Biology subjects

Pepperell, C. S.

Publications and source records attributed to Pepperell, C. S..

12 recordsLinked to original sources

bioRxiv2024

Contingencies in biofilm adaptation of Mycobacterium tuberculosis

Biofilms are structured microbial communities that offer protection from a range of environmental stressors. Studies of experimental evolution within biofilms have yielded important insights into mechanisms of biofilm formation, as well as fundamental principles governing bacterial adaptation within these structured communities. Building on research using tractable species, our lab created a model for studying biofilm adaptation in Mycobacterium tuberculosis (M. tb), a fastidious, slow growing and lethal pathogen of humans. Here, we evolved eighteen M. tb populations arising from six parental genetic backgrounds under biofilm selection to investigate diversity in mechanisms of M. tb biofilm adaptation. We found that fine-scale differences among strains at the initiation of the experiment influenced the degree of replicability in their evolution. Adaptive paths were highly parallel for some strains, whereas others evolved distinct mutations across iterations of the experiment. Our data suggest that differences in replicability arise from mutational biases and variable fitness impacts of mutations across genetic backgrounds. Comparison of our results with genomic data from M. tb populations within hosts with tuberculosis (TB) revealed that several mutations associated with biofilm selection are also among the most common to emerge during natural infection. These biofilm-associated variants are not maintained in natural M. tb populations, suggesting that biofilm selection in our model mimics selection pressures that are transiently encountered by M. tb during specific phases of infection. Overall, these results support development of biofilm directed therapies for TB and demonstrate the importance of subtle genetic variation in shaping M. tb responses to changing selection pressures. SignificanceMycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), a difficult-to-treat, persistent infection with high mortality. One cause of this persistence is the ability of M. tb to form biofilms, aggregated structures capable of resisting antibiotics and host defenses. Here, we used an evolutionary model to investigate mechanisms of M. tb biofilm formation. We found adaptation to biofilm growth to be affected by mutational biases and interactions among mutations. Our simple in vitro model appears to mimic aspects of natural infection, as identical mutations emerge in the laboratory under biofilm selection and within hosts with TB. These results expand our knowledge of M. tb biofilm development and inform our understanding of how this bacterium responds to novel selection pressures.

evolutionary biology↗

bioRxiv2023

Portrait of a generalist bacterium: pathoadaptation, metabolic specialization and extreme environments shape diversity of Staphylococcus saprophyticus

Staphylococcus saprophyticus is a Gram-positive, coagulase-negative staphylococcus found in diverse environments including soil and freshwater, meat, and dairy foods. S. saprophyticus is also an important cause of urinary tract infections (UTIs) in humans, and mastitis in cattle. However, the genetic determinants of virulence have not yet been identified, and it remains unclear whether there are distinct sub-populations adapted to human and animal hosts. Using a diverse sample of S. saprophyticus isolates from food, animals, environmental sources, and human infections, we characterized the population structure and diversity of global populations of S. saprophyticus. We found that divergence of the two major clades of S. saprophyticus is likely facilitated by barriers to horizontal gene transfer (HGT) and differences in metabolism. Using genome-wide association study (GWAS) tools we identified the first Type VII secretion system (T7SS) described in S. saprophyticus and its association with bovine mastitis. Finally, we found that in general, strains of S. saprophyticus from different niches are genetically similar with the exception of built environments, which function as a sink for S. saprophyticus populations. This work increases our understanding of the ecology of S. saprophyticus and of the genomics of bacterial generalists. Data summaryRaw sequencing data for newly sequenced S. saprophyticus isolates have been deposited to the NCBI SRA under the project accession PRJNA928770. A list of all genomes used in this work and their associated metadata are available in the supplementary material. Custom scripts used in the comparative genomics and GWAS analyses are available here: https://github.com/myoungblom/sapro_genomics. Impact statementIt is not known whether human and cattle diseases caused by S. saprophyticus represent spillover events from a generalist adapted to survive in a range of environments, or whether the capacity to cause disease represents a specific adaptation. Seasonal cycles of S. saprophyticus UTIs and molecular epidemiological evidence suggest that these infections may be environmentally-acquired rather than via transmission from person to person. Using comparative genomics and genome wide association study tools, we found that S. saprophyticus appears adapted to inhabit a wide range of environments (generalist), with isolates from animals, food, natural environments and human infections being closely related. Bacteria that routinely switch environments, particularly between humans and animals, are of particular concern when it comes to the spread of antibiotic resistance from farm environments into human populations. This work provides a framework for comparative genomic analyses of bacterial generalists and furthers our understanding of how bacterial populations move between humans, animals, and the environment.

