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Baker, K. S.

Publications and source records attributed to Baker, K. S..

At least 19 recordsLinked to original sources
bioRxiv2026

Dual ATPase-activated DNA hemimethylation and cleavage by the MANTIS defence system

Bacterial defence systems are diverse and form major barriers to horizontal gene transfer and bacteriophage (phage) infection. Though restriction-modification systems were the first such defences discovered and remain the most widespread, the basis of self/non-self discrimination in many newly identified methylation-based systems remains unclear. Here, we define the mechanism of a widely distributed BREX-related defence family, previously termed Gao_RL, which we rename MANTIS (Methyltransferase, ATPases and Nuclease Targeting Inverted-repeat Sequences). Using phage infection and plasmid transformation assays in Pseudomonas aeruginosa, biochemical reconstitution, and structural biology, we show that MANTIS establishes self-identity through adenine hemimethylation of short asymmetric DNA sequences. Unexpectedly, a AAA+ ATPase (MtsA) and a co-regulator (MtsB) are both required to recruit and activate the methyltransferase MtsC, revealing a previously unknown ATP-dependent mode of epigenetic modification. DNA recognition by the MtsABC complex then triggers restriction by a dimeric helicase-nuclease effector (MtsD), with activity dependent on target-site architecture: inverted pairs of unmodified sites support robust restriction, whereas direct repeats or single sites are restricted less efficiently. Consistent with this model, virulent phages show strand-biased depletion of MANTIS target sites, indicating evolutionary pressure to evade restriction. Together, these findings reveal how dual ATPases couple self-modification to non-self restriction and provide a framework for understanding the expanding landscape of methylation-based defence systems.

molecular biology↗
bioRxiv2026

SigA forms amyloid fibrils and supports the competition of Shigella with the microbiota

Shigella sonnei is an enteropathogen that causes bacillary dysentery. During the first step of its virulence cycle, it must outcompete the resident microbiota to establish its own niche. Here we report that SigA, the sole SPATE (Serine Protease Autotransporter of Enterobacteriaceae) family member in S. sonnei, plays an indirect but central role in this process. A genome-wide analysis showed that the SPATE family includes SigA, Pic, SepA, and Sat. We demonstrated that SigA self-assembles into amyloid fibrils (F-SigA) independently of its protease activity. F-SigA remains associated with the S. sonnei surface in vitro and in vivo. Purified F-SigA fibrils have a diameter of 17.7 {+/-} 3.2 nm, and their amyloid organization was confirmed using specific markers and biochemical methods. F-SigA is secreted into the lumen in vivo and localizes to the surface of the colonic epithelium. We found that colicin E1 (ColE1) interacts with F-SigA amyloid fibrils, and that F-SigA-ColE1 complexes display antimicrobial activity that promotes S. sonnei competition with other bacteria. Because Pic, another Shigella SPATE, also forms amyloid fibrils, we anticipate that this virulence mechanism may be relevant across a wide range of Shigella strains and enterobacteria and may serve additional roles during the Shigella virulence cycle.

microbiology↗
bioRxiv2026

Flex-It: A global standardised genotyping framework for Shigella flexneri

Shigella flexneri is the leading causative agent of shigellosis globally. The public health threat posed by S. flexneri is compounded by its emergence as a sexually transmissible infection, importance of international travel in driving dissemination, and the increasing prevalence of antimicrobial resistance (AMR). A rapid and robust computational method is needed to enhance genomic surveillance and systematically explore features of the population structure of this WHO priority pathogen, which is scalable and readily implementable across jurisdictions, particularly as vaccine development efforts are underway. Here, we present Flex-It, a genomic framework and genotyping scheme implemented in Mykrobe for S. flexneri serotypes 1-5, X & Y, compatible with previous approaches used to describe S. flexneris population structure. To develop Flex-It, we curated a retrospective dataset of 5,819 publicly available S. flexneri genomes. We characterised the global population structure for S. flexneri, exploring geographical and temporal traits, and showed the granular diversity of AMR and serotype profiles. We applied Flex-It to >13,000 genomes routinely generated by public health laboratories from Australia, the UK and the USA across a ten-year period. We found significant genotype diversity in all three locations, with the emergence of genotypes with converged resistance to all major drugs currently used for treatment. Flex-It provides an open-source, novel genotyping method that rapidly characterises S. flexneri and its ciprofloxacin resistance determinants in <1 minute from both short and long whole-genome sequencing reads. Flex-It provides the community with a standardised nomenclature to monitor the emergence and spread of S. flexneri lineages.

microbiology↗
bioRxiv2025

Defence systems drive accessory genome interactions in Pseudomonas aeruginosa.

