mSphere

Clostridioides difficile spores are essential for initiation, recurrence and transmission of the disease. The spore surface layers are composed of an outermost exosporium layer that surrounds another proteinaceous layer, the spore coat. These spore surfaces layers are responsible for initial interactions with the host and spore resistance properties contributing to transmission and recurrence of CDI. During spore-development, assembly of both layers is tightly interconnected thus studying the surface is essential for understanding the assembly of these layers and identification of potential targets for therapeutics. Several spore coat /exosporium extraction methods have utilized different extraction procedures making comparison across studies difficult and their impact on spore surface layer properties remains unclear. Here, we tested how commonly used chemical methods remove the spore coat /exosporium layers, analyzing treated-spores by phase contrast microscopy, transmission electron microscopy, western blotting, and lysozyme-triggered germination to functionally characterize the extraction efficiency of these treatment on these layers. Our results provide a systematic analysis and offer a platform for future spore coat and exosporium-related studies.

Candida albicans is one of them most common causes of fungal disease in humans and is a commensal member of the human microbiome. The ability of C. albicans to cause disease is tightly correlated with its ability to undergo a morphological transition from budding yeast to a filamentous form (hyphae and pseudohyphae). This morphological transition is accompanied by the induction of a set of well characterized hyphae-associated genes and transcriptional regulators. To date, the vast majority of data regarding this process has been based on in vitro studies of filamentation using a range of inducing conditions. Recently, we developed an in vivo imaging approach that allows the direct characterization of morphological transition during mammalian infection. Here, we couple this imaging assay with in vivo expression profiling to characterize the time course of in vivo filamentation and the accompanying changes in gene expression. We also compare in vivo observations to in vitro filamentation using a medium (RPMI 1640 tissue culture medium with 10% bovine calf serum) widely used to mimic host conditions. From these data, we make the following conclusions regarding in vivo and in vitro filamentation. First, the transcriptional programs regulating filamentation are rapidly induced in vitro and in vivo. Second, the tempo of filamentation in vivo is prolonged relative to in vitro filamentation and the period of high expression of genes associated with that process is also prolonged. Third, hyphae are adapting to changing infection environments after filamentation has reached steady-state. ImportanceCandida albicans filamentation is correlated with virulence and is an intensively studied aspect of C. albicans biology. The vast majority of studies on C. albicans filamentation are based on in vitro induction of hyphae and pseudohyphae. Here we used an in vivo filamentation assay and in vivo expression profiling to compare the tempo of morphogenesis and gene expression between in vitro and in vivo filamentation. Although the hyphal gene expression profile is induced rapidly in both conditions, it remains stably expressed over the 24hr time course in vivo while the expression of other environmentally responsive genes is dynamic. As such, it is important to regard the filamentation process as a separate growth phase of C. albicans that is as adaptable to changing growth conditions as the more familiar yeast phase.

The fungal pathogen Cryptococcus neoformans (C. neoformans) forms yeast cells of different sizes and morphological characteristics during infection. These features are usually not seen in standard laboratory in vitro conditions. Here, we describe in vivo cell morphologies when C. neoformans is grown in human plasma-like medium at 37{degrees}C-5% CO2. We observed mixed-size populations of cells less than 1 m up to 16.8 m cell diameter, increased capsule size, high chitin and DNA content in larger cells. Our findings show serum is not required for HPLM-induced C. neoformans cellular heterogeneity. Thus, this new method offers an opportunity to investigate factors of C. neoformans that mediate pathogenesis or host-pathogen interactions in a physiologically relevant setting. IMPORTANCEDescription of new in vitro culture condition using the human plasma-like medium that supports the formation of the full range of in vivo cell morphologies of C. neoformans.