microbiology↗

bioRxiv2023

Adaptation of the Mycobacterium tuberculosis transcriptome to biofilm growth

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), is a leading global cause of death from infectious disease. Biofilms are increasingly recognized as a relevant growth form during M. tb infection and may impede treatment by enabling bacterial drug and immune tolerance. M. tb has a complicated regulatory network that has been well-characterized for many relevant disease states, including dormancy and hypoxia. However, despite its importance, our knowledge of the genes and pathways involved in biofilm formation is limited. Here we characterize the biofilm transcriptomes of fully virulent clinical isolates and find that the regulatory systems underlying biofilm growth vary widely between strains and are also distinct from regulatory programs associated with other environmental cues. We used experimental evolution to investigate changes to the transcriptome during adaptation to biofilm growth and found that the application of a uniform selection pressure resulted in loss of strain-to-strain variation in gene expression, resulting in a more uniform biofilm transcriptome. The adaptive trajectories of transcriptomes were shaped by the genetic background of the M. tb population leading to convergence on a sub-lineage specific transcriptome. We identified widespread upregulation of non-coding RNA (ncRNA) as a common feature of the biofilm transcriptome and hypothesize that ncRNA function in genome-wide modulation of gene expression, thereby facilitating rapid regulatory responses to new environments. These results reveal a new facet of the M. tb regulatory system and provide valuable insight into how M. tb adapts to new environments. ImportanceUnderstanding mechanisms of resistance and tolerance in Mycobacterium tuberculosis (M. tb) can help us develop new treatments that capitalize on M. tbs vulnerabilities. Here we used transcriptomics to study both the regulation of biofilm formation in clinical isolates as well as how those regulatory systems adapt to new environments. We find that closely related clinical populations have diverse strategies for growth under biofilm conditions, and that genetic background plays a large role in determining the trajectory of evolution. These results have implications for future treatment strategies that may be informed by our knowledge of the evolutionary constraints on strain(s) from an individual infection. This work provides new information about the mechanisms of biofilm formation in M. tb and outlines a framework for population level approaches for studying bacterial adaptation.

microbiology↗

bioRxiv2023

Genotypic and phenotypic differences among phase-variable colony variants conserved across Gardnerella spp.

The Gardnerella genus, now made up of more than 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all tested Gardnerella strains, representing at least ten different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops in vivo, we characterized their differences. Beyond increased colony size, large colony variants (Lg) showed reduced vaginolysin secretion and faster growth rate relative to small colony variants (Sm). The ability to inhibit growth of Neisseria gonorrhoeae and commensal lactobacillus species varied by strain and in some instances differed between variants. Proteomics analyses indicate that 127-173 proteins are differentially expressed between variants. Further, whole genome sequencing analyses revealed an abundance of genes associated with variable poly-guanine tracts, implicating slipped strand mispairing in Gardnerella phase variation, and illuminating the potential for previously unrecognized variability within clonal populations. Collectively, these results suggest that colony variants may be primed to serve different roles in BV pathogenesis.

microbiology↗

bioRxiv2022

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests utilizing molecularly intact heme. Here we describe the mechanism underlying this trait, which we discovered using molecular genetics, phylogenetics, and population analyses. Most metronidazole-resistant strains evolved a T to G mutation, we term PnimBG, in the -10 regulatory promoter of the 5-nitroimidazole reductase nimB, resulting in the gene being constitutively transcribed. Silencing or deleting nimB eliminated metronidazole resistance. We identified the protein as a heme-dependent nitroreductase that degraded nitro-drugs to an amine lacking antimicrobial activity. We further discovered that the metronidazole-resistant PnimBGmutation was strongly associated with the Thr82Ile substitution conferring fluoroquinolone resistance in epidemic strains. Re-analysis of published genomes from global isolates confirmed that all but one encoding PnimBG also carried the Thr82Ile mutation. Our findings suggest that fluoroquinolone and metronidazole resistance co-mediated the pandemic of healthcare-associated C. difficile that are associated with poorer treatment outcomes in CDI patients receiving metronidazole.