Bacterial genomes represent dynamic ecological systems in which mutational selection and dynamic accessory genome element complements drive evolution. Emerging evidence suggests that bacterial defence systems, which protect against phages and other mobile genetic elements, interact through cooperative, competitive, and antagonistic relationships. These interactions can influence horizontal gene transfer, shape phage susceptibility, and the diversification of genome composition across environments. Recent ecological studies describe non-random co-occurrence and avoidance of defence systems, suggesting that they may emerge from ecological and evolutionary interactions, rather than by chance. This necessitates exploring whether these patterns reflect functional compatibility, shared selection pressures imposed by ecological niche, or if they merely arise from co-localisations of convenience. Understanding these patterns is key to elucidating how the accessory genome evolves and how defence systems constrain or facilitate genome plasticity. To investigate these patterns, in their genomic context, and provide a resource for future investigations, we analysed the distributions of defence systems and other accessory genome elements in a recently curated global dataset of 2,940 Pseudomonas aeruginosa genomes. Genomic defence system content varied by ecological niche, with higher numbers per genome in non-cystic fibrosis derived isolates (average n=7.9) compared to cystic fibrosis-derived isolates (average n=6.5). We identified multiple associations (n=426) and dissociations (n=50) among defence systems, and among other accessory genome elements, many of which had a plausible biological explanation. We also quantitated the relative interactions among accessory genome elements which revealed that defence systems and anti-defence systems engage in the most accessory genome interactions, compared with e.g. antimicrobial resistance genes, plasmids, phages, suggesting that systems are a major driving force in the ecological dynamics of the bacterial accessory genome. Together, these patterns provide new insights into the evolutionary forces shaping bacterial genomes, provide a valuable resource of quantitated interactions in a highly curated Pseudomonas aeruginosa dataset, and establish a framework for future mechanistic and ecological investigations of defence system interactions.

microbiology↗
bioRxiv2025

Enhanced virulence and stress tolerance are signatures of epidemiologically successful Shigella sonnei

Shigellosis is a leading cause of diarrhoeal deaths worldwide, with Shigella sonnei increasingly implicated as a dominant agent. S. sonnei is divided into five monophyletic lineages all sharing a single O-antigen, yet most contemporary infections are caused by just a few clonal sub-lineages within globally dominant Lineage 3 that are quite distinct from the widely used Lineage 2 laboratory strain 53G. Factors underlying the success of these globally dominant lineages remain poorly understood in part due to a lack of complete genome sequences and available animal models. Here, we utilise a novel reference collection of representative Lineage 1, 2 and 3 isolates with complete genome sequences, and find that epidemiologically successful S. sonnei harbour fewer genes encoding putative immunogenic components whilst key virulence-associated regions (such as the type three secretion system and O-antigen) remain highly conserved. Using a zebrafish infection model, we discover that Lineage 3 isolates are most virulent, driven by significantly increased dissemination and a greater neutrophil response. We show that Lineage 3 isolates have increased tolerance to complement-mediated killing and acidic conditions alongside upregulated expression of group four capsule synthesis genes. Consistent with these observations, infection of primary human neutrophils revealed that Lineage 3 isolates are more tolerant of phagosomal killing. Together, our findings link the epidemiological success of S. sonnei to heightened virulence and stress tolerance and highlight zebrafish as a valuable platform to illuminate factors underlying establishment of Shigella epidemiological success.

microbiology↗
bioRxiv2025

A public resource of 15 genomically characterised representative strains of Shigella sonnei

