Proteome signatures reveal homeostatic and adaptive oxidative responses by a putative co-chaperone, Wos2, to influence fungal virulence determinants in cryptococcosis.

The increasing prevalence of invasive fungal pathogens is dramatically changing the clinical landscape of infectious diseases, posing an imminent threat to public health. Specifically, Cryptococcus neoformans, the human opportunistic pathogen, expresses elaborate virulence mechanisms and is equipped with sophisticated adaptation strategies to survive in harsh host environments. This study extensively characterizes Wos2, an Hsp90 co-chaperone homolog, featuring bilateral functioning for both cryptococcal adaptation and the resulting virulence response. In this study, we evaluated the proteome and secretome signatures associated with wos2 deletion in enriched and infection-mimicking conditions to reveal Wos2-dependent regulation of the oxidative stress response through global translational reprogramming. The wos2Δ strain demonstrates 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 stress, and cell membrane stressors. We demonstrate the importance of Wos2 for intracellular lifestyle of C. neoformans during in vitro macrophage infection and provide evidence for reduced phagosomal replication levels associated with wos2Δ. Accordingly, wos2Δ featured significantly reduced virulence within impacting fungal burden in a murine model of cryptococcosis. Our study highlights a vulnerable point in the fungal chaperone network that offers a therapeutic opportunity to interfere with both fungal virulence and fitness.IMPORTANCEThe 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 increasing rates of resistance demand alternative therapeutic strategies, including an anti-virulence approach, which disarms the pathogen of critical virulence factors, empowering the host to remove the pathogens and clear the infection. To this end, we apply state-of-the-art mass spectrometry-based proteomics to evaluate the impact of a recently defined novel co-chaperone, Wos2, toward cryptococcal virulence using in vitro and in vivo models of infection. We explore global proteome and secretome remodeling driven by the protein and uncover the novel role in modulating the fungal oxidative stress response. Complementation of proteome findings with in vitro infectivity assays demonstrated the protective role of 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 infection.

Cancer cell extravasation requires iplectin-mediated delivery of MT1-MMP at invadopodia.

BACKGROUND: Invadopodia facilitate cancer cell extravasation, but the molecular mechanism whereby invadopodia-specific proteases such as MT1-MMP are called to invadopodia is unclear. METHODS: Mass spectrometry and immunoprecipitation were used to identify interactors of MT1-MMP in metastatic breast cancer cells. After identification, siRNA and small molecule inhibitors were used to assess the effect these interactors had on cellular invasiveness. The chicken embryo chorioallantoic membrane (CAM) model was used to assess extravasation and invadopodia formation in vivo. RESULTS: In metastatic breast cancer cells, MT1-MMP was found to associate with plectin, a cytolinker and scaffolding protein. Complex formation between plectin and MT1-MMP launches invadopodia formation, a subtype we termed iplectin (i = invadopodial). iPlectin delivers MT1-MMP to invadopodia and is indispensable for regulating cell surface levels of the enzyme. Genetic depletion of plectin with siRNA reduced invadopodia formation and cell invasion in vitro. In vivo extravasation efficiency assays and intravital imaging revealed iplectin to be a key contributor to invadopodia ultrastructure and essential for extravasation. Pharmacologic inhibition of plectin using the small molecule Plecstatin-1 (PST-1) abrogated MT1-MMP delivery to invadopodia and extravasation efficiency. CONCLUSIONS: Anti-metastasis therapeutic approaches that target invadopodia are possible by disrupting interactions between MT1-MMP and iplectin. CLINICAL TRIAL REGISTRATION NUMBER: NCT04608357.

Quantitative Proteomics of the Intracellular Bacterial Pathogen Salmonella enterica Serovar Typhimurium.

Mass spectrometry-based proteomics provides a wealth of information about changes in protein production and abundance under diverse conditions, as well as mechanisms of regulation, signaling cascades, interaction partners, and communication patterns across biological systems. For profiling of intracellular pathogens, proteomic profiling can be performed in the absence of a host to singularly define the pathogenic proteome or during an infection-like setting to identify dual perspectives of infection. In this chapter, we present techniques to extract proteins from the human bacterial intracellular pathogen, Salmonella enterica serovar Typhimurium, in the presence of macrophages, an important innate immune cell in host defense. We outline sample preparation, including protein extraction, digestion, and purification, as well as mass spectrometry measurements and bioinformatics analysis. The data generated from our dual perspective profiling approach provides new insight into pathogen and host protein modulation under infection-like conditions.

In Vivo Modeling of Cryptococcus neoformans Infection and Collection of Murine Samples.

