Extracellular Vesicle Analysis by Paper Spray Ionization Mass Spectrometry

Paper spray ionization mass spectrometry (PSI-MS) is a direct MS analysis technique with several reported bacterial metabolomics applications. As with most MS-based bacterial studies, all currently reported PSI-MS bacterial analyses have focused on the chemical signatures of the cellular unit. One dimension of the bacterial metabolome that is often lost in such analyses is the exometabolome (extracellular metabolome), including secreted metabolites, lipids, and peptides. A key component of the bacterial exometabolome that is gaining increased attention in the microbiology and biomedical communities is extracellular vesicles (EVs). These excreted structures, produced by cells in all domains of life, contain a variety of biomolecules responsible for a wide array of cellular functions, thus representing a core component of the bacterial secreted metabolome. Although previously examined using other MS approaches, no reports currently exist for a PSI-MS analysis of bacterial EVs, nor EVs from any other organism (exosomes, ectosomes, etc.). PSI-MS holds unique analytical strengths over other commonly used MS platforms and could thus provide an advantageous approach to EV metabolomics. To address this, we report a novel application representing, to our knowledge, the first PSI-MS analysis of EVs from any organism (using the human gut resident Oxalobacter formigenes as the experimental model, a bacterium whose EVs were never previously investigated). In this report, we show how we isolated and purified EVs from bacterial culture supernatant by EV-specific affinity chromatography, confirmed and characterized these vesicles by nanoparticle tracking analysis, analyzed the EV isolate by PSI-MS, and identified a panel of EV-derived metabolites, lipids, and peptides. This work serves as a pioneering study in the field of MS-based EV analysis and provides a new, rapid, sensitive, and economical approach to EV metabolomics.

Silver Nanoparticles Synthesized by Using Bacillus cereus SZT1 Ameliorated the Damage of Bacterial Leaf Blight Pathogen in Rice

Amongst serious biotic factors deteriorating crop yield, the most destructive pathogen of rice is Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial leaf blight (BLB) disease. This study involved targeted use of biogenic silver nanoparticles (AgNPs) to control BLB in order to cope with the disadvantages of chemical disease control. AgNPs were biologically synthesized from natively isolated Bacillus cereus strain SZT1, which was identified through 16S rRNA gene sequence analysis. Synthesis of AgNPs in bacterial culture supernatant was confirmed through UV-VIS spectroscopy. Fourier transform infrared spectroscopy (FTIR) confirmed that the existence of AgNPs was stabilized with proteins and alcoholic groups. X-ray diffraction (XRD) data revealed the crystalline nature and imaging with scanning electron microscopy (SEM) and transmission electron microscopy (TEM), showing the spherical shape of AgNPs with particle sizes ranging from 18 to 39 nm. The silver presence in AgNPs was further confirmed by energy dispersive spectra. Biogenic AgNPs showed substantial antibacterial activity (24.21 ± 1.01 mm) for Xoo. In a pot experiment, AgNPs were found to be effective weapons for BLB by significantly increasing the plant biomass with a decreased cellular concentration of reactive oxygen species and increased concentration of antioxidant enzyme activity.

Screening of protease producing bacteria from tannery wastes of leather processing industries at Hazaribag, Bangladesh

Protease enzymes are ubiquitous in nature and usually found in all living organisms. The present study was conducted for screening bacteria with the ability to produce extracellular proteases. Attempts were made to isolate protease producing bacterial strains from soil samples of tannery wastes from Hazaribag, Bangladesh and subsequent partial characterization was performed. A number of biochemical assays was performed for presumptive identification of the bacteria. Subsequent molecular identification of bacterial isolates was performed using 16S rRNA sequence analysis, which revealed that the isolated bacterium R82 to be Bacillus subtilis. The protease enzyme activity was determined 60.50 U/mL. Further characterization showed that the enzyme produced from the bacteria was extracellular and alkaline protease in nature. The optimum reaction incubation time, pH and temperature were found to be 30 min, 8.0 and 50°C, respectively. Bacterial culture supernatant was used to carry out dehairing of leather (2ʹʹ×2ʹʹ), which was achieved in 24 h. The results showed that isolated bacterial strain could be employed for strain improvement towards commercial production of protease with a promise for dehairing in leather processing industries.Jahangirnagar University J. Biol. Sci. 7(1): 23-34, 2018 (June)

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines

Pierce’s disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.

The investigation of nematocidal activity in Stenotrophomonas maltophilia G2 and characterization of a novel virulence serine protease

The Gram-negative bacterium Stenotrophomonas maltophilia G2 was isolated from a soil sample and was found to have high nematotoxic activity against a free-living nematode, Panagrellus redivivus, and a plant-parasitic nematode, Bursaphelenchus xylophilus . The analysis of virulence factors revealed that although the small molecular metabolites participated in nematode killing, the crude extracellular protein extract from the bacterial culture supernatant contributed significantly to its nematocidal activity. An extracellular protease was purified by chromatography, and its effects on degrading purified nematode cuticle and killing living nematodes were confirmed experimentally. Characterization of this purified protease revealed that the application of phenylmethylsulphonyl fluoride, an inhibitor of serine proteases, could completely abolish its proteolytic activity. The results from N-terminal amino acid sequencing showed no similarity with any known serine protease in S. maltophilia, suggesting a novel virulence serine protease was obtained. Our study is the first to show the nematocidal activity of S. maltophilia, and we identified a novel serine protease as an important pathogenicity factor.

The Emerging Human Pathogen Photorhabdus asymbiotica Is a Facultative Intracellular Bacterium and Induces Apoptosis of Macrophage-Like Cells

ABSTRACT Photorhabdus species are gram-negative entomopathogenic bacteria of the family Enterobacteriaceae. Among the different members of the genus, one species, Photorhabdus asymbiotica, is a pathogen of both insects and humans. The pathogenicity mechanisms of this bacterium are unknown. Here we show that P. asymbiotica is a facultative intracellular pathogen that is able to replicate inside human macrophage-like cells. Furthermore, P. asymbiotica was shown for the first time in an intracellular location after insect infection. We also demonstrated that among Australian and American clinical isolates, only the Australian strains were able to invade nonphagocytic human cells. In cell culture infection experiments, Australian clinical isolates as well as cell-free bacterial culture supernatant induced strong apoptosis of a macrophage cell line at 6 h postinfection. American isolates also induced cellular death, but much later than that induced by Australian ones. Mammalian cultured cells analyzed for key features of apoptosis displayed apoptotic nuclear morphology, activation of the initiator caspases 8 and 9 and the executioner caspases 3 and 7, and poly(ADP-ribose) polymerase proteolysis, suggesting activation of both the intrinsic and extrinsic apoptotic pathways.