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Cryptococcus neoformans (Cn) is an opportunistic fungal microorganism that causes life-threatening meningoencephalitis. During the infection, the microbial population is heterogeneously composed of cells with varying generational ages, with older cells accumulating during chronic infections. This is attributed to their enhanced resistance to phagocytic killing and tolerance of antifungals like fluconazole (FLC). In this study, we investigated the role of ergosterol synthesis, ATP-binding cassette (ABC) transporters, and mitochondrial metabolism in the regulation of age-dependent FLC tolerance. We find that old Cn cells increase the production of ergosterol and exhibit upregulation of ABC transporters. Old cells also show transcriptional and phenotypic characteristics consistent with increased metabolic activity, leading to increased ATP production. This is accompanied by increased production of reactive oxygen species (ROS), which results in mitochondrial fragmentation. This study demonstrates that the metabolic changes occurring in the mitochondria of old cells drive the increase in ergosterol synthesis and the upregulation of ABC transporters, leading to FLC tolerance. IMPORTANCEInfections caused by Cryptococcus neoformans cause more than 180,000 deaths annually. Estimated one-year mortality for patients receiving care ranges from 20% in developed countries to 70% in developing countries, suggesting that current treatments are inadequate. Some fungal cells can persist and replicate despite the usage of current antifungal regimens, leading to death or treatment failure. In replicative aging, older cells display a resilient phenotype, characterized by their enhanced tolerance against antifungals and resistance to killing by host cells. This study shows that age-dependent increase in mitochondrial reactive oxygen species drive changes in ABC transporters and ergosterol synthesis, ultimately leading to the heightened tolerance against fluconazole in old C. neoformans cells. Understanding the underlying molecular mechanisms of this age-associated antifungal tolerance will enable more targeted antifungal therapies for cryptococcal infections.

Quiescence, defined as the reversible exit from mitotic division and proliferative growth, is the predominant state of all microbes. Despite its prevalence, the properties and consequences of quiescence in Candida albicans, an opportunistic fungal pathogen, remain largely unexplored. In this study, we characterized the morphological, molecular, and biophysical properties of quiescent C. albicans cells and assessed the effects of quiescence on antifungal drug efficacy. Quiescent cells that were induced via carbon starvation in rich and minimal media underwent distinct morphological changes upon entry into quiescence; this included an increase in cell buoyant density, altered fluidity of the cytoplasm and nucleus, and remodeling of mitochondria. Most C. albicans cells arrested in an unbudded G1/G0 state, although a significant fraction of cells had budded morphologies and 4N DNA content, indicating that they arrested at other cell cycle phases. Both budded and unbudded quiescent cells efficiently re-entered the cell cycle upon nutrient replenishment, with time-to-quiescence exit varying depending on the total nutritional quality of the medium. Quiescence was associated with large-scale gene expression remodeling, including downregulation of ribosomal biogenesis genes and upregulation of autophagy and stress response pathways. Notably, a greater proportion of quiescent cells than proliferative cells survived exposure to the commonly used antifungal drugs micafungin, caspofungin, and amphotericin B in genetically diverse strains. Thus, quiescence is a distinct cellular state with important implications for antifungal drug efficacy in C. albicans. Author SummaryWe show that Candida albicans, a common fungal pathogen, can enter a reversible, non-dividing state when starved of carbon. Starved cells become smaller and denser, reorganize their mitochondria, change how densely packed the inside of the cell and its nucleus are, and switch on stress-protection and internal recycling programs while reducing protein synthesis activity. Most cells have ceased to actively divide, but many retained budded shapes and could restart growth when nutrients returned; the timing of recovery depended on the nutritional environment in which quiescence was initiated. Critically, quiescent cells from laboratory and clinical strains exhibited greater survival than proliferative cells when exposed to widely used fungicidal drugs including micafungin, caspofungin, and amphotericin B. These findings indicate that quiescence is an active, adaptive physiological state that helps Candida albicans survive hostile environmental conditions such as temperature stress and drug exposure. Accounting for the metabolic state of fungal cells in diagnostics and drug development may improve treatment outcomes.

