecular dynamics si...

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids a...

Membrane transporters are critical modulators of drug pharmacokinetics, efficacy, and safety. One example is the proton-dependent oligopeptide transporter PepT1, also known as SLC15A1, which is responsible for the uptake of the β-lactam antibiotics and various peptide-based prodrugs. In this study, we modeled the binding of various peptides to a ba...

Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed...

A necessary step prior to starting any membrane protein computer simulation is the creation of a well-packed configuration of protein(s) and lipids. Here, we demonstrate a method, alchembed, that can simultaneously and rapidly embed multiple proteins into arrangements of lipids described using either atomistic or coarse-grained force fields. During...

The influenza virus is surrounded by an envelope composed of a lipid bilayer and integral membrane proteins. Understanding the structural dynamics of the membrane envelope provides biophysical insights into aspects of viral function, such as the wide-ranging survival times of the virion in different environments. We have combined experimental data...

Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, mole...

Peptide transporters use a proton electrochemical gradient to move di- and tripeptides across the membrane. Despite having crystal stuctures from bacteria, little is known on how these transporters achieve a broad substrate specificity. In this study, we modelled the binding of peptides and drugs on peptide transporters based on recently published...

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Tackling the ongoing challenge of influenza infectivity would benefit greatly from a molecular understanding of why the influenza A virion exhibits seasonal patterns of infectivity and has wide-ranging survival times in different environments. A computational approach to the study of the behaviour of the virion that focuses on the poorly-understood...

The Ras family of small membrane-associated GTP-ases are important components in many different cell signalling cascades. They are thought to cluster on the cell membrane through association with cholesterol-rich nanodomains. This process remains poorly understood. Here we test the effect of adding multiple copies of NRas, one of the canonical Ras...

ABSTRACT In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but...

X-ray crystallography has provided tremendous insight into the different structural states of membrane proteins and, in particular, of ion channels. However, the molecular forces that determine the thermodynamic stability of a particular state are poorly understood. Here we analyze the different X-ray structures of an inwardly rectifying potassium...

The major facilitator superfamily (MFS) transporter lactose permease (LacY) alternates between cytoplasmic and periplasmic open conformations to co-transport a sugar molecule together with a proton across the plasma membrane. Indirect experimental evidence suggested the existence of an occluded transition intermediate of LacY, which would prevent l...

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Membrane-associated cell signalling proteins, such as Ras, have been shown to form nanoclusters, potentially enhancing signalling efficiency and fidelity [1]. It remains unclear what process is driving this behaviour, in particular whether the separation of different lipid species cause the clustering of Ras, or whether Ras affects e.g. the curvatu...

Sorting and trafficking of membrane-anchored Ras GTPases is critical for signaling and is believed to rely on their preferential portioning in ordered lipid-protein membrane domains (1). However studies in vitro have failed to quantify the preferential partitioning of full length Ras proteins into the liquid ordered phase(2), indicating that a phys...

Potassium ion channels form pores in cell membranes, allowing potassium ions through while preventing the passage of sodium ions. Despite numerous high-resolution structures, it is not yet possible to relate their structure to their single molecule function other than at a qualitative level. Over the past decade, there has been a concerted effort u...

Although extensively studied, it has proved difficult to describe in detail how potassium ion channels conduct cations and water. We present a computational study that, by using stratified umbrella sampling, examines nearly an entire conduction event of the Kv1.2/2.1 paddle chimera and thereby identifies the expected stable configurations of ions a...

Voltage-gated potassium channels form potassium-selective pores in cell membranes. They open or close in response to changes in the transmembrane potential and are essential for generating action potentials, and thus for the functioning of heart and brain. While a mechanism for how these channels close has been proposed, it is not clear what drives...

Supplementary Figures S1-S16, Supplementary Discussion, Supplementary Methods and Supplementary References

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Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transpor...

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a funct...