In vivo models provide advantages to study the progression of disease and to identify potential biomarkers to detect and monitor infections. For the human fungal pathogen Cryptococcus neoformans, murine intranasal models aim to recapitulate natural infection from inhalation of desiccated fungal cells from the environment and permit monitoring of disease over time. In this chapter, we describe the establishment of a murine model for cryptococcosis and the subsequent collection of organs, tissues, and fluids for sampling. These samples may support novel diagnostic strategies and opportunities to monitor dissemination of the fungal cells throughout the host and propose new treatment options to combat disease.

Proteomic Profiling of Samples Derived from a Murine Model Following Cryptococcus neoformans Infection.

Proteomic profiling provides in-depth information about the regulation of diverse biological processes, activation of and communication across signaling networks, and alterations to protein production, modifications, and interactions. For infectious disease research, mass spectrometry-based proteomics enables detection of host defenses against infection and mechanisms used by the pathogen to evade such responses. In this chapter, we outline protein extraction from organs, tissues, and fluids collected following intranasal inoculation of a murine model with the human fungal pathogen Cryptococcus neoformans. We describe sample preparation, followed by purification, processing on the mass spectrometer, and a robust bioinformatics analysis. The information gleaned from proteomic profiling of fungal infections supports the detection of novel biomarkers for diagnostic and prognostic purposes.

Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming.

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Natural compounds from freshwater mussels disrupt fungal virulence determinants and influence fluconazole susceptibility in the presence of macrophages in Cryptococcus neoformans.

Cryptococcus neoformans is a human fungal pathogen responsible for fatal infections, especially in patients with a depressed immune system. Overexposure to antifungal drugs due to prolonged treatment regimens and structure-similar applications in agriculture have weakened the efficacy of current antifungals in the clinic. The rapid evolution of antifungal resistance urges the discovery of new compounds that inhibit fungal virulence determinants, rather than directly killing the pathogen, as alternative strategies to overcome disease and reduce selective pressure toward resistance. Here, we evaluated the efficacy of freshwater mussel extracts (crude and clarified) against the production of well-defined virulence determinants (i.e., thermotolerance, melanin, capsule, and biofilm) and fluconazole resistance in C. neoformans. We demonstrated the extracts' influence on fungal thermotolerance, capsule production, and biofilm formation, as well as susceptibility to fluconazole in the presence of macrophages. Additionally, we measured the inhibitory activity of extracts against commercial peptidases (family representatives of cryptococcal orthologs) related to fungal virulence determinants and fluconazole resistance, and integrated these phenotypic findings with quantitative proteomics profiling. Our approach defined distinct signatures of each treatment and validated a new mechanism of anti-virulence action toward the polysaccharide capsule from a selected extract following fractionation. By understanding the mechanisms driving the antifungal activity of mussels, we may develop innovative treatment options to overcome fungal infections and promote susceptibility to fluconazole in resistant strains. IMPORTANCE: As the prevalence and severity of global fungal infections rise, along with an increasing incidence of antifungal resistance, new strategies to combat fungal pathogens and overcome resistance are urgently needed. Critically, our current methods to overcome fungal infections are limited and drive the evolution of resistance forward; however, an anti-virulence approach to disarm virulence factors of the pathogen and promote host cell clearance is promising. Here, we explore the efficacy of natural compounds derived from freshwater mussels against classical fungal virulence determinants, including thermotolerance, capsule production, stress response, and biofilm formation. We integrate our phenotypic discoveries with state-of-the-art mass spectrometry-based proteomics to identify mechanistic drivers of these antifungal properties and propose innovative avenues to reduce infection and support the treatment of resistant strains.

Soybean-SCN Battle: Novel Insight into Soybean's Defense Strategies against Heterodera glycines.

Soybean cyst nematode (SCN, Heterodera glycines, Ichinohe) poses a significant threat to global soybean production, necessitating a comprehensive understanding of soybean plants' response to SCN to ensure effective management practices. In this study, we conducted dual RNA-seq analysis on SCN-resistant Plant Introduction (PI) 437654, 548402, and 88788 as well as a susceptible line (Lee 74) under exposure to SCN HG type 1.2.5.7. We aimed to elucidate resistant mechanisms in soybean and identify SCN virulence genes contributing to resistance breakdown. Transcriptomic and pathway analyses identified the phenylpropanoid, MAPK signaling, plant hormone signal transduction, and secondary metabolite pathways as key players in resistance mechanisms. Notably, PI 437654 exhibited complete resistance and displayed distinctive gene expression related to cell wall strengthening, oxidative enzymes, ROS scavengers, and Ca2+ sensors governing salicylic acid biosynthesis. Additionally, host studies with varying immunity levels and a susceptible line shed light on SCN pathogenesis and its modulation of virulence genes to evade host immunity. These novel findings provide insights into the molecular mechanisms underlying soybean-SCN interactions and offer potential targets for nematode disease management.

