2025-04-10 | | Total: 21
Here the infection dynamics, replication, and pathogenicity of a recombinant virus containing a deletion of ORF6 (rWA1ΔORF6) on the backbone of the highly virulent SARS-CoV-2 WA1 virus (rWA1) were investigated and compared to the parental rWA1 virus. While both rWA1 and rWA1ΔORF6 viruses replicated efficiently in cultured cells, the rWA1ΔORF6 virus produced smaller plaques, suggesting reduced cell-to-cell spread. Luciferase reporter assays revealed immune suppressing effects of ORF6 on interferon and nuclear factor kappa B (NF-κB) signaling pathways. Pathogenesis assessment in cats revealed that animals inoculated with rWA1 were lethargic and presented with fever on days 2 and 4 post-infection (pi), whereas rWA1ΔORF6-inoculated animals developed subclinical infection. Additionally, animals inoculated with rWA1ΔORF6 presented reduced infectious virus shedding in nasal and oral secretions and broncho-alveolar lavage fluid when compared with the rWA1-inoculated cats. Similarly, the rWA1ΔORF6-inoculated cats presented reduced virus replication in the respiratory tract as evidenced by lower viral loads and reduced lung inflammation on day 3 and 5 pi when compared to rWA1-inoculated animals. Host gene transcriptomic analysis revealed marked differences in differentially expressed genes (DEG) in the nasal turbinate of animals infected with rWA1 when compared to rWA1ΔORF6. Importantly, type I interferon signaling was significantly upregulated in rWA1ΔORF6 infected cats when compared to rWA1-inoculated animals, which is correlated to the reduced replication of rWA1ΔORF6 in the upper and lower respiratory tracts of infected animals. Collectively, these results demonstrate that the SARS-CoV-2 ORF6 is an important virulence determinant of the virus contributing to the modulation of host antiviral immune responses.
Cyanobacteria play vital roles in aquatic ecosystems by driving photosynthesis, nitrogen fixation, carbon sequestration, and forming symbiotic relationships with diverse organisms. However, their proliferation can trigger harmful algal blooms, posing risks to aquatic biodiversity and public health. Despite their ecological significance, the interplay between cyanobacterial genomic traits and ecosystem dynamics remains poorly resolved. Here, we employed culture-independent metagenomic approaches to reconstruct cyanobacterial metagenome-assembled genomes (MAGs) from Chilika Lagoon, India, and investigate their spatiotemporal distribution and functions. Our analysis revealed distinct temporal patterns in cyanobacterial MAG abundance, with salinity emerging as the primary environmental driver of community structure and functional gene composition. Genes associated with biogeochemical cycling and toxin synthesis displayed pronounced seasonal variation, suggesting that functional genomic traits, rather than taxonomic identity govern species selection. Notably, five MAGs harboured the complete phosphate acetyltransferase-acetate kinase (Pta-Ack) pathway, a critical component of the Wood–Ljungdahl pathway, indicating an underappreciated potential for alternative carbon fixation mechanisms alongside the canonical Calvin-Benson-Bassham cycle. Furthermore, genomic variability, rather than phylogenetic relatedness was the dominant factor shaping cyanobacterial dynamics in the lagoon. This study establishes a direct link between physicochemical fluctuations and cyanobacterial functional diversity, offering critical insights into how climate-driven changes in salinity and nutrient regimes may influence aquatic ecosystems. By elucidating the genomic basis of cyanobacterial adaptation, these findings enhance our capacity to predict ecological outcomes of harmful algal blooms and inform strategies to safeguard ecosystem services in vulnerable coastal habitats.
