An anti-CRISPR that pulls apart a CRISPR-Cas complex.

In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications1. Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms2-4. Here, we characterize the anti-CRISPR AcrIF25, and we show that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications.

Diverse Antiphage Defenses Are Widespread Among Prophages and Mobile Genetic Elements.

Bacterial viruses known as phages rely on their hosts for replication and thus have developed an intimate partnership over evolutionary time. The survival of temperate phages, which can establish a chronic infection in which their genomes are maintained in a quiescent state known as a prophage, is tightly coupled with the survival of their bacterial hosts. As a result, prophages encode a diverse antiphage defense arsenal to protect themselves and the bacterial host in which they reside from further phage infection. Similarly, the survival and success of prophage-related elements such as phage-inducible chromosomal islands are directly tied to the survival and success of their bacterial host, and they also have been shown to encode numerous antiphage defenses. Here, we describe the current knowledge of antiphage defenses encoded by prophages and prophage-related mobile genetic elements.

Anti-phage defence through inhibition of virion assembly.

Bacteria have evolved diverse antiviral defence mechanisms to protect themselves against phage infection. Phages integrated into bacterial chromosomes, known as prophages, also encode defences that protect the bacterial hosts in which they reside. Here, we identify a type of anti-phage defence that interferes with the virion assembly pathway of invading phages. The protein that mediates this defence, which we call Tab (for 'Tail assembly blocker'), is constitutively expressed from a Pseudomonas aeruginosa prophage. Tab allows the invading phage replication cycle to proceed, but blocks assembly of the phage tail, thus preventing formation of infectious virions. While the infected cell dies through the activity of the replicating phage lysis proteins, there is no release of infectious phage progeny, and the bacterial community is thereby protected from a phage epidemic. Prophages expressing Tab are not inhibited during their own lytic cycle because they express a counter-defence protein that interferes with Tab function. Thus, our work reveals an anti-phage defence that operates by blocking virion assembly, thereby both preventing formation of phage progeny and allowing destruction of the infected cell due to expression of phage lysis genes.

Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa.

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages.

Development and Validation of a Brief Measure of Sexual Wellbeing for Population Surveys: The Natsal Sexual Wellbeing Measure (Natsal-SW).

Sexual wellbeing is an important aspect of population health. Addressing and monitoring it as a distinct issue requires valid measures. Our previous conceptual work identified seven domains of sexual wellbeing: security; respect; self-esteem; resilience; forgiveness; self-determination; and comfort. Here, we describe the development and validation of a measure of sexual wellbeing reflecting these domains. Based on the analysis of 40 semi-structured interviews, we operationalized domains into items, and refined them via cognitive interviews, workshops, and expert review. We tested the items via two web-based surveys (n = 590; n = 814). Using data from the first survey, we carried out exploratory factor analysis to assess and eliminate poor performing items. Using data from the second survey, we carried out confirmatory factor analysis to examine model fit and associations between the item reduced measure and external variables hypothesized to correlate with sexual wellbeing (external validity). A sub-sample (n = 113) repeated the second survey after 2 weeks to evaluate test-retest reliability. Confirmatory factor analysis indicated that a "general specific model" had best fit (RMSEA: 0.064; CFI: 0.975, TLI: 0.962), and functioned equivalently across age group, gender, sexual orientation, and relationship status. The final Natsal-SW measure comprised 13 items (from an initial set of 25). It was associated with external variables in the directions hypothesized (all p < .001), including mental wellbeing (0.454), self-esteem (0.564), body image (0.232), depression (-0.384), anxiety (-0.340), sexual satisfaction (0.680) and sexual distress (-0.615), and demonstrated good test-retest reliability (ICC = 0.78). The measure enables sexual wellbeing to be quantified and understood within and across populations.

Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa.

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages.

Complete genomes and comparative analyses of Streptomyces phages that influence secondary metabolism and sporulation.

Bacteria in the genus Streptomyces are found ubiquitously in nature and are known for the number and diversity of specialized metabolites they produce, as well as their complex developmental lifecycle. Studies of the viruses that prey on Streptomyces, known as phages, have aided the development of tools for genetic manipulation of these bacteria, as well as contributing to a deeper understanding of Streptomyces and their behaviours in the environment. Here, we present the genomic and biological characterization of twelve Streptomyces phages. Genome analyses reveal that these phages are closely related genetically, while experimental approaches show that they have broad overlapping host ranges, infect early in the Streptomyces lifecycle, and induce secondary metabolite production and sporulation in some Streptomyces species. This work expands the group of characterized Streptomyces phages and improves our understanding of Streptomyces phage-host dynamics.

F-Type Pyocins Are Diverse Noncontractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa.

