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.

Cross-Kingdom Infection of Macrophages Reveals Pathogen- and Immune-Specific Global Reprogramming and Adaptation.

The interactions between a host and microbe drive the health and disease status of the host. Of importance is the cause of dysbiosis in the presence of a pathogen, and critically, the relationship between the host and pathogen may evolve over time through response and adaptation. For immunocompromised individuals, dual infections are prevalent and contribute to disease severity and treatment options. Here, we explore the global reprogramming of host cells in response to immediate and established microbial infections with the human fungal pathogen Cryptococcus neoformans and the nosocomial bacterial pathogen Klebsiella pneumoniae. Using quantitative proteomics, we uncovered cross-kingdom protein-level changes associated with initial fungal infection, followed by a remarkable adaptation of the host and pathogen to a dormant state. This stabilization is disrupted over time upon bacterial infection, with the production of virulence-associated bacterial proteins and severely altered host response. We support our findings with the profiling of two major virulence determinants in C. neoformans, catalase and melanin, which demonstrate an interconnected regulation in response to both host defense and bacterial invasion. Overall, we report novel fungal and bacterial modulation of the host, including adaptation and stabilization, suggesting an opportunity to effectively treat dual infections by selectively targeting proteins critical to the host's infection stage. IMPORTANCE The relationship between the human microbiota and infectious disease outcome is a rapidly expanding area of study. Understanding how the host responds to changes in its symbiotic relationship with microbes provides new insight into how disruption can promote disease. In this study, we investigated the evolving relationship between innate immune cells of the host during immediate and established infections with fungal and bacterial pathogens, commonly observed within the lungs of immunocompromised individuals. We observed critical reprogramming of each biological system over time and in response to the changing environment, which influences microbial virulence. The goal of this important work is to improve our fundamental understanding of pathogenesis, as well as the regulatory relationships between hosts and microbes that drive disease outcome. We envision defining improved therapeutic treatment options for the host dependent on disease state to reduce the global impact and burden of infectious diseases, especially in the face of ever-increasing rates of antimicrobial resistance.

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions.

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism's global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

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.