Aspergillus fumigatus poses a significant threat to human well-being, in part due to the increasing emergence of strains resistant to frontline antifungal therapy. In this study, we observe that the gene, arvA, is required for A. fumigatus morphogenesis, antifungal drug susceptibility, and cell wall homeostasis. Intriguingly, our study reveals novel morphological and growth aberrations in the absence of arvA. Loss of arvA results in hyper-swollen conidia that give rise to stunted, polarity-deficient hyphae in numerous environmental conditions, indicating a pivotal role for arvA in A. fumigatus morphogenesis. Surprisingly, despite these severe in vitro morphological and cell wall defects, arvA was not required for morbidity and mortality in immunologically distinct murine models of invasive pulmonary aspergillosis (IPA). However, growth in natural calf lung surfactant was able to normalize{Delta} arvA growth with the wild-type strain suggesting lung surfactant may partially complement the severe in vitro morphological defects of arvA loss in vivo. Taken together our observations reveal arvA as a mediator of A. fumigatus antifungal drug susceptibility and highlight the complex and ill-defined pulmonary nutrient environments role in mediating A. fumigatus pathogenesis and disease progression. IMPORTANCEAspergillus fumigatus is a challenging fungal pathogen in the clinic in part due to increasing azole drug resistance. In this study, we observe that loss of the A. fumigatus gene arvA results in increased azole susceptibility and significant in vitro morphological changes highlighted by hyper-swollen conidia that yield stunted and polarity deficient hyphae. Importantly, despite these severe in vitro morphological and growth abnormalities,{Delta} arvA surprisingly retains full pathogenicity and virulence in two immunologically distinct murine models of invasive pulmonary aspergillosis. These results challenge our understanding of the in-host environment and how it mediates fungal morphogenesis and pathogenesis. These results, consequently, not only enhances our understanding of the role of arvA in A. fumigatus morphogenesis and drug susceptibility, but further emphasizes the importance of in vivo animal models in fully evaluating potential antifungal drug targets.

Functionally similar to a plant vacuole or a mammalian lysosome, the fungal vacuole plays a vital role in many cellular processes. Most studies of the vacuole have been performed in the nonpathogenic yeast Saccharomyces cerevisiae; however, recently in pathogenic fungi, the vacuole has been implicated in host invasion in both plants and mammals highlighting an important role for the vacuole in pathogenesis. Here, we report that deletion of Cryptococcus neoformans vacuolar protein 8 (VAC8) results in a fragmented vacuole morphology, impairment of vacuolar fusion, and inability to form titan cells. Additionally, absence of Vac8 results in defective growth at high temperature and in the presence of caffeine, suggesting a defect in cell wall signaling. Interestingly, despite aberrant vacuole morphology, vac8{Delta} is slightly more resistant to fluconazole treatment, and displays increased resistance to hydrogen peroxide, suggesting the irregular vacuole morphology does not impair vacuole function. Like S. cerevisiae Vac8, C. neoformans Vac8 is comprised of armadillo repeat regions which form alpha helices that fold to form a superhelix allowing for increased protein-protein interaction. Many of the known binding partners of S. cerevisiae Vac8 are not present in the C. neoformans genome, suggesting novel functions for Vac8 in this fungus. Notably, deletion of VAC8 affected some virulence traits, providing support to targeting the fungal vacuole as a potential therapeutic intervention.

Secondary infection with Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 L droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airways surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts. ImportanceThe impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, and a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially-complex solutions to better mimic physiologically relevant conditions.

Long non-coding RNAs play an important role in stress response in all forms of life; however, a tight regulation of lncRNAs is required for normal function. Abnormal expression of lncRNA is associated with uncontrolled cell growth in many forms of cancer. Recent studies have highlighted the role of lncRNAs in Aspergillus fumigatus in azole stress response and virulence. Here, using a transcriptome dataset of A. fumigatus response to stress, we identified afu-254 as an 854bp lncRNA that plays a role in modulating oxidative stress, fungal sub-MIC azole response to posaconazole and itraconazole, cell wall stress, macrophage phagocytosis and killing ex vivo, and virulence in an invertebrate model of Aspergillus infection. Importantly, afu-254 does not produce cross-azole susceptible response and plays a role in fungal azole response against posaconazole and itraconazole but not voriconazole. Furthermore, we showed that stochiometric levels of afu-254 are important for its function, and ectopic overexpression of afu-254 in the WT strain leads to an antimorph. This phenotype may stem from a higher ordered structure that is denatured with heat, indicating the presence of a non-functional isoform. In conclusion, we characterized a novel lncRNA, afu-254, that is important for stress response and virulence in the pathogenic fungus A. fumigatus. ImportanceFailure of azole treatment for invasive Aspergillus infection by both drug-resistant and drug-sensitive isolates is an area of concern and global importance. Fungal stress response is multifaceted, and long non-coding RNAs have emerged as important players in mediating it, including regulating responses to azole antifungals. Here, we have identified a long non-coding RNA, afu-254, that plays a role in modulating fungal response to oxidative stress, cell wall stress, azole stress, immune cell stress and virulence in an invertebrate model of invasive Aspergillus infection.

