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Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.

Staphylococcus aureusis an adaptable human pathogen causing life-threatening endocarditis and bacteraemia. Methicillin-resistantS. aureus(MRSA) is alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteraemia and vancomycin treatment failure is often associated with vancomycin-intermediateS. aureusstrains termed VISA. The regulatory 3’ UTR ofvigRmRNA contributes to vancomycin tolerance in the clinical VISA isolate JKD6008 and upregulates the lytic transglycosylase IsaA. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we find that thevigR3’ UTR also interacts with mRNAs involved in carbon metabolism, amino acid biogenesis, cell wall biogenesis, and virulence. ThevigR3’ UTR was found to repressdapE, a succinyl-diaminopimelate desuccinylase required for lysine and cell wall peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of thevigR3’ UTR increased VISA virulence in a wax moth larvae model, and we find that anisaAmutant is completely attenuated in the larvae model. Sequence and structural analysis of thevigR3’ UTR indicates that the UTR has expanded through the acquisition ofStaphylococcus aureusrepeat insertions (STAR repeats) that partly contribute sequence for theisaAinteraction seed and may functionalise the 3’ UTR. Our findings reveal an extended regulatory network forvigR, uncovering a novel mechanism of regulation of cell wall metabolism and virulence in a clinicalS. aureusisolate.

Acinetobacter baumannii is an important opportunistic pathogen known for its high levels of resistance to many antibiotics, particularly those considered last resorts such as colistin and carbapenems. Plasmids of this organism are increasingly associated with the spread of clinically important antibiotic resistance genes. Although A. baumannii is a ubiquitous organism, to date, most of the focus has been on studying strains recovered from clinical samples ignoring those isolated in the environment (soil, water, food, etc.). Here, we analysed the genetic structures of eight novel plasmids carried by an environmental colistin-resistant A. baumannii (strain E-072658) recovered in a recycled fibre pulp in a paper mill in Finland. It was shown that E-072658 carries a new variant of the mcr-4 colistin resistance gene (mcr-4.7) in a novel Tn3-family transposon (called Tn6926) carried by a novel plasmid p8E072658. E-072658 is also resistant to sulphonamide compounds; consistent with this, the sul2 sulphonamide resistance gene was found in a pdif module. E-072658 also carries six additional plasmids with no antibiotic resistance genes, but they contained several pdif modules shared with plasmids carried by clinical strains. Detailed analysis of the genetic structure of all eight plasmids carried by E-072658 showed a complex evolutionary history revealing genetic exchange events within the genus Acinetobacter beyond the clinical or environmental origin of the strains. This work provides evidence that environmental strains might act as a source for some of the clinically significant antibiotic resistance genes.

Infections associated with antimicrobial resistance (AMR) are poised to become the leading cause of death in the next few decades, a scenario that can be ascribed to two phenomena: antibiotic over-prescription and a lack of antibiotic drug development. The crowd-sourced initiative CO-ADD has been testing research compounds contributed by researchers around the world to find new antimicrobials to combat AMR, and during this campaign has found that metallodrugs might be a promising, yet untapped source. To this end, we submitted 18 Pd(II) and Ru(II) – pyridyl-1,2,3-triazolyl complexes which were developed as catalysts for their antimicrobial properties. It was found that the Pd-complexes possessed potent antifungal activity, especially Pd1, with MICs between 0.06 – 0.125 µg/mL against C. glabrata. The in vitro studies were extended to in vivo studies in G. mellonella larvae where it was established that the compounds were non-toxic. Here we effectively demonstrate the potential of Pd(II)-pyta complexes as antifungal agents.

A viscoelastic microfluidic device for shape-based separation of drug-treated Escherichia coli.

