mBio

All pathogens rely on dynamic gene expression for host adaptation and disease causation. This is especially important for pathogens that normally are found in the environment, like Cryptococcus neoformans, given that environmental and host conditions are dramatically different. Epigenetic modifications, like those carried out by SET-domain histone lysine methyltransferases (HKMTs), are essential for this genome expression flexibility. While transcription factors and signaling cascades have been well-studied in C. neoformanss host adaptation, the role of HKMTs in this process has not been explored. Here, we characterize the gene SET202, previously reported as a putative HKMT important for growth in host lungs. We show that set202{Delta} mutants have 50% less phagosomal permeabilization than the wild-type fungi, have phagosomal maturation defects, and poor intracellular survival. Additionally, they are defective in traits promoting virulence, including significantly smaller capsules, less melanin, and poor growth under host-like conditions. These phenotypes contributed to reduced virulence in a murine model of infection but, notably, this defect was host-temperature-dependent. In order to explain mechanistically how SET202 deletion could affect such a wide range of processes, we assessed and demonstrated, for the first time, that Set202 is a true histone-3-lysine 36 methyltransferase, supporting its role in epigenetically modifying the fungal genome to enable adaptation to the hostile host conditions. Consequently, our findings support the study of the biological function of Set202, as it can serve as a potential future therapeutic target for the treatment of infections caused by C. neoformans, and possibly other pathogenic fungi.

Taurine is an abundant sulfur-containing metabolite with diverse roles in cellular physiology across many organisms, yet its biosynthesis and biological functions remain largely unexplored in fungi. Here, we provide evidence for endogenous taurine production in the major human fungal pathogen Candida albicans and identify Csd1, a cysteine sulfinic acid decarboxylase (CSAD)-related protein, as a major determinant of this process. Loss of CSD1 nearly abolished intracellular taurine and caused extensive remodeling of sulfur metabolism, including cysteine accumulation and altered abundance of methionine-cycle metabolites. Consistent with these metabolic defects, csd1 cells exhibited impaired growth and increased sensitivity to cysteine, oxidative and osmotic stresses, elevated temperature, reactive sulfur species, and the antifungal drugs amphotericin B and caspofungin. Exogenous taurine selectively rescued a subset of these phenotypes, indicating that CSD1 loss causes both taurine-dependent and broader metabolic defects. Csd1 was also required for normal hyphal morphogenesis, and csd1 cells displayed markedly attenuated virulence in a Galleria mellonella systemic infection model. Comparative sequence analysis revealed conservation of key features of the pyridoxal 5'-phosphate-dependent catalytic machinery shared with mammalian and bacterial CSADs, together with divergence within the predicted substrate-recognition pocket. Our genetic data further suggest that taurine production in C. albicans differs from the canonical metazoan cysteine sulfinic acid pathway and may involve branched or redundant routes. Together, these findings establish endogenous taurine production as a new facet of fungal sulfur metabolism and identify Csd1-dependent metabolism as an important contributor to sulfur homeostasis, stress adaptation, morphogenesis, and pathogenic fitness in C. albicans.

Microbial weathering of minerals represents a key biogeochemical process through which microorganisms access essential nutrients locked within rock and ore substrates. In this study, we investigated the metabolic response of a model fungus during growth on lithium (Li) ore to elucidate the mechanisms underpinning biologically mediated mineral weathering and subsequent metal mobilization. Our results revealed a distinct metabolic shift in the fungus when cultivated on Li ore, characterized by altered patterns of organic acid production and energy metabolism. This shift coincided with measurable weathering of the Li ore matrix and the consequent release of soluble Li into the surrounding environment, demonstrating a direct link between fungal metabolic reprogramming and mineral breakdown. Importantly, these observations are consistent with metabolic shifts documented in previous studies of mineral weathering by this fungus, suggesting that such responses constitute a conserved and reproducible strategy employed during the colonization of mineral substrates. Although the present work was conducted using a single model microorganism under controlled conditions, the findings carry broader ecological implications. Natural microbial communities inhabiting mineral-rich environments may undergo analogous metabolic shifts when weathering minerals to acquire limiting nutrients, thereby contributing to large-scale elemental cycling and metal release. Understanding these processes not only advances fundamental knowledge of microbe-mineral interactions but also informs emerging applications in biomining and bio-based recovery of critical metals such as lithium. Collectively, this study highlights the central role of microbial metabolism in driving mineral weathering and offers a framework for predicting and harnessing similar processes within complex microbial communities. IMPORTANCEThe model fungus exhibits a distinct, reproducible shift in metabolism--particularly in organic acid production and energy pathways--when grown on Li ore, directly linking cellular metabolism to mineral breakdown. This metabolic shift promotes weathering of the ore matrix and the release of soluble lithium, demonstrating a biological route for liberating a critical metal from its mineral host. Because similar shifts occur across different minerals, natural microbial communities likely employ comparable strategies to weather minerals and acquire limiting nutrients, contributing to global elemental cycling. These findings provide a foundation for sustainable biomining and bio-based recovery of lithium and other critical metals, offering a lower-impact alternative to conventional extraction methods.