microbiology↗

bioRxiv2022

Comparative genomics of Streptococcus oralis identifies large scale homologous recombination and a genetic variant associated with infection

The viridans group streptococci (VGS) are a large consortium of commensal streptococci that colonize the human body. Many species within this group are opportunistic pathogens causing bacteremia and infective endocarditis (IE), yet little is known about why some strains cause invasive disease. Identification of virulence determinants is complicated by the difficulty of distinguishing between the closely related species of this group. Here, we analyzed genomic data from VGS isolated from patient blood cultures with invasive infections and from oral swabs of healthy volunteers and determined the best performing methods for species identification. Using whole-genome sequence data, we characterized the population structure of a diverse sample of Streptococcus oralis isolates and found evidence of frequent recombination. We used multiple genome-wide association study tools to identify candidate determinants of invasiveness. These tools gave consistent results, leading to the discovery of a single synonymous single nucleotide polymorphism (SNP) that was significantly associated with invasiveness. This SNP is within a previously undescribed gene that is conserved across the majority of VGS species. Using growth in the presence of human serum and a simulated infective endocarditis vegetation model, we were unable to identify a phenotype for the enriched allele in laboratory assays, suggesting a phenotype may be specific to natural infection. These data highlight the power of analyzing natural populations for gaining insight into pathogenicity, particularly for organisms with complex population structures like the VGS. ImportanceThe viridians group streptococci (VGS) are a large collection of closely related commensal streptococci, with many being opportunistic pathogens causing invasive diseases such as bacteremia and infective endocarditis. Little is known about virulence determinants in these species, and there is a distinct lack of genomic information available for the VGS. In this study, we collected VGS isolates from invasive infections and healthy volunteers and performed whole genome sequencing for a suite of downstream analyses. We focused on a diverse sample of Streptococcus oralis genomes and identified high rates of recombination in the population as well as a single genome variant highly enriched in invasive isolates. The variant lies within a previously uncharacterized gene, nrdM, which shares homology with the anaerobic ribonucleoside triphosphate reductase, nrdD, and is highly conserved among VGS. This work increases our knowledge of VGS genomics and indicates that differences in virulence potential among S. oralis isolates is, at least in part, genetically determined.

microbiology↗

bioRxiv2021

Rapid adaptation of a complex trait during experimental evolution of Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), is a leading cause of death due to infectious disease. TB is not traditionally associated with biofilms, but M. tb biofilms are linked with drug and immune tolerance and there is increasing recognition of their contribution to the recalcitrance of TB infections. Here we used M. tb experimental evolution to investigate this complex phenotype and identify candidate loci controlling biofilm formation. We identified novel candidate loci, adding to our understanding of the genetic architecture underlying M. tb biofilm development. Under selective pressure to grow as a biofilm, regulatory mutations rapidly swept to fixation and were associated with changes in multiple traits including extracellular matrix production, cell size, and growth rate. Genetic and phenotypic paths to enhanced biofilm growth varied according to the genetic background of the parent strain, suggesting that epistatic interactions are important in M. tb adaptation to changing environments.

microbiology↗

bioRxiv2019

Dispersal of Mycobacterium tuberculosis to indigenous populations driven by historical European trade in the South Pacific