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3' end of the F-type pyocin cluster are divergent in sequence and likely determine bactericidal specificity. We use sequence similarities among these proteins to define eleven distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly reassorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins may have emerged earlier. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as antibacterial therapeutics. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes antibiotic-resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great need for the development of alternative therapeutics. In this study, we investigate one such potential therapeutic: F-type pyocins, which are bacteriocins naturally produced by P. aeruginosa that resemble noncontractile phage tails. We show that they are potent killers of P. aeruginosa and identify their probable bactericidal specificity determinants, which opens up the possibility of engineering them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well-characterized phage tails will greatly facilitate their development into effective antibacterials.

Prophages provide a rich source of antiphage defense systems.

Temperate phages are pervasive in nature, existing within bacterial cells in a form known as prophages. In this state, survival of the phage is intricately tied to the survival of the bacterial host. As a result, prophages often encode genes that increase bacterial fitness. One important way to increase survival is to provide defense against competing phages. Recent work reviewed here reveals that prophages provide a diverse and robust reservoir of antiphage defense systems that likely play a major role in bacterial-phage dynamics.

Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type II Anti-CRISPR Proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide bacteria and archaea with an adaptive immune response against invasion by mobile genetic elements like phages, plasmids, and transposons. These systems have been repurposed as very powerful biotechnological tools for gene editing applications in both bacterial and eukaryotic systems. The discovery of natural off-switches for CRISPR-Cas systems, known as anti-CRISPR proteins, provided a mechanism for controlling CRISPR-Cas activity and opened avenues for the development of more precise editing tools. In this review, we focus on the inhibitory mechanisms of anti-CRISPRs that are active against type II CRISPR-Cas systems and briefly discuss their biotechnological applications.

Anti-CRISPR Protein AcrIIC5 Inhibits CRISPR-Cas9 by Occupying the Target DNA Binding Pocket.

Anti-CRISPR proteins inhibit CRISPR-Cas immune systems through diverse mechanisms. Previously, the anti-CRISPR protein AcrIIC5Smu was shown to potently inhibit a type II-C Cas9 from Neisseria meningitidis (Nme1Cas9). In this work, we explore the mechanism of activity of the AcrIIC5 homologue from Neisseria chenwenguii (AcrIIC5Nch) and show that it prevents Cas9 binding to target DNA. We show that AcrIIC5Nch targets the PAM-interacting domain (PID) of Nme1Cas9 for inhibition, agreeing with previous findings for AcrIIC5Smu, and newly establish that strong binding of the anti-CRISPR requires guide RNA be pre-loaded on Cas9. We determined the crystal structure of AcrIIC5Nch using X-ray crystallography and identified amino acid residues that are critical for its function. Using a protein docking algorithm we show that AcrIIC5Nch likely occupies the Cas9 DNA binding pocket, thereby inhibiting target DNA binding through a mechanism similar to that previously described for AcrIIA2 and AcrIIA4.

Exploring the influence of local alcohol availability on drinking norms and practices: A qualitative scoping review.

INTRODUCTION: High alcohol availability is related to increased alcohol consumption and harms. Existing quantitative research provides potential explanations for this relationship but there is little understanding of how people experience local alcohol availability. This is the first review to synthesise qualitative research exploring the relationship between alcohol availability and other factors in local alcohol environments. METHODS: The scoping review includes qualitative studies exploring community-level alcohol availability and other factors, facilitating the purchase and consumption of alcohol. We included studies focusing on children and adolescents as well as adults. Study findings were brought together using thematic analysis and the socio-environmental context model, which explains how certain environments may facilitate drinking. RESULTS: The review includes 34 articles. The majority of studies were conducted since 2012. Most studies were conducted in the United Kingdom, Australia and South Africa. The physical availability of alcohol and proximity to local amenities and temporal aspects, like late night opening hours, may be linked to social factors, such as normalisation of drinking and permissive drinking environments. The review highlights the importance of social and cultural factors in shaping interactions with local alcohol environments. DISCUSSION AND CONCLUSION: This qualitative scoping review advances understanding of the pathways linking alcohol availability and alcohol harms by showing that availability, accessibility and visibility of alcohol may contribute towards permissive drinking environments. Further research is needed to better understand how people experience alcohol availability in their local environment and how this can inform alcohol control policies.

Access to and quality of sexual and reproductive health services in Britain during the early stages of the COVID-19 pandemic: a qualitative interview study of patient experiences.

