Moms in Proteomics: building a supportive and unified community together.

There is a growing need to recognize, support, and promote diversity within scientific disciplines. Moms in Proteomics was founded to connect mothers in proteomics, a field exemplifying the constituent elements of science, technology, engineering, and math (STEM) from academia to industry, share stories of successes and challenges, and build a community of mothers in highly productive and influential careers.

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.

A genetic platform to investigate the functions of bacterial drug efflux pumps.

Efflux pumps are a serious challenge for the development of antibacterial agents. Overcoming efflux requires an in-depth understanding of efflux pump functions, specificities and the development of inhibitors. However, the complexities of efflux networks have limited such studies. To address these challenges, we generated Efflux KnockOut-35 (EKO-35), a highly susceptible Escherichia coli strain lacking 35 efflux pumps. We demonstrate the use of this strain by constructing an efflux platform comprising EKO-35 strains individually producing efflux pumps forming tripartite complexes with TolC. This platform was profiled against a curated diverse compound collection, which enabled us to define physicochemical properties that contribute to transport. We also show the E. coli drug efflux network is conditionally essential for growth, and that the platform can be used to investigate efflux pump inhibitor specificities and efflux pump interplay. We believe EKO-35 and the efflux platform will have widespread application for the study of drug efflux.

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.

13th annual symposium of the Canadian National Proteomics Network.

The 13th annual Canadian National Proteomics Network was held in May 2022 in Montreal, Quebec, Canada. More than 175 individuals participated in this dynamic and productive meeting either in-person or virtually. A pre-symposium organized by trainees and dedicated to highlighting the best and brightest emerging talent in proteomics across Canada preceded the main symposium, which welcomed plenary and invited speakers from around the world. The presentations covering ground-breaking science were interspersed with critical discussions on improving equity, diversity, and inclusion within the proteomics community across Canada, along with important networking opportunities for early-career researchers.

Peptidoglycomics reveals compositional changes in peptidoglycan between biofilm- and planktonic-derived Pseudomonas aeruginosa.

Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating ß-1,4-linked disaccharide of N-acetylglucosamine and N-acetylmuramic acid appended with a highly conserved stem peptide. In Gram-negative bacteria, PG is assembled in the cytoplasm and exported into the periplasm where it undergoes considerable maturation, modification, or degradation depending on the growth phase or presence of environmental stressors. These modifications serve important functions in diverse processes, including PG turnover, cell elongation/division, and antibiotic resistance. Conventional methods for analyzing PG composition are complex and time-consuming. We present here a streamlined MS-based method that combines differential analysis with statistical 1D annotation approaches to quantitatively compare PGs produced in planktonic- and biofilm-cultured Pseudomonas aeruginosa We identified a core assembly of PG that is present in high abundance and that does not significantly differ between the two growth states. We also identified an adaptive PG assembly that is present in smaller amounts and fluctuates considerably between growth states in response to physiological changes. Biofilm-derived adaptive PG exhibited significant changes compared with planktonic-derived PG, including amino acid substitutions of the stem peptide and modifications that indicate changes in the activity of amidases, deacetylases, and lytic transglycosylases. The results of this work also provide first evidence of de-N-acetylated muropeptides from P. aeruginosa The method developed here offers a robust and reproducible workflow for accurately determining PG composition in samples that can be used to assess global PG fluctuations in response to changing growth conditions or external stimuli.

Combatting the evolution of antifungal resistance in Cryptococcus neoformans.

Fungal infections are a global concern and the evolution of intrinsic resistance to current antifungals presents an alarming problem. For Cryptococcus neoformans, a human fungal pathogen of primarily immunocompromised individuals, resistance toward treatment strategies demands alternative approaches. Given the prevalence of virulence factor production during cryptococcal infection, an emerging and important field of research encompasses the development of novel antivirulence therapies proposed to improve host immune responses and promote fungal clearance. To accomplish this task, information regarding the presence and role of virulence factors, the mechanisms of action within the host, and the ability to influence fungal susceptibility to antifungals is pertinent. Research into mechanisms of antifungal resistance for C. neoformans is limited but extrapolation from successful studies in other fungal species can improve our understanding of mechanisms employed by C. neoformans and suggest targeted strategies to enhance our ability to combat the pathogen. In this Review, we highlight antifungal therapy options against Cryptococcus, explore current knowledge of underlying mechanisms promoting resistance, and present new opportunities for novel and effective strategies to overcome fungal infections and reduce, or possibly even reverse, the effects of resistance evolution.

Proteomics of host-bacterial interactions: new insights from dual perspectives.

