The pathobiology of human fungal infections.

Human fungal infections are a historically neglected area of disease research, yet they cause more than 1.5 million deaths every year. Our understanding of the pathophysiology of these infections has increased considerably over the past decade, through major insights into both the host and pathogen factors that contribute to the phenotype and severity of these diseases. Recent studies are revealing multiple mechanisms by which fungi modify and manipulate the host, escape immune surveillance and generate complex comorbidities. Although the emergence of fungal strains that are less susceptible to antifungal drugs or that rapidly evolve drug resistance is posing new threats, greater understanding of immune mechanisms and host susceptibility factors is beginning to offer novel immunotherapeutic options for the future. In this Review, we provide a broad and comprehensive overview of the pathobiology of human fungal infections, focusing specifically on pathogens that can cause invasive life-threatening infections, highlighting recent discoveries from the pathogen, host and clinical perspectives. We conclude by discussing key future challenges including antifungal drug resistance, the emergence of new pathogens and new developments in modern medicine that are promoting susceptibility to infection.

Arginine catabolism is essential to polymyxin dependence in Acinetobacter baumannii.

Polymyxins are often the only effective antibiotics against the "Critical" pathogen Acinetobacter baumannii. Worryingly, highly polymyxin-resistant A. baumannii displaying dependence on polymyxins has emerged in the clinic, leading to diagnosis and treatment failures. Here, we report that arginine metabolism is essential for polymyxin-dependent A. baumannii. Specifically, the arginine degradation pathway was significantly altered in polymyxin-dependent strains compared to wild-type strains, with critical metabolites (e.g., L-arginine and L-glutamate) severely depleted and expression of the astABCDE operon significantly increased. Supplementation of arginine increased bacterial metabolic activity and suppressed polymyxin dependence. Deletion of astA, the first gene in the arginine degradation pathway, decreased phosphatidylglycerol and increased phosphatidylethanolamine levels in the outer membrane, thereby reducing the interaction with polymyxins. This study elucidates the molecular mechanism by which arginine metabolism impacts polymyxin dependence in A. baumannii, underscoring its critical role in improving diagnosis and treatment of life-threatening infections caused by "undetectable" polymyxin-dependent A. baumannii.

Developing simple indicators of nitrogen and phosphorus removal in constructed stormwater wetlands.

Quantifying pollutant removal by stormwater wetlands requires intensive sampling which is cost-prohibitive for authorities responsible for a large number of wetlands. Wetland managers require simple indicators that provide a practical means of estimating performance and prioritising maintenance works across their asset base. We therefore aimed to develop vegetation cover and metrics derived from monitoring water level, as simple indicators of likely nutrient pollutant removal from stormwater wetlands. Over a two-year period, we measured vegetation cover and water levels at 17 wetlands and used both to predict nitrogen (N) and phosphorus (P) removal. Vegetation cover explained 48 % of variation in total nitrogen (TN) removal; with a linear relationship suggesting an approximate 9 % loss in TN removal per 10 % decrease in vegetation cover. Vegetation cover is therefore a useful indicator of TN removal. Further development of remotely-sensed data on vegetation configuration, species and condition will likely improve the accuracy of TN removal estimates. Total phosphorus (TP) removal was not predicted by vegetation cover, but was weakly related to the median water level which explained 25 % of variation TP removal. Despite weak prediction of TP removal, metrics derived from water level sensors identified faults such as excessive inflow and inefficient outflow, which in combination explained 50 % of the variation in the median water level. Monitoring water levels therefore has the potential to detect faults prior to loss of vegetation cover and therefore TN removal, as well as inform the corrective action required.

Revealing the genome of the microsporidian Vairimorpha bombi, a potential driver of bumble bee declines in North America.

