Recurrent urinary tract infections (rUTIs) are extremely common, with ~ 25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally ...dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.
Extraintestinal pathogenic Escherichia coli colonize the human gut and can spread to other body sites to induce diseases such as urinary tract infections, sepsis, and meningitis. A complete ...understanding of the infection process is hindered by both the inherent genetic diversity of E. coli and the large number of unstudied genes. Here, we focus on the uncharacterized gene rqlI, which our lab recently uncovered in a Tn-seq screen for bacterial genes required within a zebrafish model of infection. We demonstrate that the ΔrqlI mutant experiences a growth defect and increased DNA stress in low oxygen conditions. In a genetic screen for suppressor mutations in the Δrql strain, we found that the shortcomings of the Δrql mutant are attributable to the activity of RqlH, which is known in other bacteria to be a helicase of the RecQ family that contains a phosphoribosyltransferase (PRTase) domain. Disruption of rqlH rescues the ΔrqlI strain in both in vivo and in vitro assays, while the expression of RqlH alone activates the SOS response coincident with bacterial filamentation, heightened sensitivity to DNA damage, and an increased mutation rate. The analysis of truncation mutants indicates that, in the absence of RqlI, RqlH toxicity is due to its PRTase domain. Complementary studies demonstrate that the toxicity of RqlH is modulated in a context-dependent fashion by overlapping domains within RqlI. This regulation is seemingly direct, given that the two proteins physically interact and form an operon. Interestingly, RqlH and RqlI orthologs are encoded by a diverse group of bacteria, but in many of these microbes, and especially in Gram-positive organisms, rqlH is found in the absence of rqlI. In total, this work shows that RqlH and RqlI can act in a strain-specific fashion akin to a toxin-antitoxin system in which toxicity is mediated by an atypical helicase-associated PRTase domain.
Zinc is an essential cofactor for many proteins. A key mechanism of zinc homeostasis during deficiency is "zinc sparing" in which specific zinc-binding proteins are repressed to reduce the cellular ...requirement. In this report, we evaluated zinc sparing across the zinc proteome of Saccharomyces cerevisiae. The yeast zinc proteome of 582 known or potential zinc-binding proteins was identified using a bioinformatics analysis that combined global domain searches with local motif searches. Protein abundance was determined by mass spectrometry. In zinc-replete cells, we detected over 2500 proteins among which 229 were zinc proteins. Based on copy number estimates and binding stoichiometries, a replete cell contains ∼9 million zinc-binding sites on proteins. During zinc deficiency, many zinc proteins decreased in abundance and the zinc-binding requirement decreased to ∼5 million zinc atoms per cell. Many of these effects were due at least in part to changes in mRNA levels rather than simply protein degradation. Measurements of cellular zinc content showed that the level of zinc atoms per cell dropped from over 20 million in replete cells to only 1.7 million in deficient cells. These results confirmed the ability of replete cells to store excess zinc and suggested that the majority of zinc-binding sites on proteins in deficient cells are either unmetalated or mismetalated. Our analysis of two abundant zinc proteins, Fba1 aldolase and Met6 methionine synthetase, supported that hypothesis. Thus, we have discovered widespread zinc sparing mechanisms and obtained evidence of a high accumulation of zinc proteins that lack their cofactor during deficiency.
Post-transcriptional modifications can impact the stability and functionality of many different classes of RNA molecules and are an especially important aspect of tRNA regulation. It is hypothesized ...that cells can orchestrate rapid responses to changing environmental conditions by adjusting the specific types and levels of tRNA modifications. We uncovered strong evidence in support of this tRNA global regulation hypothesis by examining effects of the well-conserved tRNA modifying enzyme MiaA in extraintestinal pathogenic Escherichia coli (ExPEC), a major cause of urinary tract and bloodstream infections. MiaA mediates the prenylation of adenosine-37 within tRNAs that decode UNN codons, and we found it to be crucial to the fitness and virulence of ExPEC. MiaA levels shifted in response to stress via a post-transcriptional mechanism, resulting in marked changes in the amounts of fully modified MiaA substrates. Both ablation and forced overproduction of MiaA stimulated translational frameshifting and profoundly altered the ExPEC proteome, with variable effects attributable to UNN content, changes in the catalytic activity of MiaA, or availability of metabolic precursors. Cumulatively, these data indicate that balanced input from MiaA is critical for optimizing cellular responses, with MiaA acting much like a rheostat that can be used to realign global protein expression patterns.