INTRODUCTION: Access to quality sexual and reproductive health (SRH) services remains imperative even during a pandemic. Our objective was to understand experiences of delayed or unsuccessful access to SRH services in Britain during the early stages of the COVID-19 pandemic. METHODS: In October and November 2020 we conducted semi-structured telephone interviews with 14 women and six men reporting an unmet need for SRH services in the Natsal-COVID survey, a large-scale quasi-representative web-panel survey of sexual health and behaviour during COVID-19 (n=6654). We purposively sampled eligible participants using sociodemographic quotas. Inductive thematic analysis was used to explore service access and quality and to identify lessons for future SRH service delivery. RESULTS: Twenty participants discussed experiences spanning 10 SRH services including contraception and antenatal/maternity care. Participants reported hesitancy and self-censorship of need. Accessing telemedicine and 'socially-distanced' services required tenacity. Challenges included navigating changing information and procedures; perceptions of gatekeepers as obstructing access; and inflexible appointment systems. Concerns about reconfigured services included reduced privacy; decreased quality of interactions with professionals; reduced informal support; and fewer preventive SRH practices. However, some participants also described more streamlined services and staff efforts to compensate for disruptions. Many viewed positively the ongoing blending of telemedicine with in-person care. CONCLUSION: The COVID-19 pandemic impacted access and quality of SRH services. Participants' accounts revealed self-censorship of need, difficulty navigating shifting service configurations and perceived quality reductions. Telemedicine offers potential if intelligently combined with in-person care. We offer initial evidence-based recommendations for promoting an equitable restoration and future adaption of services.

Sexting among British adults: a qualitative analysis of sexting as emotion work governed by 'feeling rules'.

Sexting has generated considerable public and professional interest with concerns centring on young people, and potential harms to mental and sexual health. Little research thus far has explored the practice among adults and none has focused on the cultural norms relating to the emotional experience of sexting across different ages and genders. We conducted 40 semi-structured interviews with a diverse sample of adults aged 18-59 years in Britain on the role of digital technologies in participants' sexual lives. In this paper, we draw on the accounts of 34 people with experience of sexting. We identified three main themes in participants' accounts related to the emotional aspects of sexting: (1) trust, (2) desire/intimacy and (3) shame. Under each theme, we identified motivations, 'feeling rules', and examples of 'emotion work' relating to the self, the other and the dyad. We conclude that there are shared cultural norms that constitute what appropriate sexting should feel like. Interventions aiming to minimise harms arising from sexting need to build on commonly held cultural conventions regarding the 'rules of the game' concerning feelings as well as behaviours.

Balancing risk, intimacy and (non)compliance: a qualitative study of sex across household during COVID-19 social restrictions.

Government controls over intimate relationships, imposed to limit the spread of Sars-CoV-2, were unprecedented in modern times. This study draws on data from qualitative interviews with 18 participants in Natsal-COVID, a quasi-representative web-panel survey of the British population (n = 6,654 people), reporting that they had sex with someone from outside their household in the preceding four weeks; a period in which contact between households was restricted in the UK. Whilst only 10% of people reported sexual contact outside their household, among single people and those in non-cohabiting relationships, rates were much higher (Natsal-COVID). Our findings show that individuals did not take decisions to meet up with sexual partners lightly. Participants were motivated by needs-for connection, security, intimacy and a sense of normality. People balanced risks-of catching COVID-19, social judgement and punishment for rule-breaking-against other perceived risks, including to their mental health or relationships. We used situated rationality and social action theories of risk to demonstrate that people weighed up risk in socially situated ways and exhibited complex decision-making when deciding not to comply with restrictions. Understanding motivations for non-compliance is crucial to informing future public health messaging which accounts for the needs and circumstances of all population members.

Gold Nanoparticle Smartphone Platform for Diagnosing Urinary Tract Infections.

Current urinary tract infection (UTI) diagnostic methods are slow or provide limited information, resulting in prescribing antibiotic therapy before bacterial pathogen identification. Here, we adapted a gold nanoparticle colorimetric approach and developed a smartphone platform for UTI detection. We show the parallel identification of five major UTI pathogens at clinically relevant concentrations of 105 bacteria/mL using bacteria-specific and universal probes. We validated the diagnostic technology using 115 positive and 19 negative samples from patients with Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae infections. The assay successfully identified the infecting pathogen (specificity: >98% and sensitivity: 51-73%) in 3 h. Our platform is faster than culturing and can wirelessly store and transmit results at the cost of $0.38 per assay.

The Case for Targeted Parenting Interventions with Reference to Intergenerational Transmission of Parenting: Qualitative Evidence from Three Studies of Marginalised Mothers' and Fathers' Participation in Parenting Programmes.