L-cysteine biosynthesis from inorganic sulfur represents a major mechanism by which reduced sulfur is incorporated into organic compounds. Cysteine biosynthesis and regulation is well characterized in E. coli. However, the regulation of sulfur metabolism in Acinetobacter baumannii is only partly understood, with the LysR type regulator, GigC known to control some aspects of sulfur reduction. In this study, we have used transcriptomics and bioinformatic analyses to characterize a novel LysR-type transcriptional regulator encoded by ABUW_1016 (cbl), in a highly multidrug-resistant and virulent isolate of Acinetobacter baumannii. We have shown that Cbl is involved in controlling the expression of the genes required for uptake and reduction of various sulfur sources in A. baumannii. We have identified the global regulon of Cbl and proposed a model of cysteine biosynthesis and its regulation by Cbl and GigC in A. baumannii.

There are currently fewer than ten antifungal drugs in clinical development, but new fungal strains, which are resistant to most current antifungals are spreading rapidly across the world. To prevent a second resistance crisis, new classes of antifungal drugs are urgently needed. Metal complexes have proven to be promising candidates for novel antibiotics, but so far, few compounds have been explored for their potential application as antifungal agents. In this work we report the evaluation of 1039 metal-containing compounds that were screened by the Community for Open Antimicrobial Drug Discovery (CO-ADD). We show that 20.9% of all metal compounds tested have antimicrobial activity against two representative Candida and Cryptococcus strains, compared with only 1.1% of the >300,000 purely organic molecules tested through CO-ADD. We identified 90 metal compounds (8.7%) that show antifungal activity while not displaying any cytotoxicity against mammalian cell lines or haemolytic properties at similar concentrations. The structures of 21 metal complexes which display high antifungal activity (MIC ≤ 1.25 µM) are discussed and evaluated further against a broad panel of yeasts. Most of these have not been evaluated for antifungal activity. Eleven of these metal complexes were tested for toxicity in the Galleria mellonella moth larvae model, revealing that only one compound showed signs of toxicity at the highest injected concentration. Lastly, we demonstrated that the organo-Pt(II) cyclooctadiene complex Pt1 significantly reduces fungal load in an in vivo G. mellonella infection model. These findings showcase that the structural and chemical diversity of metal-based compounds can be an invaluable tool in the development of new drugs against infectious diseases.

The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the gut commensal and model organismEscherichia coli, as well as several major human pathogens including multiple serovars ofSalmonella entericaandKlebsiella pneumoniae. Essential gene sets have been determined for several members of the Enterobacteriaceae, with theE. coliKeio single-gene deletion library often regarded as a gold standard for gene essentiality studies. However, it remains unclear how gene essentiality varies between related strains and species. To investigate this, we have assembled a collection of thirteen sequenced high-density transposon mutant libraries from five genera within the Enterobacteriaceae. We first benchmark a number of gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations we develop a new classifier for gene essentiality. Using this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes, and reconstruct an ancestral essential gene set of 296 genes. Despite the presence of a large cohort of variably essential genes, surprisingly we find an absence of evidence for genus-specific essential genes. A clear example of this sporadic essentiality is given by the set of genes regulating the σEextracytoplasmic stress response, which appears to have independently become essential multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss in obligate insect endosymbionts that have emerged from within the Enterobacteriaceae. This isolates a remarkably small set of genes absolutely required for survival, and uncovers several instances of essential stress responses masked by redundancy in free-living bacteria.

[Image: see text] There are currently fewer than 10 antifungal drugs in clinical development, but new fungal strains that are resistant to most current antifungals are spreading rapidly across the world. To prevent a second resistance crisis, new classes of antifungal drugs are urgently needed. Metal complexes have proven to be promising candidates for novel antibiotics, but so far, few compounds have been explored for their potential application as antifungal agents. In this work, we report the evaluation of 1039 metal-containing compounds that were screened by the Community for Open Antimicrobial Drug Discovery (CO-ADD). We show that 20.9% of all metal compounds tested have antimicrobial activity against two representative Candida and Cryptococcus strains compared with only 1.1% of the >300,000 purely organic molecules tested through CO-ADD. We identified 90 metal compounds (8.7%) that show antifungal activity while not displaying any cytotoxicity against mammalian cell lines or hemolytic properties at similar concentrations. The structures of 21 metal complexes that display high antifungal activity (MIC ≤1.25 μM) are discussed and evaluated further against a broad panel of yeasts. Most of these have not been previously tested for antifungal activity. Eleven of these metal complexes were tested for toxicity in the Galleria mellonella moth larva model, revealing that only one compound showed signs of toxicity at the highest injected concentration. Lastly, we demonstrated that the organo-Pt(II) cyclooctadiene complex Pt1 significantly reduces fungal load in an in vivoG. mellonella infection model. These findings showcase that the structural and chemical diversity of metal-based compounds can be an invaluable tool in the development of new drugs against infectious diseases.