Acanthamoeba castellanii is a free-living amoeba (FLA) that causes fatal human infections with few effective treatments. One limitation of new drug development is the lack of accurate, high-throughput, and quantitative viability assays that score both trophozoites and cysts. A colorimetric assay using Sulforhodamine B (SRB), which measures cell adherence, has previously been adapted for A. castellanii trophozoites. In this study, we demonstrated that the SRB assay can be optimized to serve as a robust, high-throughput platform that can measure viable Acanthamoeba trophozoites, pseudocysts and cysts. We used this assay to measure the IC50 of 18 commonly used drugs and disinfectants on A. castellanii trophozoites, demonstrating that several clinically used drugs induce pseudocyst formation rather than amoeba death.

The structural, antigenic, and immunological characterization of microbial polysaccharides requires purification that often involves detergent precipitation and lyophilization. Here we examine physicochemical changes induced by lyophilization on exopolysaccharide (EPS) of the pathogenic fungus Cryptococcus neoformans. Solution 1H NMR reveals significant anomeric signal attenuation following lyophilization of native EPS while 1H ssNMR shows few changes, suggesting diminished molecular motion and consequent broadening of 1H NMR polysaccharide resonances. 13C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense rosette-like aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide binds the native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.

Extracellular vesicles (EVs) play crucial roles in fungal communication and host immune modulation, representing potential therapeutic targets for fungal infections. This study investigated the role of fungal EVs in both intra- and interspecies communication, focusing on their effects on virulence and immune responses. Co-incubation experiments were performed using EVs derived from Candida albicans and Candida auris to assess interactions with C. albicans planktonic cells and biofilms, as well as Cryptococcus neoformans and Cryptococcus gattii EVs interacting with C. neoformans cultures. EVs were observed associating with recipient cell surfaces, suggesting subsequent internalization. Functional assays revealed that EV exposure led to increased expression of Cap59, Lac1, Ure1, and Erg11 genes, correlating with reduced antifungal susceptibility in both planktonic and biofilm forms. Additionally, EVs facilitated cross-species communication, enhancing biofilm adhesion and dispersion, which underscores their role in phenotypic modulation. Macrophages stimulated with fungal EVs exhibited receptor-specific gene expression changes, notably the upregulation of galectin-3, along with a pro-inflammatory phenotype marked by increased iNOS expression and elevated cytokine levels (IL-1{beta}, IL-6, and IL-8). Collectively, these findings underscore a critical role for fungal EVs in interspecies communication, biofilm regulation, and immune modulation, offering valuable insights into fungal pathogenicity mechanisms. ImportanceCurrently, no vaccines exist to prevent fungal infections, underscoring the need for new therapies. As fungal diseases increase globally, understanding fungal biology is essential to identifying treatment targets. Fungi use EVs to communicate and evade immune responses. EVs mediate cell-cell communication, transporting proteins, polysaccharides, lipids, and nucleic acids - serving as "messages" exchanged within a fungal network. Understanding how these vesicles facilitate communication not only within a single species but also across different fungal species can shed light on their contribution to infection persistence and cross-species adaptability. Moreover, EVs may have a broader role in inter-kingdom communication, influencing how fungi interact with host immune cells. The impact of fungal EVs on human innate immune responses remains a largely underexplored area, with significant gaps in our knowledge. This study aims to examine how fungal EVs affect immune responses and whether their signaling varies across species, potentially revealing new therapeutic targets.

Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked super-resolution imaging for a broader scientific circle, lowering the cost and entry skill requirements to the field. One of its branches, ultrastructure ExM (U-ExM), has become popular among research groups studying Apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. The chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localisation. Here, we solve the in vitro cysts resistance to denaturation required for successful U-ExM of the encapsulated parasites. As the cysts main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using the pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal cell culture without interference with the epitopes of the cyst-wall associated proteins. Using StcE-enhanced U-ExM, we clarified the shape and location of the GRA2 protein important for establishing a normal cyst. Expanded cysts revealed GRA2 granules spanning across the cyst wall, with a notable presence observed outside on both sides of the CST1-positive layer. ImportanceToxoplasma gondii is an intracellular parasite capable of establishing long-term chronic infection in nearly all warm-blooded animals. During the chronic stage, parasites encapsulate into cysts in a wide range of tissues but particularly in neurons of the central nervous system and in skeletal muscle. Current anti-Toxoplasma drugs do not eradicate chronic parasites and leave behind a reservoir of infection. As the cyst is critical for both transmission and pathology of the disease, we need to understand more fully the biology of the cyst and its vulnerabilities. The advent of a new super-resolution approach called ultrastructure expansion microscopy allowed in-depth studies of the acute stage of Toxoplasma infection but not the cyst-forming stage, which resists protocol-specific denaturation. Here, we show that an additional step of enzymatic digestion using mucinase StcE allows full expansion of the Toxoplasma cysts, offering a new avenue for a comprehensive examination of the chronic stage of infection using an accessible super-resolution technique.

Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth, and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model that simulates the nutrient conditions of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate. However, changes in bile salt did not correlate with increased C. difficile susceptibility. Further, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to persistence of C. difficile in antibiotic- treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (prdB-) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. Inability to ferment glycine or leucine as electron acceptors, however, was not sufficient to limit colonization in any communities. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization of C. difficile. IMPORTANCEC. difficile is one of the leading causes of hospital-acquired infections and antibiotic-associated diarrhea. Several potential mechanisms through which the microbiota can limit C. difficile infection have been identified and are potential targets for new therapeutics. However, it is unclear which mechanisms of C. difficile inhibition represent the best targets for development of new therapeutics. These studies demonstrate that in a complex in vitro model of C. difficile infection, colonization resistance is independent of microbial bile salt metabolism. Instead, the ability of C. difficile to colonize is dependent upon its ability to metabolize proline, although proline-dependent colonization is context-dependent and is not observed in all disrupted communities. Altogether, these studies support the need for further work to understand how bile- independent mechanisms regulate C. difficile colonization.

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.

Sporulation is an integral process in the lifecycle of many fungal pathogens, including Histoplasma, a primary human pathogen that causes respiratory infections. Histoplasma conidia, or asexual spores, are the primary infectious particle but very little is known about them, in part due to the need for Biosafety Level 3 containment and inconsistency in generating viable conidia under laboratory conditions. Here, we identify media that consistently promote Histoplasma conidiation, yielding both micro- and macroconidia, and conditions that promote high levels of germination. We show that conidiation media and duration affect the proportion of macroconidia produced, and we demonstrate that Histoplasma strains vary in their response to these conidiation parameters. Finally, imaging studies of chitin, exposed chitin, and cell wall mannoproteins show that while micro- and macroconidia have similar cell wall compositions, strain type and conidiation media variation result in qualitative differences in staining. These optimized methods for Histoplasma conidial preparations will enable more detailed investigations into this understudied aspect of the biology of an important human fungal pathogen.

Pathogenic free-living amoebae (FLA) such as Acanthamoeba castellanii are present in soil and water worldwide. A. castellanii causes systemic infections with very high mortality rates, yet drugs specifically targeting this pathogen are not available. Methods to reliably generate and assay cysts, which drive infection recurrence and drug resistance, are unavailable in a high-throughput format suitable for drug screening and testing. In this study, we developed a robust and reproducible protocol for encysting A. castellanii as well as a high-throughput, quantitative cyst viability assay using fluorescent live/dead staining coupled with microscopy and automated image analysis. These methods were used to screen the cysticidal activity of 17 clinically relevant drugs and disinfectants and identified five agents, including caspofungin, as active against cysts.