The Mycobacterium tuberculosis complex lineage 4 (L4), also known as the \"Euro-American\" lineage, is the most widely dispersed of the seven human adapted lineages. L4 is comprised of ten sublineages including L4.4, which has a moderate global distribution and is the most common L4 sublineage in New Zealand. We have used a phylodynamics approach and a dataset of 236 global M. tuberculosis genomes to trace the origins and dispersal of L4.4 strains in New Zealand that are predominantly found in M[a]ori and Pacific people. We identify an L4.4.1.1 sublineage clade of European origin, likely French, that is prevalent in indigenous populations in both New Zealand and Canada. Molecular dating suggests that expansion of European trade networks in the early 19th century led to dispersal of this clade to the South Pacific. We also identify historical and social factors within the region that have contributed to the local spread and expansion of these strains, including recent Pacific migrations to New Zealand and the rapid urbanization of M[a]ori in the 20th century. Our results offer new insight into the dispersal of M. tuberculosis in the South Pacific region and provide a striking example of the role of historical European migrations in the dispersal of M. tuberculosis.\n\nAuthor SummaryTuberculosis kills more people worldwide than any other infectious disease and indigenous populations are disproportionately affected by the disease. Here, we have used a large global dataset of Mycobacterium tuberculosis bacterial genomes to trace the historical origins of tuberculosis strains in New Zealand that are most frequently found in M[a]ori and Pacific people. These strains are locally known as the Rangipo and Otara strains (both M[a]ori place names) and belong to the \"Euro-American\" lineage of M. tuberculosis. Via genome analysis, we find that these strains are closely related to M. tuberculosis strains found in indigenous populations in Canada that have a European origin. We used a molecular dating approach (a molecular clock) to infer the ages of these strains and date divergence events. The timing we infer corresponds to the introduction of these strains to Polynesia via expanding European trade networks in the South Pacific in the early 19th century and suggests that the Otara strain has migrated to New Zealand from the Pacific Islands multiple times. Our results provide insight into human social phenomena underlying the expansion and dispersal of M. tuberculosis and reassert the important role of European colonial migrations in the global dispersal of the M. tuberculosis Euro-American lineage. This work also highlights the pejorative and stigmatizing mislabelling of the New Zealand strains with indigenous M[a]ori place names, suggesting that these strains should be renamed.

evolutionary biology↗

bioRxiv2018

Multiple Exposures, Reinfection, and Risk of Progression to Active Tuberculosis

A recent study reported on a tuberculosis outbreak in a largely Inuit village. Among recently infected individuals, exposure to additional active cases was associated with an increasing probability of developing active disease within a year. Using binomial risk models, we evaluated two potential mechanisms by which multiple infections during the first year following initial infection could account for increasing disease risk with increasing exposures. In the reinfection model, multiple exposures have an independent risk of becoming an infection, and infections contribute independently to active disease. In the threshold model, disease risk follows a sigmoidal function with small numbers of exposures conferring a low risk of active disease and large numbers of exposures conferring a high risk. To determine the dynamic impact of reinfection during the early phase of infection, we performed simulations from a modified Reed-Frost model of TB dynamics following spread from an initial number of cases. We parameterized this model with the maximum likelihood estimates from the reinfection and threshold models in addition to the observed distribution of exposures among recent infections. We find that both models can plausibly account for the observed increase in disease risk with increasing exposures, but the threshold model confers a better fit than a nested model without a threshold (p=0.04). Our simulations indicate that multiple exposures during this critical time period can lead to dramatic increases in outbreak size. In order to decrease TB burden in high-prevalence settings, it may be necessary to implement measures aimed at preventing repeated exposures, in addition to preventing primary infection.

epidemiology↗

bioRxiv2017

Lineage specific histories of Mycobacterium tuberculosis dispersal in Africa and Eurasia

Mycobacterium tuberculosis (M.tb) is a globally distributed, obligate pathogen of humans that can be divided into seven clearly defined lineages. Identifying how the ancestral clone of M.tb spread and differentiated is important for identifying the ecological drivers of the current pandemic. We reconstructed M.tb migration in Africa and Eurasia, and investigated lineage specific patterns of spread. Applying evolutionary rates inferred with ancient M.tb genome calibration, we link M.tb dispersal to historical phenomena that altered patterns of connectivity throughout Africa and Eurasia: trans-Indian Ocean trade in spices and other goods, the Silk Road and its predecessors, the expansion of the Roman Empire and, the European Age of Exploration. We find that Eastern Africa and Southeast Asia have been critical in the dispersal of M.tb. Our results reveal complex relationships between spatial dispersal and expansion of M.tb populations, and delineate the independent evolutionary trajectories of bacterial sub-populations underlying the current pandemic.