Unbiased discovery of natural sequence variants that influence fungal virulence.

Isolates of Cryptococcus neoformans, a fungal pathogen that kills over 112,000 people each year, differ from a 19-Mb reference genome at a few thousand up to almost a million DNA sequence positions. We used bulked segregant analysis and association analysis, genetic methods that require no prior knowledge of sequence function, to address the key question of which naturally occurring sequence variants influence fungal virulence. We identified a region containing such variants, prioritized them, and engineered strains to test our findings in a mouse model of infection. At one locus, we identified a 4-nt variant in the PDE2 gene that occurs in common laboratory strains and severely truncates the encoded phosphodiesterase. The resulting loss of phosphodiesterase activity significantly impacts virulence. Our studies demonstrate a powerful and unbiased strategy for identifying key genomic regions in the absence of prior information and provide significant sequence and strain resources to the community.

Quantitative proteomics reveals unique responses to antimicrobial treatments in clinical Pseudomonas aeruginosa isolates.

IMPORTANCE: Pseudomonas aeruginosa is an important pathogen often associated with hospital-acquired infections and chronic lung infections in people with cystic fibrosis. P. aeruginosa possesses a wide array of intrinsic and adaptive mechanisms of antibiotic resistance, and the regulation of these mechanisms is complex. Label-free quantitative proteomics is a powerful tool to compare susceptible and resistant strains of bacteria and their responses to antibiotic treatments. Here we compare the proteomes of three isolates of P. aeruginosa with different antibiotic resistance profiles in response to five challenge conditions. We uncover unique and shared proteome changes for the widely used laboratory strain PAO1 and two isolates of the Liverpool epidemic strain of P. aeruginosa, LESlike1 and LESB58. Our data set provides insight into antibiotic resistance in clinically relevant Pseudomonas isolates and highlights proteins, including those with uncharacterized functions, which can be further investigated for their role in adaptive responses to antibiotic treatments.

Phosphoproteome Profiling of Klebsiella pneumoniae under Zinc-Limited and Zinc-Replete Conditions.

The bacterial pathogen Klebsiella pneumoniae causes nosocomial infections with the acquisition of multidrug resistance, impeding treatment options. This study investigated the effect of zinc limitation on the phosphoproteome of K. pneumoniae using quantitative mass spectrometry. New insight is provided into cellular signaling methods used by the pathogen to respond to nutrient-limited environments.

Profiling of the Klebsiella pneumoniae Phosphoproteome under Iron-Limited and Iron-Replete Conditions.

Klebsiella pneumoniae was compared across iron-limited and iron-replete conditions to assess changes within the phosphoproteome using quantitative mass spectrometry. These comparative proteomic data provide insights into cellular responses to nutrient limitation and how nutrient requirements may be exploited to provide potential antimicrobial targets.

Resolving the Temporal Splenic Proteome during Fungal Infection for Discovery of Putative Dual Perspective Biomarker Signatures.

Fungal pathogens are emerging threats to global health with the rise of incidence associated with climate change and increased geographical distribution; factors also influencing host susceptibility to infection. Accurate detection and diagnosis of fungal infections is paramount to offer rapid and effective therapeutic options. For improved diagnostics, the discovery and development of protein biomarkers presents a promising avenue; however, this approach requires a priori knowledge of infection hallmarks. To uncover putative novel biomarkers of disease, profiling of the host immune response and pathogen virulence factor production is indispensable. In this study, we use mass-spectrometry-based proteomics to resolve the temporal proteome of Cryptococcus neoformans infection of the spleen following a murine model of infection. Dual perspective proteome profiling defines global remodeling of the host over a time course of infection, confirming activation of immune associated proteins in response to fungal invasion. Conversely, pathogen proteomes detect well-characterized C. neoformans virulence determinants, along with novel mapped patterns of pathogenesis during the progression of disease. Together, our innovative systematic approach confirms immune protection against fungal pathogens and explores the discovery of putative biomarker signatures from complementary biological systems to monitor the presence and progression of cryptococcal disease.

Proteomic Profiling of Host Response in the Cereal Crop Triticum aestivum to the Mycotoxin, 15-Acetyldeoxynivalenol, Produced by the Fungal Pathogen, Fusarium graminearum.

