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Kals, M.

Publications and source records attributed to Kals, M..

10 recordsLinked to original sources

Ionic environment and nutrients affect the cysB-dependent conditional susceptibility of E. coli to mecillinam

Antibiotic efficacy is influenced by environmental context. Urinary E. coli isolates lacking a functional cysB gene have been shown to be resistant to the {beta}-lactam antibiotic mecillinam under high-osmolarity conditions, suggesting that increased fluid intake might synergize with drug treatment. In this study, we further explore E. coli susceptibility to mecillinam across growth conditions. We show that while osmolarity has a mild protective effect on cysB knockout and reference strain, it is the presence of certain salts that leads to the resistant phenotype in the mutant. Such conditional resistance is specific to mecillinam and rapidly wanes when salts are removed. In agreement with our previous work showing that slow growing cells escape mecillinam-induced bursting by maintaining a small size, for both strains we observe low susceptibility to mecillinam in scarcely nutritious pooled human urine. As we supplement urine with nutrients, we observe a sharp transition to susceptibility when growth rate surpasses 0.6 h-1. Growth in the presence of salt or uncharged osmolites decreases cell size of both reference strain and cysB knockout offering a potential explanation to the observed mild protective effects of osmolarity. Our findings give insights into the mechanism of cysB dependent susceptibility to mecillinam and, due to the dual impact of nutrients and osmolites on treatment, allow us to reformulate ideas on potential diet recommendations. While increased fluid intake will decrease urine osmolarity, it will also reduce nutrient content, limiting mecillinam efficacy. We suggest instead that reducing sodium intake during mecillinam treatment might avoid this trade-off.

microbiology↗

Microfluidic Platform for Automatic Quantification of Malaria Parasite Invasion Under Physiological Flow Conditions

Understanding the impact of forces generated by blood flow on biological processes in the circulatory system, such as the invasion of human red blood cells by malaria parasites, is currently limited by the lack of experimental systems that integrate them. Recent systematic quantification of the growth of Plasmodium falciparum, the species that causes the majority of malaria mortality, under a range of shaking conditions has shown that parasite invasion of erythrocytes is affected by the shear stress to which the interacting P. falciparum merozoites and their target red blood cells are exposed. Blood flow could similarly impact shear stress and therefore invasion in vivo, but there is currently no method to test the impact of flow-induced forces on parasite invasion. We have developed a microfluidic device with four channels, each with dimensions similar to those of a post-capillary venule, but with different flow velocities. Highly synchronised P. falciparum parasites are injected into the device, and parasite egress and invasion rates are quantified using newly developed custom video analysis, which fully automates cell type identification and trajectory tracking. The device was tested with both wild-type P. falciparum lines and lines in which genes encoding proteins involved in parasite invasion had been deleted. Deletion of Erythrocyte Binding Antigen 175 (PfEBA175) has a significant impact on invasion under flow, but not in static culture. These findings establish for the first time that flow conditions can critically affect parasite invasion in a genotype-dependent manner. The method can be applied to other biological processes affected by fluid motion, such as cell adhesion, migration, and mechanotransduction.

microbiology↗

Growing Malaria Parasites at a Critical Shaking Speed Mimicking Physiological Flow Reveals New Phenotypes for Invasion Ligands

Malaria kills over 600,000 people annually, with all the clinical symptoms being caused by the blood stage of the infection. Malaria parasites invade red blood cells (RBCs), where they grow and multiply until daughter parasites egress to invade new RBCs. This cycle happens primarily in the blood circulation, bone marrow, and spleen, where parasites and RBCs are exposed to flow-generated forces. Despite this, almost all in vitro growth assays are carried out in static conditions, which are a poor mimic for the conditions that malaria parasites encounter in the body. Therefore, we systematically tested the impact of dynamic conditions created by orbital shaking platforms on parasite growth and explored the link between growth and the wall shear stress forces generated by fluid motion. For several strains of the deadliest malaria species, Plasmodium falciparum, we showed that strikingly, for any given vessel, there is a critical shaking speed at which growth rates are reduced, corresponding to when the RBCs start to aggregate in the centre of the well, before growth increases again at higher shaking speeds. The force that parasites are exposed to at this critical shaking speed corresponds cloesly with previous measurements for the forces that RBCs and parasites are exposed to in the microvasculature. During invasion, the early attachment of the parasites to RBCs is dependent on two families of attachment proteins, the Erythrocyte Binding Antigen (PfEBA) family and the Reticulocyte Binding Protein Homologue (PfRH) family, which are thought to be largely redundant in function. We used a panel of PfEBA and PfRH knock-out lines to show for the first time that several of these ligands have greater importance in high wall shear stress conditions. This both adds new understanding to the function of these ligand families, and indicates that the concept of the critical shaking speed can reveal new parasite growth phenotypes.

