Studies on the components and functions of extracellular vesicles derived from bacteria

Abstract

All living organisms including bacteria release extracellular vesicles (EVs) into the extracellular milieu. EVs are nano-sized bilayered proteolipids containing proteins, lipids, nucleic acids, and metabolites. It has recently been proposed that EVs are involved in various pathophysiological functions, and thus the vesicles have been suggested as intercellular communicasomes. Bacterial EVs harbor proteins, lipids, nucleic acids, and other virulence factors, including lipopolysaccharides for Gram-negative bacterial EVs and lipoteichoic acids for Gram-positive bacterial EVs. The EVs have also been suggested to play several pathophysiological functions in bacteria-bacteria interactions, such as antibiotic resistance, nutrient acquisition, and killing competing bacteria, as well as in bacteria-host interactions, such as promoting inflammatory responses, adhesion to host cells, and virulence factor delivery, by stimulating the target cells directly or delivering their cargos. Mycobacterium tuberculosis releases EVs containing immunologically active molecules, and these EVs have been suggested to be applied for vaccine development and biomarker discovery. However, no comprehensive proteomic analyses have been conducted for M. tuberculosis EVs. In this study, a comprehensive proteomic analysis was performed for M. tuberculosis EVs, and a total of 287 EV proteins was identified by four LC-MS/MS analyses with high confidence. This study significantly increased the number of M. tuberculosis EV proteins compared with the previous report. More importantly, significantly extended lists of EV protein derived from the cell membrane, cell wall, and extracellular region were identified: these proteins might be associated with the pathogenesis of M. tuberculosis. Indeed, by quantitative and comparative proteomic analyses, a total of 18 and 24 proteins, respectively, were suggested to be biomarker candidates for diagnosing tuberculosis. Among the biomarker candidates, phosphate-binding protein PstS1 may be used to detect tuberculosis from the clinical specimens (urine and sputum). The comprehensive proteomic identification provides valuable information to unveil the pathogenic mechanisms of M. tuberculosis as well as for clinical applications such as vaccine development and diagnosis. Gram-negative bacteria release EVs into the extracellular environment, and these EVs contain proteins, lipids, nucleic acids, lipopolysaccharides, and other virulence factors. Among these molecules, proteins significantly contribute to the functions of Gram-negative bacterial EVs, so there have been many studies to identify EV-associated proteins. Mass spectrometry-based high-throughput proteomic analyses of Gram-negative bacterial EVs have been conducted, but integrative proteomic analyses have not been done so far. In this study, the identified EV proteins from mass spectrometry-based high-throughput proteomic analyses of Gram-negative bacterial EVs were collected, and based on EVpedia, the identified EV proteins were grouped in orthologous groups. Then, top 50 vesicular proteins most frequently identified in Gram-negative bacterial EVs were listed. Gene Ontology enrichment analysis revealed that Gram-negative bacterial EVs were enriched with proteins associated with the extracellular region, outer membrane, as well as periplasm, and were de-enriched with inner membrane proteins, when compared to the cellular proteomes of the originating bacteria. In addition, Gram-negative bacterial EV proteins were enriched with proteins involved in certain biological processes: outer membrane assembly, pathogenesis, protein folding, protein insertion into membrane, and siderophore transport. This study will shed light on future research on the field of Gram-negative bacterial EV proteomics, and contribute to the development of diagnosis and vaccines against infectious diseases caused by Gram-negative bacteria. Acinetobacter baumannii is a Gram-negative bacterium which frequently colonizes hospitalized patients. This microorganism is increasingly important for nosocomial infection, because it has acquired multidrug resistance to many clinically available antibiotics. Like other Gram-negative bacteria, A. baumannii has also been shown to secrete EVs, and the EVs can elicit innate immune responses, but little is known about the mechanism by which A. baumannii EVs mediate inflammation in the lungs. Here, EVs derived from A. baumannii were purified and detected in the lungs of mice after intranasally administered to mice. Inflammatory responses were investigated by measuring the number of inflammatory cells and the concentrations of chemokines and cytokines in the lungs. The expression of Toll-like receptors (TLRs) were determined after intranasal instillation of A. baumannii EVs. In this study, the intranasal instillation of EVs derived from A. baumannii was shown to mediate severe pulmonary inflammation. The effect is associated with the recruitment of neutrophils into the airways, and marked secretion of several chemokines and cytokines. The expression of TLR2 was highly increased by intransal administration of A. baumannii EVs, suggesting TLR2 may play roles in A. baumannii EV-mediated pulmonary inflammation. Here, a comprehensive proteomic analysis of M. tuberculosis EVs and integrative proteomic analysis of Gram-negative bacterial EVs were performed regarding the components of bacterial EVs, and pulmonary inflammation mediated by A. baumannii EVs was investigated regarding the function of bacterial EVs. These results will help us not only to reveal the pathophysiological functions of EVs, but also fasten the development of diagnostics, vaccines, and therapeutics which will advance both basic and clinical sciences.