Mass-spectrometry (MS)-based proteomics is a powerful and robust platform for studying the interactions between biological systems during health and disease. Bacterial infections represent a significant threat to global health and drive the pursuit of novel therapeutic strategies to combat emerging and resistant pathogens. During infection, the interplay between a host and pathogen determines the ability of the microbe to survive in a hostile environment and promotes an immune response by the host as a protective measure. It is the protein-level changes from either biological system that define the outcome of infection, and MS-based proteomics provides a rapid and effective platform to identify such changes. In particular, proteomics detects alterations in protein abundance, quantifies protein secretion and (or) release, measures an array of post-translational modifications that influence signaling cascades, and profiles protein-protein interactions through protein complex and (or) network formation. Such information provides new insight into the role of known and novel bacterial effectors, as well as the outcome of host cell activation. In this Review, we highlight the diverse applications of MS-based proteomics in profiling the relationship between bacterial pathogens and the host. Our work identifies a plethora of strategies for exploring mechanisms of infection from dual perspectives (i.e., host and pathogen), and we suggest opportunities to extrapolate the current knowledgebase to other biological systems for applications in therapeutic discovery.

The Canadian Fungal Research Network: current challenges and future opportunities.

Fungi critically impact the health and function of global ecosystems and economies. In Canada, fungal researchers often work within silos defined by subdiscipline and institutional type, complicating the collaborations necessary to understand the impacts fungi have on the environment, economy, and plant and animal health. Here, we announce the establishment of the Canadian Fungal Research Network (CanFunNet, https://fungalresearch.ca), whose mission is to strengthen and promote fungal research in Canada by facilitating dialogue among scientists. We summarize the challenges and opportunities for Canadian fungal research that were discussed at CanFunNet's inaugural meeting in 2019, and identify 4 priorities for our community: (i) increasing collaboration among scientists, (ii) studying diversity in the context of ecological disturbance, (iii) preserving culture collections in the absence of sustained funding, and (iv) leveraging diverse expertise to attract trainees. We have gathered additional information to support our recommendations, including a survey identifying underrepresentation of fungal-related courses at Canadian universities, a list of Canadian fungaria and culture collections, and a case study of a human fungal pathogen outbreak. We anticipate that these discussions will help prioritize fungal research in Canada, and we welcome all researchers to join this nationwide effort to enhance knowledge dissemination and funding advocacy.

Proteomics of Cryptococcus neoformans: From the Lab to the Clinic.

Fungal pathogens cause an array of diseases by targeting both immunocompromised and immunocompetent hosts. Fungi overcome our current arsenal of antifungals through the emergence and evolution of resistance. In particular, the human fungal pathogen, Cryptococcus neoformans is found ubiquitously within the environment and causes severe disease in immunocompromised individuals around the globe with limited treatment options available. To uncover fundamental knowledge about this fungal pathogen, as well as investigate new detection and treatment strategies, mass spectrometry-based proteomics provides a plethora of tools and applications, as well as bioinformatics platforms. In this review, we highlight proteomics approaches within the laboratory to investigate changes in the cellular proteome, secretome, and extracellular vesicles. We also explore regulation by post-translational modifications and the impact of protein-protein interactions. Further, we present the development and comprehensive assessment of murine models of cryptococcal infection, which provide valuable tools to define the dynamic relationship between the host and pathogen during disease. Finally, we explore recent quantitative proteomics studies that begin to extrapolate the findings from the bench to the clinic for improved methods of fungal detection and monitoring. Such studies support a framework for personalized medical approaches to eradicate diseases caused by C. neoformans.

Label-Free Quantitative Proteomics Distinguishes General and Site-Specific Host Responses to Pseudomonas aeruginosa Infection at the Ocular Surface.

Mass spectrometry-based proteomics enables the unbiased and sensitive profiling of cellular proteomes and extracellular environments. Recent technological and bioinformatic advances permit identifying dual biological systems in a single experiment, supporting investigation of infection from both the host and pathogen perspectives. At the ocular surface, Pseudomonas aeruginosa is commonly associated with biofilm formation and inflammation of the ocular tissues, causing damage to the eye. The interaction between P. aeruginosa and the immune system at the site of infection describes limitations in clearance of infection and enhanced pathogenesis. Here, the extracellular environment (eye wash) of murine ocular surfaces infected with a clinical isolate of P. aeruginosa is profiled and neutrophil marker proteins are detected, indicating neutrophil recruitment to the site of infection. The first potential diagnostic markers of P. aeruginosa-associated keratitis are also identified. In addition, the deepest murine corneal proteome to date is defined and proteins, categories, and networks critical to the host response are detected. Moreover, the first identification of bacterial proteins attached to the ocular surface is reported. The findings are validated through in silico comparisons and enzymatic profiling. Overall, the work provides comprehensive profiling of the host-pathogen interface and uncovers differences between general and site-specific host responses to infection.

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.

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.

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.

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.

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.

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.

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.