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Low-abundance members of microbial communities are difficult to study in their native habitats, including Escherichia coli, a minor but common inhabitant of the gastrointestinal tract, and key opportunistic pathogen of the urinary tract. While multi-omic analyses have detailed interactions between uropathogenic Escherichia coli (UPEC) and the bladder mediating urinary tract infection (UTI), little is known about UPEC in its pre-infection reservoir, the gastrointestinal tract, partly due to its low relative abundance (<1%). To sensitively explore the genomes and transcriptomes of diverse gut E. coli, we develop E. coli PanSelect, which uses probes designed to specifically capture E. coli's broad pangenome. We demonstrate its ability to enrich diverse E. coli by orders of magnitude, in a mock community and in human stool from a study investigating recurrent UTI (rUTI). Comparisons of transcriptomes between gut E. coli of women with and without history of rUTI suggest rUTI gut E. coli are responding to increased oxygen and nitrate, suggestive of mucosal inflammation, which may have implications for recurrent disease. E. coli PanSelect is well suited for investigations of in vivo E. coli biology in other low-abundance environments, and the framework described here has broad applicability to other diverse, low-abundance organisms., (© 2024. The Author(s).)

Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE-M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE-M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE-M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recurrent urinary tract infections (rUTIs) are a major health burden worldwide, with history of infection being a significant risk factor. While the gut is a known reservoir for uropathogenic bacteria, the role of the microbiota in rUTI remains unclear. We conducted a year-long study of women with (n = 15) and without (n = 16) history of rUTI, from whom we collected urine, blood and monthly faecal samples for metagenomic and transcriptomic interrogation. During the study 24 UTIs were reported, with additional samples collected during and after infection. The gut microbiome of individuals with a history of rUTI was significantly depleted in microbial richness and butyrate-producing bacteria compared with controls, reminiscent of other inflammatory conditions. However, Escherichia coli gut and bladder populations were comparable between cohorts in both relative abundance and phylogroup. Transcriptional analysis of peripheral blood mononuclear cells revealed expression profiles indicative of differential systemic immunity between cohorts. Altogether, these results suggest that rUTI susceptibility is in part mediated through the gut-bladder axis, comprising gut dysbiosis and differential immune response to bacterial bladder colonization, manifesting in symptoms., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Recurrent Clostridioides difficile infection (rCDI) is associated with loss of microbial diversity and microbe-derived secondary bile acids, which inhibit C. difficile germination and growth. SER-109, an investigational microbiome drug of donor-derived, purified spores, reduced recurrence in a dose-ranging, phase (P) 1 study in subjects with multiple rCDIs., Methods: In a P2 double-blind trial, subjects with clinical resolution on standard-of-care antibiotics were stratified by age (< or ≥65 years) and randomized 2:1 to single-dose SER-109 or placebo. Subjects were diagnosed at study entry by PCR or toxin testing. Safety, C. difficile-positive diarrhea through week 8, SER-109 engraftment, and bile acid changes were assessed., Results: 89 subjects enrolled (67% female; 80.9% diagnosed by PCR). rCDI rates were lower in the SER-109 arm than placebo (44.1% vs 53.3%) but did not meet statistical significance. In a preplanned analysis, rates were reduced among subjects ≥65 years (45.2% vs 80%, respectively; RR, 1.77; 95% CI, 1.11-2.81), while the <65 group showed no benefit. Early engraftment of SER-109 was associated with nonrecurrence (P < .05) and increased secondary bile acid concentrations (P < .0001). Whole-metagenomic sequencing from this study and the P1 study revealed previously unappreciated dose-dependent engraftment kinetics and confirmed an association between early engraftment and nonrecurrence. Engraftment kinetics suggest that P2 dosing was suboptimal. Adverse events were generally mild to moderate in severity., Conclusions: Early SER-109 engraftment was associated with reduced CDI recurrence and favorable safety was observed. A higher dose of SER-109 and requirements for toxin testing were implemented in the current P3 trial., Clinical Trials Registration: NCT02437487, https://clinicaltrials.gov/ct2/show/NCT02437487?term=SER-109&draw= 2&rank=4., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Urinary tract infections (UTIs) affect 15 million women each year in the United States, with > 20% experiencing frequent recurrent UTIs. A recent placebo-controlled clinical trial found a 39% reduction in UTI symptoms among recurrent UTI sufferers who consumed a daily cranberry beverage for 24 weeks. Using metagenomic sequencing of stool from a subset of these trial participants, we assessed the impact of cranberry consumption on the gut microbiota, a reservoir for UTI-causing pathogens such as Escherichia coli, which causes > 80% of UTIs., Results: The overall taxonomic composition, community diversity, carriage of functional pathways and gene families, and relative abundances of the vast majority of observed bacterial taxa, including E. coli, were not changed significantly by cranberry consumption. However, one unnamed Flavonifractor species (OTU41), which represented ≤1% of the overall metagenome, was significantly less abundant in cranberry consumers compared to placebo at trial completion. Given Flavonifractor's association with negative human health effects, we sought to determine OTU41 characteristic genes that may explain its differential abundance and/or relationship to key host functions. Using comparative genomic and metagenomic techniques, we identified genes in OTU41 related to transport and metabolism of various compounds, including tryptophan and cobalamin, which have been shown to play roles in host-microbe interactions., Conclusion: While our results indicated that cranberry juice consumption had little impact on global measures of the microbiome, we found one unnamed Flavonifractor species differed significantly between study arms. This suggests further studies are needed to assess the role of cranberry consumption and Flavonifractor in health and wellbeing in the context of recurrent UTI., Trial Registration: Clinical trial registration number: ClinicalTrials.gov NCT01776021 .