Cationic antimicrobial peptides are a large family of host defense molecules with diverse sequences and structures. Here, we present a computational and experimental pipeline for quantifying the membrane-permeabilizing effects, as well as the intracellular impacts on ribosome and DNA organization, of cationic peptides in growing Escherichia coli cells at high temporal and subcellular resolution. Applying this pipeline to 11 diverse natural peptides and synthetic peptidomimetics uncovered shared antibacterial activities, but with different kinetics that categorized them into two classes: class I, where inner membrane permeabilization predominantly correlates with growth inhibition, and class II, where it does not. With the class I peptides, intracellular ribosome and DNA reorganization, along with growth inhibition, occurred abruptly and was coupled with inner membrane permeabilization. However, this coupling led to rapid peptide absorption by the first exposed cells and poor antibacterial activity against dense cell populations. With the class II peptides, ribosome/DNA reorganization and growth inhibition occurred more gradually, as inner membrane permeabilization was either absent or delayed. This was offset by slower intracellular uptake and greater efficacy against high cell densities. These kinetic differences reveal functional trade-offs between classes that have immunological and therapeutic implications.
Lipopolysaccharide (LPS) assembly at the surfaces-exposed leaflet of the bacterial outer membrane (OM) is mediated by the OM LPS translocon. An essential transmembrane beta-barrel protein, LptD, and a cognate lipoprotein, LptE, translocate LPS selectively into the OM external leaflet via a poorly understood mechanism. Here, we characterize two additional translocon subunits, the lipoproteins LptM and LptY (formerly YedD). We use single-particle cryo-EM analysis, functional assays and molecular dynamics simulations to visualize the roles of LptM and LptY at the translocon holo-complex LptDEMY, uncovering their impact on LptD conformational dynamics. Whereas LptY binds and stabilizes the periplasmic LptD beta-taco domain that functions as LPS receptor, LptM intercalates the lateral gate of the beta-barrel domain, promoting its opening and access by LPS. Remarkably, we demonstrate a conformational switch of the LptD beta-taco/beta-barrel interface alternating between contracted and extended states. The LptD beta-strand 1, which defines the mobile side of the lateral gate, binds LPS and performs a stroke movement toward the external leaflet during the contracted-to-extended state transition. Our findings establish a detailed mechanistic framework explaining the selective transport of LPS to the membrane external leaflet.
Fluctuating conditions drive adaptive evolution, yet understanding how genomes evolve under stable conditions over extended periods remains a major challenge, since most research on microbial evolution relies on short-term experiments or phylogenetic comparisons. The Movile Cave, isolated from external influences 5.5 million years, offers a unique opportunity to explore microbial evolution under prolonged environmental stability. Here, we analyzed metagenome-assembled genomes from this cave, revealing that prokaryotes exhibit lower gene diversity and higher levels of pseudogenization compared to those from non-isolated environments, mainly affecting housekeeping functions involved in translation. Functional redundancy across genomes remained comparable to related habitats. Our results suggest that pseudogenization may serve as a fine-tuning mechanism to reduce excess redundancy. Although horizontal gene transfer is limited overall, the cave virome seems to contribute to microbial adaptation through the transfer of auxiliary metabolic genes. Movile microorganisms harbor fewer phage-defense systems than counterparts in related environments, suggesting a long-term adaptation to a relatively stable virosphere. Our findings indicate that prolonged isolation under stable selective pressures does not necessarily lead to major genomic divergence, but rather promotes adaptive gene loss. This study provides key insights into how long-term stability shapes microbial genome evolution and ecosystem function.
Previous comparative and experimental evolution studies have suggested how fungi may rapidly adapt to new environments, but direct observation of in situ selection in fungal populations is rare due to challenges with tracking populations over human time scales. We monitored a population of Penicillium solitum over eight years in a cheese cave and documented a phenotypic shift from predominantly green to white strains. Diverse mutations in the alb1 gene, which encodes the first protein in the DHN-melanin biosynthesis pathway, explained the green to white shift. A similar phenotypic shift was recapitulated with an alb1 knockout and experimental evolution in laboratory populations. The most common genetic disruption of the alb1 genomic region was caused by putative transposable element insertions upstream of the gene. White strains had substantial downregulation in global transcription, with genetically distinct white strains possessing divergent shifts in expression of different biological processes. White strains outcompeted green strains in co-culture, but this competitive advantage was only observed in the absence of light, suggesting that loss of melanin is only adaptive in dark conditions. Our results illustrate how fermented food production by humans provides opportunities for relaxed selection of key fungal traits over short time scales. Unintentional domestication of microbes by cheesemakers may provide opportunities to generate new strains for innovation in traditional cheese production.