Candida albicans (C. albicans) is a dimorphic human fungal pathogen that can cause severe oropharyngeal candidiasis (OPC, oral thrush) in susceptible hosts. During invasive infection, C. albicans hyphae invade oral epithelial cells (OECs) and secrete candidalysin, a pore-forming cytolytic peptide that is required for fungal pathogenesis at mucosal surfaces. Candidalysin induces cell damage and activates multiple MAPK-based innate signaling events that collectively drive the production of downstream inflammatory mediators. The activities of candidalysin are also dependent on the epidermal growth factor receptor (EGFR), but how these signals are integrated is undefined. Here, we identified five essential adaptor proteins as key mediators of the epithelial response to C. albicans infection on cultured OECs, including growth factor receptor bound protein 2 (Grb2), Grb2-associated-binding protein 1 (Gab1), Src homology and collagen (Shc), SH2 containing protein tyrosine phosphatase-2 (Shp2) and casitas B-lineage lymphoma (c-Cbl). All these signaling effectors were inducibly phosphorylated in response to C. albicans, in a candidalysin-dependent mechanism but additionally required EGFR phosphorylation, matrix metalloproteinases (MMPs) and cellular calcium flux. Of these, Gab1, Grb2 and Shp2 were the dominant drivers of ERK1/2 signaling and production of downstream cytokines. Together, these results identify the key adaptor proteins that drive EGFR signaling mechanisms, which determine oral epithelial responses to C. albicans.

Mitochondrial functions are critical for the ability of the fungal pathogen Cryptococcus neoformans to cause disease. However, mechanistic connections between key functions such as the mitochondrial electron transport chain (ETC) and virulence factor elaboration have yet to be thoroughly characterized. Here, we observed that inhibition of ETC complex III suppressed melanin formation, a major virulence factor. This inhibition was partially blocked upon loss of Cir1 or HapX, two transcription factors that regulate iron acquisition and use. In this regard, loss of Cir1 derepresses the expression of laccase genes as a potential mechanism to restore melanin, while HapX may condition melanin formation by controlling oxidative stress. We hypothesize that ETC dysfunction alters redox homeostasis to influence melanin formation. Consistent with this idea, inhibition of growth by hydrogen peroxide was exacerbated in the presence of the melanin substrate L-DOPA. Additionally, loss of the mitochondrial chaperone Mrj1, which influences the activity of ETC complex III and reduces ROS accumulation, also partially blocked antimycin A inhibition of melanin. The phenotypic impact of mitochondrial dysfunction was consistent with RNA-Seq analyses of WT cells treated with antimycin A or L-DOPA, or cells lacking Cir1 that revealed influences on transcripts encoding mitochondrial functions (e.g., ETC components and proteins for Fe-S cluster assembly). Overall, these findings reveal mitochondria-nuclear communication via ROS and iron regulators to control virulence factor production in C. neoformans. IMPORTANCEThere is a growing appreciation of the importance of mitochondrial functions and iron homeostasis in the ability of fungal pathogens to sense the vertebrate host environment and cause disease. Many mitochondrial functions such as heme and iron-sulfur cluster biosynthesis, and the electron transport chain (ETC), are dependent on iron. Connections between factors that regulate iron homeostasis and mitochondrial activities are known in model yeasts and are emerging for fungal pathogens. In this study, we identified connections between iron regulatory transcription factors (e.g., Cir1 and HapX) and the activity of complex III of the ETC that influence the formation of melanin, a key virulence factor in the pathogenic fungus Cryptococcus neoformans. This fungus causes meningoencephalitis in immunocompromised people and is a major threat to the HIV/AIDS population. Thus, understanding how mitochondrial functions influence virulence may support new therapeutic approaches to combat diseases caused by C. neoformans and other fungi.

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger c-di-GMP signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but 5 of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the 4 intact DGCs in an S. flexneri strain where these 4 DGCs had been deleted ({Delta}4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these 4 DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC genes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.

Many successful pathogens cause latent infections, remaining dormant within the host for years but retaining the ability to reactivate to cause symptomatic disease. The human opportunistic pathogen Cryptococcus neoformans is a ubiquitous yeast that establishes latent pulmonary infections in immunocompetent individuals upon fungal inhalation from the environment. These latent infections are frequently characterized by granulomas, or foci of chronic inflammation, that contain dormant cryptococcal cells. Immunosuppression causes these granulomas to break down and release viable fungal cells that proliferate, disseminate, and eventually cause lethal cryptococcosis. This course of C. neoformans dormancy and reactivation is understudied due to limited models, as chronic pulmonary granulomas do not typically form in most mouse models of cryptococcal infection. Here, we report that a previously characterized Cryptococcus-specific gene which is required for host-induced cell wall remodeling, MAR1, inhibits murine granuloma formation. Specifically, the mar1{Delta} loss-of-function mutant strain induces mature pulmonary granulomas at sites of infection dormancy in mice. Our data suggest that the combination of reduced fungal burden and increased immunogenicity of the mar1{Delta} mutant strain stimulates a host immune response that contains viable fungi within granulomas. Furthermore, we find that the mar1{Delta} mutant strain has slow growth and hypoxia resistance phenotypes, which may enable fungal persistence within pulmonary granulomas. Together with the conventional primary murine infection model, latent murine infection models will advance our understanding of cryptococcal disease progression and define fungal features important for persistence in the human host.