Pollinators are vital for food security and the maintenance of terrestrial ecosystems. Bumblebees are important pollinators across northern temperate, arctic, and alpine ecosystems, yet are in decline across the globe. Vairimorpha bombi is a parasite belonging to the fungal class Microsporidia that has been implicated in the rapid decline of bumblebees in North America, where it may be an emerging infectious disease. To investigate the evolutionary basis of pathogenicity of V. bombi, we sequenced and assembled its genome using Oxford Nanopore and Illumina technologies and performed phylogenetic and genomic evolutionary analyses. The genome assembly for V. bombi is 4.73 Mb, from which we predicted 1,870 protein-coding genes and 179 tRNA genes. The genome assembly has low repetitive content and low GC content. V. bombi's genome assembly is the smallest of the Vairimorpha and closely related Nosema genera, but larger than those found in the Encephalitozoon and Ordospora sister clades. Orthology and phylogenetic analysis revealed 18 core conserved single-copy microsporidian genes including the histone acetyltransferase (HAT) GCN5. Surprisingly, V. bombi was unique to the microsporidia in not encoding the second predicted HAT ESA1. The V. bombi genome assembly annotation included 265 unique genes (i.e. not predicted in other microsporidia genome assemblies), 20% of which encode a secretion signal, which is a significant enrichment. Intriguingly, of the 36 microsporidian genomes we analyzed, 26 also had a significant enrichment of secreted signals encoded by unique genes, ranging from 6 to 71% of those predicted genes. These results suggest that microsporidia are under selection to generate and purge diverse and unique genes encoding secreted proteins, potentially contributing to or facilitating infection of their diverse hosts. Furthermore, V. bombi has 5/7 conserved spore wall proteins (SWPs) with its closest relative V. ceranae (that primarily infects honeybees), while also uniquely encoding four additional SWPs. This gene class is thought to be essential for infection, providing both environmental protection and recognition and uptake into the host cell. Together, our results show that SWPs and unique genes encoding a secretion signal are rapidly evolving in the microsporidia, suggesting that they underpin key pathobiological traits including host specificity and pathogenicity.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'ß-signal' imprinted in the final ß-strand of the OMP engages the ß-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the ß-signal are repeated in other, internal ß-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the ß-signal, arranging several ß-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

Empirical evidence of climate change and urbanization impacts on warming stream temperatures.

Climate change and urbanization threaten streams and the biodiversity that rely upon them worldwide. Emissions of greenhouse gases are causing air and sea surface temperatures to increase, and even small areas of urbanization are degrading stream biodiversity, water quality and hydrology. However, empirical evidence of how increasing air temperatures and urbanization together affect stream temperatures over time and their relative influence on stream temperatures is limited. This study quantifies changes in stream temperatures in a region in South-East Australia with an urban-agricultural-forest landcover gradient and where increasing air temperatures have been observed. Using Random Forest models we identify air temperature and urbanization drive increasing stream temperatures and that their combined effects are larger than their individual effects occurring alone. Furthermore, we identify potential mitigation measures useful for waterway managers and policy makers. The results show that both local and global solutions are needed to reduce future increases to stream temperature.

Strain and temperature dependent aggregation of Candida auris is attenuated by inhibition of surface amyloid proteins.

Candida auris is a multi-drug resistant human fungal pathogen that has become a global threat to human health due to its drug resistant phenotype, persistence in the hospital environment and propensity for patient to patient spread. Isolates display variable aggregation that may affect the relative virulence of strains. Therefore, dissection of this phenotype has gained substantial interest in recent years. We studied eight clinical isolates from four different clades (I-IV); four of which had a strongly aggregating phenotype and four of which did not. Genome analysis identified polymorphisms associated with loss of cell surface proteins were enriched in weakly-aggregating strains. Additionally, we identified down-regulation of chitin synthase genes involved in the synthesis of the chitinous septum. Characterisation of the cells revealed no ultrastructural defects in cytokinesis or cell separation in aggregating isolates. Strongly and weakly aggregating strains did not differ in net surface charge or in cell surface hydrophobicity. The capacity for aggregation and for adhesion to polystyrene microspheres were also not correlated. However, aggregation and extracellular matrix formation were all increased at higher growth temperatures, and treatment with the amyloid protein inhibitor Thioflavin-T markedly attenuated aggregation. Genome analysis further indicated strain specific differences in the genome content of GPI-anchored proteins including those encoding genes with the potential to form amyloid proteins. Collectively our data suggests that aggregation is a complex strain and temperature dependent phenomenon that may be linked in part to the ability to form extracellular matrix and cell surface amyloids.

Epitopes in the capsular polysaccharide and the porin OmpK36 receptors are required for bacteriophage infection of Klebsiella pneumoniae.