Objective: To report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping., Methods: Part 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry., Results: Twelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%-4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry., Conclusion: Golodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization., Clinicaltrialsgov Identifier: NCT02310906., Classification of Evidence: This study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Cryptococcus neoformans is an opportunistic fungal pathogen that at its peak epidemic levels caused an estimated million cases of cryptococcal meningitis per year worldwide. This species can grow in diverse environmental (trees, soil and bird excreta) and host niches (intracellular microenvironments of phagocytes and free-living in host tissues). The genetic basic for adaptation to these different conditions is not well characterized, as most experimental work has relied on a single reference strain of C. neoformans . To identify genes important for yeast infection and disease progression, we profiled the gene expression of seven C. neoformans isolates grown in five representative in vitro environmental and in vivo conditions. We characterized gene expression differences using RNA-Seq (RNA sequencing), comparing clinical and environmental isolates from two of the major lineages of this species, VNI and VNBI. These comparisons highlighted genes showing lineage-specific expression that are enriched in subtelomeric regions and in lineage-specific gene clusters. By contrast, we find few expression differences between clinical and environmental isolates from the same lineage. Gene expression specific to in vivo stages reflects available nutrients and stresses, with an increase in fungal metabolism within macrophages, and an induction of ribosomal and heat-shock gene expression within the subarachnoid space. This study provides the widest view to date of the transcriptome variation of C. neoformans across natural isolates, and provides insights into genes important for in vitro and in vivo growth stages.