Deoxynivalenol (DON) is a destructive mycotoxin produced by the fungal pathogen Fusarium graminearum in the devastating cereal disease Fusarium head blight (FHB). Host resistance to FHB has been identified within some of these crops (e.g., wheat, barley, corn); however, identification of how the host reduces the production of, and tolerates, DON to lessen the effects of the disease still requires further discovery. The field of quantitative proteomics is an effective tool for measuring and quantifying host defense responses to external factors, including the presence of pathogens and toxins. Success within this area of research has increased through recent technological developments (e.g., instrument sensitivity) and the accessibility of data analysis programs. One advancement we leverage is the ability to label peptides with isobaric mass tags to allow for sample multiplexing, reducing mass spectrometer run times, and providing accurate quantification. In this protocol, we exemplify this methodology to identify protein-level responses to DON within both FHB-resistant and FHB-susceptible Triticum aestivum cultivars using tandem mass tags for quantitative labeling combined with liquid-chromatography-MS/MS (LC-MS/MS) analysis. Furthermore, this protocol can be extrapolated for the identification of host responses under various conditions, including infection and environmental fluctuations, to elucidate changes in proteomic profiling in diverse biological contexts.

Quantitative Phosphoproteome Analysis of the Interaction Between Fusarium graminearum and Triticum aestivum.

Quantitative proteomics is a powerful method for distinguishing protein abundance changes in a biological system across conditions. In addition to recent advances in computational power and bioinformatics methods, improvements to sensitivity and resolution of mass spectrometry (MS) instrumentation provide an innovative approach for studying host-pathogen interaction dynamics and posttranslational modifications. In this protocol, we provide a workflow for state-of-the-art MS-based proteomics to assess changes in phosphorylated protein abundance upon interaction between the worldwide cereal crop, Triticum aestivum (wheat), and the global cereal crop fungal pathogen, Fusarium graminearum, during infection. This protocol mimics a time course of infection of T. aestivum by F. graminearum in the greenhouse, and the harvested samples undergo Fe-NTA phosphoenrichment combined with label-free quantification (LFQ) for detection by liquid-chromatography (LC)-coupled with tandem MS/MS. Our approach provides an in-depth view of changes in phosphorylation from both the host and pathogen perspectives in a single experiment across infection time points and different host cultivars.

Prenylcysteine oxidase 1 like protein is required for neutrophil bactericidal activities.

The bactericidal function of neutrophils is dependent on a myriad of intrinsic and extrinsic stimuli. Using systems immunology approaches we identify microbiome- and infection-induced changes in neutrophils. We focus on investigating the Prenylcysteine oxidase 1 like (Pcyox1l) protein function. Murine and human Pcyox1l proteins share ninety four percent aminoacid homology revealing significant evolutionary conservation and implicating Pcyox1l in mediating important biological functions. Here we show that the loss of Pcyox1l protein results in significant reductions in the mevalonate pathway impacting autophagy and cellular viability under homeostatic conditions. Concurrently, Pcyox1l CRISPRed-out neutrophils exhibit deficient bactericidal properties. Pcyox1l knock-out mice demonstrate significant susceptibility to infection with the gram-negative pathogen Psuedomonas aeruginosa exemplified through increased neutrophil infiltrates, hemorrhaging, and reduced bactericidal functionality. Cumulatively, we ascribe a function to Pcyox1l protein in modulation of the prenylation pathway and suggest connections beween metabolic responses and neutrophil functionality.

A One Health approach to overcoming fungal disease and antifungal resistance.

The global burden of fungal disease poses a substantial threat to human, animal, and environmental health, endangering both human and livestock populations and creating vulnerabilities to food supplies world-wide. Antifungal drugs provide essential therapies to humans and animals against infections, while fungicides provide protection in agriculture. However, a limited arsenal of antifungal agents results in cross-use between agriculture and health, promoting the development of resistance, and drastically reducing our defenses against disease. Critically, antifungal resistant strains found ubiquitously within the natural environment demonstrate resistance to the same classes of antifungals used to treat human and animal infections, hindering effective treatment within the clinic. This interconnectivity supports the need for a One Health approach to combat fungal diseases and overcome antifungal resistance, ensuring that treatment and protection of a defined group does not inadvertently endanger or sacrifice other plants, animals, or humans. In this review, we present sources of antifungal resistance and discuss the integration of environmental and clinical resources to manage disease. Moreover, we explore opportunities for drug synergy and repurposing strategies, highlight fungal targets being investigated to overcome resistance, and propose technologies for the discovery of novel fungal targets. This article is categorized under: Infectious Diseases > Molecular and Cellular Physiology.