Small non-coding RNAs (sRNA) modulate diverse bacterial functions ranging from carbon metabolism to virulence gene expression. Previous research showed that the sRNA chaperone Hfq is critical for the fitness of Extraintestinal Pathogenic Escherichia coli (ExPEC), a major cause of both bloodstream and urinary tract infections (UTI). Using the reference ExPEC strain UTI89, we created deletion mutants to probe the effects of seven conserved Hfq-dependent sRNAs (DsrA, RprA, OxyS, RyhB, MicF, MicC, Spf) on resistance to oxidative stress. All of the sRNA mutants grew normally in replete lysogeny broth, but the spf and micC mutants exhibited additive effects upon challenge with reactive oxygen species generated by methyl viologen. In a murine UTI model, the spf mutant resembled the wild-type strain, whereas UTI89{Delta}micC was unable to effectively colonize the bladder despite behaving like wild type within the kidneys. This correlated with a greatly reduced ability of the micC mutant to survive within bladder epithelial cells and paralleled UTI89{Delta}micC defects in gut colonization, virulence in a sepsis model, and complement resistance. Although MicC downregulated expression of its only known target, OmpC, aberrant modulation of this porin did not entirely account for the decreased stress resistance of UTI89{Delta}micC. Rather, RNA-Seq, sRNA target predictions, and in vitro phenotypic assays revealed that MicC can impact multiple pathways linked to niche establishment, including motility, chemotaxis, and various metabolic processes. These data are consistent with MicC serving as a multifunctional regulator of ExPEC stress responses and niche-specific fitness through OmpC-dependent and - independent mechanisms. IMPORTANCEPathogenic strains of Escherichia coli are exceptionally common causes of diarrheal disease, urinary tract infection, sepsis, and meningitis. The ability of these pathogens to cause such a wide range of maladies is in part attributable to their ability to quickly adapt to and thrive within disparate and often hostile environments, including the gut, bladder, kidneys, and bloodstream. Adaptation to new environments requires rapid and precise changes in gene expression. To accomplish this feat, E. coli utilizes a suite of regulatory RNA called small RNA (sRNA). In this paper, we identified the sRNA MicC as a critical facilitator of E. coli fitness and virulence within diverse host environments via effects on the expression of multiple genes involved in bacterial motility, energy acquisition, and various other pathways. Delineating how sRNAs like MicC impact disease processes will aid the development of novel therapeutics to better combat E. coli infections.

Bacteria in the Mycobacteriales order like mycobacteria and corynebacteria surround themselves with a multilayered cell envelope. Their cytoplasmic membrane is fortified by a peptidoglycan cell wall that is decorated with branched arabinogalactan (AG) polymers. The AG glycans are further modified with mycolic acids to form an outer membrane. Biogenesis of this mycomembrane requires the transport of mycolates from their site of synthesis in the cytoplasmic membrane to the cell surface. How mycolate transport is controlled and coordinated with AG synthesis has remained unclear. Mycolate transport is mediated by the essential RND-family transporter MmpL3 in mycobacteria and a pair of related, partially redundant transporters called CmpL1 and CmpL4 in corynebacteria. The acetyltransferase TmaT has also been implicated in mycolate transport in both types of bacteria. In corynebacteria, it is required for the production of acetylated mycolates, and this modification has been proposed to promote mycolate transport via both CmpL transporters in the model organism Corynebacterium glutamicum (Cglu). Here, we reinvestigated the function of TmaT in Cglu and found that it and several factors encoded in the tmaT locus are specifically required for mycolate transport via the CmpL4 transporter pathway. Notably, one of these additional genes encodes the arabinosyltransferase AftD involved in AG biogenesis. TmaT and AftD were found to interact, and our results indicate that this interaction is required for acetylated mycolate production, mycolate transport via the CmpL4 pathway, and normal arabinan synthesis. Thus, the TmaT-AftD interaction may serve as a regulatory link connecting mycolate transport with AG biogenesis. SIGNIFICANCEMycobacteriales bacteria, including pathogens like Mycobacterium tuberculosis (Mtb), have a complex cell surface comprising an inner membrane, a cell wall modified with arabinogalactan (AG), and an outer mycomembrane made of mycolic acids linked to AG polymers. Because these surface biogenesis pathways are targeted by frontline anti-Mtb drugs, there is great interest in elucidating their underlying mechanisms. Here, we identify an interaction between factors involved in mycomembrane (TmaT) and AG biosynthesis (AftD) in the model organism Corynebacterium glutamicum. We show that this interaction is important for proper surface biogenesis and may therefore function to coordinate mycomembrane assembly with AG synthesis. This and other potential regulatory connections controlling envelope biogenesis represent attractive targets for future antibiotic development.