Background: While the international spread of multidrug-resistant (MDR) Mycobacterium tuberculosis strains is an acknowledged public health threat, a broad and more comprehensive examination of the global spread of MDR-tuberculosis (TB) using whole-genome sequencing has not yet been performed., Methods: In a global dataset of 5310 M . tuberculosis whole-genome sequences isolated from five continents, we performed a phylogenetic analysis to identify and characterise clades of MDR-TB with respect to geographic dispersion., Results: Extensive international dissemination of MDR-TB was observed, with identification of 32 migrant MDR-TB clades with descendants isolated in 17 unique countries. Relatively recent movement of strains from both Beijing and non-Beijing lineages indicated successful global spread of varied genetic backgrounds. Migrant MDR-TB clade members shared relatively recent common ancestry, with a median estimate of divergence of 13-27 years. Migrant extensively drug-resistant (XDR)-TB clades were not observed, although development of XDR-TB within migratory MDR-TB clades was common., Conclusions: Application of genomic techniques to investigate global MDR migration patterns revealed extensive global spread of MDR clades between countries of varying TB burden. Further expansion of genomic studies to incorporate isolates from diverse global settings into a single analysis, as well as data sharing platforms that facilitate genomic data sharing across country lines, may allow for future epidemiological analyses to monitor for international transmission of MDR-TB. In addition, efforts to perform routine whole-genome sequencing on all newly identified M. tuberculosis , like in England, will serve to better our understanding of the transmission dynamics of MDR-TB globally., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Tuberculosis (TB) is a global infectious threat that is intensified by an increasing incidence of highly drug-resistant disease. Whole-genome sequencing (WGS) studies of Mycobacterium tuberculosis, the causative agent of TB, have greatly increased our understanding of this pathogen. Since the first M. tuberculosis genome was published in 1998, WGS has provided a more complete account of the genomic features that cause resistance in populations of M. tuberculosis, has helped to fill gaps in our knowledge of how both classical and new antitubercular drugs work, and has identified specific mutations that allow M. tuberculosis to escape the effects of these drugs. WGS studies have also revealed how resistance evolves both within an individual patient and within patient populations, including the important roles of de novo acquisition of resistance and clonal spread. These findings have informed decisions about which drug-resistance mutations should be included on extended diagnostic panels. From its origins as a basic science technique, WGS of M. tuberculosis is becoming part of the modern clinical microbiology laboratory, promising rapid and improved detection of drug resistance, and detailed and real-time epidemiology of TB outbreaks. We review the successes and highlight the challenges that remain in applying WGS to improve the control of drug-resistant TB through monitoring its evolution and spread, and to inform more rapid and effective diagnostic and therapeutic strategies.

Immune genes are under intense, pathogen-induced pressure, which causes these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of "Intracellular Pathogen Response" or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens, including Nematocida parisii microsporidia and the Orsay virus. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. In summary, the species-specific pals-22 and pals-25 genes act as a switch to regulate a program of gene expression, growth, and defense against diverse natural pathogens in C. elegans., Competing Interests: The authors have declared that no competing interests exist.

Accurate orthologue identification is a vital component of bacterial comparative genomic studies, but many popular sequence-similarity-based approaches do not scale well to the large numbers of genomes that are now generated routinely. Furthermore, most approaches do not take gene synteny into account, which is useful information for disentangling paralogues. Here, we present SynerClust, a user-friendly synteny-aware tool based on synergy that can process thousands of genomes. SynerClust was designed to analyse genomes with high levels of local synteny, particularly prokaryotes, which have operon structure. SynerClust's run-time is optimized by selecting cluster representatives at each node in the phylogeny; thus, avoiding the need for exhaustive pairwise similarity searches. In benchmarking against Roary, Hieranoid2, PanX and Reciprocal Best Hit, SynerClust was able to more completely identify sets of core genes for datasets that included diverse strains, while using substantially less memory, and with scalability comparable to the fastest tools. Due to its scalability, ease of installation and use, and suitability for a variety of computing environments, orthogroup clustering using SynerClust will enable many large-scale prokaryotic comparative genomics efforts.

The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 μm cells and large titan cells (> 10 μm and up to 100 μm). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 μm), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways., Competing Interests: The authors have declared that no competing interests exist.

Cryptococcus species are the causative agents of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals. Initial work on the molecular epidemiology of this fungal pathogen utilized genotyping approaches to describe the genetic diversity and biogeography of two species, Cryptococcus neoformans and Cryptococcus gattii. Whole genome sequencing of representatives of both species resulted in reference assemblies enabling a wide array of downstream studies and genomic resources. With the increasing availability of whole genome sequencing, both species have now had hundreds of individual isolates sequenced, providing fine-scale insight into the evolution and diversification of Cryptococcus and allowing for the first genome-wide association studies to identify genetic variants associated with human virulence. Sequencing has also begun to examine the microevolution of isolates during prolonged infection and to identify variants specific to outbreak lineages, highlighting the potential role of hyper-mutation in evolving within short time scales. We can anticipate that further advances in sequencing technology and sequencing microbial genomes at scale, including metagenomics approaches, will continue to refine our view of how the evolution of Cryptococcus drives its success as a pathogen.