Shigella sonnei is rapidly emerging as the dominant agent of shigellosis, an enteric disease responsible for a significant burden of morbidity and mortality worldwide. Whole-genome sequencing of S. sonnei isolated over the last three decades has revealed phylogenomic diversity within the population and the emergence of multiple lineages associated with distinct epidemiological patterns such as resistance to critical antimicrobials and/or transmission within different groups. However most experimental work on S. sonnei biology and pathogenicity has focused on a single laboratory strain (53G), which is phylogenetically distant from currently circulating strains. Here we introduce a set of 15 phylogenetically diverse and epidemiologically relevant S. sonnei isolates made available through publicly accessible culture collections as a resource for laboratory science. We present their complete whole-genome sequences, including the pINV invasion plasmid (missing from a large proportion of public genome data due to loss during laboratory culture). Finally, the characterisation and comparison of these complete genome sequences highlight evidence for ongoing adaptive evolution in S. sonnei, featuring the accumulation of insertion sequences, gene pseudogenisation and structural variation. Significance as a BioResource to the communityGenomic analysis of Shigella has historically been challenging due to presence of hundreds of repetitive sequence elements (which can cause fragmented assemblies) and loss of the pINV invasion plasmid (essential to virulence) during laboratory culture. Furthermore, most experimental work on S. sonnei pathogenicity uses a lab strain that is phylogenetically distant from circulating isolates. To support S. sonnei experimental and in silico research and increase its relevance to current clinical problems, we report here the complete, high-quality genome sequences of 15 S. sonnei isolates, each selected to represent distinct sub-clades of epidemiological interest. We also make the corresponding strains publicly available in national reference culture collections. Data summaryAll sequencing reads and complete assemblies have been deposited into the National Center for Biotechnology Information (NCBI) database (accessions to be determined). Genome assemblies and Bakta annotations used in the analysis can be found in Figshare (https://doi.org/10.6084/m9.figshare.28302986) together with Mauve multiple-sequence alignments for the chromosome and pINV plasmid sequences, and genome-scale metabolic models produced for each strain. Pure cultures of all strains were deposited in the publicly accessible National Collection of Type Cultures (NCTC, UK) or the "Collection de lInstitut Pasteur" (CIP, France) (accessions to be determined).

microbiology↗
bioRxiv2024

Brain-wide projections of mouse dopaminergic zona incerta neurons

The zona incerta (ZI) supports diverse behaviors including binge feeding, sleep/wake cycles, nociception, and hunting. This diversity of functions can be attributed to the heterogenous neurochemicals, cytoarchitecture, and efferent connections that characterize the ZI. The ZI is predominantly GABAergic, but we recently identified a subset of medial ZI GABA cells that co-express dopamine (DA), as marked by the enzyme tyrosine hydroxylase (TH). While the role of GABA within the ZI is well studied, little is understood about the function of ZI DA cells. To identify potential roles of ZI DA cells we mapped the efferent fiber projections from Th-cre ZI cells. We first validated a Th-cre;L10-Egfp mouse line and found that medial Egfp ZI cells were more likely to co-express TH-immunoreactivity (TH-ir). We thus delivered a cre-dependent virus into the medial ZI of Th-cre or Th-cre;L10-Egfp mice and selected two injection cases for full brain mapping. We selected the cases with the lowest (17%) and highest (53%) percentage of colocalization between TH-ir and virus transfected cells labelled with DsRed. Overall, DsRed-labelled fibers were observed throughout the brain and were most prominent within motor-related regions of the midbrain (MBmot), notably the periaqueductal grey area and superior colliculus. We also observed considerable DsRed-labelled fibers within the polymodal cortex associated regions of the thalamus (DORpm), including the paraventricular thalamic nucleus and nucleus of reunions. Overall, ZI DA cells displayed a similar connectivity profile to ZI GABA cells, suggesting that ZI DA cells may perform synergistic or opposing functions at the same target sites. List of RRIDsAB_2201528, AB_10013483, AB_11177031, AB_2340593, AB_2315778, SCR_016477 Three key points- Tyrosine hydroxylase immunoreactivity was more prominent in the medial zona incerta - Intersection points from two Th-cre injection cases revealed common target regions - Dopaminergic zona incerta cells project predominantly to motor-related brain regions

neuroscience↗
bioRxiv2024

Global diversity and evolution of Salmonella Panama, an understudied serovar causing gastrointestinal and invasive disease worldwide: a genomic epidemiology study