Background: The axenisation of phototrophic eukaryotic microalgae has been studied for over a century, with antibiotics commonly employed to achieve axenic cultures. However, this approach often yields inconsistent outcomes and may contribute to the emergence of antibiotic-resistant microbes. A comprehensive review of microalgal species and the methods used to achieve axeny could provide insights into potentially effective workflows and identify gaps for future exploration. Methods: Scholarly databases were systematically searched, supplemented by citation network analysis and AI-assisted tools, to collect studies on achieving axenic phototrophic eukaryotic microalgae cultures. Data about microalgal species, axenisation workflows, outcomes, and related factors (e.g., sampling locations, axenisation confirmation methods) were summarised. Network component analysis was used to identify clusters of commonly reported methods for diatoms, dinoflagellates, and green algae. A scoring framework was developed to assess the quality and reliability of evidence presented in the studies. Results: Emerging patterns suggest that workflows involving filtration, washing, and micropicking are frequently reported for diatoms; micropicking, subculturing, and flow cytometry for dinoflagellates; and anoxy, photosensitisation, and streak plating for green algae. Evidence from the literature indicates that a combination of microscopy (e.g., epifluorescence), cell counting (e.g., agar plating), and sequencing (16S and/or 18S) could enhance confidence in confirming axeny. Conclusion: While antibiotics dominate current practices, alternative pathways for achieving axenic cultures are identifiable through network component analysis. Higher confidence in these methods depends on improved experimental designs and high-quality reporting.
Pacific Islanders have hyperendemic rates of Chlamydia trachomatis (Ct) sexually transmitted infections (STIs) and remain underrepresented in research. Using metagenomic shotgun sequencing, we investigated the impact of azithromycin (AZ) treatment on microbial communities, function and the resistomes of paired vaginal, endocervical and rectal microbiomes among three cohorts of women in Fiji: Ct-infected women treated at baseline who cleared or had persistent infection at follow-up compared to uninfected, untreated women at either time point. Several species were significantly associated with Ct persistence: endocervical Neisseria gonorrhoeae (Ng); and rectal Ng and species associated with biofilm formation. Women with Ct clearance and persistent were also significantly more likely to have high-risk (hr)HPV types at follow-up compared to uninfected, untreated women. Three distinct microbiome phylotypes were identified based on Bray-Curtis hierarchical clustering with evidence for genital-to-rectal and rectal-to-genital transmission among all cohorts. The AZ resistance gene ermB in L. iners (Ln) was significantly higher in the endocervix of both treatment cohorts at follow-up compared to baseline, while the tetracycline resistance gene tetM was highly prevalent among all cohorts at both time points. G. vaginalis (Gv) had a high prevalence of the AZ resistance gene ermX in the rectum of all cohorts. These Gv strains were associated with moderate/high biofilm formation. Both Ln and Gv increased in abundance post-treatment, which could perpetuate Ct persistence and increase the risk for other STIs. Nonsynonymous mutations in the Ct rplV gene were highly prevalent in the Ct persistent cohort in all anatomic sites at both time points and mapped to mutations associated with AZ resistance. Our results reveal perturbing effects of azithromycin treatment with increased risk of hrHPV, Ng and other STIs, and azithromycin resistance among co-infecting pathogens with potential homotypic resistance in Ct that may impact successful treatment of Ct, indicating the need for novel therapeutic strategies to treat Ct and protect and restore the microbiome.
Studies of antimicrobial therapeutics have traditionally neglected the contribution of the host in determining the course of treatment and its outcome. One critical host element, which shapes the dynamics of treatment is the innate immune system. Studies of chemotherapeutics and complementary therapies such as bacteriophage (phage), are commonly performed with mice that purposely have an ablated innate immune system. Here, we generate a mathematical and computer-simulation model of the joint action of antibiotics, phage, and phagocytes. Our analysis of this model highlights the need for future studies to consider the role of the innate immune system of a host in determining treatment outcomes. Critically, our model predicts that the conditions under which resistance to the treatment agent(s) will emerge are much narrower than commonly anticipate. We also generate a second model to predict the dynamics of treatment when multiple phages are used. This model provides support for the application of cocktails to treat infections rather than individual phages. Overall, this study provides hypotheses that can readily be tested experimentally with both in vitro and in vivo experiments.