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically human immunodeficiency virus/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterize the major subdivisions, their relative diversity, and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii ( serotype AA) and of hybrids with C. neoformans var. neoformans (serotype AD) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil reveal that the previously "African" VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions; demonstrating the highly dispersive nature of this pathogen., (Copyright © 2017 Rhodes et al.)

Cryptococcus neoformans is an opportunistic fungal pathogen that causes approximately 625,000 deaths per year from nervous system infections. Here, we leveraged a unique, genetically diverse population of C. neoformans from sub-Saharan Africa, commonly isolated from mopane trees, to determine how selective pressures in the environment coincidentally adapted C. neoformans for human virulence. Genome sequencing and phylogenetic analysis of 387 isolates, representing the global VNI and African VNB lineages, highlighted a deep, nonrecombining split in VNB (herein, VNBI and VNBII). VNBII was enriched for clinical samples relative to VNBI, while phenotypic profiling of 183 isolates demonstrated that VNBI isolates were significantly more resistant to oxidative stress and more heavily melanized than VNBII isolates. Lack of melanization in both lineages was associated with loss-of-function mutations in the BZP4 transcription factor. A genome-wide association study across all VNB isolates revealed sequence differences between clinical and environmental isolates in virulence factors and stress response genes. Inositol transporters and catabolism genes, which process sugars present in plants and the human nervous system, were identified as targets of selection in all three lineages. Further phylogenetic and population genomic analyses revealed extensive loss of genetic diversity in VNBI, suggestive of a history of population bottlenecks, along with unique evolutionary trajectories for mating type loci. These data highlight the complex evolutionary interplay between adaptation to natural environments and opportunistic infections, and that selection on specific pathways may predispose isolates to human virulence., (© 2017 Desjardins et al.; Published by Cold Spring Harbor Laboratory Press.)

Cardiomyocytes acquire their primary specialized function (contraction) before exiting the cell cycle. In this regard, proliferation and differentiation must be precisely coordinated for proper cardiac morphogenesis. Here, we have investigated the complex transcriptional mechanisms employed by cardiomyocytes to coordinate antagonistic cell-cycle and differentiation gene programs through the molecular dissection of the core cardiac transcription factor, MEF2. Knockdown of individual MEF2 proteins, MEF2A, -C, and -D, in primary neonatal cardiomyocytes resulted in radically distinct and opposite effects on cellular homeostasis and gene regulation. MEF2A and MEF2D were absolutely required for cardiomyocyte survival, whereas MEF2C, despite its major role in cardiac morphogenesis and direct reprogramming, was dispensable for this process. Inhibition of MEF2A or -D also resulted in the activation of cell-cycle genes and down-regulation of markers of terminal differentiation. In striking contrast, the regulation of cell-cycle and differentiation gene programs by MEF2C was antagonistic to that of MEF2A and -D. Computational analysis of regulatory regions from MEF2 isoform-dependent gene sets identified the Notch and Hedgehog signaling pathways as key determinants in coordinating MEF2 isoform-specific control of antagonistic gene programs. These results reveal that mammalian MEF2 family members have distinct transcriptional functions in cardiomyocytes and suggest that these differences are critical for proper development and maturation of the heart. Analysis of MEF2 isoform-specific function in neonatal cardiomyocytes has yielded insight into an unexpected transcriptional regulatory mechanism by which these specialized cells utilize homologous members of a core cardiac transcription factor to coordinate cell-cycle and differentiation gene programs., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