BackgroundNontyphoidal Salmonella (NTS) is a globally important bacterial pathogen, typically associated with foodborne gastrointestinal infection. Some NTS serovars can also colonise normally sterile sites in humans to cause invasive NTS (iNTS) disease. One understudied Salmonella enterica serovar which is responsible for a significant number of cases of iNTS disease is Panama. Despite global dissemination, numerous outbreaks, and a reported association with iNTS disease, S. enterica serovar Panama (S. Panama) has not been investigated in detail. MethodsUsing combined epidemiological and whole genome sequencing data we analysed 836 S. Panama genomes derived from historical collections, national surveillance datasets, and publicly available data. The collection represents all inhabited continents and includes isolates collected between 1931 and 2019. Maximum likelihood and Bayesian phylodynamic approaches were used to determine population structure & evolutionary history, and to infer geo-temporal dissemination. A combination of different bioinformatic approaches utilising short-read and long-read data were used to characterise geographic and clade-specific trends in antimicrobial resistance (AMR), and genetic markers for invasiveness. FindingsWe identified the presence of multiple geographically linked S. Panama clades, and regional trends in antimicrobial resistance profiles. Most isolates were pan-susceptible to antibiotics and belonged to clades circulating in the United States of America, Latin America, and the Caribbean. Multidrug resistant (MDR) isolates in our collection belonged to two phylogenetic clades circulating in Europe and Asia/Oceania, which exhibited the highest invasiveness indices based on the conservation of 196 extra-intestinal predictor genes. InterpretationThis first large-scale phylogenetic analysis of S. Panama revealed important information about population structure, AMR, global ecology, and genetic markers of invasiveness of the identified genomic subtypes. Our findings provide an important baseline for understanding S. Panama infection in the future. The presence of MDR clades with an elevated invasiveness index should be monitored by ongoing surveillance as such clades may pose an increased public health risk. FundingThe project was supported by both a Global Challenges Research Fund (GCRF) data & resources grant BBS/OS/GC/000009D and the BBSRC Core Capability Grant to the Earlham Institute BB/CCG1720/1. JCDH received a Wellcome Trust Investigator award (grant number 222528/Z/21/Z). CVP was supported by the John Lennon Memorial Scholarship from the University of Liverpool and a Fee Bursary Award from the Institute of Integrative Biology at the University of Liverpool. FXW was awarded the Institut Pasteur; Sante publique France; the Fondation Le Roch-Les Mousquetaires; and the French governments Investissement dAvenir programme, Laboratoire dExcellence "Integrative Biology of Emerging Infectious Diseases" (grant number ANR-10-LABX-62-IBEID). MDS was supported by a BBSRC grant (KSB: BB/V009184/1). RJBengtsson was supported by an MRC grant (KSB: MR/R020787/1). RJBennett was supported by a BBSRC DTP (BB/M011186/1). DJI was supported by a National Health and Medical Research Council (NHMRC) Emerging Leadership Fellowship (GNT1195210). BPH was supported by NHMRC Leadership Fellowship (GNT1196103). KSB and EVR are affiliated to the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Gastrointestinal Infections at University of Liverpool in partnership with the United Kingdom Health Security Agency, in collaboration with University of Warwick. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health and Social Care or UKHSA. MAC is affiliated to the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) (NIHR200892) in Genomics and Enabling Data at University of Warwick in partnership with the UK Health Security Agency (UKHSA), in collaboration with Universities of Cambridge and Oxford. MAC is based at UKHSA. The views expressed are those of the author(s) and not necessarily those of the NIHR, the Department of Health and Social Care or the UK Health Security Agency. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. No payment was received by any pharmaceutical company or other agency to write this article. The authors were not precluded from accessing data in the study, and accept responsibility to submit for publication. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSalmonella Panama has consistently been reported as a frequently isolated Salmonella serovar in national surveillance datasets, causing both sporadic cases and larger outbreaks. However, this picture has been masked due to most of the focus being placed on the top two serovars associated with nontyphoidal Salmonella (NTS) and invasive NTS disease: Typhimurium and Enteritidis. Previous works on S. Panama have determined transmission to include human faeces and breast milk, as well as non-human sources such as environmental reservoirs and animals (reptiles and pigs used for food). In contrast to most of the Salmonella serovars causing gastroenteritis, S. Panama has also been associated with a range of systemic infections including septicaemia, meningitis, and osteomyelitis, mostly affecting infants. The patterns of antimicrobial resistance of this pathogen were not well known, with studies reporting a mixture of different antimicrobial resistance profiles. Added value of this studyHere we conducted a large-scale study of 836 globally relevant S. Panama isolates to understand global population structure and disease ecology. The S. Panama genomes used in this study were sourced from a combination of historical collections (including the first ever isolated strain of this serovar dating from 1931), national surveillance datasets, and publicly available data. Some of these isolates had been linked to travel, allowing an inferred location for understanding population structure. Overall, this assembled S. Panama collection spanned a range of 88 years and covered 45 countries and regions of all inhabited continents. We applied bacterial phylodynamic approaches to determine population structure, evolutionary history, and clade-specific trends of invasiveness and antimicrobial resistance correlated with geo-temporal parameters. Implications of all the available evidenceThis study revealed the population structure and global ecology of S. Panama, indicating that multidrug resistant S. Panama circulates in European and Asian regions, and presents an increased number of genetic markers for extra-intestinal invasiveness. The transmission and expansion of antimicrobial-resistant S. Panama strains presented in this study highlight the significance of supporting control and monitoring efforts from an international perspective.