The increasing prevalence of invasive fungal pathogens are dramatically changing the clinical landscape of infectious diseases and are an imminent burden to public health that lack the resources (i.e., robust antifungals) to tackle this threat. Specifically, the human opportunistic pathogen, Cryptococcus neoformans, expresses elaborate virulence mechanisms and is equipped with sophisticated adaptation strategies to survive in harsh host environments. In this study, we extensively characterize Wos2, an Hsp90 co-chaperone homologue, featuring bilateral functioning for both cryptococcal adaptation and virulence strategies. Here, we evaluated the proteome and secretome signatures of Wos2 in enriched and infection-mimicking conditions to reveal a Wos2-dependent regulation of oxidative stress response. The wos2{Delta} strain reports defective intracellular and extracellular antioxidant protection systems measurable through a decreased abundance of critical antioxidant enzymes and reduced growth in the presence of peroxide stress. Additional Wos2-associated stress phenotypes were observed upon fungal challenge with heat shock, osmotic, and cell wall stressors. We demonstrate the importance of Wos2 for C. neoformans intracellular lifestyle during in vitro macrophage infection and provide evidence for wos2{Delta} reduced phagosomal replication levels. Accordingly, wos2{Delta} featured significantly reduced virulence in a murine model of cryptococcosis. Our study highlights a vulnerable point in the fungal chaperone network that offers a powerful druggable opportunity to interfere with both virulence and fitness. Author SummaryThe global impact of fungal pathogens, both emerging and emerged, is undeniable and the alarming increase in antifungal resistance rates hampers our ability to protect the global population from deadly infections. For cryptococcal infections, a limited arsenal of antifungals and resistance demands alternative therapeutic strategies, including an anti-virulence approach, which disarms the pathogen of critical virulence factors, empowering the host to remove the pathogen and clear the infection. To this end, we apply state-of-the-art mass spectrometry-based proteomics to interrogate the impact of a recently defined novel co-chaperone, Wos2, towards cryptococcal virulence using in vitro and in vivo models of infection. We defined global proteome and secretome remodeling driven by the protein and uncovered a novel role in modulating the fungal oxidative stress response. Complementation of the proteome findings with in vitro infectivity assays demonstrated a protective role for Wos2 within the macrophage phagosome, influencing fungal replication and survival. These results underscore differential cryptococcal survivability and weakened patterns of dissemination in the absence of wos2. Overall, our study establishes Wos2 as an important contributor to fungal pathogenesis and warrants further research into critical proteins within global stress response networks as potential druggable targets to reduce fungal virulence and clear the infection.

Candida albicans is one of the most common causes of superficial and invasive fungal disease in humans. Its ability to cause disease has been closely linked to its ability to undergo a morphological transition from budding yeast to filamentous forms (hyphae and pseudohyphae). The ability of C. albicans strains isolated from patients to undergo filamentation varies significantly. In addition, the filamentation phenotypes of mutants involving transcription factors that positively regulate hyphal morphogenesis can also vary from strain to strain. Here, we characterized the virulence, in vitro and in vivo filamentation, and in vitro and in vivo hypha-associated gene expression profiles of four poorly filamenting C. albicans isolates and their corresponding deletion mutants of the repressor of filamentation NRG1. The two most virulent strains, 57055 and 78048, show robust in vivo filamentation while remaining predominately yeast phase exposed to RPMI+10% bovine calf serum at 37{degrees}C; the two low virulence strains (94015 and 78042) do not filament well under either condition. Deletion of NRG1 increases hyphae formation in the SC5314 derivative SN250 but only pseudohyphae are formed in the clinical isolates in vivo. Deletion of NRG1 modestly increased the virulence of 78042 which was accompanied by increased expression of hyphae-associated genes without an increase in filamentation. Strikingly, deletion of NRG1 in 78048 reduced filamentation, expression of candidalysin (ECE1) and virulence in vivo without dramatically altering establishment of infection. Thus, the function of NRG1 varies significantly within this set of C. albicans isolates and can actually suppress filamentation in vivo. ImportanceClinical isolates of the human fungal pathogen Candida albicans show significant variation in their ability to undergo in vitro filamentation and in the function of well-characterized transcriptional regulators of filamentation. Here, we show that Nrg1, a key repressor of filamentation and filament specific gene expression in standard reference strains, has strain dependent functions, particularly during infection. Most strikingly, loss of NRG1 function can reduce filamentation, hypha-specific gene expression such as the toxin candidalysin, and virulence in some strains. Our data emphasize that the functions of seemingly fundamental and well-conserved transcriptional regulators such as Nrg1 are contextual with respect to both environment and genetic background.

Many mammalian viruses encounter bacteria and bacterial molecules over the course of infection. Previous work has shown that the microbial ecology of the gut plays an integral role in poliovirus and coxsackievirus infection, where bacterial glycans can facilitate virus-receptor interactions, enhance viral replication, and stabilize viral particles. However, how airway bacteria alter respiratory viral infection is less understood. Therefore, we investigated whether a panel of airway bacteria affect rhinovirus stability. We found that Pseudomonas aeruginosa, an opportunistic airway pathogen, protects human rhinovirus 14 from acid or heat inactivation. Further investigation revealed that P. aeruginosa rhamnolipids, glycolipids with surfactant properties, are necessary and sufficient for stabilization of rhinovirus virions. Taken together, this work demonstrates that specific molecules produced by an opportunistic airway pathogen can influence a respiratory virus. ImportanceBacteria can enhance viral stability and infection for enteric members of the Picornaviridae such as poliovirus and coxsackievirus; however, whether bacteria influence respiratory picornaviruses is unknown. In this study, we examined impacts of airway bacteria on rhinovirus, a major etiological agent of the common cold. We found that P. aeruginosa protects human rhinovirus 14 from both acid and heat inactivation through rhamnolipids. Overall, this work demonstrates bacterial effects on respiratory virus through specific bacterial molecules.