To kill bacteria, bacteriophages (phages) must first bind to a receptor, triggering the release of the phage DNA into the bacterial cell. Many bacteria secrete polysaccharides that had been thought to shield bacterial cells from phage attack. We use a comprehensive genetic screen to distinguish that the capsule is not a shield but is instead a primary receptor enabling phage predation. Screening of a transposon library to select phage-resistant Klebsiella shows that the first receptor-binding event docks to saccharide epitopes in the capsule. We discover a second step of receptor binding, dictated by specific epitopes in an outer membrane protein. This additional and necessary event precedes phage DNA release to establish a productive infection. That such discrete epitopes dictate two essential binding events for phages has profound implications for understanding the evolution of phage resistance and what dictates host range, two issues critically important to translating knowledge of phage biology into phage therapies.

Comparing genomic variant identification protocols for Candida auris.

Genomic analyses are widely applied to epidemiological, population genetic and experimental studies of pathogenic fungi. A wide range of methods are employed to carry out these analyses, typically without including controls that gauge the accuracy of variant prediction. The importance of tracking outbreaks at a global scale has raised the urgency of establishing high-accuracy pipelines that generate consistent results between research groups. To evaluate currently employed methods for whole-genome variant detection and elaborate best practices for fungal pathogens, we compared how 14 independent variant calling pipelines performed across 35 Candida auris isolates from 4 distinct clades and evaluated the performance of variant calling, single-nucleotide polymorphism (SNP) counts and phylogenetic inference results. Although these pipelines used different variant callers and filtering criteria, we found high overall agreement of SNPs from each pipeline. This concordance correlated with site quality, as SNPs discovered by a few pipelines tended to show lower mapping quality scores and depth of coverage than those recovered by all pipelines. We observed that the major differences between pipelines were due to variation in read trimming strategies, SNP calling methods and parameters, and downstream filtration criteria. We calculated specificity and sensitivity for each pipeline by aligning three isolates with chromosomal level assemblies and found that the GATK-based pipelines were well balanced between these metrics. Selection of trimming methods had a greater impact on SAMtools-based pipelines than those using GATK. Phylogenetic trees inferred by each pipeline showed high consistency at the clade level, but there was more variability between isolates from a single outbreak, with pipelines that used more stringent cutoffs having lower resolution. This project generated two truth datasets useful for routine benchmarking of C. auris variant calling, a consensus VCF of genotypes discovered by 10 or more pipelines across these 35 diverse isolates and variants for 2 samples identified from whole-genome alignments. This study provides a foundation for evaluating SNP calling pipelines and developing best practices for future fungal genomic studies.

Two-speed genome evolution drives pathogenicity in fungal pathogens of animals.

The origins and evolution of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that Bsal has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific Bd, with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded (n = 177) in Bsal, most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally, Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both Bd and Bsal. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

Species-level dataset is required for setting biodiversity conservation priorities for freshwater macroinvertebrates in Melbourne streams.

Family-level identification of freshwater macroinvertebrates is often used to monitor the health of streams due to the lower cost and higher accuracy of identification compared to identifying species. While data on the presence of families from stream monitoring programs can also be used for biodiversity conservation planning, the ability of family-level datasets to accurately reflect regional biodiversity patterns for freshwater macroinvertebrates in Australia remains untested. This study compares family-level and species-level datasets for freshwater insects identified using morphological features and collected over 16 years from 140 sites in Greater Melbourne, Australia. Similar to the results of other studies, our results show a strong positive relationship between family- and species-level taxon richness. However, using the planning software Marxan to compare conservation priorities in our study region, we found that a data analysis of the family-level dataset underestimated the minimum sampling effort required to accurately reflect species diversity. It also identified sub-optimal conservation priority sites and overlooked regionally rare species. We recommend that aquatic macroinvertebrate monitoring programs aimed at understanding regional biodiversity patterns and conservation priorities should routinely include species-level identification, which is now becoming feasible with advances in molecular methods.

Improving early detection of colorectal cancer in Aotearoa New Zealand; how do the direct access criteria perform?