genomics↗
bioRxiv2023

Connecting single neuron transcriptomes to the projectome in mouse visual cortex

The mammalian brain is composed of diverse neuron types that play different functional roles. Recent single-cell RNA sequencing approaches have led to a whole brain taxonomy of transcriptomically-defined cell types, yet cell type definitions that include multiple cellular properties can offer additional insights into a neurons role in brain circuits. While the Patch-seq method can investigate how transcriptomic properties relate to the local morphological and electrophysiological properties of cell types, linking transcriptomic identities to long-range projections is a major unresolved challenge. To address this, we collected coordinated Patch-seq and whole brain morphology data sets of excitatory neurons in mouse visual cortex. From the Patch-seq data, we defined 16 integrated morphoelectric-transcriptomic (MET)-types; in parallel, we reconstructed the complete morphologies of 300 neurons. We unified the two data sets with a multi-step classifier, to integrate cell type assignments and interrogate cross-modality relationships. We find that transcriptomic variations within and across MET-types correspond with morphological and electrophysiological phenotypes. In addition, this variation, along with the anatomical location of the cell, can be used to predict the projection targets of individual neurons. We also shed new light on infragranular cell types and circuits, including cell-type-specific, interhemispheric projections. With this approach, we establish a comprehensive, integrated taxonomy of excitatory neuron types in mouse visual cortex and create a system for integrated, high-dimensional cell type classification that can be extended to the whole brain and potentially across species.

neuroscience↗
bioRxiv2023

Bacterial defences interact synergistically by disrupting phage cooperation

The constant arms race between bacteria and their phages has resulted in a large diversity of bacterial defence systems1,2, with many bacteria carrying several systems3,4. In response, phages often carry counter-defence genes5-9. If and how bacterial defence mechanisms interact to protect against phages with counter-defence genes remains unclear. Here, we report the existence of a novel defence system, coined MADS (Methylation Associated Defence System), which is located in a strongly conserved genomic defence hotspot in Pseudomonas aeruginosa and distributed across Gram-positive and Gram-negative bacteria. We find that the natural co-existence of MADS and a Type IE CRISPR-Cas adaptive immune system in the genome of P. aeruginosa SMC4386 provides synergistic levels of protection against phage DMS3, which carries an anti-CRISPR (acr) gene. Previous work has demonstrated that Acr-phages need to cooperate to overcome CRISPR immunity, with a first sacrificial phage causing host immunosuppression to enable successful secondary phage infections10,11. Modelling and experiments show that the co-existence of MADS and CRISPR-Cas provides strong and durable protection against Acr-phages by disrupting their cooperation and limiting the spread of mutants that overcome MADS. These data reveal that combining bacterial defences can robustly neutralise phage with counter-defence genes, even if each defence on its own can be readily by-passed, which is key to understanding how selection acts on defence combinations and their coevolutionary consequences.

microbiology↗
bioRxiv2022

Shigella serotypes associated with carriage in humans establish persistent infection in zebrafish