The emergence and spread of drug-resistant Mycobacterium tuberculosis (DR-TB) are critical global health issues. Eastern Europe has some of the highest incidences of DR-TB, particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. To better understand the genetic composition and evolution of MDR- and XDR-TB in the region, we sequenced and analyzed the genomes of 138 M. tuberculosis isolates from 97 patients sampled between 2010 and 2013 in Minsk, Belarus. MDR and XDR-TB isolates were significantly more likely to belong to the Beijing lineage than to the Euro-American lineage, and known resistance-conferring loci accounted for the majority of phenotypic resistance to first- and second-line drugs in MDR and XDR-TB. Using a phylogenomic approach, we estimated that the majority of MDR-TB was due to the recent transmission of already-resistant M. tuberculosis strains rather than repeated de novo evolution of resistance within patients, while XDR-TB was acquired through both routes. Longitudinal sampling of M. tuberculosis from 34 patients with treatment failure showed that most strains persisted genetically unchanged during treatment or acquired resistance to fluoroquinolones. HIV+ patients were significantly more likely to have multiple infections over time than HIV- patients, highlighting a specific need for careful infection control in these patients. These data provide a better understanding of the genomic composition, transmission, and evolution of MDR- and XDR-TB in Belarus and will enable improved diagnostics, treatment protocols, and prognostic decision-making., (Copyright © 2017 Wollenberg et al.)

Background: Candida auris, a multidrug-resistant yeast that causes invasive infections, was first described in 2009 in Japan and has since been reported from several countries., Methods: To understand the global emergence and epidemiology of C. auris, we obtained isolates from 54 patients with C. auris infection from Pakistan, India, South Africa, and Venezuela during 2012-2015 and the type specimen from Japan. Patient information was available for 41 of the isolates. We conducted antifungal susceptibility testing and whole-genome sequencing (WGS)., Results: Available clinical information revealed that 41% of patients had diabetes mellitus, 51% had undergone recent surgery, 73% had a central venous catheter, and 41% were receiving systemic antifungal therapy when C. auris was isolated. The median time from admission to infection was 19 days (interquartile range, 9-36 days), 61% of patients had bloodstream infection, and 59% died. Using stringent break points, 93% of isolates were resistant to fluconazole, 35% to amphotericin B, and 7% to echinocandins; 41% were resistant to 2 antifungal classes and 4% were resistant to 3 classes. WGS demonstrated that isolates were grouped into unique clades by geographic region. Clades were separated by thousands of single-nucleotide polymorphisms, but within each clade isolates were clonal. Different mutations in ERG11 were associated with azole resistance in each geographic clade., Conclusions: C. auris is an emerging healthcare-associated pathogen associated with high mortality. Treatment options are limited, due to antifungal resistance. WGS analysis suggests nearly simultaneous, and recent, independent emergence of different clonal populations on 3 continents. Risk factors and transmission mechanisms need to be elucidated to guide control measures., (Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Fluoroquinolones (FQs) are effective second-line drugs for treating antibiotic-resistant tuberculosis (TB) and are being considered for use as first-line agents. Because FQs are used to treat a range of infections, in a setting of undiagnosed TB, there is potential to select for drug-resistant Mycobacterium tuberculosis mutants during FQ-based treatment of other infections, including pneumonia. Here we present a detailed characterization of ofloxacin-resistant M. tuberculosis samples isolated directly from patients in Taiwan, which demonstrates that selection for FQ resistance can occur within patients who have not received FQs for the treatment of TB. Several of these samples showed no mutations in gyrA or gyrB based on PCR-based molecular assays, but genome-wide next-generation sequencing (NGS) revealed minority populations of gyrA and/or gyrB mutants. In other samples with PCR-detectable gyrA mutations, NGS revealed subpopulations containing alternative resistance-associated genotypes. Isolation of individual clones from these apparently heterogeneous samples confirmed the presence of the minority drug-resistant variants suggested by the NGS data. Further NGS of these purified clones established evolutionary links between FQ-sensitive and -resistant clones derived from the same patient, suggesting de novo emergence of FQ-resistant TB. Importantly, most of these samples were isolated from patients without a history of FQ treatment for TB. Thus, selective pressure applied by FQ monotherapy in the setting of undiagnosed TB infection appears to be able to drive the full or partial emergence of FQ-resistant M. tuberculosis, which has the potential to confound diagnostic tests for antibiotic susceptibility and limit the effectiveness of FQs in TB treatment., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Proper formation of the mammalian heart requires precise spatiotemporal transcriptional regulation of gene programs in cardiomyocytes. Sophisticated regulatory networks have evolved to not only integrate the activities of distinct transcription factors to control tissue-specific gene programs but also, in many instances, to incorporate multiple members within these transcription factor families to ensure accuracy and specificity in the system. Unsurprisingly, perturbations in this elaborate transcriptional circuitry can lead to severe cardiac abnormalities. Myocyte enhancer factor-2 (MEF2) transcription factor belongs to the evolutionarily conserved cardiac gene regulatory network. Given its central role in muscle gene regulation and its evolutionary conservation, MEF2 is considered one of only a few core cardiac transcription factors. In addition to its firmly established role as a differentiation factor, MEF2 regulates wide variety of, sometimes antagonistic, cellular processes such as cell survival and death. Vertebrate genomes encode multiple MEF2 family members thereby expanding the transcriptional potential of this core transcription factor in the heart. This review highlights the requirement of the MEF2 family and their orthologs in cardiac development in diverse animal model systems. Furthermore, we describe the recently characterized role of MEF2 in direct reprogramming and genome-wide cardiomyocyte gene regulation. A thorough understanding of the regulatory functions of the MEF2 family in cardiac development and cardiogenomics is required in order to develop effective therapeutic strategies to repair the diseased heart., Competing Interests: The authors declare no conflict of interest.