Triazole antifungals inhibit the lanosterol C14-demethylase Cyp51A/B and are the first-line treatment for invasive aspergillosis caused by Aspergillus fumigatus. We previously proposed that triazoles impose a secondary constraint on ergosterol biosynthesis through negative feedback regulation of HMG-CoA reductase (Hmg1) through its sterol sensing domain (SSD), and SSD mutations compromise this regulation to produce resistance. The molecular mechanism underpinning this feedback remained unknown. In this study, we show that voriconazole drives accelerated protein degradation of Hmg1 and that SSD mutation abolishes this regulated degradation. Triazole-induced Hmg1 degradation is proteasome-dependent and partially requires the ERAD E3 ubiquitin ligase, HrdA, and INSIG ortholog, InsA, both conserved regulators of HMG-CoA Reductase abundance. We provide genetic evidence that the initiating negative feedback signal is not triazole acting directly on Hmg1 but lanosterol accumulation resulting from Cyp51A/B inhibition. To test whether depleting Hmg1 protein could re-sensitize resistant strains, we utilized a GFP-targeted degradation system designed to target Hmg1 for degradation without engaging its SSD. Combining this system with voriconazole treatment reduced the MIC at least four-fold in both conidia and established hyphae in susceptible and resistant strains. Together, our findings support a model wherein triazole-induced lanosterol accumulation drives HrdA/InsA-dependent accelerated degradation of Hmg1 and establish that pharmacologically inducing this secondary mechanism can enhance triazole activity in both susceptible and resistant isolates.

The opportunistic pathogen Pseudomonas aeruginosa secretes numerous proteases that disrupt host defenses. Among them, the lysyl endopeptidase PrpL has been implicated in virulence, but its role in inflammatory responses has remained unclear. This study shows that purified PrpL activates the AP-1 transcription factors following instillation into mouse lungs, and drives robust production of IL-1{beta}, IL-6, and TNF, as well as clinical symptoms. A proteolytically inactive mutant of PrpL fails to elicit these responses. Structural analysis of the complex of PrpL and its natural inhibitory propeptide (PrpLPP) using X-ray crystallography revealed the inhibitory mechanism of PrpLPP, which guided the identification of a pre-existing dipeptide inhibitor (LasBi) that blocked PrpL activity and restricted AP-1-driven cytokine induction and clinical symptoms in vivo. This study shows that the alkaline protease AprA degrades PrpLPP and functions redundantly with LasB to liberate mature, active PrpL. Our findings: 1) establish PrpL as a key virulence factor that triggers AP-1-mediated inflammatory signaling, 2) provide structural and functional insights into PrpL inhibition, and 3) identify AprA as a novel upstream PrpL activator. Together, these results highlight PrpL as a promising anti-virulence therapeutic target. Given the functional redundancy of inflammatory effectors produced by P. aeruginosa, strategies aimed at mitigating P. aeruginosa-induced lung inflammation through PrpL inhibition would likely need to be combined with approaches targeting additional pro-inflammatory bacterial factors. Author SummaryWe identify PrpL as a critical Pseudomonas aeruginosa protease that activates the inflammatory transcription factor AP-1 and drives potent lung inflammation. Structural analysis of a natural PrpL inhibitor guided development of an existing dipeptide inhibitor that significantly reduced the production of proinflammatory cytokines and clinical responses in vivo. Moreover, discovery of the AprA protease as an additional PrpL activator refines the activation model and suggests potential upstream therapeutic strategies. These findings highlight PrpL as both a driver of pathogenesis and a promising therapeutic target for treating P. aeruginosa infections.

