Cryptococcus neoformans is an encapsulated yeast responsible for approximately a quarter of a million deaths worldwide annually despite therapy, and upwards of 11% of HIV/AIDS-related deaths, rivaling the impact of tuberculosis and malaria. However, the most effective antifungal agent, amphotericin B, requires intravenous delivery and has significant renal and hematopoietic toxicity, making it difficult to utilize, especially in resource-limited settings. The present studies describe a new nanoparticle crystal encapsulated formulation of amphotericin B known as encochleated amphotericin B (CAmB) that seeks to provide an oral formulation that is low in toxicity and cost. Using a 3-day delayed model of murine cryptococcal meningoencephalitis and a large inoculum of a highly virulent strain of serotype A C. neoformans, CAmB, in combination with flucytosine, was found to have efficacy equivalent to parental amphotericin B deoxycholate with flucytosine and superior to oral fluconazole without untoward toxicity. Transport of fluorescent CAmB particles to brain as well as significant brain levels of amphotericin drug was demonstrated in treated mice, and immunological profiles were similar to those of mice treated with conventional amphotericin B. Additional toxicity studies using a standardized rat model showed negligible toxicity after a 28-day treatment schedule. These studies thus offer the potential for an efficacious oral formulation of a known fungicidal drug against intrathecal cryptococcal disease.IMPORTANCECryptococcus neoformans is a significant global fungal pathogen that kills an estimated quarter of a million HIV-infected individuals yearly and has poor outcomes despite therapy. The most effective therapy, amphotericin B, is highly effective in killing the fungus but is available only in highly toxic, intravenous formulations that are unavailable in most of the developing world, where cryptococcal disease in most prevalent. For example, in Ethiopia, reliance on the orally available antifungal fluconazole results in high mortality, even when initiated as preemptive therapy at the time of HIV diagnosis. Thus, alternative agents could result in significant saving of lives. Toward this end, the present work describes the development of a new formulation of amphotericin B (CAmB) that encapsulates the drug as a crystal lipid nanoparticle that facilitates oral absorption and prevents toxicity. Successful oral absorption of the drug was demonstrated in a mouse model that, in combination with the antifungal flucytosine, provided efficacy equal to a parental preparation of amphotericin B plus flucytosine. These studies demonstrate the potential for CAmB in combination with flucytosine to provide an effective oral formulation of a well-known, potent fungicidal drug combination.

Cryptococcal meningoencephalitis is an urgent global health problem. Induction regimens using 14 days of amphotericin B deoxycholate (dAmB) are considered the standard of care but may not be suitable for resource-poor settings. We investigated the efficacy of conventional and abbreviated regimens of dAmB for cryptococcal meningoencephalitis in both murine and rabbit models of cryptococcal meningoencephalitis. We examined the extent to which immunological effectors contribute to the antifungal effect. We bridged the results to humans as a first critical step to define regimens suitable for further study in clinical trials. There were significant differences in the murine plasma-versus-cerebrum dAmB concentration-time profiles. dAmB was detectable in the cerebrum throughout the experimental period, even following the administration of only three doses of 3 mg/kg. dAmB induced a fungistatic effect in the cerebrum with a 2- to 3-log10 CFU/g reduction compared with controls. The effect of 3 days of therapy was the same as that of daily therapy for 14 days. There was no evidence of increased numbers of CD3(+) CD4(+) or CD3(+) CD8(+) cells in treated mice to account for the persistent antifungal effect of an abbreviated regimen. The administration of dAmB at 1 mg/kg/day for 3 days was the same as daily therapy in rabbits. The bridging studies suggested that a human regimen of 0.7 mg/kg/day for 3 days resulted in nearly maximal antifungal activity in both the cerebrum and cerebrospinal fluid. An abbreviated regimen (3 days of therapy) of dAmB appears to be just as effective as conventional induction therapy for cryptococcal meningoencephalitis.