Acinetobacter baumannii is a critically important pathogen known for its widespread antibiotic resistance and ability to persist in hospital-associated environments. Whilst the majority of A. baumannii infections are hospital-acquired, infections from outside the hospital have been reported with high mortality. Despite this, little is known about the natural environmental reservoir(s) of A. baumannii and the virulence potential underlying non-clinical strains. Here, we report the complete genome sequences of six diverse strains isolated from environments such as river, soil, and industrial sites around the world. Phylogenetic analyses showed that four of these strains were unrelated to representative nosocomial strains and do not share a monophyletic origin, whereas two had sequence types belonging to the global clone lineages GC1 and GC2. Further, the majority of these strains harboured genes linked to virulence and stress protection in nosocomial strains. These genotypic properties correlated well with in vitro virulence phenotypic assays testing resistance to abiotic stresses, serum survival, and capsule formation. Virulence potential was confirmed in vivo, with most environmental strains able to effectively kill Galleria mellonella greater wax moth larvae. Using phenomic arrays and antibiotic resistance profiling, environmental and nosocomial strains were shown to have similar substrate utilisation patterns although environmental strains were distinctly more sensitive to antibiotics. Taken together, these features of environmental A. baumannii strains suggest the existence of a strain-specific distinct gene pools for niche specific adaptation. Furthermore, environmental strains appear to be equally virulent as contemporary nosocomial strains but remain largely antibiotic sensitive.

A special-care neonatal unit from a large public hospital in Malawi was noted as having more frequent, difficult-to-treat infections, and a suspected outbreak of multi-drug-resistant Klebsiella pneumoniae was investigated using genomic characterisation. All K. pneumoniae bloodstream infections (BSIs) from patients in the neonatal ward (n=62), and a subset of K. pneumoniae BSI isolates (n=38) from other paediatric wards in the hospital, collected over a 4 year period were studied. After whole genome sequencing, the strain sequence types (STs), plasmid types, virulence and resistance genes were identified. One ST340 clone, part of clonal complex 258 (CC258) and an ST that drives hospital outbreaks worldwide, harbouring numerous resistance genes and plasmids, was implicated as the likely cause of the outbreak. This study contributes molecular information necessary for tracking and characterizing this important hospital pathogen in sub-Saharan Africa.

Functional genomics of bacteria commonly aims at establishing genotype-phenotype links in microorganisms of industrial, technological and biomedical relevance. In this regard, random transposon mutagenesis coupled to high-throughput next-generation sequencing approaches, termed transposon-insertion sequencing (TIS), has emerged as a robust, genome-wide alternative to perform functional genome analysis. Though these approaches have been used in a large number of studies involving pathogenic and clinically relevant bacteria, they have received little attention in the fields of commensal and potentially beneficial bacteria, including probiotic microorganisms. In this chapter, we describe the implementation of the TIS method Transposon-Directed Insertion Sequencing to describe the set of essential genes in a representative strain of a genus encompassing several commensal and potentially probiotic bacteria and discuss considerations when applying similar methodological approaches to other Bifidobacterium species/strains of interest.

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion-site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria., L.M.N. was supported by Medical Research Council (MRC) Grant MR/N023250/1 and a Marie Curie Fellowship (PIIF-GA-2013-625318). A.F. was supported by MRC Grants MR/K001930/1 and MR/N023250/1 and Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/N002539/1. R.C.D.F. and D.A.I.M. were supported by the MRC Career Development Award MR/M009505/1. D.A.-J. acknowledges support by the MINECO Contract CTQ2016-76941-R, Fundación Biofísica Bizkaia, the Basque Excellence Research Centre (BERC) program and IT709-13 of the Basque Government, and Fundación BBVA. M.A.S.-P. was supported by the MINECO under the “Juan de la Cierva Postdoctoral program” (position FJCI-2015-25725).