Shigella represents a paraphyletic group of human-adapted Escherichia coli lineages that converged towards the same enteropathogenic pathovar. In low-income countries, Shigella is typically transmitted by ingestion of contaminated water and food, while in high-income countries sexual transmission among men who have sex with men (MSM) is the dominant route of transmission. Although more than 40 different serotypes of Shigella have been reported globally, within the MSM community the majority of reported cases are attributed to three Shigella serotypes: the Shigella sonnei unique serotype (Ss) and the Shigella flexneri serotypes 2a and 3a. Here, using a zebrafish infection model, we demonstrate that Shigella can establish persistent infection in vivo. In this case, bacteria are not cleared by the host immune system and become tolerant to therapeutic doses of antibiotics. We show that persistence is dependent on the Shigella O-Antigen, a key constituent of the bacterial cell surface and determinant of serotype classification. Representative isolates of three Shigella serotypes associated with global dissemination and MSM transmission (S. sonnei Ss, S. flexneri 2a and 3a) all persist in zebrafish, while a serotype not associated with MSM transmission (S. flexneri 5a) does not. Strikingly, Shigella serotypes which establish persistent infection fail to promote macrophage cell death in vivo. We conclude that zebrafish can be a valuable platform to illuminate host and pathogen factors underlying the establishment of persistent infection with Shigella in humans. HighlightsO_LIShigella can establish persistent infection in zebrafish C_LIO_LIEstablishment of persistent infection in vivo depends on Shigella serotype C_LIO_LIThe same serotypes establishing persistent infection in zebrafish are prevalent in a patient group in whom persistent infection is observed C_LIO_LIShigella serotypes which establish persistent infection fail to promote macrophage cell death in vivo C_LI In BriefIn high-income countries, sexual transmission among men who have sex with men (MSM) is the dominant cause of domestic Shigella transmission. Torraca et al. discover that three serotypes of Shigella associated with MSM transmission also persist in zebrafish. These results indicate that Shigella serotypes associated with MSM transmission can establish carriage in the host, and highlight the use of zebrafish infection to study Shigella persistent infection in humans.

microbiology↗
bioRxiv2022

The BRAIN Initiative Cell Census Data Ecosystem: A User's Guide

Characterizing cellular diversity at different levels of biological organization across data modalities is a prerequisite to understanding the function of cell types in the brain. Classification of neurons is also required to manipulate cell types in controlled ways, and to understand their variation and vulnerability in brain disorders. The BRAIN Initiative Cell Census Network (BICCN) is an integrated network of data generating centers, data archives and data standards developers, with the goal of systematic multimodal brain cell type profiling and characterization. Emphasis of the BICCN is on the whole mouse brain and demonstration of prototypes for human and non-human primate (NHP) brains. Here, we provide a guide to the cellular and spatial approaches employed, and to accessing and using the BICCN data and its extensive resources, including the BRAIN Cell Data Center (BCDC) which serves to manage and integrate data across the ecosystem. We illustrate the power of the BICCN data ecosystem through vignettes highlighting several BICCN analysis and visualization tools. Finally, we present emerging standards that have been developed or adopted by the BICCN toward FAIR (Wilkinson et al. 2016a) neuroscience. The combined BICCN ecosystem provides a comprehensive resource for the exploration and analysis of cell types in the brain.

bioinformatics↗
bioRxiv2022

Temporal GWAS identifies a widely distributed putative adhesin contributing to pathogen success in Shigella spp.

Shigella has emerged as a successful pathogen posing a threat to human health worldwide in the recent years. The rise of Shigella over the years has variably been attributed to AMR genes, virulence, and bacterial competition factor but no method has taken a function-agnostic approach to look for factors associated with modern variants of the pathogens. To address this gap, here we combined historical and modern isolate collections to identify such factors through a novel approach, termed temporal GWAS (tGWAS). Our analyses identified a novel putative adhesin gene, which we called stv that was associated with time of isolation and concentrated in expanding lineages of Shigella spp, as well as widely distributed in other bacterial species. We confirmed that stv is carried on a small 2689bp plasmid and in silico analyses revealed that Stv contained a new protein domain that was in combination with other domains in a manner suggestive of a secreted bacterial toxin. However, an all-proteome AlphaFold interaction screen indicated a high likelihood of interactions with fimbrial proteins, despite fimbrae thought to be defunct in Shigella sp. Collectively, these findings suggest that Stv is a novel protein domain with a likely role in Shigella success that is also widely distributed in other species.

genomics↗
bioRxiv2022

Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution.