Microorganisms including fungi adapt to profound changes in their local environment during human infections. After exposure to high temperature and other stress conditions, the opportunistic fungal pathogen Cryptococcus neoformans enacts changes in metabolism, cell wall structure, and transmembrane transport that allow it to survive and proliferate in a mammalian host. This stress response program is regulated by the HECT E3-ubiquitin ligase Rsp5 which is required for growth at high salinity, pH and temperature. However, the complete set of Rsp5 substrates that direct these molecular changes remains incompletely understood. Here we demonstrate that C. neoformans Rsp5 confers increased tolerance to temperature and salt stress in part through regulation of the trehalose biosynthesis pathway. Two enzymes in the trehalose biosynthesis pathway, Tps1 and Tps2, are differentially ubiquitinated by Rsp5 after exposure to stress conditions. We directly measured trehalose production after exposure to high temperature and found that a C. neoformans strain lacking Rsp5 is unable to induce trehalose production. Quantitative proteomic analysis of the C. neoformans response to high salinity identified Rsp5-dependent and independent adaptations to osmotic stress, and that Rsp5-dependent ubiquitination does not alter the abundance of Tps1 or Tps2. These results demonstrate that regulation of trehalose biosynthesis is one of the cellular mechanisms by which Rsp5-dependent ubiquitination in C. neoformans facilitates survival in response to stressors encountered in the human infection environment. ImportanceCryptococcus neoformans is an opportunistic fungal pathogen that kills over 180,000 people every year with few effective treatment options. As a yeast that normally lives in the environment, C. neoformans has to survive large changes in its physical environment, including elevated body temperature, when it causes human infections. Here we show how C. neoformans uses a protein modification to regulate production of a fungal-specific metabolic pathway important for survival at human body temperature. Unraveling how environmental fungi tolerate and survive temperature and other stressors will help to understand how they cause disease and identify new and better ways to treat these deadly infections.

Fluconazole is the most commonly used antifungal today. A result of this has been the inevitable selection of fluconazole resistant organisms. This is an especially acute problem in the pathogenic yeast Candida glabrata. Elevated minimal inhibitory concentrations (MICs) for fluconazole in C. glabrata are frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding gene PDR1. These mutant Pdr1 regulators drive constitutively high expression of target genes like CDR1 that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure of C. glabrata to fluconazole induced expression of both Pdr1 and CDR1, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation and a hyperactive form of this phosphatase can increase resistance to the echinocandin caspofungin but does not show a similar elevation for fluconazole resistance. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease in CDR1 expression and fluconazole resistance, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin resistance together via the control of transcription factor activity. ImportanceWhile drug resistant microorganisms are a problem in treatment of all infectious disease, this is an especially acute problem with fungi due to the existence of only 3 classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeast Candida glabrata, mutant forms of a transcription factor called Pdr1 are commonly associated with fluconazole resistance and poor clinical outcomes. Here we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug resistance. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally elevated fluconazole resistance. Previous studies showed that calcineurin is required for resistance to the echinocandin class of antifungal drugs and our data demonstrate this protein phosphatase is also required for azole drug resistance. Calcineurin plays a central role in resistance to two of the three major classes of antifungal drugs in C. glabrata.