The fungal cell wall is a critically important structure that represents a permeability barrier and protective shield. We probed Candida albicans and Cryptococcus neoformans with liposomes containing amphotericin B (AmBisome), with or without 15-nm colloidal gold particles. The liposomes have a diameter of 60 to 80 nm, and yet their mode of action requires them to penetrate the fungal cell wall to deliver amphotericin B to the cell membrane, where it binds to ergosterol. Surprisingly, using cryofixation techniques with electron microscopy, we observed that the liposomes remained intact during transit through the cell wall of both yeast species, even though the predicted porosity of the cell wall (pore size, ~5.8 nm) is theoretically too small to allow these liposomes to pass through intact. C. albicans mutants with altered cell wall thickness and composition were similar in both their in vitro AmBisome susceptibility and the ability of liposomes to penetrate the cell wall. AmBisome exposed to ergosterol-deficient C. albicans failed to penetrate beyond the mannoprotein-rich outer cell wall layer. Melanization of C. neoformans and the absence of amphotericin B in the liposomes were also associated with a significant reduction in liposome penetration. Therefore, AmBisome can reach cell membranes intact, implying that fungal cell wall viscoelastic properties are permissive to vesicular structures. The fact that AmBisome can transit through chemically diverse cell wall matrices when these liposomes are larger than the theoretical cell wall porosity suggests that the wall is capable of rapid remodeling, which may also be the mechanism for release of extracellular vesicles.IMPORTANCE AmBisome is a broad-spectrum fungicidal antifungal agent in which the hydrophobic polyene antibiotic amphotericin B is packaged within a 60- to 80-nm liposome. The mode of action involves perturbation of the fungal cell membrane by selectively binding to ergosterol, thereby disrupting membrane function. We report that the AmBisome liposome transits through the cell walls of both Candida albicans and Cryptococcus neoformans intact, despite the fact that the liposome is larger than the theoretical cell wall porosity. This implies that the cell wall has deformable, viscoelastic properties that are permissive to transwall vesicular traffic. These observations help explain the low toxicity of AmBisome, which can deliver its payload directly to the cell membrane without unloading the polyene in the cell wall. In addition, these findings suggest that extracellular vesicles may also be able to pass through the cell wall to deliver soluble and membrane-bound effectors and other molecules to the extracellular space.

Living organisms are constantly exposed to DNA damage stress caused by endogenous and exogenous events. Eukaryotic cells have evolutionarily conserved DNA damage checkpoint surveillance systems. We previously reported that a unique transcription factor, Bdr1, whose expression is strongly induced by the protein kinase Rad53 governs DNA damage responses by controlling the expression of DNA repair genes in the basidiomycetous fungus Cryptococcus neoformans However, the regulatory mechanism of the Rad53-dependent DNA damage signal cascade and its function in pathogenicity remain unclear. Here, we demonstrate that Rad53 is required for DNA damage response and is phosphorylated by two phosphatidylinositol 3-kinase (PI3K)-like kinases, Tel1 and Mec1, in response to DNA damage stress. Transcriptome analysis revealed that Rad53 regulates the expression of several DNA repair genes in response to gamma radiation. We found that expression of CHK1, another effector kinase involved in the DNA damage response, is regulated by Rad53 and that CHK1 deletion rendered cells highly susceptible to DNA damage stress. Nevertheless, BDR1 expression is regulated by Rad53, but not Chk1, indicating that DNA damage signal cascades mediated by Rad53 and Chk1 exhibit redundant and distinct functions. We found that perturbation of both RAD53 and CHK1 attenuated the virulence of C. neoformans, perhaps by promoting phagosome maturation within macrophage, reducing melanin production, and increasing susceptibility to oxidative stresses. Furthermore, deletion of both RAD53 and CHK1 increased susceptibility to certain antifungal drugs such as amphotericin B. This report provides insight into the regulatory mechanism of fungal DNA damage repair systems and their functional relationship with fungal virulence and antifungal drug susceptibility.IMPORTANCE Genome instability is detrimental for living things because it induces genetic disorder diseases and transfers incorrect genome information to descendants. Therefore, living organisms have evolutionarily conserved signaling networks to sense and repair DNA damage. However, how the DNA damage response pathway is regulated for maintaining the genome integrity of fungal pathogens and how this contributes to their pathogenicity remain elusive. In this study, we investigated the DNA damage response pathway in the basidiomycete pathogen Cryptococcus neoformans, which causes life-threatening meningoencephalitis in immunocompromised individuals, with an average of 223,100 infections leading to 181,100 deaths reported annually. Here, we found that perturbation of Rad53- and Chk1-dependent DNA damage response pathways attenuated the virulence of C. neoformans and increased its susceptibility to certain antifungal drugs, such as amphotericin B and flucytosine. Therefore, our work paves the way to understanding the important role of human fungal DNA damage networks in pathogenesis and antifungal drug susceptibility.