Heat stress poses a significant constraint on the annual production of L. edodes, a challenge that has been intensified by global warming. Previous studies have established a close relationship between intracellular IAA content and heat tolerance in L. edodes. However, the specific changes in the IAA synthesis pathway and the target genes regulated by IAA under heat stress remain unclear. We employed targeted metabolomics and transcriptomics analysis to investigate the alterations in IAA synthesis pathways and gene expression in both heat-tolerant and heat-sensitive strains at various time points during heat stress. Our findings revealed that IAA is primarily synthesized via the TAM and IPYA pathways in L. edodes. Heat-sensitive strain Y3357 exhibited excessive accumulation of tryptamine after heat stress. Silencing of the key genes of IAA synthesis include tryptophan decarboxylase (TDC) and tryptophan transaminase (TAA) in strain S606 could reduce the thermotolerance of L. edodes mycelia. Transcriptome analysis revealed that heat-tolerant strain S606 had an earlier response to protein folding and mitochondrial gene expression compared to the heat-sensitive strain Y3357. Additionally, most genes in the MAPK signaling pathway were up-regulated after 10-24 hours of heat stress, with auxin response elements (AREs) identified in their promoters. These results suggest that the excessive tryptamine accumulation is the newly discovered limiting factor for thermotolerance, and the expression levels of key genes in the IAA synthesis pathway could directly influence hyphal thermotolerance. This study provides a new perspective on the mechanism by which IAA and its synthesis precursors affects thermotolerance of L. edodes.
The genus Hanseniaspora includes apiculate yeasts commonly found in fruit- and fermentation-associated environments. Their genetic diversity and evolutionary adaptations remain largely unexplored despite their ecological and enological significance. This study investigated the phylogenetic relationships, genome structure, selection patterns, and phenotypic diversity of Hanseniaspora species isolated from wine environments, focusing on Hanseniaspora uvarum, the most abundant non-Saccharomyces yeast in wine fermentation. A total of 151 isolates were sequenced, including long-read genomes for representatives of the main phylogenetic clades. Comparative genomics revealed ancestral chromosomal rearrangements between the slow- (SEL) and fast-evolving (FEL) lineages that could have contributed to their evolutionary split, as well as significant loss of genes associated with mRNA splicing, chromatid segregation and signal recognition particle protein targeting specifically in the FEL lineage. Pangenome analysis within H. uvarum identified extensive copy number variation (CNV), particularly in genes related to xenobiotic tolerance, nutrient transport and metabolism. Investigation into the selective landscape following the FEL/SEL divergence identified diversifying selection in 229 genes in the FEL lineage, with significant enrichment in genes within the lysine biosynthetic pathway, suggesting a key role for this amino acid in early FEL adaptation. In H. uvarum, signatures of recent positive selection were detected in genes linked to sulphur assimilation, sterol biosynthesis and glycerol production, indicating potential adaptation to the stresses imposed by grape and wine fermentation. Furthermore, phenotypic screening of 113 isolates revealed substantial intraspecific diversity, with specific species exhibiting enhanced ethanol, osmotic, copper, SO₂, and cold tolerance. These findings provide novel insights into the genomic evolution and functional diversity of Hanseniaspora, expanding our understanding of yeast adaptation to wine fermentation and laying the foundation for targeted gene investigations within this important genus.