The emergence and spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) and the associated Coronavirus disease (COVID-19) pandemic have affected millions globally. Like other respiratory viruses, a significant complication of COVID-19 infection is secondary bacterial co-infection, which is seen in approximately 25% of severe cases. The most common organism isolated from co-infection is the Gram-positive bacterium Staphylococcus aureus. Here, we developed an in vitro co-infection model where both CoV-2 and S. aureus replication kinetics can be examined. We demonstrate CoV-2 infection does not alter how S. aureus attaches to or grows in host epithelial cells. In contrast, the presence of replicating S. aureus enhances the replication of CoV-2 by 10-15-fold. We identify this pro-viral activity is due to the S. aureus iron-regulated surface determinant A (IsdA) and this effect is mimicked across different SARS CoV-2 permissive cell lines infected with multiple viral variants. Analysis of co-infected cells demonstrated an IsdA dependent modification of host transcription. Using chemical inhibition, we determined S. aureus IsdA modifies host Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) signalling, ultimately leading to increased viral replication. These findings provide key insight into the molecular interactions that occur between host cells, CoV-2 and S. aureus during co-infection. ImportanceBacterial co-infection is a common and significant complication of respiratory viral infection, including in patients with COVID-19, and leads to increased morbidity and mortality. The relationship between virus, bacteria and host is largely unknown, which makes it difficult to design effective treatment strategies. In the present study we created a model of co-infection between SARS CoV-2 and Staphylococcus aureus, the most common species identified in COVID-19 patients with co-infection. We demonstrate that the S. aureus protein IsdA enhances the replication of SARS CoV-2 in vitro by modulating host cell signal transduction pathways. The significance of this finding is in identifying a bacterial component that enhances CoV-2 pathogenesis, which could be a target for the development of co-infection specific therapy in the future. In addition, this protein can be used as a tool to decipher the mechanisms by which CoV-2 manipulates the host cell, providing a better understanding of COVID-19 virulence.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B. pertussis, the causal agent of human whooping cough, and B. bronchiseptica, which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B. bronchiseptica, which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B. pertussis, which evolved from a B. bronchiseptica-like ancestor but now only survives only in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1, ctaCDFGE1, and cyoABCD1. To test the hypothesis that the three cytochrome oxidases encoded within the B. pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B. bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo. No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B. pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B. bronchiseptica producing only the three B. pertussis-conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also showed that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on respiration requirements for bacteria that colonize the respiratory tract, the environment at the surface of the ciliated epithelium, and the evolution of virulence in bacterial pathogens. AUTHOR SUMMARYCytochrome oxidases, critical components for aerobic respiration, have been shown to be vital for pathogenesis and tissue tropism in several bacterial species. However, the majority of the research has focused on facultative anaerobes and infections of microoxic to anaerobic host environments, like the gut. We sought to understand the role of cytochrome oxidases during respiratory infection by Bordetella bronchiseptica, an obligate aerobe, performing the first analysis of cytochrome oxidases in an extracellular respiratory pathogen that we know of. By comparing B. bronchiseptica to the closely related B. pertussis, a strictly human-specific pathogen and the causative agent of whooping cough, we found three cytochrome oxidases that are important for growth and survival within the mammalian respiratory tract. We also found that a bo3-type cytochrome oxidase, predicted to have a low affinity for oxygen and therefore best suited to ambient air levels of oxygen, was sufficient for both the establishment of infection and persistence in the respiratory tract in mice. Our findings reveal the importance of low affinity cytochrome oxidases in respiratory pathogens, and emphasize the need to study the physiology of diverse pathogens.

[NiFe]-hydrogenases have a bimetallic NiFe(CN)2CO cofactor in their large, catalytic subunit. The 136 Da Fe(CN)2CO group of this cofactor is assembled on a distinct HypC-HypD scaffold complex prior to delivery to the apo-catalytic subunit, but the intracellular source of the iron ion is unresolved. Native mass spectrometric (native MS) analysis of HypCD complexes defined the [4Fe-4S] cluster associated with HypD and identified +26 - 28 Da and +136 Da modifications specifically associated with HypC. A HypCC2A variant dissociated from its complex with native HypD lacked all modifications. HypC dissociated from HypCD complexes isolated from Escherichia coli strains deleted for the iscS or iscU genes, encoding core components of the Isc iron-sulfur cluster biogenesis machinery, specifically lacked the +136 Da modification; however, it was retained on HypC isolated from suf mutants. The presence or absence of the +136 Da modification on the HypCD complex correlated with the hydrogenase enzyme activity profiles of the respective mutant strains. Notably, the [4Fe-4S] cluster on HypD was identified in all HypCD complexes analyzed. These results suggest that the iron of the Fe(CN)2CO group on HypCD derives from the Isc machinery, while either the Isc or the Suf machinery can deliver the [4Fe-4S] cluster to HypD.