Cryptococcal Meningitis (CM) is uniformly fatal if not treated, and treatment options are limited. We previously reported on the activity of APX2096, the prodrug of the novel Gwt1 inhibitor APX2039, in a mouse model of CM. Here, we investigated the efficacy of APX2039 in mouse and rabbit models of CM. In the mouse model, the controls had a mean lung fungal burden of 5.95 log10 CFU/g, whereas those in the fluconazole-, amphotericin B-, and APX2039-treated mice were 3.56, 4.59, and 1.50 log10 CFU/g, respectively. In the brain, the control mean fungal burden was 7.97 log10 CFU/g, while the burdens were 4.64, 7.16, and 1.44 log10 CFU/g for treatment with fluconazole, amphotericin B, and APX2039, respectively. In the rabbit model of CM, the oral administration of APX2039 at 50 mg/kg of body weight twice a day (BID) resulted in a rapid decrease in the cerebrospinal fluid (CSF) fungal burden, and the burden was below the limit of detection by day 10 postinfection. The effective fungicidal activity (EFA) was -0.66 log10 CFU/mL/day, decreasing from an average of 4.75 log10 CFU/mL to 0 CFU/mL, over 8 days of therapy, comparing favorably with good clinical outcomes in humans associated with reductions of the CSF fungal burden of -0.4 log10 CFU/mL/day, and, remarkably, 2-fold the EFA of amphotericin B deoxycholate in this model (-0.33 log10 CFU/mL/day). A total drug exposure of the area under the concentration-time curve from 0 to 24 h (AUC0-24) of 25 to 50 mg · h/L of APX2039 resulted in near-maximal antifungal activity. These data support the further preclinical and clinical evaluation of APX2039 as a new oral fungicidal monotherapy for the treatment of CM. IMPORTANCE Cryptococcal meningitis (CM) is a fungal disease with significant global morbidity and mortality. The gepix Gwt1 inhibitors are a new class of antifungal drugs. Here, we demonstrated the efficacy of APX2039, the second member of the gepix class, in rabbit and mouse models of cryptococcal meningitis. We also analyzed the drug levels in the blood and cerebrospinal fluid in the highly predictive rabbit model and built a mathematical model to describe the behavior of the drug with respect to the elimination of the fungal pathogen. We demonstrated that the oral administration of APX2039 resulted in a rapid decrease in the CSF fungal burden, with an effective fungicidal activity of -0.66 log10 CFU/mL/day, comparing favorably with good clinical outcomes in humans associated with reductions of -0.4 log10 CFU/mL/day. The drug APX2039 had good penetration of the central nervous system and is an excellent candidate for future clinical testing in humans for the treatment of CM.

