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.

Examining the Impacts of CO2 Concentration and Genetic Compatibility on Perennial Ryegrass-Epichloë festucae var lolii Interactions.

Perennial ryegrass (Lolium perenne) is the most cultivated cool-season grass worldwide with crucial roles in carbon fixation, turfgrass applications, and fodder for livestock. Lolium perenne forms a mutualism with the strictly vertically transmitted fungal endophyte, Epichloë festucae var lolii. The fungus produces alkaloids that protect the grass from herbivory, as well as conferring protection from drought and nutrient stress. The rising concentration of atmospheric CO2, a proximate cause of climatic change, is known to have many direct and indirect effects on plant growth. There is keen interest in how the nature of this plant-fungal interaction will change with climate change. Lolium perenne is an obligately outcrossing species, meaning that the genetic profile of the host is constantly being reshuffled. Meanwhile, the fungus is asexual implying both a relatively constant genetic profile and the potential for incompatible grass-fungus pairings. In this study, we used a single cultivar, "Alto", of L. perenne. Each plant was infected with one of four strains of the endophyte: AR1, AR37, NEA2, and Lp19 (the "common strain"). We outcrossed the Alto mothers with pollen from a number of individuals from different ryegrass cultivars to create more genetic diversity in the hosts. We collected seed such that we had replicate maternal half-sib families. Seed from each family was randomly allocated into the two levels of the CO2 treatment, 400 and 800 ppm. Elevated CO2 resulted in an c. 18% increase in plant biomass. AR37 produced higher fungal concentrations than other strains; NEA2 produced the lowest fungal concentrations. We did not find evidence of genetic incompatibility between the host plants and the fungal strains. We conducted untargeted metabolomics and quantitative proteomics to investigate the grass-fungus interactions between and within family and treatment groups. We identified a number of changes in both the proteome and metabalome. Taken together, our data set provides new understanding into the intricacy of the interaction between endophyte and host from multiple molecular levels and suggests opportunity to promote plant robustness and survivability in rising CO2 environmental conditions through application of bioprotective epichloid strains.

Iron Limitation in Klebsiella pneumoniae Defines New Roles for Lon Protease in Homeostasis and Degradation by Quantitative Proteomics.

Nutrient adaptation is key in limiting environments for the promotion of microbial growth and survival. In microbial systems, iron is an essential component for many cellular processes, and bioavailability varies greatly among different conditions. In the bacterium, Klebsiella pneumoniae, the impact of iron limitation is known to alter transcriptional expression of iron-acquisition pathways and influence secretion of iron-binding siderophores, however, a comprehensive view of iron limitation at the protein level remains to be defined. Here, we apply a mass-spectrometry-based quantitative proteomics strategy to profile the global impact of iron limitation on the cellular proteome and extracellular environment (secretome) of K. pneumoniae. Our data define the impact of iron on proteins involved in transcriptional regulation and emphasize the modulation of a vast array of proteins associated with iron acquisition, transport, and binding. We also identify proteins in the extracellular environment associated with conventional and non-conventional modes of secretion, as well as vesicle release. In particular, we demonstrate a new role for Lon protease in promoting iron homeostasis outside of the cell. Characterization of a Lon protease mutant in K. pneumoniae validates roles in bacterial growth, cell division, and virulence, and uncovers novel degradation candidates of Lon protease associated with improved iron utilization strategies in the absence of the enzyme. Overall, we provide evidence of unique connections between Lon and iron in a bacterial system and suggest a new role for Lon protease in the extracellular environment during nutrient limitation.

Decoding communication patterns of the innate immune system by quantitative proteomics.

The innate immune system is a collective network of cell types involved in cell recruitment and activation using a robust and refined communication system. Engagement of receptor-mediated intracellular signaling initiates communication cascades by conveying information about the host cell status to surrounding cells for surveillance and protection. Comprehensive profiling of innate immune cells is challenging due to low cell numbers, high dynamic range of the cellular proteome, low abundance of secreted proteins, and the release of degradative enzymes (e.g., proteases). However, recent advances in mass spectrometry-based proteomics provides the capability to overcome these limitations through profiling the dynamics of cellular processes, signaling cascades, post-translational modifications, and interaction networks. Moreover, integration of technologies and molecular datasets provide a holistic view of a complex and intricate network of communications underscoring host defense and tissue homeostasis mechanisms. In this Review, we explore the diverse applications of mass spectrometry-based proteomics in innate immunity to define communication patterns of the innate immune cells during health and disease. We also provide a technical overview of mass spectrometry-based proteomic workflows, with a focus on bottom-up approaches, and we present the emerging role of proteomics in immune-based drug discovery while providing a perspective on new applications in the future.