Project description:Detection of species-specific proteotypic peptides for accurate and easy characterization of infectious non-tuberculous mycobacteria such as Mycobacterium fortuitum is essential. Therefore, we carried out an in-depth global proteomic experiment using M. fortuitum ATCC 6841 strain followed by a proteome database search and spectral library generation. The lysate was subjected to in-solution proteomic sample preparation and fractionated using an offline C18 StageTip. Each fraction was acquired in technical triplicates using a 180 min data-dependent acquisition (DDA) method in Orbitrap Fusion Tribrid (Thermo Scientific) mass spectrometer. The resulting raw DDA data were searched against the M. fortuitum proteome database using Proteome Discoverer and FragPipe. The resulting peptide spectrum matches were converted into a spectral library using BiblioSpec.
Project description:Biofilm formation by the environmental and clinical contaminant Mycobacterium fortuitum causes economic losses and serious threat to human health, as a consequence of its increasing contribution to nosocomial infections. There are no reports that elucidate physiological adaptations taking place during its planktonic to biofilm transition. The present study was hence carried out considering the global proteome of the mycobacterium. This is the first description of a global proteomic investigation into M. fortuitum biofilm. Scrutiny of biological functions in the two states provided insights into the phenotypic switch, and fundamental pathways associated with M. fortuitum pathobiology.
Project description:Lung disease due to non-tuberculous mycobacteria (NTM) is rising in incidence. While both 2D cell culture and animal models exist for NTM infections, a major knowledge gap is the early responses of human alveolar and innate immune cells to NTM within the human lung microenvironment. Here we describe development of a humanized, 3D, alveolus lung-on-a-chip (ALoC) model of Mycobacterium fortuitum infection that incorporates only primary human cells such as pulmonary vascular endothelial cells in a vascular channel, and type I and II alveolar cells and monocyte-derived macrophages in an alveolar channel along an air-liquid interface. M. fortuitum introduced into the alveolar channel primarily infected macrophages, with rare bacteria inside alveolar cells. Bulk-RNA sequencing of infected chips revealed marked upregulation of transcripts for cytokines, chemokines and secreted protease inhibitors (SERPINs). Our results demonstrate how a humanized ALoC system can identify critical early immune and epithelial responses to M. fortuitum infection. We envision potential application of the ALoC to other NTM and for studies of new antibiotics
Project description:Biofilm formation by the environmental and clinical contaminant Mycobacterium fortuitum causes economic losses and serious threat to human health, as a consequence of its increasing contribution to nosocomial infections. There are no reports that elucidate physiological adaptations taking place during its planktonic to biofilm transition. The present study was hence carried out considering the global proteome of the mycobacterium. This is the first description of a global proteomic investigation into M. fortuitum biofilm. Scrutiny of biological functions in the two states provided insights into the phenotypic switch, and fundamental pathways associated with M. fortuitum pathobiology.