AIM: Colorectal cancer (CRC) is a common malignancy in New Zealand, and there is increasing pressure on investigative resources for diagnosis. The national direct access referral guidelines from the Ministry of Health (MoH) guide who should be referred for investigation, but their performance in detecting CRC and other significant diseases has not been reported previously. This paper describes the yield, by direct access criterion, of all referrals through the direct access pathway to the Canterbury District Health Board (CDHB) during 2018. METHODS: First referrals received through the direct access colonoscopy/computed tomography colonography (CTC) pathway for 2018 were audited. Patients were assigned to symptom groups corresponding to the MoH direct access criteria, and demographic data were captured. Diagnostic outcomes were collected through analysis of all endoscopy, CT colonography and histology reports in the 18 months following referral for primary analysis, with further follow-up through to May 2021 to detect missed pathology. RESULTS: Three thousand two hundred referrals were analysed, and 88.5% underwent colorectal investigation. 128 CRC were diagnosed, 176 advanced polyps, 49 cases of inflammatory bowel disease (IBD) and there were 56 other significant findings. The yield by category for the direct access criteria varied between 0-15.0%, and one urgent criterion had a CRC yield lower than two semi-urgent categories. For patients whose symptoms met at least one of the criteria, excluding those referred with suspected IBD, the combined CRC yield was 4.9%, compared with 1.8% in those who did not meet criteria. The sensitivity and specificity of the criteria for CRC (excluding IBD) was 90% and 23% respectively. There were no CRC detected during the extended follow-up period. CONCLUSION: In this referred population, the MoH direct access colonoscopy/CTC criteria varied significantly in their CRC yield, with an arbitrary distinction between urgent and semi-urgent categories. The low specificity of the criteria means the number needed to investigate to detect one CRC was one in 22. Improved diagnostic algorithms are urgently required to improve both the sensitivity and specificity, thereby more appropriately allocating finite resources to those patients who are most in need of investigation.

LPS O Antigen Plays a Key Role in Klebsiella pneumoniae Capsule Retention.

Despite the importance of encapsulation in bacterial pathogenesis, the biochemical mechanisms and forces that underpin retention of capsule by encapsulated bacteria are poorly understood. In Gram-negative bacteria, there may be interactions between lipopolysaccharide (LPS) core and capsule polymers, between capsule polymers with retained acyl carriers and the outer membrane, and in some bacteria, between the capsule polymers and Wzi, an outer membrane protein lectin. Our transposon studies in Klebsiella pneumoniae B5055 identified additional genes that, when insertionally inactivated, resulted in reduced encapsulation. Inactivation of the gene waaL, which encodes the ligase responsible for attaching the repeated O antigen of LPS to the LPS core, resulted in a significant reduction in capsule retention, measured by atomic force microscopy. This reduction in encapsulation was associated with increased sensitivity to human serum and decreased virulence in a murine model of respiratory infection and, paradoxically, with increased biofilm formation. The capsule in the WaaL mutant was physically smaller than that of the Wzi mutant of K. pneumoniae B5055. These results suggest that interactions between surface carbohydrate polymers may enhance encapsulation, a key phenotype in bacterial virulence, and provide another target for the development of antimicrobials that may avoid resistance issues associated with growth inhibition. IMPORTANCE Bacterial capsules, typically comprised of complex sugars, enable pathogens to avoid key host responses to infection, including phagocytosis. These capsules are synthesized within the bacteria, exported through the outer envelope, and then secured to the external surface of the organism by a force or forces that are incompletely described. This study shows that in the important hospital pathogen Klebsiella pneumoniae, the polysaccharide capsule is retained by interactions with other surface sugars, especially the repeated sugar molecule of the LPS molecule in Gram-negative bacteria known as "O antigen." This O antigen is joined to the LPS molecule by ligation, and loss of the enzyme responsible for ligation, a protein called WaaL, results in reduced encapsulation. Since capsules are essential to the virulence of many pathogens, WaaL might provide a target for new antimicrobial development, critical to the control of pathogens like K. pneumoniae that have become highly drug resistant.

Riparian buffers: Disrupting the transport of E. coli from rural catchments to streams.