Indirect transmission via a contaminated environment can occur for a number of pathogens, even those typically thought of as being directly transmitted, such as influenza virus, norovirus, bovine tuberculosis, or foot-and-mouth disease virus (FMDV). Indirect transmission facilitates spread from multiple sources beyond the infectious host, complicating the epidemiology and control of these diseases. This study carried out a series of transmission experiments to determine the dose-response relationship between environmental contamination and transmission of FMDV in cattle from measurements of viral shedding and rates of environmental contamination and survival. Seven out of ten indirect exposures resulted in successful transmission. The basic reproduction number for environmental transmission of FMDV in this experimental setting was estimated at 1.65, indicating that environmental transmission alone could sustain an outbreak. Importantly, detection of virus in the environment prior to the appearance of clinical signs in infected cattle and successful transmission from these environments highlights there is a risk of environmental transmission even before foot-and-mouth disease (FMD) is clinically apparent in cattle. Estimated viral decay rates suggest that FMDV remained viable in this environment for up to 14 days, emphasizing the requirement for stringent biosecurity procedures following outbreaks of FMD and the design of control measures that reflect the biology of a pathogen.IMPORTANCE Effective control of a disease relies on comprehensive understanding of how transmission occurs, in order to design and apply effective control measures. Foot-and-mouth disease virus (FMDV) is primarily spread by direct contact between infected and naive individuals, although the high levels of virus shed by infected animals mean that virus can also be spread through contact with contaminated environments. Using a series of transmission experiments, we demonstrate that environmental transmission alone would be sufficient to sustain an outbreak. Key observations include that a risk of transmission exists before clinical signs of foot-and-mouth disease (FMD) are apparent in cattle and that survival of virus in the environment extends the transmission risk period. This study highlights the role a contaminated environment can play in the transmission of FMDV and presents approaches that can also be applied to study the transmission of other pathogens that are able to survive in the environment.

Cryptococcus neoformans is a pathogenic fungus that is responsible for up to half a million cases of meningitis globally, especially in immunocompromised individuals. Common fungistatic drugs, such as fluconazole, are less toxic for patients but have low efficacy for initial therapy of the disease. Effective therapy against the disease is provided by the fungicidal drug amphotericin B; however, due to its high toxicity and the difficulty in administering its intravenous formulation, it is imperative to find new therapies targeting the fungus. The antiparasitic drug bithionol has been recently identified as having potent fungicidal activity. In this study, we used a combined gene dosing and drug affinity responsive target stability (GD-DARTS) screen as well as protein modeling to identify a common drug binding site of bithionol within multiple NAD-dependent dehydrogenase drug targets. This combination genetic and proteomic method thus provides a powerful method for identifying novel fungicidal drug targets for further development.

Helicobacter pylori has an impressive ability to persist chronically in the human stomach. Similar characteristics are associated with biofilm formation in other bacteria. The H. pylori biofilm process, however, is poorly understood. To gain insight into this mode of growth, we carried out comparative transcriptomic analysis between H. pylori biofilm and planktonic cells, using the mouse-colonizing strain SS1. Optimal biofilm formation was obtained with a low concentration of serum and 3 days of growth, conditions that caused both biofilm and planktonic cells to be ∼80% coccoid. Transcriptome sequencing (RNA-seq) analysis found that 8.18% of genes were differentially expressed between biofilm and planktonic cell transcriptomes. Biofilm-downregulated genes included those involved in metabolism and translation, suggesting these cells have low metabolic activity. Biofilm-upregulated genes included those whose products were predicted to be at the cell envelope, involved in regulating a stress response, and surprisingly, genes related to formation of the flagellar apparatus. Scanning electron microscopy visualized flagella that appeared to be a component of the biofilm matrix, supported by the observation that an aflagellated mutant displayed a less robust biofilm with no apparent filaments. We observed flagella in the biofilm matrix of additional H. pylori strains, supporting that flagellar use is widespread. Our data thus support a model in which H. pylori biofilm involves a multigene stress-biased response and that flagella play an important role in H. pylori biofilm formation.IMPORTANCE Biofilms, communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances, pose a substantial health risk and are key contributors to many chronic and recurrent infections. Chronicity and recalcitrant infections are also common features associated with the ulcer-causing human pathogen H. pylori However, relatively little is known about the role of biofilms in H. pylori pathogenesis, as well as the biofilm structure itself and the genes associated with this mode of growth. In the present study, we found that H. pylori biofilm cells highly expressed genes related to cell envelope and stress response, as well as those encoding the flagellar apparatus. Flagellar filaments were seen in high abundance in the biofilm. Flagella are known to play a role in initial biofilm formation, but typically are downregulated after that state. H. pylori instead appears to have coopted these structures for nonmotility roles, including a role building a robust biofilm.