We used single-molecule nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of Superfamily 1 helicase PcrA as it translocates on, or unwinds, several kb-long DNA molecules. We recorded over 2 million enzyme steps under various assisting and opposing forces in diverse ATP and ADP conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA experiences in vivo, such as when the helicase encounters bound proteins or duplex DNA; we show how PcrAs kinetics change with such stimuli. SPRNT allows for direct association of the underlying DNA sequence with observed enzyme kinetics. Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base-pairs being unwound. Instead, the sequence of the single stranded DNA on which the PcrA walks determines much of the kinetics of unwinding.

biophysics↗
bioRxiv2021

A tale of two plasmids: contributions of plasmid associated phenotypes to epidemiological success among Shigella

Dissemination of antimicrobial resistance (AMR) genes by horizontal gene transfer (HGT) mediated through plasmids is a major global concern. Genomic epidemiology studies have shown varying success of different AMR plasmids during outbreaks, but the underlying reasons for these differences are unclear. Here, we investigated two Shigella plasmids (pKSR100 and pAPR100) that circulated in the same transmission network but had starkly contrasting epidemiological outcomes to identify plasmid features that may have contributed to the differences. We used plasmid comparative genomics to reveal divergence between the two plasmids in genes encoding AMR, SOS response alleviation, and conjugation. Experimental analyses revealed that these genomic differences corresponded with reduced conjugation rates for the epidemiologically successful pKSR100, but more extensive AMR, reduced fitness costs, and a reduced SOS response in the presence of antimicrobials, compared with the less successful pAPR100. The discrepant phenotypes between the two plasmids are consistent with the hypothesis that plasmid associated phenotypes contribute to determining the epidemiological outcome of AMR HGT and suggest that phenotypes relevant in responding to antimicrobial pressure and fitness impact may be more important than those around conjugation in this setting. Plasmid phenotypes could thus be valuable tools in conjunction with genomic epidemiology for predicting AMR dissemination.

microbiology↗
bioRxiv2021

Informing shigellosis prevention and control through pathogen genomics

Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMIC), are increasingly antimicrobial resistant and have no licensed vaccine. We performed genomic analyses of 1246 systematically collected shigellae from seven LMIC to inform control and identify factors that could limit the effectiveness of current approaches. We found that S. sonnei contributes [&ge;]20-fold more disease than other Shigella species relative to its genomic diversity and highlight existing diversity and adaptative capacity among S. flexneri that may generate vaccine escape variants in <6 months. Furthermore, we show convergent evolution of resistance against the current recommended antimicrobial among shigellae. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, and other pathogens.

microbiology↗
bioRxiv2020

Attention and prediction modulations in expected and unexpected visuospatial trajectories.

Humans are constantly exposed to a rich tapestry of visual information in a potentially changing environment. To cope with the computational burden this engenders, our perceptual system must use prior context to simultaneously prioritise stimuli of importance and suppress irrelevant surroundings. This study investigated the influence of prediction and attention in visual perception by investigating event-related potentials (ERPs) often associated with these processes, N170 and N2pc for prediction and attention, respectively. A contextual trajectory paradigm was used which violated visual predictions and neglected to predetermine areas of spatial interest, to account for the potentially unpredictable nature of a real-life visual scene. Participants (N=36) viewed a visual display of cued and non-cued shapes rotating in a five-step predictable trajectory, with the fifth and final position of either the cued or non-cued shape occurring in a predictable or unpredictable spatial location. To investigate the predictive coding theory of attention we used factors of attention and prediction, whereby attention was manipulated as either cued or non-cued conditions, and prediction manipulated in either predictable or unpredictable conditions. Results showed both enhanced N170 and N2pc amplitudes to unpredictable compared to predictable stimuli. Stimulus cueing status also increased N170 amplitude, but this did not interact with stimulus predictability. The N2pc amplitude was not affected by stimulus cueing status. In accordance with previous research these results suggest the N170 is in part a visual prediction error response with respect to higher-level visual processes, and furthermore the N2pc may index attention reorientation. The results demonstrate prior context influences the sensitivity of the N170 and N2pc electrophysiological responses. These findings add further support to the role of N170 as a prediction error signal and suggest that the N2pc may reflect attentional reorientation in response to unpredicted stimulus locations.

neuroscience↗