Abstract
Francisella tularensis is a Gram-negative, facultative intracellular bacterium, causing a severe disease called tularemia. It secretes unusually shaped nanotubular outer membrane vesicles (OMV) loaded with a number of virulence factors and immunoreactive proteins. In the present study, the vesicles were purified from a clinical isolate of subsp. holarctica strain FSC200. We here provide a comprehensive proteomic characterization of OMV using a novel approach in which a comparison of OMV and membrane fraction is performed in order to find proteins selectively enriched in OMV vs. membrane. Only these proteins were further considered to be really involved in the OMV function and/or their exceptional structure. OMV were also isolated from bacteria cultured under various cultivation conditions simulating the diverse environments of F. tularensis life cycle. These included conditions mimicking the milieu inside the mammalian host during inflammation: oxidative stress, low pH, and high temperature (42°C); and in contrast, low temperature (25°C). We observed several-fold increase in vesiculation rate and significant protein cargo changes for high temperature and low pH. Further proteomic characterization of stress-derived OMV gave us an insight how the bacterium responds to the hostile environment of a mammalian host through the release of differentially loaded OMV. Among the proteins preferentially and selectively packed into OMV during stressful cultivations, the previously described virulence factors connected to the unique intracellular trafficking of Francisella were detected. Considerable changes were also observed in a number of proteins involved in the biosynthesis and metabolism of the bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids. Data are available via ProteomeXchange with identifier PXD013074.
Highlights
Francisella tularensis is a Gram-negative, facultative intracellular bacterium, causing a severe disease known as tularemia
Bacteria were cultivated in the complex medium – brain heart infusion (BHI), which is known to support the bacterium to switch to the so-called host-adapted phenotype (Hazlett et al, 2008; Zarrella et al, 2011; Holland et al, 2017)
LPS and the OmpA family protein FopA were here used as vesicular markers, while proteins from the Francisella pathogenicity island (FPI) IglA, IglB, and IglC peaked in the medium density region of the gradient (Figure 1F) showing that they are probably not secreted via outer membrane vesicles (OMV)
Summary
Francisella tularensis is a Gram-negative, facultative intracellular bacterium, causing a severe disease known as tularemia. Being one of the most infectious pathogenic bacterium (as few as 10 bacteria can initiate the disease), F. tularensis has been classified as a potential biological warfare agent by the Working Group on Civilian Biodefense (Dennis et al, 2001). F. tularensis is transmitted to humans via inhalation, ingestion of contaminated food or water, bites by arthropods, or through direct contact with infected animals. Three different F. tularensis subspecies are distinguished which differ in their geographical distribution, virulence, and severity of the disease. Tularensis (type A) is found almost exclusively in North America and is characterized by the highest virulence, severity of the disease, and mortality. Holarctica (type B) is spread along the north hemisphere and exhibits moderate virulence. The last subsp. mediasiatica exhibits comparable virulence and is found in central Asia (Oyston, 2008)
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