A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on twitching motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens

The stigmatisation of source isolation: a literature review

Background: Isolation precautions in patients with multi-drug-resistant bacteria and other communicable infectious agents can be associated with adverse effects. Patients’ perspectives of isolation suggest that the imposed environment and procedures create barriers to their physical, social and emotional needs.Aims: The purpose of this paper is to review the literature to uncover any reliable evidence supporting the assertion that stigma is a significant characteristic of the experience of source isolation in healthcare settings.Methods: The methodological framework of Arksey and O’Malley was applied to this review. A total of 14 papers identified from 189 abstracts screened were included in the review.Results: The research reviewed suggests a clear association between stigmatisation and isolation in which stigma does have a direct negative effect on patients placed in hospital isolation. None of the studies found evidence to the contrary.Conclusions: The implications of this literature review for policy-makers and healthcare professionals suggest that when isolation or other forms of constraint are implemented and in use, patients must be provided with strengthened forms of support, including social and emotional support, and given access to healthcare of optimal quality to prevent the associated adverse effects of isolation as much as possibl

Physiological Characteristics of Female Soccer Players and Health and Performance Considerations: A Narrative Review

From Springer Nature via Jisc Publications RouterHistory: accepted 2021-03-22, registration 2021-03-22, online 2021-04-12, pub-electronic 2021-04-12, pub-print 2021-07Publication status: PublishedAbstract: Female soccer has seen a substantial rise in participation, as well as increased financial support from governing bodies over the last decade. Thus, there is an onus on researchers and medical departments to develop a better understanding of the physical characteristics and demands, and the health and performance needs of female soccer players. In this review, we discuss the current research, as well as the knowledge gaps, of six major topics: physical demands, talent identification, body composition, injury risk and prevention, health and nutrition. Data on female talent identification are scarce, and future studies need to elucidate the influence of relative age and maturation selection across age groups. Regarding the physical demands, more research is needed on the pattern of high-intensity sprinting during matches and the contribution of soccer-specific movements. Injuries are not uncommon in female soccer players, but targeting intrinsically modifiable factors with injury prevention programmes can reduce injury rates. The anthropometric and physical characteristics of female players are heterogeneous and setting specific targets should be discouraged in youth and sub-elite players. Menstrual cycle phase may influence performance and injury risk; however, there are few studies in soccer players. Nutrition plays a critical role in health and performance and ensuring adequate energy intake remains a priority. Despite recent progress, there is considerably less research in female than male soccer players. Many gaps in our understanding of how best to develop and manage the health and performance of female soccer players remain

The RNA-binding protein TbDRBD3 regulates the stability of a specific subset of mRNAs in trypanosomes

In trypanosomes, the apparent lack of regulation of RNA polymerase II-dependent transcription initiation poses a challenge to understand how these eukaryotes adjust gene expression to adapt to the contrasting environments they find during their life cycles. Evidence so far indicates that mRNA turnover and translation are the major control points in which regulation is exerted in trypanosomes. However, very little is known about which proteins are involved, and how do they regulate the abundance and translation of different mRNAs in different life stages. In this work, an RNA-binding protein, TbDRBD3, has been identified by affinity chromatography, and its function addressed using RNA interference, microarray analysis and immunoprecipitation of mRNA–protein complexes. The results obtained indicate that TbDRBD3 binds to a subset of developmentally regulated mRNAs encoding membrane proteins, and that this association promotes the stabilization of the target transcripts. These observations raise the possibility that TbDRBD3-mRNA complexes act as a post-transcriptional operon, and provide a framework to interpret how trypanosomes regulate gene expression in the absence of transcriptional control

Spliced Leader Trapping Reveals Widespread Alternative Splicing Patterns in the Highly Dynamic Transcriptome of Trypanosoma brucei

Trans-splicing of leader sequences onto the 5′ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5′splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5′ splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/

Differential Trypanosome Surface Coat Regulation by a CCCH Protein That Co-Associates with procyclin mRNA cis-Elements

The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET) that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i) that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii) that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii) their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP) is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes. © 2009 Walrad et al

Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host

This work was supported by a Wellcome Trust Programme grant to KM and by a Wellcome Trust Strategic award to the Centre for Immunity, Infection and Evolution at the University of Edinburgh. SM was supported by a studentship from the Medical Research Council, UK.The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically 'slender forms' to transmissible 'stumpy forms' through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms), irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.Publisher PDFPeer reviewe

Genomic reconstruction of the SARS-CoV-2 epidemic in England.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.1.1.7/Alpha lineage. The Alpha variant grew when other lineages declined during the second national lockdown and regionally tiered restrictions between November and December 2020. A third more stringent national lockdown suppressed the Alpha variant and eliminated nearly all other lineages in early 2021. Yet a series of variants (most of which contained the spike E484K mutation) defied these trends and persisted at moderately increasing proportions. However, by accounting for sustained introductions, we found that the transmissibility of these variants is unlikely to have exceeded the transmissibility of the Alpha variant. Finally, B.1.617.2/Delta was repeatedly introduced in England and grew rapidly in early summer 2021, constituting approximately 98% of sampled SARS-CoV-2 genomes on 26 June 2021

High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile

Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. IMPORTANCE Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been hampered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection. FOOTNOTE