microbiology↗

Antibiotics change the growth rate heterogeneity and morphology of bacteria

A better understanding of the system-level effects of antibiotics is necessary to fight the rise of antibiotic resistance. Utilising the Multipad Agarose Plate (MAP), we monitor the growth rate and cell morphology of three clinically relevant species (E.coli, S.aureus and P.aeruginosa) after exposure to 11 different concentrations of 13 antibiotics (for a total of 24 microbe-antibiotic combinations). As the drug dose approaches the MIC and regardless of the mode of action, our results show a consistent increase in growth rate heterogeneity. Remarkably, drugs that affect protein synthesis consistently show the opposite trend, reducing heterogeneity. We hypothesize that growth rate heterogeneity under antibiotic treatment might therefore depend on the functional distance of the target from ribosomal activity, which is key in determining growth rate. Low heterogeneity is desirable from a clinical perspective, as the opposite is often associated to persistence and antibiotic survival. For all of the antibiotics and species tested, we also find a striking and non-trivial correlation between morphological alterations and growth inhibition. This observation allows us to introduce a new morphological parameter, MOR50, that enables the estimation of minimum inhibitory concentration (MIC) for antibiotic susceptibility testing (AST) with a single snapshot after 2.5 hours of incubation. In addition to introducing a novel, resource-efficient, rapid AST method, our findings shed new light on the effects of antibiotic perturbations on bacteria at the system level that might inform treatment design.

microbiology↗

Optical tweezers reveal that PfEBA and PfRH ligands, not PfMSP1, play a central role in Plasmodium-falciparum merozoite-erythrocyte attachment

Malaria pathogenesis and parasite multiplication both depend on the ability of Plasmodium falciparum merozoites to invade human erythrocytes. Invasion is a complex multi-step process that is known to involve multiple P. falciparum proteins but dissecting the precise role of individual proteins has to date been limited by the availability of quantifiable phenotypic assays. In this study, we apply a new approach to assigning function to invasion proteins by using optical tweezers to directly manipulate recently egressed merozoites and erythrocytes and quantify the strength of attachment between them, as well as the frequency with which such attachments occur. Using a range of inhibitors, antibodies, and genetically modified P. falciparum strains, we quantitated the contribution of individual P. falciparum proteins to these merozoite-erythrocyte attachment phenotypes for the first time. Most of the interactions investigated did not affect the force needed to pull merozoites and erythrocytes apart, including loss of the major P. falciparum merozoite surface protein PfMSP1 and PfGAP45, part of the glideosome actinomyosin motor complex. The only factors that significantly reduced the strength of merozoite-erythrocyte attachment were ones that disrupted the function of members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families. While these assays also reinforced the known redundancy within these families, with the deletion of some ligands not impacting detachment force, it appears that the PfEBA/PfRH families play a central role in merozoite attachment, not the major merozoite surface protein PfMSP1. Author summaryMalaria is a devastating disease caused by a parasitic infection. The deadliest species is Plasmodium falciparum, which causes more than 600,000 deaths annually. The parasites life cycle is complex, but all the symptoms of malaria are caused when the parasites replicate in human red blood cells. Replication depends on the invasion of the red blood cells by the parasites which is a complex process involving multiple molecular interactions and multiple steps. Invasion begins with the attachment of the parasite to the red blood cell, making this step of particular interest in the development of new therapeutics. We assessed which interactions are key to the strength of attachment using an optical tweezer assay, which allowed us to directly measure the binding force between individual parasites and red blood cells whilst using a range of molecular and genetic tools that target specific interactions known to have a role in invasion. This showed that loss of a protein commonly thought to be critical to the early stages of binding (PfMSP1) had no effect on attachment strength, whereas disruptions of several members from two families of proteins (the Erythrocyte Binding Like protein family and the reticulocyte binding-like protein family) affect attachment strength.