Candida albicans is a common human fungal pathogen that is also a commensal of the oral cavity and gastrointestinal tract. C. albicans pathogenesis is linked to its transition from budding yeast to filamentous morphologies including hyphae and pseudohyphae. The centrality of this virulence trait to C. albicans pathobiology has resulted in extensive characterization a wide range factors associated with filamentation with a strong focus on transcriptional regulation. The vast majority of these experiments have used in vitro conditions to induce the yeast-to-filament transition. Taking advantage of in vivo approaches to quantitatively characterize both morphology and gene expression during filamentation during mammalian infection, we have investigated the dynamics of these two aspects of filamentation in vivo and compared them to in vitro filament induction with "host-like" tissue culture media supplemented with serum at mammalian body temperature. Although filamentation shares many common features in the two conditions, we have found two significant differences. First, alternative carbon metabolism genes are expressed early during in vitro filamentation and late in vivo, suggesting significant differences in glucose availability. Second, C. albicans begins a hyphae-to-yeast transition after 4hr incubation while we find little evidence of hyphae-to-yeast transition in vivo up to 24hr post infection. We show that the low rate of in vivo hyphae-to-yeast transition is likely due to very low expression of PES1, a key driver of lateral yeast in vitro, and that heterologous expression of PES1 is sufficient to trigger lateral yeast formation in vivo. ImportanceCandida albicans filamentation is correlated with virulence and is an intensively studied aspect of C. albicans biology. The vast majority of studies on C. albicans filamentation are based on in vitro induction of hyphae and pseudohyphae. Here we used an in vivo filamentation assay and in vivo expression profiling to compare the tempo of morphogenesis and gene expression between in vitro and in vivo filamentation. Although the hyphal gene expression profile is induced rapidly in both conditions, it remains stably expressed over a 12hr time course in vivo while it peaks after 4hr in vitro and is reduced. This reduced hyphal gene expression in vitro correlates with reduced hyphae and increased hyphae-to-yeast transition. In contrast, there is little evidence of evidence of hyphae-to-yeast transition in vivo.

Candida albicans is a diploid human fungal pathogen that displays significant genomic and phenotypic heterogeneity over a range of virulence traits and in the context of a variety of environmental niches. Here, we show that the effects of Rob1 on biofilm and filamentation virulence traits is dependent on both the specific environmental condition and the clinical strain of C. albicans. The C. albicans reference strain SC5314 is a ROB1 heterozygote with two alleles that differ by a single nucleotide polymorphism at position 946 resulting in a serine or proline containing isoform. An analysis of 224 sequenced C. albicans genomes indicates that SC5314 is the only ROB1 heterozygote documented to date and that the dominant allele contains a proline at position 946. Remarkably, the ROB1 alleles are functionally distinct and the rare ROB1946Sallele supports increased filamentation in vitro and increased biofilm formation in vitro and in vivo, suggesting it is a phenotypic gain-of-function allele. SC5314 is amongst the most highly filamentous and invasive strains characterized to date. Introduction of the ROB1946Sallele into a poorly filamenting clinical isolate increases filamentation and conversion of an SC5314 laboratory strain to a ROB1946S homozygote increases in vitro filamentation and biofilm formation. In a mouse model of oropharyngeal infection, the predominant ROB1946P allele establishes a commensal state while the ROB1946S phenocopies the parent strain and invades into the mucosae. These observations provide an explanation for the distinct phenotypes of SC5314 and highlight the role of heterozygosity as a driver of C. albicans phenotypic heterogeneity. ImportanceCandida albicans is a commensal fungus that colonizes human oral cavity and gastrointestinal tracts but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogenous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These finding explain, in part, the outlier phenotype of the reference strain and highlight the role of heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.