Background: Bacterial resistance, exacerbated by multidrug-resistant Gram-negative (GN) pathogens, poses a public health threat due to their impermeable envelopes, which block many antibiotics. Objectives: We aimed to develop a high-throughput screening (HTS) method to identify small molecules targeting GN bacterial envelopes and assess their antibacterial potential. Methods: Envelope disruption in Escherichia coli and Pseudomonas aeruginosa was assessed using a beta-galactosidase (LacZ)/CPRG reporter assay in LB at 37 degrees C. The assay was validated through screening the LOPAC1280 and KD24761 compound libraries. Concentration-response relationships, permeabilisation constants (K50), co-permeabilisation assays, minimal inhibitory concentration (MIC) measurements, and bacterial microscopy post-MICs were performed. Results: The assay demonstrated robust performance, evidenced by high Z-prime factor and signal-to-noise (S/N) ratios. Screening identified 57 active compounds (1.2 percent of the library), including beta-lactams and three non-antibiotic molecules-suloctidil, isorotenone, and alexidine-that exhibited concentration-dependent antibacterial activity. Alexidine showed the most potent activity, with the lowest K50 (2.7 x 10^-3 mM) and MICs of 0.004 mM for Escherichia coli and 0.015 mM for Pseudomonas aeruginosa. Suloctidil and isorotenone induced spherical cell morphology, while alexidine induced a filamentous phenotype, indicative of envelope disruption. The assay also identified antibiotics for monotherapy and combination therapy, with ampicillin, alexidine, and suloctidil enhancing chloramphenicol's efficacy against Escherichia coli MG1655. Conclusions: The LacZ/CPRG reporter assay effectively identified compounds targeting bacterial envelopes, including novel molecules with antibacterial activity against GN pathogens, making it a promising tool for antibiotic discovery or combination therapy.
Avian Pathogenic Escherichia coli (APEC), a major bacterial pathogen of poultry, is comprised of a diverse range of high-risk clonal groups. However, phenotypic interactions with the avian host cell and how they may differ between lineages remains poorly understood. Therefore, the ability of predominant and outbreak-associated APEC clonal groups to invade and survive within avian host cells, as well as virulence within the Galleria mellonella infection model was investigated. The molecular characterisation of APEC isolated from an outbreak of colibacillosis in turkey poults in the UK, identified APEC sequence type (ST)-101 as the dominant clonal group, carrying a high number of virulence factors. As such, ST-101 was compared as an outbreak-associated lineage to a range of predominant APEC high-risk clonal groups (ST-23, ST-140, ST-95, ST-117). Utilising in vitro cell culture models, APEC isolates displayed comparable adhesion to 8E11 chicken epithelial gut and HD11 chicken macrophage cell lines. However, a trend of increased invasion of the 8E11 cells, and intracellular survival within HD11 macrophages was observed for ST-95, ST-101, and ST-140 APEC, relative to ST-23 and ST-117, suggestive of pronounced phenotypic differences between clonal groups. However, in HD11 cell assays, no difference in magnitude of elicited immune response was observed between lineages, indicating lineages had differing capacities to resist phagocyte killing. In vivo virulence in the Galleria mellonella infection model was also observed to differ between APEC genotypes, with ST-117 inducing the highest mortality, despite the comparatively lower epithelial invasion and intramacrophage survival to other lineages. Collectively, this suggests a distinct phenotypic profile associated with high-risk clonal groups within APEC, potentially allowing the future development of broad-spectrum disease management strategies.
Staphylococcus aureus is a leading cause of healthcare-associated pneumonia, contributing significantly to morbidity and mortality worldwide. As a ubiquitous colonizer of the upper respiratory tract, S. aureus must undergo substantial metabolic adaptation to achieve persistent infection in the distinctive microenvironment of the lung. We observed that fumC, which encodes the enzyme that converts fumarate to malate, is highly conserved with low mutation rates in S. aureus isolates from chronic lung infections. Fumarate, a pro-inflammatory metabolite produced by macrophages during infection, is regulated by the host fumarate hydratase (FH) to limit inflammation. Here, we demonstrate that fumarate, which accumulates in the chronically infected lung, is detrimental to S. aureus, blocking primary metabolic pathways such as glycolysis and oxidative phosphorylation (OXPHOS). This creates a metabolic bottleneck that drives staphylococcal FH (FumC) activity for airway adaptation. FumC not only degrades fumarate but also directs its utilization into critical pathways including the tricarboxylic acid (TCA) cycle, gluconeogenesis and hexosamine synthesis to maintain metabolic fitness and form a protective biofilm. Itaconate, another abundant immunometabolite in the infected airway enhances FumC activity, in synergy with fumarate. In a mouse model of pneumonia, a ΔfumC mutant displays significant attenuation compared to its parent and complemented strains, particularly in fumarate- and itaconate-replete conditions. Our findings underscore the pivotal role of immunometabolites in promoting S. aureus pulmonary adaptation.