Invasive pulmonary aspergillosis (IPA) is a life-threatening infection caused by species in the ubiquitous fungal genus Aspergillus. While leukocyte-generated reactive oxygen species (ROS) are critical for the clearance of fungal conidia from the lung and resistance to IPA, the processes that govern ROS-dependent fungal cell death remain poorly defined. Using a flow cytometric approach that monitors two independent cell death markers, an endogenous histone H2A:mRFP nuclear integrity reporter and Sytox Blue cell impermeable (live/dead) stain, we observed that loss of A. fumigatus cytochrome c (cycA) results in reduced susceptibility to cell death from hydrogen peroxide (H2O2) treatment. Consistent with these observations in vitro, loss of cycA confers resistance to both NADPH-oxidase -dependent and -independent killing by host leukocytes. Fungal ROS resistance is partly mediated in part by Bir1, a homolog to survivin in humans, as Bir1 overexpression results in decreased ROS-induced conidial cell death and reduced killing by innate immune cells in vivo. We further report that overexpression of the Bir1 N-terminal BIR domain in A. fumigatus conidia results in altered expression of metabolic genes that functionally converge on mitochondrial function and cytochrome c (cycA) activity. Together, these studies demonstrate that cycA in A. fumigatus contributes to cell death responses that are induced by exogenous H2O2 and by host leukocytes. ImportanceAspergillus fumigatus can cause a life-threatening infection known as invasive pulmonary aspergillosis (IPA), which is marked by fungus-attributable mortality rates of 20%-30%. Individuals at risk of IPA harbor genetic mutations or incur pharmacologic defects that impair myeloid cell numbers and/or function, exemplified by bone marrow transplant recipients, patients that receive corticosteroid therapy, or patients with Chronic Granulomatous Disease (CGD). However, treatments for Aspergillus infections remains limited, and resistance to the few existing drug classes is emerging. Recently, the World Health Organization (WHO) classified A. fumigatus as a critical priority fungal pathogen. Our research identifies an important aspect of fungal biology that impacts susceptibility to leukocyte killing. Furthering our understanding of mechanisms that mediate the outcome of fungal-leukocyte interactions will increase our understanding of both the underlying fungal biology governing cell death and innate immune evasion strategies utilized during mammalian infection pathogenesis. Consequently, our studies are a critical step toward leveraging these mechanisms for novel therapeutic advances.

The composition of phospholipid membranes is critical to regulating the activity of membrane proteins for cellular functions. Cardiolipin is a unique phospholipid present within the bacterial membrane and mitochondria of eukaryotes and plays a role in maintaining the function and stabilization of membrane proteins. Here, we report that, in the human pathogen Staphylococcus aureus, cardiolipin is required for full activity of the SaeRS two-component system (TCS). Deletion of the cardiolipin synthase genes, cls1, and cls2, reduces the basal activity of SaeRS and other TCSs. Cardiolipin is an indispensable requisite for Sae activation mediated by human neutrophil peptides (HNPs) in the stationary growth phase but not mandatory for Sae induction in the exponential growth phase. Ectopic expression with cls2, but not with cls1, in the cls1 cls2 double mutant fully restores Sae activity. Elimination of cardiolipin from the membranes results in decreased kinase activity of the sensor protein SaeS. Purified SaeS protein directly binds to cardiolipin as well as phosphatidylglycerol. A strain lacking cls2 or cls1cls2 renders S. aureus less cytotoxic to human neutrophils and less virulent in a mouse model of infection. Our findings suggest that cardiolipin enables a pathogen to confer virulence by modulating the kinase activity of SaeS and other sensor kinases upon infection.