molecular biology↗

Multipad Agarose Plate (MAP): A Rapid and High-Throughput Approach for Antibiotic Susceptibility Testing

We describe a phenotypic antibiotic susceptibility testing (AST) method that can provide an eightfold speedup in turnaround time compared to the current clinical standard by leveraging advances in microscopy and single-cell imaging. A newly developed growth plate containing 96 agarose pads, termed the Multipad Agarose Plate (MAP), can be assembled at low costs. Pads can be prepared with dilution series of antibiotics. Bacteria are seeded on the pads and automatically imaged using brightfield microscopy, with a fully automated segmentation pipeline quantifying microcolony formation and growth rate. Using a test set of nine antibiotics with very different targets, we demonstrate that accurate minimum inhibitory concentration (MIC) measurements can be performed based on the growth rate of microcolonies within three hours of incubation with the antibiotic. Faster, reliable and high throughput methods for AST, such as MAP, could improve patient care by expediting treatment initiation and alleviating the burden of antimicrobial resistance.

microbiology↗

Genetic determinants of plasma protein levels in the Estonian population

The proteome holds great potential as an intermediate layer between the genome and phenome. Previous protein quantitative trait locus studies have focused mainly on describing the effects of common genetic variations on the proteome. Here, we assessed the impact of the common and rare genetic variations as well as the copy number variants (CNVs) on 326 plasma proteins measured in up to 500 individuals. We identified 184 cis and 94 trans signals for 157 protein traits, which were further fine-mapped to credible sets for 101 cis and 87 trans signals for 151 proteins. Rare genetic variation contributed to the levels of 7 proteins, with 5 cis and 14 trans associations. CNVs were associated with the levels of 11 proteins (7 cis and 5 trans), examples including a 3q12.1 deletion acting as a hub for multiple trans associations; and a CNV overlapping NAIP, a sensor component of the NAIP-NLRC4 inflammasome which is affecting pro-inflammatory cytokine interleukin 18 levels. In summary, this work presents a comprehensive resource of genetic variation affecting the plasma protein levels and provides the interpretation of identified effects.

genomics↗

Omics-informed CNV calls reduce false positive rate and improve power for CNV-trait associations

Copy number variations (CNV) are believed to play an important role in a wide range of complex traits but discovering such associations remains challenging. Whilst whole genome sequencing (WGS) is the gold standard approach for CNV detection, there are several orders of magnitude more samples with available genotyping microarray data. Such array data can be exploited for CNV detection using dedicated software (e.g., PennCNV), however these calls suffer from elevated false positive and negative rates. In this study, we developed a CNV quality score that weights PennCNV calls (pCNV) based on their likelihood of being true positive. First, we established a measure of pCNV reliability by leveraging evidence from multiple omics data (WGS, transcriptomics and methylomics) obtained from the same samples. Next, we built a predictor of omics-confirmed pCNVs, termed omics-informed quality score (OQS), using only PennCNV software output parameters. Promisingly, OQS assigned to pCNVs detected in close family members was up to 35% higher than the OQS of pCNVs not carried by other relatives (P < 3.0-10-90), outperforming other scores. Finally, in an association study of four anthropometric traits in 89,516 Estonian Biobank samples, the use of OQS led to a relative increase in the trait variance explained by CNVs of up to 34% compared to raw pCNVs or previous quality scores. Overall, we put forward a flexible framework to improve any CNV detection method leveraging multi-omics evidence, applied it to improve PennCNV calls and demonstrated its utility by improving the statistical power for downstream association analyses.

bioinformatics↗

Advantages of genotype imputation with ethnically matched reference panel for rare variant association analyses