Marine alveolates (MALVs) are diverse, primarily parasitic micro-eukaryotes that significantly impact marine ecosystems. The life cycles of most MALVs remain elusive and the role of sexual reproduction in these organisms is a key question that may determine their ecological success. In this study we focus on a widespread dinoflagellate parasite of bloom-forming dinoflagellates, Amoebophrya. After infection, we identified two distinct spores, differing in size, ultrastructure, swimming behavior, lifespan, gene expression, and metabolite composition. The smaller spores serve as infectious propagules, equipped with an apical complex for host invasion. They exhibit a distinct, shorter, and straighter swimming pattern, likely optimized for an extended lifespan while enhancing dispersion and chance for host encounters. Transcriptomic analysis reveals that these smaller spores are primed for efficient protein synthesis upon initiating a new infection. Conversely, the larger spores cannot infect new hosts and are characterized by the expression of meiotic genes, underscoring their sexual nature. They have a shorter lifespan, exhibit more tortuous movement, along display condensed chromosomes, signaling readiness for mating. Interestingly, infected hosts already express meiotic genes, and a single infected host only produces progeny of the same spore type, suggesting that cell fate is determined prior to spore release. Our study provides one of the first formal demonstrations of a sexually specialized cell in MALVs. Isolating compatible strains for cross-breeding and understanding how environmental conditions favor each reproductive route are the next key questions for elucidating the ecological success of MALVs in marine waters.
The invasive red imported fire ant, Solenopsis invicta, poses significant ecological and economic challenges, necessitating sustainable alternatives to conventional pest control. This study evaluates the biocontrol potential of Cunninghamella echinulata isolate SMH-1, an entomopathogenic fungus, through integrated morphological, biochemical, and metabolomic analyses. Light and scanning electron microscopy elucidated infection dynamics, revealing conidial germination, cuticular penetration, and systemic hyphal proliferation, culminating in complete cadaver colonization. At 1×108 conidia/mL, SMH-1 achieved 100% mortality in S. invicta workers within 7 days, with virulence maintained across thermal gradients (10–30°C). Acetone extracts of SMH-1 induced dose-dependent lethality, reaching 100% mortality at 1,000 mg/L. Nineteen significantly differentiated metabolites were identified from phospholipids category through UPLC/MS analysis of isolate SMH-1. The derived compounds also showed strong potential against S. invicta, among which dihydrocoumarin caused 100% mortality over 12-days of exposure. The bioactivity of isolate SMH-1 (Conidia), (extracts) and (dihydrocoumarin) against S. invicta revealed significantly differentiated metabolites as (Sulfolithocholylglycine, PE(18_1(9Z)_0_0), CE(12:0), LysoPI(18:1(9Z)/0:0) and Hydroxydestruxin B), (Tropine, lignoceric acid, 9-deoxy-9-methylene-16,16-dimethyl -PGE2, Flecainide and Jubanine B), and (3-O-alpha-D-Glucopyranuronosyl-D-xylose, Rilmakalim, 2-Amino-3-methyl-1-butanol, 2,4-Dimethyl-1,3-oxazole-5-carboxylic acid and Caffeinol), respectively. Citrate cycle (TCA) was significantly impacted by differential expression of pyruvate, pyruvic acid, succinic acid and fumaric acid. These findings underscore SMH-1’s multifaceted biocontrol potential, combining direct pathogenicity, thermo-tolerance, and metabolite-mediated toxicity. By delineating host-pathogen interactions and metabolic disruptions, this work advances C. echinulata isolate SMH-1 as a sustainable candidate for integrated S. invicta management, offering an eco-friendly alternative to synthetic insecticides.
