CRISPR immune diversity in simulated and natural microbial populations

Coevolution between microbes and their viruses influences the trajectories of these communities through gene transfer and predation. When these communities are a part of the human microbiome, these interactions can also have significant impacts on the health of the human host. The CRISPR adaptive immune system is one of the ways in which microbes defend against viral infection, and it also holds a record of acquired immunity, allowing us to read a history of microbe-viral interactions. In this work, we examine the emergence, impact, and applications of diverse CRISPR immune alleles in microbial populations. Using a mathematical model of CRISPR-mediated host-virus coevolution to simulate microbial populations, we observe the emergence of multiple coexisting CRISPR alleles in a single population, which we call distributed immunity. We find that distributed immunity is most likely to occur in communities with more potential spacers and relatively low viral mutation rates, and that it is linked to increased stability for the host population, while the viral population is driven to lower densities or even to extinction. To see if this phenomenon is also present in natural microbial populations, we examined CRISPR diversity in two human-associated communities: the vaginal microbiomes of pregnant women and the lung microbiomes of cystic fibrosis patients. To investigate the vaginal microbiome, we developed a network-based methodology to identify and extract CRISPR spacers from all species present in samples taken from pregnant women at high and low risk of preterm birth. This approach yielded over 20 different CRISPR types, with spacer content varying among individuals. Coexisting alleles linked to shifts in the abundance of the matched element were detected in one Lactobacillus species in one of the samples, demonstrating the potential of our approach. In the cystic fibrosis lung microbiome, we used this method to identify CRISPRs in four patients infected with the major cystic fibrosis pathogen Pseudomonas aeruginosa. Spacer content was completely different between patients, but no variation was detected within a patient. Finally, we examined spacer diversity in a large global dataset of P. aeruginosa and used the thousands of spacers identified as a tracking tool to monitor dynamics of viral populations. This approach, which we refer to as prototyping, revealed a panmictic P. aeruginosa phage population and holds promise as a tool for tracking mobile elements and personalizing phage therapy treatments

Evolutionary causes and consequences of diversified CRISPR immune profiles in natural populations

Abstract Host-pathogen co-evolution is a significant force which shapes the ecology and evolution of all types of organisms, and such interactions are driven by resistance and immunity mechanisms of the host. Diversity of resistance and immunity can affect the co-evolutionary trajectory of both host and pathogen. The microbial CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is one host immunity mechanism which offers a tractable model for examining the dynamics of diversity in an immune system. In the present article, we review CRISPR variation observed in a variety of natural populations, examine the forces which can push CRISPRs towards high or low diversity, and investigate the consequences of various levels of diversity on microbial populations

Emergence and Spread of the SARS-CoV-2 Omicron Variant in Alberta Communities Revealed by Wastewater Monitoring

Wastewater monitoring of SARS-CoV-2 allows for early detection and monitoring of COVID-19 burden in communities and can track specific variants of concern. Targeted assays enabled relative proportions of SARS-CoV-2 Omicron and Delta variants to be determined across 30 municipalities covering >75% of the province of Alberta (pop. 4.5M) in Canada, from November 2021 to January 2022. Larger cities like Calgary and Edmonton exhibited a more rapid emergence of Omicron relative to smaller and more remote municipalities. Notable exceptions were Banff, a small international resort town, and Fort McMurray, a more remote northern city with a large fly-in worker population. The integrated wastewater signal revealed that the Omicron variant represented close to 100% of SARS-CoV-2 burden prior to the observed increase in newly diagnosed clinical cases throughout Alberta, which peaked two weeks later. These findings demonstrate that wastewater monitoring offers early and reliable population-level results for establishing the extent and spread of emerging pathogens including SARS-CoV-2 variants.Alberta Healt

Evaluation of Excess Significance Bias in Animal Studies of Neurological Diseases

Animal studies generate valuable hypotheses that lead to the conduct of preventive or therapeutic clinical trials. We assessed whether there is evidence for excess statistical significance in results of animal studies on neurological disorders, suggesting biases. We used data from meta-analyses of interventions deposited in Collaborative Approach to Meta-Analysis and Review of Animal Data in Experimental Studies (CAMARADES). The number of observed studies with statistically significant results (O) was compared with the expected number (E), based on the statistical power of each study under different assumptions for the plausible effect size. We assessed 4,445 datasets synthesized in 160 meta-analyses on Alzheimer disease (n = 2), experimental autoimmune encephalomyelitis (n = 34), focal ischemia (n = 16), intracerebral hemorrhage (n = 61), Parkinson disease (n = 45), and spinal cord injury (n = 2). 112 meta-analyses (70%) found nominally (p≤0.05) statistically significant summary fixed effects. Assuming the effect size in the most precise study to be a plausible effect, 919 out of 4,445 nominally significant results were expected versus 1,719 observed (p<10-9). Excess significance was present across all neurological disorders, in all subgroups defined by methodological characteristics, and also according to alternative plausible effects. Asymmetry tests also showed evidence of small-study effects in 74 (46%) meta-analyses. Significantly effective interventions with more than 500 animals, and no hints of bias were seen in eight (5%) meta-analyses. Overall, there are too many animal studies with statistically significant results in the literature of neurological disorders. This observation suggests strong biases, with selective analysis and outcome reporting biases being plausible explanations, and provides novel evidence on how these biases might influence the whole research domain of neurological animal literature. © 2013 Tsilidis et al