Candida albicans, a benign commensal yeast in the healthy gut, is increasingly being recognized as a tumor-promoting component of the gut mycobiome that is linked to worse patient outcomes in colorectal cancer (CRC). Emerging evidence suggests that in the tumor microenvironment, C. albicans drives tumor progression in its pathogenic hyphal form with the secretion of its peptide toxin, candidalysin, enhancing tumor cell evolution. CRC cells differentially express transglutaminase 2 (TG2), which mediates covalent binding to C. albicans and can promote tumor adaptability and metastases. However, mechanistic insight into how C. albicans infection leads to CRC progression is incomplete. CRC lines that express high (SW480) and low (LoVo, HCT 116) TG2 were infected with C. albicans and examined for effects on epithelial to mesenchymal transition (EMT) and cell migration. Parallel studies were also conducted using a candidalysin-deficient C. albicans strain. C. albicans invasion stimulated TG2 activity that allowed covalent crosslinking of the hyphae adhesin, Hwp1, to SW480 cells in a candidalysin-dependent manner. Independent of TG2 expression, infection of SW480, LoVo, and HCT 116 cells induced EMT-like changes including downregulation of E-cadherin, altered N-glycosylation of N-cadherin, and variable Snail1 expression. Infection increased MGAT5 activity, which raised levels of branched {beta}(1-6) N-glycans. Infection promoted CRC cell motility that was blocked by pharmacological inhibition of MGAT5. Thus, C. albicans engages CRC cells through separable mechanisms: a candidalysin-TG2-Hwp1 axis that may stabilize fungal attachment in TG2-high tumor cells, and a TG2-independent program of epithelial plasticity, altered N-glycosylation and migration-associated behavior that requires validation in vivo.

The eukaryotic microbes known as oomycetes express both cytosolic and mitochondrial sets of enzymes for the last six steps in glycolysis, unlike canonical eukaryotes where glycolysis is exclusively cytosolic. Linked to glycolysis through 3-phosphoglycerate is another pathway with an atypical mitochondrial location in oomycetes, phosphorylated serine biosynthesis (PSB), which is cytosolic in most other eukaryotes. Previous studies confirmed the mitochondrial location of these enzymes, which are encoded by nuclear genes. However, information was lacking about their contributions to metabolism and fitness. We addressed this using Phytophthora infestans, a pathogen of potato and tomato. Single-gene knockouts of the mitochondrial forms of phosphoglycerate kinase and enolase had little effect on growth or pathogenicity, but these traits were severely impaired in strains deleted for both genes. Metabolomic analysis of the double knockouts revealed changes in levels of glycolytic and TCA cycle intermediates, adenylate pools, vitamins, and other compounds important to cellular function. Blocking the PSB pathway by knocking out phosphoserine aminotransferase also compromised growth but caused fewer metabolic changes, and the results suggested that the main role of the pathway is to generate 3-phosphoglycerate for glycolysis and not for making serine. Most of these enzymes appear to have been acquired by lateral gene transfer into the stramenopile lineage, which besides oomycetes include diatoms and brown algae. We conclude that at least in oomycetes, metabolism has adapted such that these enzymes are now required for fitness. IMPORTANCEThe non-sexual movement of genetic material between species (horizontal gene transfer, HGT) has driven evolution in both prokaryotes and eukaryotes. One impact of HGT on the eukaryotic microbes known as oomycetes has been the acquisition of a glycolytic pathway in mitochondria alongside the standard version that resides in the cytosol. This is an uncommon outcome of HGT involving metabolic genes, which usually results in replacement of the ancestral pathway. We show that the mitochondrial glycolytic pathway, and another novel pathway that shares a metabolic intermediate with glycolysis in that organelle, make important contributions to metabolism and are required for fitness. This study helps illustrate how oomycete metabolism has adapted to the acquisition of enzymes with different evolutionary origins. Also, since oomycetes cause diseases on many important crop plants and animals, these novel features of metabolism may provide targets for their chemical control.

Pseudogymnoascus destructans is a psychrophilic ascomycete fungus that causes White-Nose Syndrome in North American hibernating bats. P. destructans invades the tissue present in bats' noses and wing membranes, causing the characteristic white nose and cupping erosion in the wings. P. destructans exhibits characteristics of a hemibiotrophic fungal pathogen, in which P. destructans can invade the host cells without inducing damage. During this stage, P. destructans hyphae assume a morphology similar to fungal plant pathogens during the biotrophic stage of infection. However, the mechanism by which P. destructans regulates its morphology and virulence is not fully understood. The phosphatase calcineurin is a key regulator of fungal stress response and virulence and could play a role in P. destructans virulence. Calcineurin can be inhibited using FK506 or Cyclosporin A (CSA). FK506 interacts with FKBP12, while CSA interacts with Cyclosporin A, and the protein-drug complex then inhibits calcineurin activity. Here, we show that P. destructans is intrinsically resistant to one calcineurin inhibitor (FK506), possibly because P. destructans FKBP12 has a lysine instead of the conserved arginine at position 89, which has been shown to reduce the affinity of the FKBP12-FK506 complex for calcineurin. Using cyclosporin A, we found that calcineurin is important for fungal growth. Additionally, calcineurin inhibition increases voriconazole inhibition. In contrast, calcineurin inhibition was antagonistic to the anti-cell wall drug micafungin. Taken together, calcineurin's role in fungal growth and response to voriconazole is conserved in P. destructans but may play a unique role in P. destructans response to micafungin.