A series of hybrid compounds that incorporated anthranilic acid with activated 1H-indoles through a glyoxylamide linker were designed to target bacterial RNA polymerase holoenzyme formation using computational docking. Synthesis, in vitro transcription inhibition assays, and biological testing of the hybrids identified a range of potent anti-transcription inhibitors with activity against a range of pathogenic bacteria with MICs as low as 3.1 μM. A structure activity relationship study identified the key structural components necessary for inhibition of both bacterial growth and transcription. Correlation of in vitro transcription inhibition activity with in vivo mechanism of action was established using fluorescence microscopy and resistance passaging using Gram-positive bacteria showed no resistance development over 30 days. Furthermore, no toxicity was observed from the compounds in a wax moth larvae model, establishing a platform for the development of a series of new antibacterial drugs with an established mode of action.

Acinetobacter baumannii is a ubiquitous Gram-negative bacterium, that is associated with significant disease in immunocompromised individuals. The success of A. baumannii is partly attributable to its high level of antibiotic resistance. Further, A. baumannii expresses a broad arsenal of putative zinc efflux systems that are likely to aid environmental persistence and host colonization, but detailed insights into how the bacterium deals with toxic concentrations of zinc are lacking. In this study we present the transcriptomic responses of A. baumannii to toxic zinc concentrations. Subsequent mutant analyses revealed a primary role for the resistance-nodulation-cell division heavy metal efflux system CzcCBA, and the cation diffusion facilitator transporter CzcD in zinc resistance. To examine the role of zinc at the host-pathogen interface we utilized a murine model of zinc deficiency and challenge with wild-type and czcA mutant strains, which identified highly site-specific roles for zinc during A. baumannii infection. Overall, we provide novel insight into the key zinc resistance mechanisms of A. baumannii and outline the role these systems play in enabling the bacterium to survive in diverse environments.

Increasing antimicrobial resistance and limited alternative treatments have led to fluoroquinolone-resistant Shigella strain inclusion on the WHO global priority pathogens list. In this study we characterized multiple Shigella isolates from Malawi with whole genome sequence analysis, identifying the acquirable fluoroquinolone resistance determinant qnrS1.

Bacterial coordination of stress resistance mechanisms in harsh environments is key to long-term survival and evolutionary success. In many Gram-negative pathogens, both general- and specific-stress response are controlled by alternative sigma factors such as RpoS. The critically important pathogen Acinetobacter baumannii is notoriously recalcitrant to external stressors, yet it lacks RpoS, so the molecular control of its resilience remains unclear. Here, we used transposon insertion sequencing to characterize the molecular responses of Acinetobacter baumannii to two biologically-important metals stressors, zinc and copper, and discovered that the transcriptional regulator DksA acts as a major regulatory stress-protection switch. We mapped the highly pleiotropic nature of DksA using transcriptomics and phenomics and found that it controls ribosomal protein expression, metabolism of gluconeogenic substrates and survival in stresses that cause oxidative damage. A. baumannii strains lacking DksA were no longer virulent in both murine and Galleria mellonella in vivo models. In vitro, DksA mutants exhibited increased sensitivity to human serum and antibiotics yet promoted biofilm and capsule formation. Our study provides detailed insight into the unique role that DksA plays in stress protection and virulence for A. baumannii and lays the groundwork for understanding of RpoS-independent regulatory general stress response.

