Importance of flagella in acute and chronic Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa is an environmental microorganism and a causative agent of diverse acute and chronic, biofilm-associated infections. Advancing research-based knowledge on its adaptation to conditions within the human host is bound to reveal novel strategies and targets for therapeutic intervention. Here, we investigated the traits that P. aeruginosa PA14 as well as a virulence attenuated ΔlasR mutant need to survive in selected murine infection models. Experimentally, the genetic programs that the bacteria use to adapt to biofilm-associated versus acute infections were dissected by passaging transposon mutant libraries through mouse lungs (acute) or mouse tumours (biofilm-infection). Adaptive metabolic changes of P. aeruginosa were generally required during both infection processes. Counter-selection against flagella expression was observed during acute lung infections. Obviously, avoidance of flagella-mediated activation of host immunity is advantageous for the wildtype bacteria. For the ΔlasR mutant, loss of flagella did not confer a selective advantage. Apparently, other pathogenesis mechanisms are active in this virulence attenuated strain. In contrast, the infective process of P. aeruginosa in the chronic biofilm model apparently required expression of flagellin. Together, our findings imply that the host immune reactions against the infectious agent are very decisive for acuteness and duration of the infectious disease. They direct disease outcome.

Signaling through Lrg1, Rho1 and Pkc1 Governs Candida albicans Morphogenesis in Response to Diverse Cues.

The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is Candida albicans. C. albicans mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of C. albicans filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 C. albicans homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of LRG1 or introduction of a RHO1 mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector PKC1 results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a pkc1Δ/pkc1Δ mutant, adenylyl cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in C. albicans morphogenesis, and illuminate functional relationships that govern activation of a central transducer of signals that control environmental response and virulence programs.

Refining the Application of Microbial Lipids as Tracers of Staphylococcus aureus Growth Rates in Cystic Fibrosis Sputum.

Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effects of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of seven pediatric CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. The microbial community composition was determined by counting rRNA with NanoString DNA analysis, and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two branched-chain ("anteiso") fatty acids (a-C15:0 and a-C17:0) using gas chromatography-mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae; hence, their isotopic enrichment was interpreted as a growth proxy for Staphylococcus aureus Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients; thus, deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small nonmicrobial background source detected in some patient samples, a-C15:0 fatty acid still appears to be a relatively robust proxy for CF pathogens, revealing a median generation time of ∼1.5 days, similar to prior observations. Contrary to our expectation, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two straightforward "best practices" for application of stable-isotope probing to CF sputum metabolites: (i) parallel determination of microbial community composition in CF sputum using culture-independent tools and (ii) assessing background levels of the diagnostic metabolite.IMPORTANCE In chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop a new workflow for the application of stable-isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways.

Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate different environmental and intracellular signals, including metabolic status, to decide the fate of cellular morphology.

Analysis of Candida albicans mutants defective in the Cdk8 module of mediator reveal links between metabolism and biofilm formation.

Candida albicans biofilm formation is a key virulence trait that involves hyphal growth and adhesin expression. Pyocyanin (PYO), a phenazine secreted by Pseudomonas aeruginosa, inhibits both C. albicans biofilm formation and development of wrinkled colonies. Using a genetic screen, we identified two mutants, ssn3Δ/Δ and ssn8Δ/Δ, which continued to wrinkle in the presence of PYO. Ssn8 is a cyclin-like protein and Ssn3 is similar to cyclin-dependent kinases; both proteins are part of the heterotetrameric Cdk8 module that forms a complex with the transcriptional co-regulator, Mediator. Ssn3 kinase activity was also required for PYO sensitivity as a kinase dead mutant maintained a wrinkled colony morphology in the presence of PYO. Furthermore, similar phenotypes were observed in mutants lacking the other two components of the Cdk8 module-Srb8 and Srb9. Through metabolomics analyses and biochemical assays, we showed that a compromised Cdk8 module led to increases in glucose consumption, glycolysis-related transcripts, oxidative metabolism and ATP levels even in the presence of PYO. In the mutant, inhibition of respiration to levels comparable to the PYO-treated wild type inhibited wrinkled colony development. Several lines of evidence suggest that PYO does not act through Cdk8. Lastly, the ssn3 mutant was a hyperbiofilm former, and maintained higher biofilm formation in the presence of PYO than the wild type. Together these data provide novel insights into the role of the Cdk8 module of Mediator in regulation of C. albicans physiology and the links between respiratory activity and both wrinkled colony and biofilm development.

