Secreted aspartic proteinases (Sap) play a role in the virulence of pathogenic Candida spp. Candida parapsilosis possesses three genes encoding these enzymes: SAPP1, SAPP2, and SAPP3. We analyzed the expression of the SAPP1 and SAPP2 genes and the production of Sapp1p and Sapp2p proteinases in the presence of different nitrogen sources. While the SAPP2 transcript was present under all of the conditions tested, expression of SAPP1 was induced only by the presence of exogenous protein as the sole nitrogen source. The concentration of Sapp1 p in the medium upon induction was at least one order of magnitude higher than the concentration of Sapp2p in all media tested in this study. Enzymological characterization of purified Sapp1 p and Sapp2p demonstrated that Sapp2p has a more restricted substrate specificity and significantly lower catalytic activity than Sapp1p. Homology models of Sapp1p and Sapp2p revealed structural motifs that may be responsible for the differences between these two enzymes. Our results indicate that C. parapsilosis secretes a low level of Sapp2p proteinase with narrow substrate specificity and low proteolytic activity under most conditions, while expression and secretion of a higher amount of catalytically efficient Sapp1p enzymes is triggered in the presence of exogenous protein serving as a nitrogen source.

Absolute quantification of proteins in tissue is important for numerous fields of study. Liquid chromatography–mass spectrometry (LC–MS) methods are the norm but typically involve lengthy sample preparation including tissue homogenization, which results in the loss of information relating to spatial distribution. Here, we propose liquid extraction surface analysis (LESA) mass spectrometry (MS) of stable isotope labeled mimetic tissue models for the spatially resolved quantification of intact ubiquitin in rat and mouse brain tissue. Measured ubiquitin concentrations are in agreement with values found in the literature. Images of rat and mouse brain tissue demonstrate spatial variation in the concentration of ubiquitin and demonstrate the utility of spatially resolved quantitative measurement of proteins in tissue. Although we have focused on ubiquitin, the method has the potential for broader application to the absolute quantitation of any endogenous protein or protein-based drug in tissue.

Trauma is one of the leading causes of death in people under the age of 49 and complications due to wound infection are the primary cause of death in the first few days after injury. The ESKAPE pathogens are a group of bacteria that are a leading cause of hospital-acquired infections and a major concern in terms of antibiotic resistance. Here, we demonstrate a novel and highly accurate approach for the rapid identification of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) directly from infected wounds in 3D in vitro skin models. Wounded skin models were inoculated with bacteria and left to incubate. Bacterial proteins were identified within minutes, directly from the wound, by liquid extraction surface analysis mass spectrometry. This approach was able to distinguish closely related strains and, unlike genomic approaches, can be modified to provide dynamic information about pathogen behaviour at the wound site. In addition, since human skin proteins were also identified, this method offers the opportunity to analyse both host and pathogen biomarkers during wound infection in near real-time. Trauma is one of the leading causes of death in people under the age of 49 1. Complications due to infection are the primary cause of death in patients who survive the first few days after a traumatic injury. In recent military conflicts, over half of the total injuries sustained were the result of trauma to the extremities 2. Over 25% of those patients suffered complications due to infection (rising to 50% for those requiring intensive care), either in the bones (osteomyelitis) or deep-wound infection 2,3. Similar rates of infection are associated with civilian trauma 2. Infectious complications result in significantly higher amputation rates, which are also of greater severity. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are a group of opportunistic pathogens which account for most hospital-acquired infections, and their antibiotic resistance is rising 4,5. Rapid diagnosis and treatment of wound infection is therefore of utmost importance. Current diagnosis involves visual inspection of the wound for signs of infection (inflammation), followed by collection of swabs or tissues, microbial culture and identification in a clinical laboratory, which can take hours or even days. Mass spectrometry (MS) has emerged as a powerful technique for identification and classification of microorganisms. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS has been in development for that purpose since the late 1990s 6,7 and is now well-established in the clinic 8. In the analysis, patient-derived samples are cultured and individual colonies are smeared onto a MALDI target plate. A matrix is applied and the sample is subjected to MALDI-TOF MS. Identification is achieved by spectral fingerprinting...

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Yeasts constitute an oft-neglected class of pathogens among which the resistance to first-line treatments, attributed in part to mutations in efflux pumps, is rapidly emerging. Their thick, chitinreinforced cell walls render cell lysis difficult, complicating their analysis and identification by methods routinely used for bacteria, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Liquid extraction surface analysis mass spectrometry (LESA-MS) has previously been applied to the analysis of intact proteins from Gram-positive and Gram-negative bacterial colonies sampled directly on solid nutrient media. To date, a similar analysis of yeast colonies has not proved possible. Here we demonstrate the rapid release of intact yeast proteins for LESA-MS by electroporation using a home-built high-voltage device designed to lyse cells grown in colonies on agar media. Detection and identification of previously inaccessible proteins from baker's yeast Saccharomyces cerevisiae, as well as two clinically relevant yeast species (Candida glabrata and Cryptococcus neoformans), is shown. The electroporation approach also has the potential to be translated to other mass spectrometric analysis techniques, including MALDI and various ambient ionization methods.

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