Successful treatment of Mycobacterium tuberculosis infection typically requires a complex regimen administered over at least 6 months. Interestingly, many of the antibiotics used to treat M. tuberculosis are prodrugs that require intracellular activation. Here, we describe three small molecules, active against both replicating and non-replicating M. tuberculosis, that require activation by Baeyer-Villiger monooxygenases (BVMOs). Two molecules require BVMO EthA (Rv3854c) for activation and the third molecule requires the BVMO MymA (Rv3083). While EthA is known to activate the antitubercular drug ethionamide, this is the first description of MymA as an activating enzyme of a prodrug. Furthermore, we found that MymA also plays a role in activating ethionamide, with loss of MymA function resulting in ethionamide-resistant M. tuberculosis. These findings suggest overlap in function and specificity of the BVMOs in M. tuberculosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs such as D-cycloserine. Here we used the whole-genome sequences from 498 strains of M. tuberculosis to identify new resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss of function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss of function conferred resistance to D-cycloserine, and susceptibility to the drug was partially restored by complementation of ald. Clinical strains with mutations in ald and alr exhibited increased resistance to D-cycloserine when cultured in vitro. Incorporation of D-cycloserine resistance in novel molecular diagnostics could allow for targeted use of this toxic drug among patients with susceptible infections.

We report the whole-genome sequencing of two extensively drug-resistant tuberculosis strains belonging to the Euro-American S lineage. The RSA 114 strain showed single-nucleotide polymorphisms predicted to have drug efflux activity., (Copyright © 2016 Malinga et al.)

Background: Mycobacterium africanum, made up of lineages 5 and 6 within the Mycobacterium tuberculosis complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system., Methodology/principal Findings: We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes., Conclusions: This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity.

Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, Blastomyces, and two species of the related genus Emmonsia, typically pathogens of small mammals. Compared to related species, Blastomyces genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for gypsy elements, are variable in total size between isolates, and are least expanded in the avirulent B. dermatitidis strain ER-3 as compared with the virulent B. gilchristii strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the Blastomyces lineage. While gene content is highly conserved between Blastomyces and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of B. dermatitidis during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in Blastomyces, and upregulation of dioxygenases in vivo suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively in vivo, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in in vitro studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of Blastomyces to its host environment. These results reveal the dynamics of genome evolution and of factors contributing to virulence in Blastomyces.