Research often relies on well-studied orthologs within related species, with researchers using a well-studied gene or protein to allow prediction of the function of the ortholog. In the opportunistic pathogen Candida albicans, orthologs are usually compared with Saccharomyces cerevisiae, and this approach has been very fruitful. Many transcription factors (TFs) do similar jobs in the two species, but many do not, and typically changes in function are driven not by modifications in the structures of the TFs themselves but in the connections between the transcription factors and their regulated genes. This strategy of changing TF function has been termed transcription factor rewiring. In this study, we specifically looked for rewired transcription factors, or Candida-specific TFs, that might play a role in drug resistance. We investigated 30 transcription factors that were potentially rewired or were specific to the Candida clade. We found that the Adr1 transcription factor conferred resistance to drugs like fluconazole, amphotericin B, and terbinafine when activated. Adr1 is known for fatty acid and glycerol utilization in Saccharomyces, but our study reveals that it has been rewired and is connected to ergosterol biosynthesis in Candida albicans.

Pseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds, and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP-expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP-expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both nonself P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture.IMPORTANCE Biofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight into how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.

Most viruses that replicate in the cytoplasm of host cells form neo-organelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral replication proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Despite the importance of inclusion complexes in viral replication, there are key gaps in the knowledge of how these organelles form and mediate their functions. Reoviruses are nonenveloped, double-stranded RNA (dsRNA) viruses that serve as tractable experimental models for studies of dsRNA virus replication and pathogenesis. Following reovirus entry into cells, replication occurs in large cytoplasmic structures termed inclusions that fill with progeny virions. Reovirus inclusions are nucleated by viral nonstructural proteins, which in turn recruit viral structural proteins for genome replication and particle assembly. Components of reovirus inclusions are poorly understood, but these structures are generally thought to be devoid of membranes. We used transmission electron microscopy and three-dimensional image reconstructions to visualize reovirus inclusions in infected cells. These studies revealed that reovirus inclusions form within a membranous network. Viral inclusions contain filled and empty viral particles and microtubules and appose mitochondria and rough endoplasmic reticulum (RER). Immunofluorescence confocal microscopy analysis demonstrated that markers of the ER and ER-Golgi intermediate compartment (ERGIC) codistribute with inclusions during infection, as does dsRNA. dsRNA colocalizes with the viral protein σNS and an ERGIC marker inside inclusions. These findings suggest that cell membranes within reovirus inclusions form a scaffold to coordinate viral replication and assembly.

Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold.

The apicomplexan parasite Cryptosporidium is a leading global cause of diarrheal disease, and the infection poses a particularly grave threat to young children and those with weakened immune function. Infection occurs by ingestion of meiotic spores called oocysts, and transmission relies on fecal shedding of new oocysts. The entire life cycle thus occurs in a single host and features asexual as well as sexual forms of replication. Here, we identify and locus tag two Apetala 2-type (AP2) transcription factors and demonstrate that they are exclusively expressed in male and female gametes, respectively. To enable functional studies of essential genes in Cryptosporidium parvum, we develop and validate a small-molecule-inducible gene excision system, which we apply to the female factor AP2-F to achieve conditional gene knockout. Analyzing this mutant, we find the factor to be dispensable for asexual growth and early female fate determination in vitro but to be required for oocyst shedding in infected animals in vivo. Transcriptional analyses conducted in the presence or absence of AP2-F revealed that the factor controls the transcription of genes encoding crystalloid body proteins, which are exclusively expressed in female gametes. In C. parvum, the organelle is restricted to sporozoites, and its loss in other apicomplexan parasites leads to blocked transmission. Overall, our development of conditional gene ablation in C. parvum provides a robust method for genetic analysis in this parasite that enabled us to identify AP2-F as an essential regulator of transcription required for oocyst shedding and transmission. IMPORTANCE The parasite Cryptosporidium infects millions of people worldwide each year, leading to life-threatening diarrheal disease in young children and immunosuppressed individuals. There is no vaccine and only limited treatment. Transmission occurs via the fecal-oral route by an environmentally resilient spore-like oocyst. Infection takes place in the intestinal epithelium, where parasites initially propagate asexually before transitioning to male and female gametes, with sex leading to the formation of new oocysts. The essential role of sexual development for continuous infection and transmission makes it an attractive target for therapy and prevention. To study essential genes and potential drug targets across the life cycle, we established inducible gene excision for C. parvum. We determined that the female-specific transcription factor AP2-F is not required for asexual growth and early female development in vitro but is necessary for oocyst shedding in vivo. This work enhances the genetic tools available to study Cryptosporidium gene function.