Regulated proteolysis of Candida albicans Ras1 is involved in morphogenesis and quorum sensing regulation.

In Candida albicans, a fungal pathogen, the small G-protein Ras1 regulates many important behaviors including white-opaque switching, biofilm formation, and the induction and maintenance of hyphal growth. Like other Ras proteins, Ras1 is activated upon guanine triphosphate binding, and its activity is further modulated by post-translational lipid modifications. Here, we report that the levels of membrane-associated, full-length Ras1 were higher in hyphae than in yeast, and that yeast contained a shorter, soluble Ras1 species that resulted from cleavage. Deletion of the putative cleavage site led to more rapid induction of hyphal growth and delayed hypha-to-yeast transitions. The cleaved Ras1 species was less able to activate its effector, adenylate cyclase (Cyr1), unless tethered to the membrane by a heterologous membrane-targeting domain. Ras1 cleavage was repressed by cAMP-signalling, indicating the presence of a positive feedback loop in which Cyr1 and cAMP influence Ras1. The C. albicans quorum sensing molecule farnesol, which inhibits Cyr1 and represses filamentation, caused an increase in the fraction of Ras1 in the cleaved form, particularly in nascent yeast formed from hyphae. This newly recognized mode of Ras regulation may control C. albicans Ras1 activity in important ways.

Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus.

The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host as part of its ability to cause disease. The transcription factor SrbA plays a significant role in this process by regulating genes involved in hypoxia and low-iron adaptation, antifungal drug responses and virulence. SrbA is a direct transcriptional regulator of genes encoding key enzymes in the ergosterol biosynthesis pathway, including erg25A and erg25B, and ΔsrbA accumulates C4-methyl sterols, suggesting a loss of Erg25 activity [C4-sterol methyl oxidase (SMO)]. Characterization of the two genes encoding SMOs in Aspergillus fumigatus revealed that both serve as functional C4-demethylases, with Erg25A serving in a primary role, as Δerg25A accumulates more C4-methyl sterol intermediates than Δerg25B. Single deletion of these SMOs revealed alterations in canonical ergosterol biosynthesis, indicating that ergosterol may be produced in an alternative fashion in the absence of SMO activity. A Δerg25A strain displayed moderate susceptibility to hypoxia and the endoplasmic reticulum stress-inducing agent DTT, but was not required for virulence in murine or insect models of invasive aspergillosis. Inducing expression of erg25A partially restored the hypoxia growth defect of ΔsrbA. These findings implicated Aspergillus fumigatus SMOs in the maintenance of canonical ergosterol biosynthesis and indicated an overall involvement in the fungal stress response.

SREBP coordinates iron and ergosterol homeostasis to mediate triazole drug and hypoxia responses in the human fungal pathogen Aspergillus fumigatus.

Sterol regulatory element binding proteins (SREBPs) are a class of basic helix-loop-helix transcription factors that regulate diverse cellular responses in eukaryotes. Adding to the recognized importance of SREBPs in human health, SREBPs in the human fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus are required for fungal virulence and susceptibility to triazole antifungal drugs. To date, the exact mechanism(s) behind the role of SREBP in these observed phenotypes is not clear. Here, we report that A. fumigatus SREBP, SrbA, mediates regulation of iron acquisition in response to hypoxia and low iron conditions. To further define SrbA's role in iron acquisition in relation to previously studied fungal regulators of iron metabolism, SreA and HapX, a series of mutants were generated in the ΔsrbA background. These data suggest that SrbA is activated independently of SreA and HapX in response to iron limitation, but that HapX mRNA induction is partially dependent on SrbA. Intriguingly, exogenous addition of high iron or genetic deletion of sreA in the ΔsrbA background was able to partially rescue the hypoxia growth, triazole drug susceptibility, and decrease in ergosterol content phenotypes of ΔsrbA. Thus, we conclude that the fungal SREBP, SrbA, is critical for coordinating genes involved in iron acquisition and ergosterol biosynthesis under hypoxia and low iron conditions found at sites of human fungal infections. These results support a role for SREBP-mediated iron regulation in fungal virulence, and they lay a foundation for further exploration of SREBP's role in iron homeostasis in other eukaryotes.

