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We present the discovery of possibly the youngest Galactic supernova remnant (SNR) with associated pulsar-wind nebula (PWN), which we name Perun (G329.9−0.5). Perun was serendipitously discovered in the Australian Square Kilometre Array Pathfinder–Evolutionary Map of the Universe survey at 943 MHz, and subsequent follow-up observations were conducted with the Australia Telescope Compact Array observatory at 5500 and 9000 MHz. We combine these with additional radio observations from the MeerKAT, Molonglo Observatory Synthesis Telescope, and Murchison Widefield Array telescopes, infrared (IR) observations from the SpitzerSpace Telescope , and X-ray observations from the Chandra X-ray observatory to perform a multifrequency analysis. The radio morphology shows a small angular size shell (D  = 70 arcsec) with a luminous, central PWN. We measure a total spectral index of |$\alpha = -0.49\pm 0.05$|⁠ , which should be typical for a young, composite SNR. Crucial evidence for Perun's SNR classification comes from the detection of linear fractional polarization at radio frequencies of |$\sim$| 7 per cent–10 per cent with both radial and tangential orientations, similar to the young SNR G1.9+0.3. We use data from the Southern Galactic Plane Survey to perform an H  i analysis and estimate a favoured distance range of 6–9 kpc, and thus a favoured age range of |$\sim$| 70–500 yr. We find no high-energy emission in Fermi- Large Area Telescope data. We detect Perun's outer shell in 24  |$\mu$| m indicating the possible presence of [O  iv ] and [Fe  iii ] emission, also typical for young SNRs. Overall, these observations and analysis confirm Perun as a young, Galactic SNR with a prominent PWN. [ABSTRACT FROM AUTHOR]

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 wos2 Δ 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 wos2 Δ. Accordingly, wos2 Δ 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., Competing Interests: The authors declare no conflict of interest.

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., (© 2023. His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food.)

Phosphorylation is a key post-translational modification central to the biological behavior of proteins. This reversible modification specifically regulates cell signaling mechanisms to control survival and growth. Moreover, microbial pathogens, including both fungi and bacteria, rely on this modification to coordinate protein production and functioning during infection and dissemination within a host. Understanding phosphorylation and its involvement with effector proteins and complex networks are now possible with the recent technological advancements of mass spectrometry. Herein, we describe a phosphopeptide enrichment strategy optimized for the invasive mycosis-causing fungal pathogen Cryptococcus neoformans. Our protocol details proper sample preparation for efficient lysis and protein extraction with minimal phosphorylation losses followed by outlined steps for enrichment, instrumentation handling, and data analysis to permit deep profiling of the global phosphoproteome. The high-throughput versatility of bottom-up proteomics combined with our sample preparation approach facilitates opportunities for in-depth phosphorylation mapping and novel biological discoveries., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)