Metapopulation Structure of CRISPR-Cas Immunity in Pseudomonas aeruginosa and Its Viruses

ABSTRACT Viruses that infect the widespread opportunistic pathogen Pseudomonas aeruginosa have been shown to influence physiology and critical clinical outcomes in cystic fibrosis (CF) patients. To understand how CRISPR-Cas immune interactions may contribute to the distribution and coevolution of P. aeruginosa and its viruses, we reconstructed CRISPR arrays from a highly sampled longitudinal data set from CF patients attending the Copenhagen Cystic Fibrosis Clinic in Copenhagen, Denmark (R. L. Marvig, L. M. Sommer, S. Molin, and H. K. Johansen, Nat Genet 47:57–64, 2015, https://doi.org/10.1038/ng.3148). We show that new spacers are not added to or deleted from CRISPR arrays over time within a single patient but do vary among patients in this data set. We compared assembled CRISPR arrays from this data set to CRISPR arrays extracted from 726 additional publicly available P. aeruginosa sequences to show that local diversity in this population encompasses global diversity and that there is no evidence for population structure associated with location or environment sampled. We compare over 3,000 spacers from our global data set to 98 lytic and temperate viruses and proviruses and find a subset of related temperate virus clusters frequently targeted by CRISPR spacers. Highly targeted viruses are matched by different spacers in different arrays, resulting in a pattern of distributed immunity within the global population. Understanding the multiple immune contexts that P. aeruginosa viruses face can be applied to study of P. aeruginosa gene transfer, the spread of epidemic strains in cystic fibrosis patients, and viral control of P. aeruginosa infection. IMPORTANCE Pseudomonas aeruginosa is a widespread opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis patients. Microbe-virus interactions play a critical role in shaping microbial populations, as viral infections can kill microbial populations or contribute to gene flow among microbes. Investigating how P. aeruginosa uses its CRISPR immune system to evade viral infection aids our understanding of how this organism spreads and evolves alongside its viruses in humans and the environment. Here, we identify patterns of CRISPR targeting and immunity that indicate P. aeruginosa and its viruses evolve in both a broad global population and in isolated human “islands.” These data set the stage for exploring metapopulation dynamics occurring within and between isolated “island” populations associated with CF patients, an essential step to inform future work predicting the specificity and efficacy of virus therapy and the spread of invasive viral elements and pathogenic epidemic bacterial strains

Chemical Approaches To Analyzing RNA Structure Transcriptome‐Wide

RNA molecules can fold into complex two- and three-dimensional shapes that are critical for their function. Chemical probes have long been utilized to interrogate RNA structure and are now considered invaluable resources in the goal of relating structure to function. Recently, the power of deep sequencing and careful chemical probe design have merged, permitting researchers to obtain a holistic understanding of how RNA structure can be utilized to control RNA biology transcriptome-wide. Within this review, we outline the recent advancements in chemical probe design for interrogating RNA structures inside cells and discuss the recent advances in our understanding of RNA biology through the lens of chemical probing

Fermentation products in the cystic fibrosis airways induce aggregation and dormancy-associated expression profiles in a CF clinical isolate of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a well-known dominant opportunistic pathogen in cystic fibrosis (CF) with a wide range of metabolic capacities. However, P. aeruginosa does not colonize the airways alone, and benefits from the metabolic products of neighboring cells-especially volatile molecules that can travel between different parts of the airways easily. Here, we present a study that investigates the metabolic, gene expression profiles and phenotypic responses of a P. aeruginosa clinical isolate to fermentation products lactic acid and 2,3-butanediol, metabolites that are produced by facultative anaerobic members of the CF polymicrobial community and potential biomarkers of disease progression. Although previous studies have successfully investigated the metabolic and transcriptional profiles of P. aeruginosa, most have used common lab reference strains that may differ in important ways from clinical isolates. Using transcriptomics and metabolomics with gas chromatography time of flight mass spectrometry, we observe that fermentation products induce pyocyanin production along with the expression of genes involved in P. aeruginosa amino acid utilization, dormancy and aggregative or biofilm modes of growth. These findings have important implications for how interactions within the diverse CF microbial community influence microbial physiology, with potential clinical consequences