Acinetobacter baumannii is a high priority Gram negative opportunistic pathogen known for its high rates of multidrug resistance (MDR). Minocycline (MIN), a tetracycline class antibiotic, is one of the most effective antibiotics for treating A. baumannii infections in patients. Unfortunately, MIN resistance is spreading internationally and has begun to emerge in the United States. While efflux pumps are correlated with MIN resistant A. baumannii, clinical data suggests alternative mechanisms of MIN resistance. To explore the genetic basis for MIN resistance in A. baumannii we employed a machine learning model to predict genetic resistance correlates from clinical isolates. Mutations in ruvB, a DNA repair protein, were strongly correlated with MIN resistant clinical strains of A. baumannii .Consistent with the prediction, tn26 insertion in ruvB in A. baumannii strain AB5075, and deletion of ruvB in strain ATCC 19606, increased MIN minimum inhibitory concentrations to a level that exceeds the MIN resistance breakpoint. RuvB complexes with RuvA and RuvC to resolve Holliday junctions during recombination. However, only ruvB mutants showed the resistance phenotype; neither ruvA nor ruvC mutants were MIN resistant, suggesting loss of the activity of the complex was not the basis for resistance. We observed ruvB mutants produced increased biomass during planktonic growth relative to the other two ruv mutants. Upon examination, the ruvB::tn26 mutant had a 451% increase in biomass and 360% thicker biofilms relative to wildtype. We determined the disruption of ruvB lead to thicker biofilms and enriched in extracellular DNA (eDNA), and DNase I treatment collapsed the enhanced biofilm phenotype and markedly reduced tetracycline class MICs. FLAG-RuvA accumulated within the biofilm matrix in the absence of RuvB, supporting a model in which RuvA contributes to stabilization of eDNA-rich structures. In a murine pneumonia model, ruvB disruption did not significantly alter survival or pulmonary burden in untreated infection but reduced bacterial dissemination and increased minocycline resistance. Together, these findings reveal an unexpected connection between Holliday junction processing, eDNA-rich biofilm architecture, and antibiotic resistance in A. baumannii.

Natural transformation (NT), a major mechanism of horizontal gene transfer, promotes bacterial genome plasticity and contributes to the dissemination of antibiotic resistance. NT requires the development of competence, a transient and tightly regulated physiological state that is induced by environmental cues. Although competence has recently been demonstrated in the human pathogen Staphylococcus aureus, the regulatory architecture and temporal coordination governing its development remain poorly understood. Using a highly sensitive luciferase-based transcriptional reporter, we demonstrate that NT gene expression in S. aureus is transient, thereby defining a temporal window during which DNA uptake and recombination can occur. We further identify two classes of NT genes with distinct regulatory requirements and activation kinetics: Class I genes require both SigH and ComK1 and are activated approximately 2.5 hours later than Class II genes, which are regulated exclusively by ComK1. Mechanistically, we demonstrate that ComK1 activates sigH transcription, establishing a hierarchical regulatory cascade that explains its essential role in Class I gene expression. Finally, time-resolved transcriptomic analyses identify the global regulator CodY as an early regulator of competence that differentially controls comK1 and sigH transcription, thereby linking metabolic adaptation to competence development. Together, our findings define the regulatory architecture that governs competence development in S. aureus and reveal how a hierarchical cascade integrates environmental and metabolic cues to restrict horizontal gene transfer to a precise developmental window. This work provides a mechanistic framework for understanding how bacteria coordinate physiological adaptation with genome diversification through NT. Author summaryBacteria constantly evolve by acquiring new genetic information from their environment. One important mechanism driving this process is natural transformation, which allows bacterial cells to take up and integrate exogenous DNA into their genomes. However, this process is not continuously active: bacteria must first enter a specialized physiological state called competence. How pathogens control the development of competence remain poorly understood. In this study, we investigate how the human pathogen Staphylococcus aureus regulates natural transformation. Using a highly sensitive reporter system that allows us to monitor gene expression over time, we show that competence is activated only during a limited period, creating a defined window during which bacteria can acquire new DNA sequences. We uncover a hierarchical regulatory cascade involving the transcription factors ComK1 and SigH that controls the sequential activation of natural transformation genes. We further identify the metabolic regulator CodY as an upstream regulator that connects bacterial physiological state with the initiation of competence. Our findings reveal that natural transformation is not a random or continuous process, but a precisely timed developmental program controlled by multiple regulatory layers. By defining how S. aureus coordinates environmental sensing, metabolism, and horizontal gene transfer, this work provides new insight into how bacterial pathogens generate genetic diversity and adapt to changing environments, including conditions that may promote the emergence of antibiotic resistance.