The cardiomyocyte cell cycle is a poorly understood process. Mammalian cardiomyocytes permanently withdraw from the cell cycle shortly after birth but can re-enter the cell cycle and proliferate when subjected to injury within a brief temporal window in the neonatal period. Thus, investigating the mechanisms of cell cycle regulation in neonatal cardiomyocytes may provide critical insight into the molecular events that prevent adult myocytes from proliferating in response to injury or stress. MEF2D is a key transcriptional mediator of pathological remodeling in the adult heart downstream of various stress-promoting insults. However, the specific gene programs regulated by MEF2D in cardiomyocytes are unknown. By performing genome-wide transcriptome analysis using MEF2D-depleted neonatal cardiomyocytes, we found a significant impairment in the cell cycle, characterized by the up-regulation of numerous positive cell cycle regulators. Expression of Pten, the primary negative regulator of PI3K/Akt, was significantly reduced in MEF2D-deficient cardiomyocytes and found to be a direct target gene of MEF2D. Consistent with these findings mutant cardiomyocytes showed activation of the PI3K/Akt survival pathway. Paradoxically, prolonged deficiency of MEF2D in neonatal cardiomyocytes did not trigger proliferation but instead resulted in programmed cell death, which is likely mediated by the E2F transcription factor. These results demonstrate a critical role for MEF2D in cell cycle regulation of post-mitotic, neonatal cardiomyocytes in vitro., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents., Methods and Findings: We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937-1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974-1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988-1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe., Conclusions: In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.

Unlabelled: Cryptococcus gattii is a fungal pathogen of humans, causing pulmonary infections in otherwise healthy hosts. To characterize genomic variation among the four major lineages of C. gattii (VGI, -II, -III, and -IV), we generated, annotated, and compared 16 de novo genome assemblies, including the first for the rarely isolated lineages VGIII and VGIV. By identifying syntenic regions across assemblies, we found 15 structural rearrangements, which were almost exclusive to the VGI-III-IV lineages. Using synteny to inform orthology prediction, we identified a core set of 87% of C. gattii genes present as single copies in all four lineages. Remarkably, 737 genes are variably inherited across lineages and are overrepresented for response to oxidative stress, mitochondrial import, and metal binding and transport. Specifically, VGI has an expanded set of iron-binding genes thought to be important to the virulence of Cryptococcus, while VGII has expansions in the stress-related heat shock proteins relative to the other lineages. We also characterized genes uniquely absent in each lineage, including a copper transporter absent from VGIV, which influences Cryptococcus survival during pulmonary infection and the onset of meningoencephalitis. Through inclusion of population-level data for an additional 37 isolates, we identified a new transcontinental clonal group that we name VGIIx, mitochondrial recombination between VGII and VGIII, and positive selection of multidrug transporters and the iron-sulfur protein aconitase along multiple branches of the phylogenetic tree. Our results suggest that gene expansion or contraction and positive selection have introduced substantial variation with links to mechanisms of pathogenicity across this species complex., Importance: The genetic differences between phenotypically different pathogens provide clues to the underlying mechanisms of those traits and can lead to new drug targets and improved treatments for those diseases. In this paper, we compare 16 genomes belonging to four highly differentiated lineages of Cryptococcus gattii, which cause pulmonary infections in otherwise healthy humans and other animals. Half of these lineages have not had their genomes previously assembled and annotated. We identified 15 ancestral rearrangements in the genome and over 700 genes that are unique to one or more lineages, many of which are associated with virulence. In addition, we found evidence for recent transcontinental spread, mitochondrial genetic exchange, and positive selection in multidrug transporters. Our results suggest that gene expansion/contraction and positive selection are diversifying the mechanisms of pathogenicity across this species complex., (Copyright © 2015 Farrer et al.)