Aspergillus fumigatus mitochondrial electron transport chain mediates oxidative stress homeostasis, hypoxia responses and fungal pathogenesis.

We previously observed that hypoxia is an important component of host microenvironments during pulmonary fungal infections. However, mechanisms of fungal growth in these in vivo hypoxic conditions are poorly understood. Here, we report that mitochondrial respiration is active in hypoxia (1% oxygen) and critical for fungal pathogenesis. We generated Aspergillus fumigatus alternative oxidase (aoxA) and cytochrome C (cycA) null mutants and assessed their ability to tolerate hypoxia, macrophage killing and virulence. In contrast to ΔaoxA, ΔcycA was found to be significantly impaired in conidia germination, growth in normoxia and hypoxia, and displayed attenuated virulence. Intriguingly, loss of cycA results in increased levels of AoxA activity, which results in increased resistance to oxidative stress, macrophage killing and long-term persistence in murine lungs. Thus, our results demonstrate a previously unidentified role for fungal mitochondrial respiration in the pathogenesis of aspergillosis, and lay the foundation for future research into its role in hypoxia signalling and adaptation.

In vivo hypoxia and a fungal alcohol dehydrogenase influence the pathogenesis of invasive pulmonary aspergillosis.

Currently, our knowledge of how pathogenic fungi grow in mammalian host environments is limited. Using a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA) and (1)H-NMR metabolomics, we detected ethanol in the lungs of mice infected with Aspergillus fumigatus. This result suggests that A. fumigatus is exposed to oxygen depleted microenvironments during infection. To test this hypothesis, we utilized a chemical hypoxia detection agent, pimonidazole hydrochloride, in three immunologically distinct murine models of IPA (chemotherapeutic, X-CGD, and corticosteroid). In all three IPA murine models, hypoxia was observed during the course of infection. We next tested the hypothesis that production of ethanol in vivo by the fungus is involved in hypoxia adaptation and fungal pathogenesis. Ethanol deficient A. fumigatus strains showed no growth defects in hypoxia and were able to cause wild type levels of mortality in all 3 murine models. However, lung immunohistopathology and flow cytometry analyses revealed an increase in the inflammatory response in mice infected with an alcohol dehydrogenase null mutant strain that corresponded with a reduction in fungal burden. Consequently, in this study we present the first in vivo observations that hypoxic microenvironments occur during a pulmonary invasive fungal infection and observe that a fungal alcohol dehydrogenase influences fungal pathogenesis in the lung. Thus, environmental conditions encountered by invading pathogenic fungi may result in substantial fungal metabolism changes that influence subsequent host immune responses.

HacA-independent functions of the ER stress sensor IreA synergize with the canonical UPR to influence virulence traits in Aspergillus fumigatus.

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacA(i), or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireA(Δ10). Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus.

Hypoxia and fungal pathogenesis: to air or not to air?