Strains of Extraintestinal Pathogenic Escherichia c oli (ExPEC) exhibit an array of virulence strategies and are a major cause of urinary tract infections, sepsis and meningitis. Efforts to ...understand ExPEC pathogenesis are challenged by the high degree of genetic and phenotypic variation that exists among isolates. Determining which virulence traits are widespread and which are strain-specific will greatly benefit the design of more effective therapies. Towards this goal, we utilized a quantitative genetic footprinting technique known as transposon insertion sequencing (Tn-seq) in conjunction with comparative pathogenomics to functionally dissect the genetic repertoire of a reference ExPEC isolate. Using Tn-seq and high-throughput zebrafish infection models, we tracked changes in the abundance of ExPEC variants within saturated transposon mutant libraries following selection within distinct host niches. Nine hundred and seventy bacterial genes (18% of the genome) were found to promote pathogen fitness in either a niche-dependent or independent manner. To identify genes with the highest therapeutic and diagnostic potential, a novel Trait Enrichment Analysis (TEA) algorithm was developed to ascertain the phylogenetic distribution of candidate genes. TEA revealed that a significant portion of the 970 genes identified by Tn-seq have homologues more often contained within the genomes of ExPEC and other known pathogens, which, as suggested by the first axiom of molecular Koch's postulates, is considered to be a key feature of true virulence determinants. Three of these Tn-seq-derived pathogen-associated genes--a transcriptional repressor, a putative metalloendopeptidase toxin and a hypothetical DNA binding protein--were deleted and shown to independently affect ExPEC fitness in zebrafish and mouse models of infection. Together, the approaches and observations reported herein provide a resource for future pathogenomics-based research and highlight the diversity of factors required by a single ExPEC isolate to survive within varying host environments.
Extraintestinal pathogenic
(ExPEC) strains are typically benign within the mammalian gut but can disperse to extraintestinal sites to cause diseases like urinary tract infections and sepsis. As ...occupation of the intestinal tract is often a prerequisite for ExPEC-mediated pathogenesis, we set out to understand how ExPEC colonizes this niche. A screen using transposon sequencing (Tn-seq) was performed to search for genes within ExPEC isolate F11 that are important for growth in intestinal mucus, which is thought to be a major source of nutrients for
in the gut. Multiple genes that contribute to ExPEC fitness in mucus broth were identified, with genes that are directly or indirectly associated with fatty acid beta-oxidation pathways being especially important. One of the identified mucus-specific fitness genes encodes the rhomboid protease GlpG.
, we found that the disruption of
had polar effects on the downstream gene
, which encodes a transcriptional repressor of factors that catalyze glycerol degradation. Mutation of either
or
impaired ExPEC growth in mucus and on plates containing the long-chain fatty acid oleate as the sole carbon source. In contrast, in a mouse gut colonization model in which the natural microbiota is unperturbed, the disruption of
but not
significantly reduced ExPEC survival. This work reveals a novel biological role for a rhomboid protease and highlights new avenues for defining mechanisms by which ExPEC strains colonize the mammalian gastrointestinal tract.
Plants are a rich source of bioactive compounds and a number of plant-derived antiplasmodial compounds have been developed into pharmaceutical drugs for the prevention and treatment of malaria, a ...major public health challenge. However, identifying plants with antiplasmodial potential can be time-consuming and costly. One approach for selecting plants to investigate is based on ethnobotanical knowledge which, though having provided some major successes, is restricted to a relatively small group of plant species. Machine learning, incorporating ethnobotanical and plant trait data, provides a promising approach to improve the identification of antiplasmodial plants and accelerate the search for new plant-derived antiplasmodial compounds. In this paper we present a novel dataset on antiplasmodial activity for three flowering plant families - Apocynaceae, Loganiaceae and Rubiaceae (together comprising c. 21,100 species) - and demonstrate the ability of machine learning algorithms to predict the antiplasmodial potential of plant species. We evaluate the predictive capability of a variety of algorithms - Support Vector Machines, Logistic Regression, Gradient Boosted Trees and Bayesian Neural Networks - and compare these to two ethnobotanical selection approaches - based on usage as an antimalarial and general usage as a medicine. We evaluate the approaches using the given data and when the given samples are reweighted to correct for sampling biases. In both evaluation settings each of the machine learning models have a higher precision than the ethnobotanical approaches. In the bias-corrected scenario, the Support Vector classifier performs best - attaining a mean precision of 0.67 compared to the best performing ethnobotanical approach with a mean precision of 0.46. We also use the bias correction method and the Support Vector classifier to estimate the potential of plants to provide novel antiplasmodial compounds. We estimate that 7677 species in Apocynaceae, Loganiaceae and Rubiaceae warrant further investigation and that at least 1300 active antiplasmodial species are highly unlikely to be investigated by conventional approaches. While traditional and Indigenous knowledge remains vital to our understanding of people-plant relationships and an invaluable source of information, these results indicate a vast and relatively untapped source in the search for new plant-derived antiplasmodial compounds.