In previous studies we determined that the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase in Cryptococcus neoformans, is essential for fungal pathogenesis. Heat-killed fbp1{Delta} cells (HK-fbp1) can confer vaccine-induced immunity against lethal challenge with clinically important invasive fungal pathogens, e.g., C. neoformans, C. gattii, and Aspergillus fumigatus. In this study, we found that either CD4+ T cells or CD8+ T cells are sufficient to confer protection against lethal challenge of C. neoformans in HK-fbp1 induced-immunity. Given the potent effect of HK-fbp1 as a preventative vaccine, we further tested the potential efficacy of administering HK-fbp1 cells as a therapeutic agent for treating animals after infection. Remarkably, administration of HK-fbp1 provided robust host protection against pre-existing C. neoformans infection. The mice infected with wild type H99 cells and then treated with HK-fbp1 showed significant reduction of fungal CFU in the infected lung, and no dissemination of fungal cells to the brain and spleen. we find that early treatment is critical for the effective use of HK-fbp1 as a therapeutic agent. Immune analysis revealed that early treatment with HK-fbp1 cells elicited Th1 biased protective immune responses that help block fungal dissemination and promote better host protection. Our data thus suggest that HK-fbp1 is both an effective prophylactic vaccine candidate against C. neoformans infection in both immunocompetent and immunocompromised populations, as well as a potential novel therapeutic strategy to treat early stage cryptococcosis. ImportanceInvasive fungal infections, e.g., cryptococcosis, are often life threatening and difficult to treat with very limited therapeutic options. There is no vaccine available in clinical use to prevent or treat fungal infections. Our previous studies demonstrated that heat-killed fbp1{Delta} cells (HK-fbp1) in Cryptococcus neoformans can be harnessed to confer protection against a challenge by the virulent parental strain, even in immunocompromised animals, such as the ones lacking CD4+ T cells. In this study, we further determined that T cells are required for vaccine-induced protection against homologous challenge and that either CD4+ or CD8+ cells are sufficient. This finding is particularly important for the potential utility of this vaccine candidate in the context of HIV/AIDS-induced immune deficiency, the main risk factor for cryptococcosis in humans. Furthermore, in addition to the utility of HK-fbp1 as a prophylactic vaccine, we found that HK-fbp1 administration can inhibit disease dissemination when animals are treated at an early-stage during Cryptococcus infection. Our findings could significantly expand the utility of HK-fbp1 not only as prophylactic vaccine but also as a novel therapy against cryptococcosis. Conceptually, therapeutic administration of HK-fbp1 could have an advantage over small molecule antifungal drugs in that it is expected to have minimal side effects and lower cost. In all, our studies showed that HK-fbp1 strain can be used both preventively and therapeutically to elicit robust host protection against cryptococcosis.

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily causes pulmonary infections, with the potential to cause life-threatening infections including meningoencephalitis in immunocompromised individuals. Key virulence factors including polysaccharide capsule and melanin, facilitate immune evasion and tissue invasion in the host. Recently, titan cell formation has been defined as another important virulence factor of C. neoformans and plays a pivotal role in disease progression. The cyclic AMP-protein kinase A (cAMP-PKA) pathway in C. neoformans has been shown to be an important regulator of virulence-associated processes, including capsule formation, melanin biosynthesis and titan cell formation. However, the upstream signals, critical for the activation of cAMP-PKA pathway in the context of titan cell formation remain poorly understood. In this study, we demonstrate that the central carbon metabolic pathway, glycolysis, is critical for cAMP-dependent titan cell formation. Pharmacological and genetic perturbation of glycolysis significantly attenuated titan cell formation. Remarkably, exogenous addition of cAMP completely reversed the titan cell defects, observed during glycolysis perturbation. Interestingly, melanin deficient strains exhibited a significant attenuation in titan cell formation establishing a novel link between dimorphic switching and melanin biosynthesis in C. neoformans. These findings establish a novel regulatory axis wherein central carbon metabolism orchestrates morphogenetic switching in C. neoformans by regulating the activity of the well-conserved cAMP-PKA pathway. We also demonstrate, for the first time, that melanin biosynthesis which is under the regulatory control of cAMP-PKA pathway, is critical for titan cell formation, providing new insights into the metabolic control of C. neoformans dimorphism. SummaryA leading opportunistic fungal pathogen, Cryptococcus neoformans possess a significant threat to human health. Titan cell formation is one of the established virulence factors of C. neoformans. However, the mechanisms underlying the regulation of titan cell formation remains largely uncharacterized. Our study demonstrates that glycolysis regulates titan cell formation in a cAMP-PKA pathway dependent manner and melanin biosynthesis, which is under the direct regulatory control of cAMP-PKA pathway, is critical for titan cell formation. This study offers crucial insights into the metabolic regulation of dimorphic transitions in the human fungal pathogen, C. neoformans.