Genotype imputation has become a standard procedure prior genome-wide association studies (GWASs). For common and low-frequency variants, genotype imputation can be performed sufficiently accurately with publicly available and ethnically heterogeneous reference datasets like 1000 Genomes Project (1000G) and Haplotype Reference Consortium panels. However, the imputation of rare variants has been shown to be significantly more accurate when ethnically matched reference panel is used. Even more, greater genetic similarity between reference panel and target samples facilitates the detection of rare (or even population-specific) causal variants. Notwithstanding, the genome-wide downstream consequences and differences of using ethnically mixed and matched reference panels have not been yet comprehensively explored.\n\nWe determined and quantified these differences by performing several comparative evaluations of the discovery-driven analysis scenarios. A variant-wise GWAS was performed on seven complex diseases and body mass index by using genome-wide genotype data of [~]37,000 Estonians imputed with ethnically mixed 1000G and ethnically matched imputation reference panels. Although several previously reported common (minor allele frequency; MAF > 5%) variant associations were replicated in both resulting imputed datasets, no major differences were observed among the genome-wide significant findings or in the fine-mapping effort. In the analysis of rare (MAF < 1%) coding variants, 46 significantly associated genes were identified in the ethnically matched imputed data as compared to four genes in the 1000G panel based imputed data. All resulting genes were consequently studied in the UK Biobank data.\n\nThese associations provide a solid example of how rare variants can be efficiently analysed to discover novel, potentially functional genetic variants in relevant phenotypes. Furthermore, our work serves as proof of a cost-efficient study design, demonstrating that the usage of ethnically matched imputation reference panels can enable substantially improved imputation of rare variants, facilitating novel high-confidence findings in rare variant GWAS scans.\n\nAuthor summaryOver the last decade, genome-wide association studies (GWASs) have been widely used for detecting genetic biomarkers in a wide range of traits. Typically, GWASs are carried out using chip-based genotyping data, which are then combined with a more densely genotyped reference panel to infer untyped genetic variants in chip-typed individuals. The latter method is called genotype imputation and its accuracy depends on multiple factors. Publicly available and ethnically heterogeneous imputation reference panels (IRPs) such as 1000 Genomes Project (1000G) are sufficiently accurate for imputation of common and low-frequency variants, but custom ethnically matched IRPs outperform these in case of rare variants. In this work, we systematically compare downstream association analysis effects on eight complex traits in [~]37,000 Estonians imputed with ethnically mixed and ethnically matched IRPs. We do not observe major differences in the single variant analysis, where both imputed datasets replicate previously reported significant loci. But in the gene-based analysis of rare protein-coding variants we show that ethnically matched panel clearly outperforms 1000G panel based imputation, providing 10-fold increase in significant gene-trait associations. Our study demonstrates empirically that imputed data based on ethnically matched panel is very promising for rare variant analysis - it captures more population-specific variants and makes it possible to efficiently identify novel findings.

genomics↗

Translating genotype data of 44,000 biobank participants into clinical pharmacogenetic recommendations: challenges and solutions

PurposeBiomedical databases combining electronic medical records, phenotypic and genomic data constitute a powerful resource for the personalization of treatment. To leverage the wealth of information provided, algorithms are required that systematically translate the contained information into treatment recommendations based on existing genotype-phenotype associations.\n\nMethodsWe developed and tested algorithms for translation of pre-existing genotype data of over 44,000 participants of the Estonian biobank into pharmacogenetic recommendations. We compared the results obtained by whole genome sequencing, whole exome sequencing and genotyping using microarrays, and evaluated the impact of pharmacogenetic reporting based on drug prescription statistics in the Nordic countries and Estonia.\n\nResultsOur most striking result was that the performance of genotyping arrays is similar to that of whole genome sequencing, whereas exome sequencing is not suitable for pharmacogenetic predictions. Interestingly, 99.8% of all assessed individuals had a genotype associated with increased risks to at least one medication, and thereby the implementation of pharmacogenetic recommendations based on genotyping affects at least 50 daily drug doses per 1000 inhabitants.\n\nConclusionWe find that microarrays are a cost-effective solution for creating pre-emptive pharmacogenetic reports, and with slight modifications, existing databases can be applied for automated pharmacogenetic decision support for clinicians.

pharmacology and toxicology↗