Extraintestinal pathogenic
(ExPEC) acts as a commensal within the mammalian gut but can induce pathology upon dissemination to other host environments such as the urinary tract and bloodstream. ExPEC ...genomes are likely shaped by evolutionary forces encountered within the gut, where the bacteria spend much of their time, provoking the question of how their extraintestinal virulence traits arose. The principle of coincidental evolution, in which a gene that evolved in one niche happens to be advantageous in another, has been used to argue that ExPEC virulence factors originated in response to selective pressures within the gut ecosystem. As a test of this hypothesis, the fitness of ExPEC mutants lacking canonical virulence factors was assessed within the intact murine gut in the absence of antibiotic treatment. We found that most of the tested factors, including cytotoxic necrotizing factor type 1 (CNF1), Usp, colibactin, flagella, and plasmid pUTI89, were dispensable for gut colonization. The deletion of genes encoding the adhesin PapG or the toxin HlyA had transient effects but did not interfere with longer-term persistence. In contrast, a mutant missing the type 1 pilus-associated adhesin FimH displayed somewhat reduced persistence within the gut. However, this phenotype varied dependent on the presence of specific competing strains and was partially attributable to aberrant flagellin expression in the absence of
These data indicate that FimH and other key ExPEC-associated factors are not strictly required for gut colonization, suggesting that the development of extraintestinal virulence traits is not driven solely by selective pressures within the gut.
We report on new geoarchaeological finds in a recently discovered cave-chamber (Liang Bawah, “Cave Underneath”) positioned below Liang Bua on the island of Flores, Indonesia, where the type specimen ...for Homo floresiensis was recovered from Late Pleistocene sediment. At the rear of Liang Bua, a 23-m-long shaft, inclined at 60°, leads to a lower chamber measuring 23m×24m×5m high (about half the size of Liang Bua). Stone artefacts and bones were found shallowly buried in rubble at the base of the shaft, and around a 5-m-high mud mound that fills the northwest sector of Liang Bawah. We recovered 17 stone artefacts made from chert and volcanics, and more than 220 well-preserved bone elements belonging to endemic giant rats, pigs, primates, small murid rodents, bats and introduced species. Multi-collector inductively coupled plasma mass spectrometer (MC-ICPMS) analysis of uranium and thorium in carbonate coatings on four bones yielded ages of ~240–180ka (endemic giant rat femur), ~110–60ka (unidentified phalanx), ~33–23ka (pig skull fragment), and ~7–3ka (giant rat femur), which overlap with the ~95 to 17ka occupation of Liang Bua by H. floresiensis. The ~33–23ka age of the pig skull fragment indicates that Sus sp. may have dispersed into island Southeast Asia earlier than previously recognised. The passageway at the rear of Liang Bawah, and a currently buried front entrance, represent two possible transport paths for cultural and faunal material to the cave-chamber. Analysis of the geomorphic evolution of Liang Bawah shows that it may have been a Late Pleistocene depocentre for material transported from the occupation chamber of Liang Bua, and a repository for human subsistence refuse, or pit-fall trap, via the rear passage. These physical attributes, and the antiquity of the faunal remains found thus far, indicate that Liang Bawah could contain an archive of the Late Pleistocene and, potentially, remains of H. floresiensis.
•Discovery of Liang Bawah and geoarchaeological finds below Liang Bua•U–Th ages of bones in Liang Bawah extend back to ~180,000years ago•Pleistocene pig skull provides evidence for oldest Sus in island Southeast Asia•Liang Bawah is a pit-fall trap that may contain remains of H. floresiensis
Zinc homeostasis is essential for all organisms. The Zap1 transcriptional activator regulates these processes in the yeast
. During zinc deficiency, Zap1 increases expression of zinc transporters and ...proteins involved in adapting to the stress of zinc deficiency. Transcriptional activation by Zap1 can also repress expression of some genes, e.g.,
. In zinc-replete cells,
mRNA is produced with a short transcript leader that is efficiently translated. During deficiency, Zap1-dependent expression of an RNA with a longer transcript leader represses the
promoter. This long leader transcript (LLT) is not translated due to the presence of small open reading frames upstream of the
coding region. In this study, we show that the
LLT RNA also plays a second function, i.e., repression of the adjacent
gene. In generating the LLT transcript, RNA polymerase II transcribes
through the
promoter. Production of the LLT RNA correlates with the decreased expression of
mRNA and mutations that prevent synthesis of the LLT RNA or terminate it before the
promoter renders
mRNA expression and Gis2 protein accumulation constitutive. Thus, we have discovered an unusual regulatory mechanism that uses a bicistronic RNA to control two genes simultaneously.
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