evolutionary biology↗

bioRxiv2017

Signatures of selection at drug resistance loci in Mycobacterium tuberculosis

Tuberculosis (TB) is the leading cause of death by an infectious disease, and global TB control efforts are increasingly threatened by drug resistance in Mycobacterium tuberculosis (M. tb). Unlike most bacteria, where lateral gene transfer is an important mechanism of resistance acquisition, resistant M. tb arises solely by de novo chromosomal mutation. Using whole genome sequencing data from two natural populations of M. tb, we characterized the population genetics of known drug resistance loci using measures of diversity, population differentiation, and convergent evolution. We found resistant sub-populations to be less diverse than susceptible sub-populations, consistent with ongoing transmission of resistant M. tb. A subset of resistance genes (\"sloppy targets\") were characterized by high diversity and multiple rare variants; we posit that a large genetic target for resistance and relaxation of purifying selection contribute to high diversity at these loci. For \"tight targets\" of selection, the path to resistance appeared narrower, evidenced by single favored mutations that arose numerous times on the phylogeny and segregated at markedly different frequencies in resistant and susceptible sub-populations. These results suggest that diverse genetic architectures underlie drug resistance in M. tb, and combined approaches are needed to identify causal mutations. Extrapolating from patterns observed in well-characterized genes, we identified novel candidate variants involved in resistance. The approach outlined here can be extended to identify resistance variants for new drugs, to investigate the genetic architecture of resistance, and, when phenotypic data are available, to find candidate genetic loci underlying other positively selected traits in clonal bacteria.\n\nImportanceMycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), is a significant burden on global health. Antibiotic treatment imposes strong selective pressure on M. tb populations. Identifying the mutations that cause drug resistance in M. tb is important for guiding TB treatment and halting the spread of drug resistance. Whole genome sequencing (WGS) of M. tb isolates can be used to identify novel mutations mediating drug resistance and to predict resistance patterns faster than traditional methods of drug susceptibility testing. We have used WGS from natural populations of drug resistant M. tb to characterize effects of selection for advantageous mutations on patterns of diversity at genes involved in drug resistance. The methods developed here can be used to identify novel advantageous mutations, including new resistance loci, in M. tb and other clonal pathogens.

microbiology↗

bioRxiv2017

Adaptation Of Staphylococcus saprophyticus At The Host-Pathogen Interface

Human-pathogenic bacteria are found in a variety of niches, including free-living, zoonotic, and microbiome environments. Identifying bacterial adaptions that enable invasive disease is an important means of gaining insight into the molecular basis of pathogenesis and understanding pathogen emergence. Staphylococcus saprophyticus, a leading cause of urinary tract infections, can be found in the environment, food, animals, and the human microbiome. We identified a selective sweep in the gene encoding the Aas adhesin, a key virulence factor that binds host fibronectin. We hypothesize that the mutation under selection (aas_2206A>C) facilitates colonization of the urinary tract, an environment where bacteria are subject to strong shearing forces. The mutation appears to have enabled emergence and expansion of a human pathogenic lineage of S. saprophyticus. These results demonstrate the power of evolutionary genomic approaches in discovering the genetic basis of virulence and emphasize the pleiotropy and adaptability of bacteria occupying diverse niches.\n\nImportanceStaphylococcus saprophyticus is an important cause of urinary tract infections (UTI) in women, which are common, can be severe, and are associated with significant impacts to public health. In addition to being a cause of human UTI, S. saprophyticus can be found in the environment, in food, and associated with animals. After discovering that UTI strains of S. saprophyticus are for the most part closely related to each other, we sought to determine whether these strains are specially adapted to cause disease in humans. We found evidence suggesting that a mutation in the gene aas is advantageous in the context of human infection. We hypothesize that the mutation allows S. saprophyticus to survive better in the human urinary tract. These results show how bacteria found in the environment can evolve to cause disease.

microbiology↗