Soft tissue sarcomas (STS) are a group of rare and heterogeneous cancers. While large-scale genomic and epigenomic profiling of STS have been undertaken, proteomic analysis has thus far been limited. Here we utilise sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) for proteomic profiling of formalin fixed paraffin embedded (FFPE) specimens from a cohort of STS patients (n=36) across four histological subtypes (leiomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma). We quantified 2951 proteins across all cases and show that there is a significant enrichment of gene sets associated with smooth muscle contraction in leiomyosarcoma, RNA splicing regulation in synovial sarcoma and leukocyte activation in undifferentiated pleomorphic sarcoma. We further identified a subgroup of STS cases (independent of histological subtype) that have a distinct expression profile in a panel of 133 proteins, with worse survival outcomes when compared to the rest of the cohort. Our study highlights the value of comprehensive proteomic characterisation as a means to identify histotype-specific STS profiles that describe key biological pathways of clinical and therapeutic relevance; as well as for discovering new prognostic biomarkers in this group of rare and difficult-to-treat diseases.

Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

Gram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gram-negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram-negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-κB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice-induced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram-negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.

Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines.

Summary: When grown as a biofilm in laboratory flow chambers Pseudomonas aeruginosa can develop mushroom‐shaped multicellular structures consisting of distinct subpopulations in the cap and stalk portions. We have previously presented evidence that formation of the cap portion of the mushroom‐shaped structures in P. aeruginosa biofilms occurs via bacterial migration and depends on type IV pili ( Mol Microbiol 50: 61–68). In the present study we examine additional factors involved in the formation of this multicellular substructure. While pilA mutants, lacking type IV pili, are deficient in mushroom cap formation, pilH and chpA mutants, which are inactivated in the type IV pili‐linked chemosensory system, showed only minor defects in cap formation. On the contrary, fliM mutants, which are non‐flagellated, and cheY mutants, which are inactivated in the flagellum‐linked chemotaxis system, were largely deficient in cap formation. Experiments involving DNase treatment of developing biofilms provided evidence that extracellular DNA plays a role in cap formation. Moreover, mutants that are deficient in quorum sensing‐controlled DNA release formed microcolonies upon which wild‐type bacteria could not form caps. These results constitute evidence that type IV pili, flagellum‐mediated motility and quorum sensing‐controlled DNA release are involved in the formation of mature multicellular structures in P. aeruginosa biofilms.