Over the last 3 decades, the frequency of life-threatening human fungal infections has increased as advances in medical therapies, solid-organ and hematopoietic stem cell transplantations, an increasing geriatric population, and HIV infections have resulted in significant rises in susceptible patient populations. Although significant advances have been made in understanding how fungi cause disease, the dynamic microenvironments encountered by fungi during infection and the mechanisms by which they adapt to these microenvironments are not fully understood. As inhibiting and preventing in vivo fungal growth are main goals of antifungal therapies, understanding in vivo fungal metabolism in these host microenvironments is critical for the improvement of existing therapies or the design of new approaches. In this minireview, we focus on the emerging appreciation that pathogenic fungi like Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus are exposed to oxygen-limited or hypoxic microenvironments during fungal pathogenesis. The implications of these in vivo hypoxic microenvironments for fungal metabolism and pathogenesis are discussed with an aim toward understanding the potential impact of hypoxia on invasive fungal infection outcomes.

The small GTPase RacA mediates intracellular reactive oxygen species production, polarized growth, and virulence in the human fungal pathogen Aspergillus fumigatus.

Aspergillus fumigatus is the predominant mold pathogen in immunocompromised patients. In this study, we present the first characterization of the small GTPase RacA in A. fumigatus. To gain insight into the function of racA in the growth and pathogenesis of A. fumigatus, we constructed a strain that lacks a functional racA gene. The ΔracA strain showed significant morphological defects, including a reduced growth rate and abnormal conidiogenesis on glucose minimal medium. In the ΔracA strain, apical dominance in the leading hyphae is lost and, instead, multiple axes of polarity emerge. Intriguingly, superoxide production at the hyphal tips was reduced by 25% in the ΔracA strain. Treatment of wild-type hyphae with diphenylene iodonium, an inhibitor of NADPH oxidase, resulted in phenotypes similar to that of the ΔracA strain. These data suggest that ΔracA strain phenotypes may be due to a reduction or alteration in the production of reactive oxygen species. Most surprisingly, despite these developmental and growth abnormalities, the ΔracA strain retained at least wild-type virulence in both an insect model and two immunologically distinct murine models of invasive pulmonary aspergillosis. These results demonstrate that in vitro growth phenotypes do not always correlate with in vivo virulence and raise intriguing questions about the role of RacA in Aspergillus virulence.

Trehalose 6-phosphate phosphatase is required for cell wall integrity and fungal virulence but not trehalose biosynthesis in the human fungal pathogen Aspergillus fumigatus.

The trehalose biosynthesis pathway is critical for virulence in human and plant fungal pathogens. In this study, we tested the hypothesis that trehalose 6-phosphate phosphatase (T6PP) is required for Aspergillus fumigatus virulence. A mutant of the A. fumigatus T6PP, OrlA, displayed severe morphological defects related to asexual reproduction when grown on glucose (1%) minimal media. These defects could be rescued by addition of osmotic stabilizers, reduction in incubation temperature or increase in glucose levels (> 4%). Subsequent examination of the mutant with cell wall perturbing agents revealed a link between cell wall biosynthesis and trehalose 6-phosphate (T6P) levels. As expected, high levels of T6P accumulated in the absence of OrlA resulting in depletion of free inorganic phosphate and inhibition of hexokinase activity. Surprisingly, trehalose production persisted in the absence of OrlA. Further analyses revealed that A. fumigatus contains two trehalose phosphorylases that may be responsible for trehalose production in the absence of OrlA. Despite a normal growth rate under in vitro growth conditions, the orlA mutant was virtually avirulent in two distinct murine models of invasive pulmonary aspergillosis. Our results suggest that further study of this pathway will lead to new insights into regulation of fungal cell wall biosynthesis and virulence.

TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.

Detection of Low Oxygen Microenvironments in a Murine Model of Invasive Pulmonary Aspergillosis Using Pimonidazole.

Infection tissue microenvironments are dynamic, complex, and play a critical role in host-microbe interaction outcomes. A crucial parameter of the infection site microenvironment is oxygen. Both host and microbial cell physiology is significantly impacted by the availability of oxygen. When oxygen tensions drop to levels that do not meet the metabolic demands of the cell, a hypoxia response ensues. In numerous host-microbe studies, it has now been observed that the host and microbial hypoxia response plays a critical role in disease outcomes. However, in most pathosystems, spatial and temporal oxygen dynamics throughout the infection remain ill defined. Here, we detail a protocol for detecting low oxygen environments in tissue in a murine model of invasive pulmonary aspergillosis. The protocol utilizes mice immune compromised with a high dose of steroid and challenged via the aerosol route with conidia of the major human fungal pathogen Aspergillus fumigatus. Qualitative analysis of oxygen levels at the site of infection in the murine lung is accomplished with pimonidazole-mediated adduct detection via immunohistochemistry. The protocol is adaptable to other host-microbe interaction models.