Biocides, such as antiseptics and disinfectants, are used ubiquitously for hygiene in households and for life-saving infection control in hospitals. An increasing concern is that the widespread use of biocides may contribute to the emergence and spread of multidrug-resistant bacteria. We performed transposon directed insertion site sequencing (TraDIS) to identify genes and key cellular pathways of the multidrug resistant nosocomial pathogen Acinetobacter baumannii, that affect host fitness during exposure to a panel of ten structurally-diverse and clinically-relevant biocides: silver nitrate, benzalkonium, cetyltrimethylammonium bromide (CTAB), chlorhexidine, triclosan, chloroxylenol, polyvidone iodine, bleach, glutaraldehyde and ethanol. Multiple genes encoding proteins localised either in the cell envelope or in the cytoplasm were shown to affect biocide susceptibility. These proteins are involved in multiple processes including fatty acid biogenesis, multidrug efflux, the tricarboxylic acid cycle, cell respiration and cell division, suggesting that these biocides may have intracellular targets in addition to their known effects on the cell envelope. Based on the importance of cell respiration genes for A. baumannii fitness on biocides, we proposed and confirmed that apart from triclosan, the other 9 biocides at sub-inhibitory concentration can dissipate the membrane potential and lead to A. baumannii tolerance to antibiotics that have intracellular targets. Our results support the concern that residual biocides in clinical or community environments can promote the development of antibiotic resistance in pathogenic bacteria.

Aim Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts. Methods We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. Results At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. Conclusions Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.

Galleria mellonella has risen to fame as an invertebrate model organism given its ethical advantages, low maintenance costs, rapid reproduction time, short life cycle, high number of progeny, tolerance for human body temperatures, innate immune system and similarities to mammalian host models. It is increasingly being utilised to evaluate in vivo toxicity and efficacy of chemical compounds and antimicrobials, modelling microbial (bacterial, fungal, viral) pathogenicity and assessing host-pathogen interaction during infection. During this molecular age of genomic, transcriptomic, proteomic, and genetic manipulation approaches, our understanding of microbial pathogenicity and host-pathogen interactions has deepened from high-throughput molecular studies performed in G. mellonella. In this review, we describe the use of G. mellonella in a broad range of studies involving omics, drug resistance, functional analysis and host-microbial community relationships. The future of G. mellonella in the molecular age is bright, with a multitude of new approaches and uses for this model from clinical to biotechnological on the horizon.

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, soil and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Although metal ion homeostasis in the hospital pathogen Acinetobacter baumannii is known to contribute to pathogenesis, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. Further, we show that the CzcCBA heavy metal efflux system also contributes to cadmium efflux. Analysis of the A. baumannii metallome under cadmium stress showed zinc depletion and copper enrichment, which are likely to influence cellular fitness. Overall, this work expands our understanding of the role of membrane transporters in A. baumannii metal ion homeostasis.IMPORTANCECadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, which includes Acinetobacter baumannii. Here we show that A. baumannii utilises a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux, to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be the primary means by which cadmium exerts toxicity upon the bacterium.

BackgroundSplicing factor proline and glutamine rich (SFPQ, also known as polypyrimidine tract-binding protein-associated-splicing factor, PSF) is a RNA-DNA binding protein with roles in key cellular pathways such as DNA transcription and repair, RNA processing and paraspeckle formation. Dysregulation of SFPQ is emerging as a common pathological feature of multiple neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Increased retention of SFPQ intron nine and nuclear loss of the protein have been linked to multiple genetic subtypes of ALS. Consequently, SFPQ dysregulation has been hypothesised to be a common pathological feature of this highly heterogeneous disease.MethodsThis study provides a comprehensive assessment of SFPQ pathology in large ALS patient cohorts. SFPQ gene expression and intron nine retention were examined in multiple neuroanatomical regions and blood from ALS patients and control individuals using RNA sequencing (RNA-Seq) and quantitative PCR (RT-qPCR). SFPQ protein levels were assessed by immunoblotting of patient and control motor cortex and SFPQ expression pattern was examined by immunofluorescent staining of patient and control spinal cord sections. Finally, whole-genome sequencing data from a large cohort of sporadic ALS patients was analysed for genetic variation in SFPQ.ResultsSFPQ intron nine retention was significantly increased in ALS patient motor cortex. Total SFPQ mRNA expression was significantly downregulated in ALS patient motor cortex but not ALS patient blood, indicating tissue specific SFPQ dysregulation. At the protein level, nuclear expression of SFPQ in both control and patient spinal motor neurons was highly variable and nuclear depletion of SFPQ was not a consistent feature in our ALS cohort. However, we did observe SFPQ-positive cytoplasmic ubiquitinated protein aggregates in ALS spinal motor neurons. In addition, our genetic screen of ALS patients identified two novel, and two rare sequence variants in SFPQ not previously reported in ALS.ConclusionsThis study shows that dysregulation of SFPQ is a feature of ALS patient central nervous system tissue. These findings confirm SFPQ pathology as a feature of ALS and indicate that investigations into the functional consequences of this pathology will provide insight into the biology of ALS.