Background.  Five neuroinvasive Bacillus cereus infections (4 fatal) occurred in hospitalized patients with acute myelogenous leukemia (AML) during a 9-month period, prompting an investigation by infection control and public health officials. Methods.  Medical records of case-patients were reviewed and a matched case-control study was performed. Infection control practices were observed. Multiple environmental, food, and medication samples common to AML patients were cultured. Multilocus sequence typing was performed for case and environmental B cereus isolates. Results.  All 5 case-patients received chemotherapy and had early-onset neutropenic fevers that resolved with empiric antibiotics. Fever recurred at a median of 17 days (range, 9-20) with headaches and abrupt neurological deterioration. Case-patients had B cereus identified in central nervous system (CNS) samples by (1) polymerase chain reaction or culture or (2) bacilli seen on CNS pathology stains with high-grade B cereus bacteremia. Two case-patients also had colonic ulcers with abundant bacilli on autopsy. No infection control breaches were observed. On case-control analysis, bananas were the only significant exposure shared by all 5 case-patients (odds ratio, 9.3; P = .04). Five environmental or food isolates tested positive for B cereus, including a homogenized banana peel isolate and the shelf of a kitchen cart where bananas were stored. Multilocus sequence typing confirmed that all case and environmental strains were genetically distinct. Multilocus sequence typing-based phylogenetic analysis revealed that the organisms clustered in 2 separate clades. Conclusions.  The investigation of this neuroinvasive B cereus cluster did not identify a single point source but was suggestive of a possible dietary exposure. Our experience underscores the potential virulence of B cereus in immunocompromised hosts.

The myocyte enhancer factor 2 (MEF2) transcription factor requires interactions with co-factors for precise regulation of its target genes. Our lab previously reported that the mammalian MEF2A isoform regulates the cardiomyocyte costamere, a critical muscle-specific focal adhesion complex involved in contractility, through its transcriptional control of genes encoding proteins localized to this cytoskeletal structure. To further dissect the transcriptional mechanisms of costamere gene regulation and identify potential co-regulators of MEF2A, a bioinformatics analysis of transcription factor binding sites was performed using the proximal promoter regions of selected costamere genes. One of these predicted sites belongs to the early growth response (EGR) transcription factor family. The EGR1 isoform has been shown to be involved in a number of pathways in cardiovascular homeostasis and disease, making it an intriguing candidate MEF2 coregulator to further characterize. Here, we demonstrate that EGR1 interacts with MEF2A and is a potent and specific repressor of MEF2 transcriptional activity. Furthermore, we show that costamere gene expression in cardiomyocytes is dependent on EGR1 transcriptional activity. This study identifies a mechanism by which MEF2 activity can be modulated to ensure that costamere gene expression is maintained at levels commensurate with cardiomyocyte contractile activity.

Obligate intracellular pathogens depend on their host for growth yet must also evade detection by host defenses. Here we investigate host adaptation in two Microsporidia, the specialist Edhazardia aedis and the generalist Vavraia culicis, pathogens of disease vector mosquitoes. Genomic analysis and deep RNA-Seq across infection time courses reveal fundamental differences between these pathogens. E. aedis retains enhanced cell surface modification and signalling capacity, upregulating protein trafficking and secretion dynamically during infection. V. culicis is less dependent on its host for basic metabolites and retains a subset of spliceosomal components, with a transcriptome broadly focused on growth and replication. Transcriptional profiling of mosquito immune responses reveals that response to infection by E. aedis differs dramatically depending on the mode of infection, and that antimicrobial defensins may play a general role in mosquito defense against Microsporidia. This analysis illuminates fundamentally different evolutionary paths and host interplay of specialist and generalist pathogens.

Skeletal muscle differentiation requires precisely coordinated transcriptional regulation of diverse gene programs that ultimately give rise to the specialized properties of this cell type. In Drosophila, this process is controlled, in part, by MEF2, the sole member of an evolutionarily conserved transcription factor family. By contrast, vertebrate MEF2 is encoded by four distinct genes, Mef2a, -b, -c, and -d, making it far more challenging to link this transcription factor to the regulation of specific muscle gene programs. Here, we have taken the first step in molecularly dissecting vertebrate MEF2 transcriptional function in skeletal muscle differentiation by depleting individual MEF2 proteins in myoblasts. Whereas MEF2A is absolutely required for proper myoblast differentiation, MEF2B, -C, and -D were found to be dispensable for this process. Furthermore, despite the extensive redundancy, we show that mammalian MEF2 proteins regulate a significant subset of nonoverlapping gene programs. These results suggest that individual MEF2 family members are able to recognize specific targets among the entire cohort of MEF2-regulated genes in the muscle genome. These findings provide opportunities to modulate the activity of MEF2 isoforms and their respective gene programs in skeletal muscle homeostasis and disease., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)