High levels of E. coli and associated faecal microbes in waterways as a result of agricultural and residential land use can pose environmental, human health, and economic risks. This study aims to understand the impacts of land use, climatic variables, and riparian buffers on in-stream E. coli concentrations. Flow, temperature, and E. coli were monitored during three sampling campaigns within eleven independent catchments. These catchments have varying land use and extents of riparian buffer coverage. Results showed that catchments with predominantly agricultural and residential land uses (average = 349.7 MPN/100 mL) had higher E. coli concentrations than predominantly forested catchments (average = 111.8 MPN/100 mL). However, there were no statistically significant differences in E. coli concentrations between the agricultural and residential land uses. Riparian buffers appear to reduce E. coli concentrations in streams, as indicated by significant negative correlations between in-stream E. coli concentrations with the riparian buffer areal coverage (Pearson's r = -0.95, Spearman's ρ = -0.90) and the ratio of buffer length to stream length (Pearson's r = -0.87, Spearman's ρ = -0.90). We find that riparian buffers potentially disrupt transport pathways that govern E. coli movement, which in-turn can affect the concentration-discharge relationship. This reinforces the importance of protecting and restoring riparian buffers along drainage lines in agricultural and rural-residential catchments to improve downstream microbial water quality.

Optimised Pre-Processing of Raman Spectra for Colorectal Cancer Detection Using High-Performance Computing.

Spectral pre-processing is an essential step in data analysis for biomedical diagnostic applications of Raman spectroscopy, allowing the removal of undesirable spectral contributions that could mask biological information used for diagnosis. However, due to the specificity of pre-processing for a given sample type and the vast number of potential pre-processing combinations, optimisation of pre-processing via a manual "trial and error" format is often time intensive with no guarantee that the chosen method is optimal for the sample type. Here we present the use of high-performance computing (HPC) to trial over 2.4 million pre-processing permutations to demonstrate the optimisation on the pre-processing of human serum Raman spectra for colorectal cancer detection. The effect of varying pre-processing order, using extended multiplicative scatter correction, spectral smoothing, baseline correction, binning and normalization was considered. Permutations were assessed on their ability to detect patients with disease using a random forest (RF) algorithm trained with 102 patients (510 spectra) and independently tested with a set of 439 patients (1317 spectra) in a primary care patient cohort. Optimising via HPC enables improved performance in diagnostic abilities, with sensitivity increasing by 14.6%, specificity increasing by 6.9%, positive predictive value increasing by 3.4%, and negative predictive value increasing by 2.4% when compared to a standard pre-processing optimisation. Ultimate values of these metrics are very important for diagnostic adoption, and once diagnostics demonstrate good accuracy these types of optimisations can make a significant difference to roll-out of a test and demonstrating advantages over existing tests. We also provide tips/recommendations for pre-processing optimisation without the use of HPC. From the HPC permutations, recommendations for appropriate parameter constraints for conducting a more basic pre-processing optimisation are also detailed, thus helping model development for researchers not having access to HPC.

Evaluation of Dynamic Contrast-Enhanced MRI Measures of Lung Congestion and Endothelial Permeability in Heart Failure: A Prospective Method Validation Study.

BACKGROUND: Methods for accurate quantification of lung fluid in heart failure (HF) are needed. Dynamic contrast-enhanced (DCE)-MRI may be an appropriate modality. PURPOSE: DCE-MRI evaluation of fraction of fluid volume in the interstitial lung space (ve ) and vascular permeability (Ktrans ). STUDY TYPE: Prospective, single-center method validation. POPULATION: Seventeen evaluable healthy volunteers (HVs), 12 participants with HF, and 3 with acute decompensated HF (ADHF). FIELD STRENGTH/SEQUENCE: T1 mapping (spoiled gradient echo variable flip angle acquisition) followed by dynamic series (three-dimensional spoiled gradient-recalled echo acquisitions [constant echo time, repetition time, and flip angle at 1.5 T]). ASSESSMENT: Three whole-chest scans were acquired: baseline (Session 1), 1-week later (Session 2), following exercise (Session 3). Extended Tofts model quantified ve and Ktrans (voxel-wise basis); total lung median measures were extracted and fitted via repeat measure analysis of variance (ANOVA) model. Patient tolerability of the scanning protocol was assessed. STATISTICAL TESTS: This was constructed as an experimental medicine study. PRIMARY ENDPOINTS: Ktrans and ve at baseline (HV vs. HF), change in Ktrans and ve following exercise, and following lung congestion resolution (ADHF). Ktrans and ve were fitted separately using ANOVA. Secondary endpoint: repeatability, that is, within-participant variability in ve and Ktrans between sessions (coefficient of variation estimated via mixed effects model). RESULTS: There was no significant difference in mean Ktrans between HF and HV (P ≤ 0.17): 0.2216 minutes-1 and 0.2353 minutes-1 (Session 1), 0.2044 minutes-1 and 0.2567 minutes-1 (Session 2), 0.1841 minutes-1 and 0.2108 minutes-1 (Session 3), respectively. ve was greater in the HF group (all scans, P ≤ 0.02). Results were repeatable between Sessions 1 and 2; mean values for HF and HV were 0.4946 and 0.3346 (Session 1), 0.4353 and 0.3205 (Session 2), respectively. There was minimal difference in Ktrans or ve between scans for participants with ADHF (small population precluded significance testing). Scanning was well tolerated. DATA CONCLUSION: While no differences were detected in Ktrans , ve was greater in chronic HF patients vs. HV, augmented beyond plasma and intracellular volume. DCE-MRI is a valuable diagnostic and physiologic tool to evaluate changes in fluid volume in the interstitial lung space associated with symptomatic HF. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2.