A suspected outbreak of multi-drug resistant (MDR) Klebsiella pneumoniae in a Malawian neonatal unit was investigated using whole-genome sequencing. Strain-types, virulence and resistance genes of K. pneumoniae isolated from patients from the hospital over a four-year period were identified. A MDR ST340 clone was implicated as the likely outbreak cause.

Resistance to currently available antifungal drugs has quietly been on the rise but overshadowed by the alarming spread of antibacterial resistance. There is a striking lack of attention to the threat of drug resistant fungal infections, with only a handful of new drugs currently in development. Given that metal complexes have proven to be useful new chemotypes in the fight against diseases such as cancer, malaria, and bacterial infections, it stands to reason to explore their possible utility in treating fungal infections. Herein we report a series of cobalt(III) Schiff base complexes with broad spectrum antifungal activity. Some of these complexes (1-3) show minimum inhibitory concentrations (MIC) in the low micro- to nanomolar range against a series of Candida and Cryptococcus yeasts. Additionally, we demonstrate that these compounds show no cytotoxicity against both bacterial and human cells. Finally, we report first in vivo toxicity data on these compounds in Galleria mellonella, showing that doses as high as 266 mg/kg are tolerated without adverse effects, paving the way for further in vivo studies of these complexes.

Resistance to currently available antifungal drugs has quietly been on the rise but overshadowed by the alarming spread of antibacterial resistance. There is a striking lack of attention to the threat of drug‐resistant fungal infections, with only a handful of new drugs currently in development. Given that metal complexes have proven to be useful new chemotypes in the fight against diseases such as cancer, malaria, and bacterial infections, it is reasonable to explore their possible utility in treating fungal infections. Herein we report a series of cobalt(III) Schiff base complexes with broad‐spectrum antifungal activity. Some of these complexes show minimum inhibitory concentrations (MIC) in the low micro‐ to nanomolar range against a series of Candida and Cryptococcus yeasts. Additionally, we demonstrate that these compounds show no cytotoxicity against both bacterial and human cells. Finally, we report the first in vivo toxicity data on these compounds in Galleria mellonella, showing that doses as high as 266 mg kg−1 are tolerated without adverse effects, paving the way for further in vivo studies of these complexes., Metals vs. fungi: Given that metal complexes have proven to be useful new chemotypes in the fight against diseases such as cancer, malaria, and bacterial infections, it is reasonable to explore their possible utility in treating fungal infections. Herein we report a series of cobalt(III) Schiff base complexes with broad‐spectrum antifungal activity against Candida and Cryptococcus yeasts, with MICs in the low micro‐ to nanomolar range.

Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacteria that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used in this species. Here we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses and transport were under-represented. Comparison of the essential gene sets of Pf-5 and P. aeruginosa PA14, an opportunistic human pathogen, provides insight into the biological processes important for their different lifestyles. Key differences include cytochrome c biogenesis, formation of periplasmic disulfide bonds, lipid biosynthesis, ribonuclease activity, lipopolysaccharides and cell surface structures. Comparison of the Pf-5 in silico predicted and in vitro determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles can improve our understanding of the biological processes important for survival and growth in different environmental niches.ImportanceEssential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens, but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analysis of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of P. aeruginosa PA14, an opportunistic human pathogen, provides insight into differences in gene essentiality which may be linked to their different lifestyles.

Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3–4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the ‘no observable adverse effect level’ (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models., Peptide antibiotics often display a very narrow therapeutic index. Here, the authors present an optimized peptide antibiotic with broad-spectrum in vitro activities, in vivo efficacy in multiple disease models against multidrug-resistant Gram-negative infections, and reduced toxicity.