Ergosterol, the major sterol in fungal membranes, is critical for defining membrane fluidity and regulating cellular processes. Although ergosterol synthesis has been well defined in model yeast, little is known about sterol organization in the context of fungal pathogenesis. We identified a retrograde sterol transporter, Ysp2, in the opportunistic fungal pathogen Cryptococcus neoformans. We found that the lack of Ysp2 under host-mimicking conditions leads to abnormal accumulation of ergosterol at the plasma membrane, invagination of the plasma membrane, and malformation of the cell wall, which can be functionally rescued by inhibiting ergosterol synthesis with the antifungal drug fluconazole. We also observed that cells lacking Ysp2 mislocalize the cell surface protein Pma1 and have abnormally thin and permeable capsules. As a result of perturbed ergosterol distribution and its consequences, ysp2∆ cells cannot survive in physiologically relevant environments such as host phagocytes and are dramatically attenuated in virulence. These findings expand our knowledge of cryptococcal biology and underscore the importance of sterol homeostasis in fungal pathogenesis. IMPORTANCE Cryptococcus neoformans is an opportunistic fungal pathogen that kills over 100,000 people worldwide each year. Only three drugs are available to treat cryptococcosis, and these are variously limited by toxicity, availability, cost, and resistance. Ergosterol is the most abundant sterol in fungi and a key component in modulating membrane behavior. Two of the drugs used for cryptococcal infection, amphotericin B and fluconazole, target this lipid and its synthesis, highlighting its importance as a therapeutic target. We discovered a cryptococcal ergosterol transporter, Ysp2, and demonstrated its key roles in multiple aspects of cryptococcal biology and pathogenesis. These studies demonstrate the role of ergosterol homeostasis in C. neoformans virulence, deepen our understanding of a pathway with proven therapeutic importance, and open a new area of study.

The relevance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF8 in the pathogenesis of COVID-19 is unclear. Virus natural isolates with deletions in ORF8 were associated with wild milder disease, suggesting that ORF8 might contribute to SARS-CoV-2 virulence. This manuscript shows that ORF8 is involved in inflammation and in the activation of macrophages in two experimental systems: humanized K18-hACE2 transgenic mice and organoid-derived human airway cells. These results identify ORF8 protein as a potential target for COVID-19 therapies.

The influenza A virus (IAV) neuraminidase (NA) is essential for virion release from cells and decoy receptors and an important target of antiviral drugs and antibodies. Adaptation to a new host sialome and escape from the host immune system are forces driving the selection of mutations in the NA gene. Phylogenetic analysis shows that until 2015, 16 amino acid substitutions in NA became fixed in the virus population after introduction in the human population of the pandemic IAV H1N1 (H1N1pdm09) in 2009. The accumulative effect of these substitutions, in the order in which they appeared, was analyzed using recombinant proteins and viruses in combination with different functional assays. The results indicate that NA activity did not evolve to a single optimum but rather fluctuated within a certain bandwidth. Furthermore, antigenic and enzymatic properties of NA were intertwined, with several residues affecting multiple properties. For example, the substitution K432E in the second sialic acid binding site, next to the catalytic site, was shown to affect catalytic activity, substrate specificity, and the pH optimum for maximum activity. This substitution also altered antigenicity of NA, which may explain its selection. We propose that the entanglement of NA phenotypes may be an important determining factor in the evolution of NA.IMPORTANCE Since its emergence in 2009, the pandemic H1N1 influenza A virus (IAV) has caused significant disease and mortality in humans. IAVs contain two envelope glycoproteins, the receptor-binding hemagglutinin (HA) and the receptor-destroying neuraminidase (NA). NA is essential for virion release from cells and decoy receptors, is an important target of antiviral drugs, and is increasingly being recognized as an important vaccine antigen. Not much is known, however, about the evolution of this protein upon the emergence of the novel pandemic H1N1 virus, with respect to its enzymatic activity and antigenicity. By reconstructing the evolutionary path of NA, we show that antigenic and enzymatic properties of NA are intertwined, with several residues affecting multiple properties. Understanding the entanglement of NA phenotypes will lead to better comprehension of IAV evolution and may help the development of NA-based vaccines.