Population genetics and microevolution of clinical Candida glabrata reveals recombinant sequence types and hyper-variation within mitochondrial genomes, virulence genes, and drug targets.

Candida glabrata is the second most common etiological cause of worldwide systemic candidiasis in adult patients. Genome analysis of 68 isolates from 8 hospitals across Scotland, together with 83 global isolates, revealed insights into the population genetics and evolution of C. glabrata. Clinical isolates of C. glabrata from across Scotland are highly genetically diverse, including at least 19 separate sequence types that have been recovered previously in globally diverse locations, and 1 newly discovered sequence type. Several sequence types had evidence for ancestral recombination, suggesting transmission between distinct geographical regions has coincided with genetic exchange arising in new clades. Three isolates were missing MATα1, potentially representing a second mating type. Signatures of positive selection were identified in every sequence type including enrichment for epithelial adhesins thought to facilitate fungal adhesin to human epithelial cells. In patent microevolution was identified from 7 sets of recurrent cases of candidiasis, revealing an enrichment for nonsynonymous and frameshift indels in cell surface proteins. Microevolution within patients also affected epithelial adhesins genes, and several genes involved in drug resistance including the ergosterol synthesis gene ERG4 and the echinocandin target FKS1/2, the latter coinciding with a marked drop in fluconazole minimum inhibitory concentration. In addition to nuclear genome diversity, the C. glabrata mitochondrial genome was particularly diverse, with reduced conserved sequence and conserved protein-encoding genes in all nonreference ST15 isolates. Together, this study highlights the genetic diversity within the C. glabrata population that may impact virulence and drug resistance, and 2 major mechanisms generating this diversity: microevolution and genetic exchange/recombination.

Tall tails: cryo-electron microscopy of phage tail DNA ejection conduits.

The majority of phages, viruses that infect prokaryotes, inject their genomic material into their host through a tubular assembly known as a tail. Despite the genomic diversity of tailed phages, only three morphological archetypes have been described: contractile tails of Myoviridae-like phages; short non-contractile tails of Podoviridae-like phages; and long and flexible non-contractile tails of Siphoviridae-like phages. While early cryo-electron microscopy (cryo-EM) work elucidated the organisation of the syringe-like injection mechanism of contractile tails, the intrinsic flexibility of the long non-contractile tails prevented high-resolution structural determination. In 2020, four cryo-EM structures of Siphoviridae-like tail tubes were solved and revealed common themes and divergences. The central tube is structurally conserved and homologous to the hexameric rings of the tail tube protein (TTP) also found in contractile tails, bacterial pyocins, and type VI secretion systems. The interior surface of the tube presents analogous motifs of negatively charged amino acids proposed to facilitate ratcheting of the DNA during genome ejection. The lack of a conformational change upon genome ejection implicates the tape measure protein in triggering genome release. A distinctive feature of Siphoviridae-like tails is their flexibility. This results from loose inter-ring connections that can asymmetrically stretch on one side to allow bending and flexing of the tube without breaking. The outer surface of the tube differs greatly and may be smooth or rugged due to additional Ig-like domains in TTP. Some of these variable domains may contribute to adsorption of the phage to prokaryotic and eukaryotic cell surfaces affecting tropism and virulence.