Acinetobacter baumannii can cause prolonged infections that disproportionately affect immunocompromised populations. Our understanding of A. baumannii respiratory pathogenesis relies on an acute murine infection model with limited clinical relevance that employs an unnaturally high number of bacteria and requires the assessment of bacterial load at 24-36 hours post-infection. Here, we demonstrate that low intranasal inoculums in immunocompromised mice with a tlr4 mutation leads to reduced inflammation, allowing for persistent infections lasting at least 3 weeks. Using this "chronic infection model," we determined the adhesin InvL is an imperative virulence factor required during later stages of infection, despite being dispensable in the early phase. We also demonstrate that the chronic model enables the distinction between antibiotics that, although initially reduce bacterial burden, either lead to complete clearance or result in the formation of bacterial persisters. To illustrate how our model can be applied to study polymicrobial infections, we inoculated mice with an active A. baumannii infection with Staphylococcus aureus or Klebsiella pneumoniae. We found that S. aureus exacerbates the infection, while K. pneumoniae enhances A. baumannii clearance. In all, the chronic model overcomes some limitations of the acute pulmonary model, expanding our capabilities to study of A. baumannii pathogenesis and lays the groundwork for the development of similar models for other important opportunistic pathogens.

To predict the outcomes of disseminated fungal disease, a deeper understanding of host-pathogen interactions at the site of infection is needed to identify targets for clinical intervention and diagnostic development. Cryptococcus neoformans is the causative agent of cryptococcosis, the largest infectious killer of individuals living with HIV. Cryptococcal infection begins in the lungs, with loss of immunological control leading to disseminated central nervous system disease and death. Using advanced mass spectrometry-based proteomic techniques, in vivo infection models, and patient-derived clinical strains, we explored the proteomic profiles of C. neoformans infections related to differences in strain virulence. Our findings reveal that non-lethal latent infection produces a proteomic response that differs significantly from the response caused by lethal infections, and that the proteomic profiles of typical and hypervirulent infections are markedly similar despite differences in time-to-death. Overall, the mouse pulmonary proteomic response in latent infection is defined by enrichment of proteins and pathways involved in extracellular matrix organization, cell adhesion, and structural changes, while the lethal infection is dominated by host-defense, translation, and metabolic processes. These results provide clinically relevant information on how infections caused by different Cryptococcus strains may produce significantly different outcomes. We also identified abundant fungal proteins that could be future drug targets in latent and lethal cryptococcal infection. IMPORTANCECryptococcus neoformans is a fungal pathogen that causes substantial morbidity and mortality in immunocompromised individuals. The initial infection begins in the lungs after exposure to inhaled spores after which local immune cells respond by either killing or containing the fungal cells. Immunosuppression weakens the immune system and allows fungal cells in the lungs to escape through the circulatory system and invade the central nervous system and cause fatal disease. However, differences between fungal strains influence the severity of disease manifestation. Our group has previously described genetic differences that contribute to strain-specific disease manifestations. In this study, we expanded our analysis to investigate the proteomic differences between strains of C. neoformans to identify candidate proteins and pathways that contribute to disease manifestation. We found that latent infection differs significantly from lethal disease from both the host and pathogen proteomic perspectives and have identified several fungal protein targets for future study.

The Aspergillus fumigatus unfolded protein response (UPR) is a two-component relay consisting of the ER-bound IreA protein, which splices and activates the mRNA of the transcription factor HacA. Spliced hacA accumulates under conditions of acute ER stress in vitro, and UPR null mutants are hypovirulent in a murine model of invasive pulmonary infection. In this report, we demonstrate that a hacA deletion mutant is completely unable to establish infection in a model of fungal keratitis, a corneal infection and an important cause of ocular morbidity and unilateral blindness worldwide. Contrary to our initial prediction, however, we demonstrate that hacA splicing is not increased above baseline conditions in the cornea, nor is the expression of genes classically associated with UPR activation, such as protein chaperones. We employed transcriptomics on wild-type and{Delta} hacA strains in gelatin media, as a proxy for the corneal environment, and found that hacA supports the expression of numerous primary and secondary metabolic processes that likely promote adaptation to nutrient limitation. Taken together, our results support a model in which the cornea, similar to growth on protein in vitro, is a source of sub-acute ER stress for A. fumigatus, but one nevertheless that requires the UPR pathway for proper adaptation. The data also suggest that this pathway could be a target for novel antifungals that improve visual outcomes for fungal keratitis patients. AUTHOR SUMMARYFungal keratitis has emerged as a leading cause of ocular morbidity and unilateral blindness worldwide. Relative to other infectious contexts, however, little is known about the fungal genes or pathways that regulate invasive growth and virulence in the corneal environment. In this report, we demonstrate that genetic disruption of the Aspergillus fumigatus unfolded protein response (UPR) abolishes the ability of the mold to establish infection in a mouse model of FK. Despite this critical role for virulence, however, we did not detect a concerted activation of the pathway beyond levels observed on standard medium, suggesting that the host environment is not an acute source of endoplasmic reticulum stress. Transcriptomic profiling of the wild-type and UPR-deficient strains under host-relevant nutrient conditions revealed a critical role for the pathway in regulating primary and secondary metabolism, cell wall biology, and mitochondrial function, all of which likely modulate fungal growth within and interactions with the host. These results expand our understanding of UPR regulation and function in this important mold pathogen and suggest the pathway could serve as a target for novel antifungals that improve visual outcomes in the setting of fungal keratitis.