Differentiated extracts from freshwater and terrestrial mollusks inhibit virulence factor production in Cryptococcus neoformans.

The human fungal pathogen, Cryptococcus neoformans, is responsible for deadly infections among immunocompromised individuals with the evolution of antifungal resistance driving the solution to discover new compounds that inhibit fungal virulence factors rather than kill the pathogen. Recently, exploration into natural sources (e.g., plants, invertebrates, microbes) of antifungal agents has garnered attention by integrating a One Health approach for new compound discovery. Here, we explore extracts from three mollusk species (freshwater and terrestrial) and evaluate effects against the growth and virulence factor production (i.e., thermotolerance, melanin, capsule, and biofilm) in C. neoformans. We demonstrate that clarified extracts of Planorbella pilsbryi have a fungicidal effect on cryptococcal cells comparable to fluconazole. Similarly, all extracts of Cipangopaludina chinensis affect cryptococcal thermotolerance and impair biofilm and capsule production, with clarified extracts of Cepaea nemoralis also conveying the latter effect. Next, inhibitory activity of extracts against peptidases related to specific virulence factors, combined with stress assays and quantitative proteomics, defined distinct proteome signatures and proposed proteins driving the observed anti-virulence properties. Overall, this work highlights the potential of compounds derived from natural sources to inhibit virulence factor production in a clinically important fungal pathogen.

The ER Protein Translocation Channel Subunit Sbh1 Controls Virulence of Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans is distinguished by a cell-wall-anchored polysaccharide capsule that is critical for virulence. Biogenesis of both cell wall and capsule relies on the secretory pathway. Protein secretion begins with polypeptide translocation across the endoplasmic reticulum (ER) membrane through a highly conserved channel formed by three proteins: Sec61, Sbh1, and Sss1. Sbh1, the most divergent, contains multiple phosphorylation sites, which may allow it to regulate entry into the secretory pathway in a species- and protein-specific manner. Absence of SBH1 causes a cell-wall defect in both Saccharomyces cerevisiae and C. neoformans, although other phenotypes differ. Notably, proteomic analysis showed that when cryptococci are grown in conditions that mimic aspects of the mammalian host environment (tissue culture medium, 37°C, 5% CO2), a set of secretory and transmembrane proteins is upregulated in wild-type, but not in Δsbh1 mutant cells. The Sbh1-dependent proteins show specific features of their ER targeting sequences that likely cause them to transit less efficiently into the secretory pathway. Many also act in cell-wall biogenesis, while several are known virulence factors. Consistent with these observations, the C. neoformans Δsbh1 mutant is avirulent in a mouse infection model. We conclude that, in the context of conditions encountered during infection, Sbh1 controls the entry of virulence factors into the secretory pathway of C. neoformans, and thereby regulates fungal pathogenicity. IMPORTANCE Cryptococcus neoformans is a yeast that causes almost 200,000 deaths worldwide each year, mainly of immunocompromised individuals. The surface structures of this pathogen, a protective cell wall surrounded by a polysaccharide capsule, are made and maintained by proteins that are synthesized inside the cell and travel outwards through the secretory pathway. A protein called Sbh1 is part of the machinery that determines which polypeptides enter this export pathway. We found that when Sbh1 is absent, both C. neoformans and the model yeast S. cerevisiae show cell-wall defects. Lack of Sbh1 also changes the pattern of secretion of both transmembrane and soluble proteins, in a manner that depends on characteristics of their sequences. Notably, multiple proteins that are normally upregulated in conditions similar to those encountered during infection, including several needed for cryptococcal virulence, are no longer increased. Sbh1 thereby regulates the ability of this important pathogen to cause disease.

Influence of circadian clocks on adaptive immunity and vaccination responses.

The adaptive immune response is under circadian control, yet, why adaptive immune reactions continue to exhibit circadian changes over long periods of time is unknown. Using a combination of experimental and mathematical modeling approaches, we show here that dendritic cells migrate from the skin to the draining lymph node in a time-of-day-dependent manner, which provides an enhanced likelihood for functional interactions with T cells. Rhythmic expression of TNF in the draining lymph node enhances BMAL1-controlled ICAM-1 expression in high endothelial venules, resulting in lymphocyte infiltration and lymph node expansion. Lymph node cellularity continues to be different for weeks after the initial time-of-day-dependent challenge, which governs the immune response to vaccinations directed against Hepatitis A virus as well as SARS-CoV-2. In this work, we present a mechanistic understanding of the time-of-day dependent development and maintenance of an adaptive immune response, providing a strategy for using time-of-day to optimize vaccination regimes.