Virophages and Polinton-like Viruses (PLVs) are related viral elements classified within the supergroup Polisuviricotina. While virophages typically depend on large dsDNA viruses (Nucleocytoviricota) for replication in eukaryotic hosts, the replication strategies of most PLVs are unknown. While both virophages and PLVs can exist as stand-alone entities or be integrated into host genomes, their co-occurrence within a single eukaryotic genome is rare. We investigated the chromosome-scale nuclear genome assembly of the halophilic protist Halocafeteria seosinensis and discovered 41 endogenous PLVs and 36 virophage sequences, most of which are full-length elements; together they comprise 6.2% of the genome. These viral elements belong to six PLV and seven virophage subtypes. Notably, we found genes shared between H. seosinensis PLVs and virophages, suggesting active genetic exchange between them. We also observed supraparasitism, with MULE DNA transposons and LINE retrotransposons frequently embedded within the viral genomes. Our study reveals dynamic interactions between viral elements and host mobile DNA in a halophilic protist, expanding our understanding of viral diversity in extreme environments. SIGNIFICANCEVirophages and polinton-like viruses (PLVs) are enigmatic viral elements found in many aquatic habitats. Our understanding of their diversity stems mainly from analysis of environmental sequence data, but they have also been found integrated into the genomes of cultured protists. The evolutionary impacts of such integrations are poorly understood. In this study, we reveal the co-occurrence of multiple PLV and virophage subtypes that together constitute a substantial fraction of the genome of the halophilic protist Halocafeteria seosinensis. These distantly related viral lineages coexist within the same genome and share a common gene pool, forming chimeric arrangements with host transposons and with one another. The H. seosinensis genome thus serves as a dynamic arena for viral gene exchange and genome remodelling, reshaping host genome architecture and potentially conferring immunity to giant viruses.

Although Burkholderia cenocepacia exhibits biosurfactant-dependent phenotypes and possesses an rhl operon, the chemical identity of its surface-active product has long remained unresolved. The rhl operon of B. cenocepacia harbors an additional gene, rhlM, predicted to encode a methyltransferase, and recently linked to methylated rhamnolipid synthesis in a few Burkholderiaceae. Here, we hypothesized that this biosurfactant remained cryptic due to low expression under standard laboratory conditions. Using a chromosomal Prhl-luxCDABE fusion reporter, we screened diverse nutritional conditions and identified a defined medium containing galactose and urea that strongly induced rhl transcription. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of B. cenocepacia K56-2 extracts identified the biosurfactants as methylated dirhamnolipid congeners, predominantly L-rhamnosyl-L-rhamnosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoate methyl ester (RR-C14-C14Me). Extending our analysis across the Burkholderia cepacia complex (Bcc) revealed that methylated rhamnolipid production is widespread and restricted to rhl-positive species. Comparative genomic and protein structural analyses further demonstrated high conservation of both the rhlM-containing operon architecture and the RhlM protein across the Bcc. Together, our findings identify the long-standing cryptic biosurfactant of B. cenocepacia as methylated rhamnolipids and establish rhamnolipid methylation as a conserved hallmark across the Bcc, shifting the paradigm of rhamnolipid structural diversity from an isolated exception to a widespread rule.