There are no approved vaccines against the life-threatening Middle East respiratory syndrome coronavirus (MERS-CoV). Attenuated vaccines have proven their potential to induce strong and long-lasting immune responses. We have previously described that severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a virulence factor. Based on this knowledge, a collection of mutants carrying partial deletions spanning the C-terminal domain of the E protein (rMERS-CoV-E*) has been generated using a reverse genetics system. One of these mutants, MERS-CoV-E*Δ2in, was attenuated and provided full protection in a challenge with virulent MERS-CoV after a single immunization dose. The MERS-CoV-E*Δ2in mutant was stable as it maintained its attenuation after 16 passages in cell cultures and has been selected as a promising vaccine candidate.IMPORTANCE The emergence of the new highly pathogenic human coronavirus SARS-CoV-2 that has already infected more than 80 million persons, killing nearly two million of them, clearly indicates the need to design efficient and safe vaccines protecting from these coronaviruses. Modern vaccines can be derived from virus-host interaction research directed to the identification of signaling pathways essential for virus replication and for virus-induced pathogenesis, in order to learn how to attenuate these viruses and design vaccines. Using a reverse genetics system developed in our laboratory, an infectious cDNA clone of MERS-CoV was engineered. Using this cDNA, we sequentially deleted several predicted and conserved motifs within the envelope (E) protein of MERS-CoV, previously associated with the presence of virulence factors. The in vitro and in vivo evaluation of these deletion mutants highlighted the relevance of predicted linear motifs in viral pathogenesis. Two of them, an Atg8 protein binding motif (Atg8-BM), and a forkhead-associated binding motif (FHA-BM), when deleted, rendered an attenuated virus that was evaluated as a vaccine candidate, leading to full protection against challenge with a lethal dose of MERS-CoV. This approach can be extended to the engineering of vaccines protecting against the new pandemic SARS-CoV-2.

Nonencapsulated Streptococcus pneumoniae (NESp) is an emerging human pathogen that colonizes the nasopharynx and is associated with noninvasive diseases such as otitis media (OM), conjunctivitis, and nonbacteremic pneumonia. Since capsule expression was previously thought to be necessary for establishment of invasive pneumococcal disease (IPD), serotype-specific polysaccharide capsules are targeted by currently licensed pneumococcal vaccines. Yet, NESp expressing oligopeptide binding proteins AliC and AliD have been isolated during IPD. Thus, we hypothesize AliC and AliD are major NESp virulence determinants that facilitate persistence and development of IPD. Our study reveals that NESp expressing AliC and AliD have intensified virulence compared to isogenic mutants. Specifically, we demonstrate AliC and AliD enhance murine nasopharyngeal colonization and pulmonary infection and are required for OM in a chinchilla model. Furthermore, AliC and AliD increase pneumococcal survival in chinchilla whole blood and aid in resistance to killing by human leukocytes. Comparative proteome analysis revealed significant alterations in protein levels when AliC and AliD were absent. Virulence-associated proteins, including a pneumococcal surface protein C variant (CbpAC), were significantly downregulated, while starvation response indicators were upregulated in the double mutant relative to wild-type levels. We also reveal that differentially expressed CbpAC was essential for NESp adherence to epithelial cells, virulence during OM, reduction of C3b deposition on the NESp surface, and binding to nonspecific IgA. Altogether, the rise in NESp prevalence urges the need to understand how NESp establishes disease and persists in a host. This study highlights the roles of AliC, AliD, and CbpAC in the pathogenesis of NESp.IMPORTANCE Despite the effective, widespread use of licensed pneumococcal vaccines over many decades, pneumococcal infections remain a worldwide burden resulting in high morbidity and mortality. NESp subpopulations are rapidly rising in the wake of capsule-targeted vaccine strategies, yet there is very little knowledge on NESp pathogenic potential and virulence mechanisms. Although NESp lacks a protective capsule, NESp lineages expressing AliC and AliD have been associated with systemic infections. Furthermore, higher antibiotic resistance rates and transformation efficiencies associated with emerging NESp threaten treatment strategies needed to control pneumococcal infections and transmission. Elucidating how NESp survives within a host and establishes disease is necessary for development of broadened pneumococcal prevention methods. Our study identifies virulence determinants and host survival mechanisms employed by NESp with a high pathogenic potential. Moreover, our study also identifies virulence determinants shared by NESp and encapsulated strains that may serve as broad prevention and therapeutic targets.

Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome-wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes, and echinocandins. We show the stepwise accumulation of copy number variations and novel mutations in genes both known and unknown in antifungal drug resistance. Echinocandin resistance was accompanied by a codon deletion in FKS1 hot spot 1 and a substitution in FKS1 "novel" hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was linked to a mutation in transcription factor TAC1b and overexpression of the drug efflux pump Cdr1, a segmental duplication of chromosome 1 containing ERG11, and a whole chromosome 5 duplication, which contains TAC1b The latter was associated with increased expression of ERG11, TAC1b, and CDR2 but not CDR1 The simultaneous emergence of nonsense mutations in ERG3 and ERG11 was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross-resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential for and diversity of MDR development in C. auris, even in a clade until now not associated with MDR (clade II), stressing its clinical importance and the urge for future research.IMPORTANCECandida auris is a recently discovered human fungal pathogen and has shown an alarming potential for developing multi- and pan-resistance toward all classes of antifungals most commonly used in the clinic. Currently, C. auris has been globally recognized as a nosocomial pathogen of high concern due to this evolutionary potential. So far, this is the first study in which the stepwise progression of multidrug resistance (MDR) in C. auris is monitored in vitro Multiple novel mutations in known resistance genes and genes previously not or vaguely associated with drug resistance reveal rapid MDR evolution in a C. auris clade II isolate. Additionally, this study shows that in vitro experimental evolution can be a powerful tool to discover new drug resistance mechanisms, although it has its limitations.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma; however, most infected individuals never develop this malignancy. Strain-specific microbial factors, such as the oncoprotein CagA, as well as environmental conditions, such as iron deficiency, augment cancer risk. Importantly, dysbiosis of the gastric microbiota is also associated with gastric cancer. To investigate the combinatorial effects of these determinants in an in vivo model of gastric cancer, Mongolian gerbils were infected with the carcinogenic cag+H. pylori strain 7.13 or a 7.13 cagA isogenic mutant, and microbial DNA extracted from gastric tissue was analyzed by 16S rRNA sequencing. Infection with H. pylori significantly increased gastric inflammation and injury, decreased α-diversity, and altered microbial community structure in a cagA-dependent manner. The effect of iron deficiency on gastric microbial communities was also investigated within the context of infection. H. pylori-induced injury was augmented under conditions of iron deficiency, but despite differences in gastric pathology, there were no significant differences in α- or β-diversity, phyla, or operational taxonomic unit (OTU) abundance among infected gerbils maintained on iron-replete or iron-depleted diets. However, when microbial composition was stratified based solely on the severity of histologic injury, significant differences in α- and β-diversity were present among gerbils harboring premalignant or malignant lesions compared to gerbils with gastritis alone. This study demonstrates that H. pylori decreases gastric microbial diversity and community structure in a cagA-dependent manner and that as carcinogenesis progresses, there are corresponding alterations in community structure that parallel the severity of disease.IMPORTANCE Microbial communities are essential for the maintenance of human health, and when these communities are altered, hosts can become susceptible to inflammation and disease. Dysbiosis contributes to gastrointestinal cancers, and specific bacterial species are associated with this phenotype. This study uses a robust and reproducible animal model to demonstrate that H. pylori infection induces gastric dysbiosis in a cagA-dependent manner and further that dysbiosis and altered microbial community structure parallel the severity of H. pylori-induced gastric injury. Ultimately, such models of H. pylori infection and cancer that can effectively evaluate multiple determinants simultaneously may yield effective strategies for manipulating the gastric microbiota to prevent the development of gastric cancer.