Aquaporins are small, integral membrane channels that facilitate the transport of water across cellular membranes and, in the case of aquaglyceroporins, can also conduct specific neutral solutes, such as glycerol. These proteins are conserved across biological kingdoms, yet their roles in fungal virulence remain relatively understudied. In Cryptococcus neoformans, an opportunistic fungal pathogen, we examined the organisms single aquaporin, Aqp1, and uncovered unanticipated influences on cellular morphology. Loss of Aqp1 resulted in smaller cells, whereas its presence promoted the formation of enlarged titan-like cells. This shift in size was closely linked to intracellular redox physiology. Consequently, the overexpression of the cryptococcal aquaporin increased sensitivity to oxidative stress and led to the largest titan-like cells; antioxidant supplementation suppressed this enlargement, consistent with a ROS-dependent regulatory mechanism. Additionally, Aqp1 overexpression produced vacuolar abnormalities in titan-like cells, suggesting that excessive water influx strained intracellular organization during rapid cell expansion. These findings position Aqp1 at a functional crossroads connecting membrane transport, oxidative balance, and size control, and they support a model in which an aquaporin contributes to the morphological plasticity that allows C. neoformans to adapt to environmental pressures.

Bacillus anthracis is a gram-positive spore-forming bacterium that causes lethal inhalation anthrax. The use of B. anthracis as a bioweapon is predicated in its ability to form dormant and resistant infective spores that can be used as agents. B. anthracis spores contain large concentrations of dipicolinic acid complexed with cations, especially calcium (Ca-DPA). Following phagocytosis by alveolar macrophages, spores germinate inside the phagolysosome and excrete the Ca-DPA depot into the phagosomal space. In this study, we assessed the effects of DPA on B. anthracis spore biology and cytotoxicity. We generated B. anthracis mutants with defects in DPA synthesis ({Delta}spoVFA, {Delta}spoVFB, {Delta}spoVFAB) or transport ({Delta}spoVV). To increase the viability of DPA-less spores, we also constructed double mutants ({Delta}sleB{Delta}spoVFA, {Delta}sleB{Delta}spoVFB, {Delta}sleB{Delta}spoVFAB and{Delta} sleB{Delta}spoVV) by deleting the cortex lytic sleB gene. We found that single- and double-mutant DPA-less spores were profoundly compromised in dormancy, viability, germination, and heat resistance. Contrary to expectations, each DPA synthesis mutant exhibited distinct viability and resistance phenotypes. Even with compromised stability, DPA-less B. anthracis spores, with the exception of the {Delta}sleB{Delta}spoVV double mutant, were as cytotoxic as wild-type spores. In summary, DPA is required to sustain normal B. anthracis spore biology but is not required for macrophage-targeted virulence. Furthermore, the SpoVV transporter and SleB lytic protein seem to have redundant roles in anthrax spore cytotoxicity beyond DPA accumulation. ImportanceBacillus anthracis causes deadly pulmonary anthrax and has been used as a weapon for bioterrorism. B. anthracis form spores that germinate and establish infection. During germination, B. anthracis spores release large amounts of calcium complexed with dipicolinic acid (DPA). In this study, we deleted the B. anthracis genes that are required to synthesize and transport DPA into spores. We found that B. anthracis DPA-less mutant spores exhibited differential biological effects that were DPA independent. Furthermore, we found that DPA was not required for anthrax cytotoxicity. Finally, we found that proteins involved in DPA synthesis, transport, and cortex lysis have biological and virulence functions that extend beyond DPA accumulation.