Respiratory syncytial virus (RSV) disease burden is greatest between six weeks and 6 months of life, with young age the most common risk factor among hospitalised children. A robust innate immune response in the airway epithelium is crucial for mitigating RSV-associated disease, but early-life immune responses to infection remain largely unexplored. RNA-seq analysis of RSV-infected primary nasal epithelial cell cultures from healthy infants at birth and one year revealed diminished expression of interferon epsilon (IFNE), a poorly characterised type I IFN, in newborns versus one-year. We hypothesised, therefore, that IFNE plays an important role during infant RSV infection. We found that IFNE is endogenously expressed in airway epithelial cell lines. Recombinant human IFNε (rhIFNε) induced an antiviral state against an RSV clinical isolate and related Sendai virus, but not SARS-CoV-2 under the conditions tested. The antiviral potency of rhIFNε was diminished relative to rhIFN beta (rhIFNβ1) or rhIFN lambda-1 (rhIFNλ1), as evidenced by IC50 data. Importantly, rhIFNε induced similar ISGs as rhIFNβ1 but demonstrated a transient temporal expression profile that differed from both rhIFNβ1 and rhIFNλ1. These results suggest that lower IFNε expression at birth may contribute to increased susceptibility to severe RSV-associated disease, offering insights into potential therapeutic interventions.
Unrelated pathogens, including viruses and bacteria, use a common DDVF-like short linear motif (SLiM) to interact with cellular kinases of the RSK (p90 S6 ribosomal kinase) family. Such a "DDVF" SLiM occurs in the leader (L) protein encoded by picornaviruses of the genus Cardiovirus, including Theiler's murine encephalomyelitis virus (TMEV), Boone cardiovirus (BCV), and Encephalomyocarditis virus (EMCV). The L-RSK complex is targeted to the nuclear pore, where RSK triggers FG-nucleoporins hyperphosphorylation, thereby causing nucleocytoplasmic trafficking disruption. In this work, we identified a second SLiM in the L proteins of TMEV and BCV, which enables the L-RSK complex to interact with RAE1 at the level of the nuclear pore complex. AlphaFold predictions suggest that the RAE1-interacting SLiM of L proteins is analogous to that found in unrelated viral proteins such as ORF6 of SARS-CoV-1/2, ORF10 of Kaposi sarcoma-associated herpes virus (KSHV), and the matrix (M) protein of vesicular stomatitis virus (VSV). Co-immunoprecipitations confirmed the interaction between BCV L and RAE1 and competition experiments revealed that L can compete with ORF6 for RAE1 binding, suggesting that BCV and TMEV L proteins interact with RAE1 via the same docking site as M, ORF6, or ORF10. This RAE1 binding SLiM tentatively named "M-acidic", is predicted to occur in other viral proteins such as Rift valley fever virus NSs as well as in cell proteins such as NXF1. BCV and TMEV L proteins use a combination of two independent SLiMs to hijack cellular kinases and retarget those kinases toward the nuclear pore complex.
Intricate communication networks and sensing systems underpin the complexity of microbe-host interactions, enabling spatiotemporal control of optimised microbe-host consortia in a diverse range of ecosystems. A central component of these complex interactomes has been the signalling events that enable recognition of host and microbe, whether that niche be clinical or environmental, human or plant. Coumarins have emerged as significant plant derived signalling molecules shaping microbiome dynamics and pathogen behaviours from a broad spectrum of ecosystems. Here we explored the role of natural and synthetic coumarin compounds in signal interference and control of pathogenesis in bacterial and fungal pathogens, uncovering an important ‘hydroxylation-motif’ in the specific inhibition of two Pseudomonas aeruginosa interspecies and interkingdom communication molecules. Characterisation of the anti-biofilm activity of coumarins revealed changes in exopolysaccharide production independent of the initial attachment phenotype. Molecular modelling provides an insight into the receptor binding dynamics of three closely related natural coumarins, suggesting an intricate and highly specific host-microbe interaction at the species level. As the very real threat of antimicrobial resistance continues to shadow our horizons, phytochemicals such as coumarins have potential to deliver an ecological solution to dysbiosis in the host-microbe interaction.