Signaling through the calcium-activated calcineurin phosphatase promotes fungal survival of stressful conditions including those imposed by antifungal medications. Calcium is an essential secondary messenger that regulates diverse cellular processes in eukaryotes; however, it is also profoundly toxic and cytoplasmic concentrations must be tightly controlled. In fungi, the vacuole serves as a major calcium reservoir with the H+-exchanger Vcx1p and P-type ATPase Pmc1p sequestering intracellular calcium. Upon stimulation, these stores can be released through the Yvc1p ion channel to create transient cytoplasmic pulses that activate calcium-dependent responses. Despite the importance of calcineurin signaling in sustaining fungal viability upon antifungal insult, the contribution of many other proteins responsible for intracellular calcium homeostasis during antifungal exposure remains poorly understood. Here, we investigated whether Yvc1p, Vcx1p, and Pmc1p influence Candida albicans capacity to endure exposure to the first-line echinocandin antifungals. Our results demonstrate that loss of Pmc1p function confers high-levels of tolerance, with pmc1?/? mutant cells sustaining less damage, surviving and capable of proliferation in the presence of supra-MIC concentrations of the echinocandins. Moreover, this phenotype is dependent upon elevated signaling through the calcineurin pathway. Finally, we demonstrate that at least a subset of drugs previously identified as echinocandin antagonists activate calcineurin signaling in a Pmc1-dependent manner to drive echinocandin tolerance. These findings reveal how genetic or pharmacological modulation of calcium homeostasis can have profound consequences on the outcome of C. albicans - echinocandin interaction.

Changes in gene copy number are an extremely important source of variation and are frequently observed during the acquisition of drug resistance. The opportunistic human fungal pathogen Candida albicans frequently adapts to antifungal drugs via large copy number variations (CNVs) that amplify hundreds of genes simultaneously. Despite the recurrent amplification of CNVs across diverse clinical isolates, the genes that contribute to drug resistance are not known. Additionally, by amplifying many genes, CNVs might result in cross-adaptation or fitness trade-offs to multiple environments, which has major implications for how CNVs are expected to contribute to adaptation in complex environments like a mammalian host. We use CRISPR-activation to systematically assay the fitness effects of individually overexpressing [~]800 genes in four genetically diverse isolates across eight physiologically relevant environments. We identify 198 genes with significant fitness effects in at least one environment in one or more genetic backgrounds. We identify novel genes with positive fitness effects in two different classes of antifungal drug and observe frequent gene-by-environment interactions for the fitness effects of gene overexpression. Additive fitness effects of individual gene overexpression are a significant predictor of the fitness of multiple isolates with CNVs and can explain fitness trade-offs observed between classes of antifungal drug for the CNV isolates. These findings identify genes that increase fitness in drug and those that create vulnerabilities in CNV isolates and can help inform treatment of isolates adapting to antifungal drug via CNVs.

Host cell invasion by malaria parasites requires specific molecular interactions with host receptors. Plasmodium vivax merozoite invasion of reticulocytes is mediated by P. vivax Duffy binding protein (PvDBP) and its homolog, P. vivax erythrocyte binding protein (PvEBP). Here, we identify and characterize two novel P. vivax merozoite proteins, PvMP45 and PvMP36, which co- localize with PvDBP and PvEBP in the micronemes and bind reticulocyte receptors. PvMP45 and PvMP36 share high sequence identity with their P. knowlesi homologs, PkMP45 and PkMP36, which form a complex with other invasion related proteins. Field studies reveal that naturally acquired antibodies against PvMP36, PvEBP and PvDBP are associated with protection against clinical P. vivax malaria. We demonstrate that naturally acquired antibodies to PvEBP bind Fcy receptors and likely mediate protection by enabling opsonic phagocytosis. In addition, we show that combining antibodies against PvDBP and PvMP36 results in an additive invasion inhibitory effect against P. vivax blood stages. These results suggest that combining PvDBP, PvEBP and PvMP36 in a multivalent blood stage vaccine could elicit diverse immune mechanisms against P. vivax to achieve high efficacy. ImportanceAll the clinical symptoms of malaria are attributed to the blood stage of malaria parasites during which merozoites invade and multiply within red blood cells. A clear understanding of the host- parasite interactions that enable invasion can open paths for development of novel methods to block parasite growth and prevent malaria. Here, we identify and characterize two novel invasion related proteins from P. vivax merozoites that form an invasion complex and bind host RBC receptors. We demonstrate that antibodies targeting these proteins can block RBC invasion by P. vivax and naturally acquired antibodies that develop following P. vivax infection against one of these proteins are associated with protection against P. vivax malaria. These studies not only expand our understanding of the molecular mechanisms that enable host cell invasion by P. vivax but open new avenues for development of vaccines to protect against P. vivax malaria.