A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus.

At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA null mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA null mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds.

Regulation of hypoxia adaptation: an overlooked virulence attribute of pathogenic fungi?

Over the past two decades, the incidence of fungal infections has dramatically increased. This is primarily due to increases in the population of immunocompromised individuals attributed to the HIV/AIDS pandemic and immunosuppression therapies associated with organ transplantation, cancer, and other diseases where new immunomodulatory therapies are utilized. Significant advances have been made in understanding how fungi cause disease, but clearly much remains to be learned about the pathophysiology of these often lethal infections. Fungal pathogens face numerous environmental challenges as they colonize and infect mammalian hosts. Regardless of a pathogen's complexity, its ability to adapt to environmental changes is critical for its survival and ability to cause disease. For example, at sites of fungal infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments to which both the pathogen and host cells must adapt in order to survive. However, our current knowledge of how pathogenic fungi adapt to and survive in hypoxic conditions during fungal pathogenesis is limited. Recent studies have begun to observe that the ability to adapt to various levels of hypoxia is an important component of the virulence arsenal of pathogenic fungi. In this review, we focus on known oxygen sensing mechanisms that non-pathogenic and pathogenic fungi utilize to adapt to hypoxic microenvironments and their possible relation to fungal virulence.

Parallel evolutionary paths to produce more than one Pseudomonas aeruginosa biofilm phenotype.

Studying parallel evolution of similar traits in independent within-species lineages provides an opportunity to address evolutionary predictability of molecular changes underlying adaptation. In this study, we monitored biofilm forming capabilities, motility, and virulence phenotypes of a plethora of phylogenetically diverse clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa. We also recorded biofilm-specific and planktonic transcriptional responses. We found that P. aeruginosa isolates could be stratified based on the production of distinct organismal traits. Three major biofilm phenotypes, which shared motility and virulence phenotypes, were produced repeatedly in several isolates, indicating that the phenotypes evolved via parallel or convergent evolution. Of note, while we found a restricted general response to the biofilm environment, the individual groups of biofilm phenotypes reproduced biofilm transcriptional profiles that included the expression of well-known biofilm features, such as surface adhesive structures and extracellular matrix components. Our results provide insights into distinct ways to make a biofilm and indicate that genetic adaptations can modulate multiple pathways for biofilm development that are followed by several independent clinical isolates. Uncovering core regulatory pathways that drive biofilm-associated growth and tolerance towards environmental stressors promises to give clues to host and environmental interactions and could provide useful targets for new clinical interventions.

Aspergillus fumigatus metabolism: clues to mechanisms of in vivo fungal growth and virulence.

Aspergillus fumigatus is a saprophytic fungus commonly found in soil and compost piles. In immunocompromised patients it takes on a sinister form as a potentially lethal opportunistic human pathogen. We currently have a limited understanding of the in vivo growth mechanisms used by A. fumigatus during invasive pulmonary aspergillosis (IPA). The ability of A. fumigatus to adapt to various microenvironments encountered during growth in the human host may explain why A. fumigatus is the most frequently occurring opportunistic pathogenic mold. The transcriptional and metabolic responses to changing microenvironments found in the mammalian lung require the activation of pathways implicated in resistance to unique stresses. Thus, the production of primary metabolites in vivo may give clues to the critical pathways used by A. fumigatus to cause disease in human hosts. We recently have identified primary metabolites in the mammalian lung typically associated with fungal growth under hypoxic environments suggesting that A. fumigatus may encounter low oxygen tensions during IPA. These and other studies on A. fumigatus metabolism are the focus of this review.