Caspofungin is an echinocandin antifungal that inhibits glucan synthesis in the fungal cell wall. A Candida parapsilosis bloodstream isolate resistant to echinocandins was recovered from a patient who had undergone allogeneic hematopoietic stem cell transplantation. The FKS1 gene, encoding the target glucan synthase, contained a heterozygous mutation resulting in an I1380T amino acid change, in addition to the naturally occurring P660A polymorphism. When expressed at the equivalent position in the Fks1p protein of C. lusitaniae, P642A and I1359T, alone and in combination, led to 6-, 12-, and ≥256-fold increases in the minimal inhibitory concentration (MIC) of caspofungin, respectively. The caspofungin concentration needed to inhibit 50% of glucan synthase activity was increased 3-, 37-, and 270-fold, respectively. At high drug concentrations, and also in drug-free medium, infrared spectroscopy revealed a decrease in β-glucan content and an increase in chitin in the cell wall of the I1359T Fks1p mutants. Atomic force microscopy showed cell wall damage and cell swelling in both susceptible and resistant strains under caspofungin exposure. Analysis of susceptibility to cell-wall stressors and key factors in cell wall integrity (CWI) and high-osmolarity glycerol (HOG) pathways showed that all strains activated these pathways under caspofungin stress. In the I1359T Fks1p mutants, Mkc1p was constitutively activated even without caspofungin. Deletion of MKC1 restored caspofungin susceptibility, indicating that activation of the CWI pathway is a key molecular determinant of resistance in vitro to caspofungin in these mutants.
Chikungunya virus (CHIKV) is an Alphavirus transmitted by Aedes mosquitoes, causing fever, rash and arthralgia. The function of the CHIKV non-structural protein 3 (nsP3) remains enigmatic. Building on previous studies (Gao et al, 2019), we generated a panel of mutants in a conserved and surface-exposed cluster in the nsP3 alphavirus unique domain (AUD) and tested their replication phenotype using a sub-genomic replicon (SGR) in mammalian and mosquito cells. Three mutants that replicated poorly in mammalian cells showed no defect in mosquito cells. These mutants were temperature-sensitive, rather than species-specific, as they exhibited no replication defect in mammalian cells at sub-physiological temperature (28°C). Similar effects were observed in the context of infectious CHIKV and the closely related O’Nyong Nyong virus. This analysis also revealed that the wildtype SGR replicated more efficiently at 28°C compared to 37°C. This was not due to either impaired interferon responses as the enhancement was observed in Vero cells, or to a defect in eIF2α phosphorylation as treatment with ISRIB, an inhibitor of global translation attenuation, did not compensate for replication defects at 37°C. The phenotype did correlate with enhanced recruitment of stress granule proteins (G3BP, eIF4G and TIA-1) into cytoplasmic sites of genome replication at 28°C. As cells in the periphery will be at sub-physiological temperatures, and will be the first cells infected in the mammalian host following a mosquito bite, we propose that alphaviruses such as CHIKV have evolved mechanisms to both promote viral genome replication and concomitantly limit antiviral responses in these cells. Importance Chikungunya virus (CHIKV) is a re-emerging arbovirus, transmitted by Aedes mosquitos and posing epidemic threats. Arboviruses must be able to replicate efficiently in both the mosquito vector and the mammalian host, at different temperatures. Following a mosquito bite the first cells infected will be in the skin and at sub-physiological temperature (less than 37°C). We show that mutants in one of the CHIKV proteins (nsP3) could not replicate at 37°C, but replicated efficiently in mammalian cells at 28°C. We also showed that wildtype CHIKV replicated more efficiently at 28°C in comparison to 37°C in mammalian cells. We investigated the mechanism behind this observation and showed that at sub-physiological temperatures proteins present in cytoplasmic stress granules were more efficiently recruited to sites of virus replication. We propose that CHIKV has evolved mechanisms to promote their replication in mammalian cells at sub-physiological temperatures to facilitate infection of mammals via a mosquito bite.