Azoles remain the most common antifungal therapy worldwide. However, Nakaseomyces glabratus (previously named Candida glabrata) has a high intrinsic tolerance against azole drugs. The organism can also accrue additional chromosomal mutations to elevate its resistant level during treatment. These genetic alterations often result in overexpression of the ABC transmembrane transporter Cdr1, which has been shown to directly transport drugs out of the fungal cells. Another resistant mechanism is the upregulation of the ergosterol biosynthesis pathway, which is the direct target of azoles. Although the mechanisms of azole resistance in N. glabratus are well defined, knowledge of their regulation remains limited. Here, we show that the protein kinase Fpk1 is required for optimal azole response in vitro and in an in vivo mouse infection model. Loss of Fpk1 gene or its kinase function significantly enhances azole sensitivity in both azole-susceptible and -resistant clinical isolates. Fpk1 function is required for optimal expression of Cdr1 upon azole challenge. It also influences the intracellular trafficking of ergosterol, without affecting its biosynthesis. Together, our data demonstrates the important role of Fpk1 function in the N. glabratus azole response and characterizes it as a new regulator of the efflux pump and ergosterol biosynthesis pathways. IMPORTANCEAntifungal treatment against life-threatening bloodstream Candida infection remains limited to azoles, echinocandins, and polyenes. Among them, azoles are the most prescribed therapy worldwide. However, the pathogenic yeast Nakaseomyces glabrataus has a high level of resistance against azoles (> 10%) (1). This often complicates treatment and increases mortality and morbidity rates. Therefore, understanding the mechanism of azole resistance would reinforce the treatment strategy and bolster future therapy development. Here, we identify the protein kinase Fpk1 as an important regulator of the drug efflux plump and ergosterol biosynthesis pathways. Disruption of the Fpk1 function significantly enhances the azole efficacy in vitro and in a mouse model of Candida systemic infection. Protein kinases are druggable targets, and our data presents Fpk1 as a viable candidate for future antifungal development.

Bacterial flagellar filaments are often modified by glycans, but the structural basis and physiological significance of flagellin glycosylation remain poorly understood in many species. Vibrio alginolyticus produces lateral flagella for surface-associated motility in viscous environments, with LafA forming its filament as flagellin. A maf homolog encoding a putative flagellin glycosylation factor is located immediately downstream of lafA, suggesting that the lateral filament is glycosylated. To investigate this possibility, we purified the lateral flagellar filament from V. alginolyticus and determined the structure at 2.37 [A] resolution by electron cryomicroscopy. Upon model building of LafA in the map, we identified additional densities connected to five serine residues, possibly corresponding to O-linked pseudaminic acid modifications. Mass spectrometric analyses identified these modifications as pseudaminic acid attached to Ser148, Ser173, Ser183, Ser189, and Ser197. Deletion of maf abolished lateral flagella formation and motility, and these defects were restored by complementation with maf. These results demonstrate that the Vibrio lateral flagellar filament is extensively modified by pseudaminic acid and that Maf is required for filament formation. Our findings provide the first structural insight into the glycosylation of Vibrio lateral flagellar filament and establish a framework for understanding the role of flagellin glycosylation in surface-associated motility.

Haemophilus influenzae is a Gram-negative bacterium that causes pneumonia, otitis media, and invasive infections such as meningitis and bacteremia. A vaccine that confers protection against disease caused by H. influenzae serotype b is currently available and is highly effective, but infections caused by non-b serotypes and non-typeable strains are still prevalent and increasing, suggesting a need for a broadly cross-protective vaccine that will protect against infection by all H. influenzae strains. To address the need for broad cross-protection, this study focused on the surface lipoprotein component of the bipartite bacterial transferrin receptor, transferrin binding protein B (TbpB), which is universally present H. influenzae, irrespective of encapsulation status, and is essential for bacterial colonization and pathogenesis. To prevent vaccine escape, we assessed the sequence and structural diversity among TbpB variants derived from local and international H. influenzae isolates and observed that these sequences cluster independently of encapsulation status, suggesting that conferring protection against all H. influenzae strains regardless of capsule type or presence of a capsule using a TbpB-based vaccine is feasible. In addition, our diversity analysis also revealed that the C-terminal lobe (C-lobe) of TbpB contains several large, highly variable loops. After immunizing mice with a trivalent vaccine consisting of a representative set of TbpB variants, we found that the resulting antiserum demonstrated modest cross-reactivity against heterologous TbpB variants. However, immunizing with a loopless C-lobe (LCL) that lacks the large, highly variable loops we had identified elicited broad cross-reactivity against a panel of intact TbpB variants. This suggests that while the intact TbpB may only generate a moderately cross-reactive antibody response, a vaccine consisting of a single LCL may be able to elicit a sufficiently cross-reactive humoral response against diverse TbpB variants, and in turn, confer broad cross-protection targeting both encapsulated and non-typeable H. influenzae.