The idea that how you were parented is key to how you parent your own children is widely recognisable. It is present in popular cultural references, underpins much policy on families and parenting in the UK, and is supported by a substantive body of academic literature. We explore this concept of intergenerational transmission of parenting, understanding it as the context in which parenting interventions have been implemented. We draw on interview data from three Scottish samples of marginalised parents (n = 54) to explore how participants think their own parenting behaviours have been shaped by their experience of being parented and how they talk about participation in a parenting intervention in relation to this. We find that how these parents have been parented is salient in considering their own parenting behaviour, and is a key context for their engagement with the intervention. We make the case for parenting interventions targeted at marginalised parents, arguing that they are acceptable to, and useful for, these parents and may, potentially, be effective in breaking cycles of negative parenting. Policy-makers should not shy away from implementing targeted parenting programmes as part of endeavours to address negative parenting.

Intimate physical contact between people from different households during the COVID-19 pandemic: a mixed-methods study from a large, quasi-representative survey (Natsal-COVID).

OBJECTIVES: Physical distancing as a non-pharmaceutical intervention aims to reduce interactions between people to prevent SARS-CoV-2 transmission. Intimate physical contact outside the household (IPCOH) may expand transmission networks by connecting households. We aimed to explore whether intimacy needs impacted adherence to physical distancing following lockdown in Britain in March 2020. METHODS: The Natsal-COVID web-panel survey (July-August 2020) used quota-sampling and weighting to achieve a quasi-representative population sample. We estimate reporting of IPCOH with a romantic/sexual partner in the 4 weeks prior to interview, describe the type of contact, identify demographic and behavioural factors associated with IPCOH and present age-adjusted ORs (aORs). Qualitative interviews (n=18) were conducted to understand the context, reasons and decision making around IPCOH. RESULTS: Of 6654 participants aged 18-59 years, 9.9% (95% CI 9.1% to 10.6%) reported IPCOH. IPCOH was highest in those aged 18-24 (17.7%), identifying as gay or lesbian (19.5%), and in steady non-cohabiting relationships (56.3%). IPCOH was associated with reporting risk behaviours (eg, condomless sex, higher alcohol consumption). IPCOH was less likely among those reporting bad/very bad health (aOR 0.54; 95% CI 0.32 to 0.93) but more likely among those with COVID-19 symptoms and/or diagnosis (aOR 1.34; 95% CI 1.10 to 1.65). Two-thirds (64.4%) of IPCOH was reported as being within a support bubble. Qualitative interviews found that people reporting IPCOH deliberated over, and made efforts to mitigate, the risks. CONCLUSIONS: Given 90% of people did not report IPCOH, this contact may not be a large additional contributor to SARS-CoV-2 transmission, although heterogeneity exists within the population. Public health messages need to recognise how single people and partners living apart balance sexual intimacy and relationship needs with adherence to control measures.

A Filamentous Bacteriophage Protein Inhibits Type IV Pili To Prevent Superinfection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in a variety of settings. Many P. aeruginosa isolates are infected by filamentous Pf bacteriophage integrated into the bacterial chromosome as a prophage. Pf virions can be produced without lysing P. aeruginosa. However, cell lysis can occur during superinfection, which occurs when Pf virions successfully infect a host lysogenized by a Pf prophage. Temperate phages typically encode superinfection exclusion mechanisms to prevent host lysis by virions of the same or similar species. In this study, we sought to elucidate the superinfection exclusion mechanism of Pf phage. Initially, we observed that P. aeruginosa that survive Pf superinfection are transiently resistant to Pf-induced plaquing and are deficient in twitching motility, which is mediated by type IV pili (T4P). Pf utilize T4P as a cell surface receptor, suggesting that T4P are suppressed in bacteria that survive superinfection. We tested the hypothesis that a Pf-encoded protein suppresses T4P to mediate superinfection exclusion by expressing Pf proteins in P. aeruginosa and measuring plaquing and twitching motility. We found that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), promoted resistance to Pf infection and suppressed twitching motility by binding the T4P protein PilC. Because T4P play key roles in biofilm formation and virulence, the ability of Pf phage to modulate T4P via PfsE has implications in the ability of P. aeruginosa to persist at sites of infection. IMPORTANCE Pf bacteriophage (phage) are filamentous viruses that infect Pseudomonas aeruginosa and enhance its virulence potential. Pf virions can lyse and kill P. aeruginosa through superinfection, which occurs when an already infected cell is infected by the same or similar phage. Here, we show that a small, highly conserved Pf phage protein (PA0721, PfsE) provides resistance to superinfection by phages that use the type IV pilus as a cell surface receptor. PfsE does this by inhibiting assembly of the type IV pilus via an interaction with PilC. As the type IV pilus plays important roles in virulence, the ability of Pf phage to modulate its assembly has implications for P. aeruginosa pathogenesis.