A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia

Lights, Camera, Actin: Divergent roles of beta- and gamma-cytoplasmic actin in vaccinia virus infection

Intracellular pathogens require access to host cells for their replication and spread. The host actin cytoskeleton represents a physical barrier to them, although many have evolved ways to circumvent, or hijack, this system to their advantage. Vaccinia virus (VACV) can manipulate the host actin cytoskeleton to facilitate dissemination. It expedites its cellular egress by nucleating actin beneath its particles, creating filamentous actin (F-actin) comets that propel virions across the cell surface. Tagging VACV proteins with fluorescent markers is used to study virus-host interactions, and define host molecular mechanisms, particularly within dynamic actin pathways. To this end, we developed a novel, optimised protocol for generating recombinant VACV. We then used this to create a recombinant VACV expressing Lifeact-GFP, a fluorescent marker that can highlight F-actin on infection, enabling live tracking of VACV comets via real-time fluorescence microscopy. F-actin comprises two cytoplasmic isoforms: β- and γ-actin. Despite differing only by four N-terminal amino acids, recent studies outlined their distinct localisations and functions in cell lines and whole organisms. We performed a detailed study of their roles in VACV actin-based motility. Initiation of comet formation appears to have an essential requirement for β-actin. Conversely, speed of virus movement was enhanced when γ-actin was silenced, indicating a moderating effect on the rate of actin polymerisation by this isoform. We aimed to define the site of β-actin dependency for VACV actin-based motility by biochemical pull-down assays. This represents the first investigation of the role of actin isoforms in pathogen motility, implicating the importance of their relative distribution in initiating VACV-induced actin comets. Further studies may underpin the importance of β- over γ-actin in other organisms using actin-based motility, providing a route to curb actin-assisted spread of intracellular pathogens

Proteomic profiling of <it>Pseudomonas aeruginosa </it>AES-1R, PAO1 and PA14 reveals potential virulence determinants associated with a transmissible cystic fibrosis-associated strain

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas aeruginosa </it>is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire <it>P. aeruginosa </it>from the environment, person-person transmissible strains have been identified in CF clinics worldwide. The molecular basis for transmissibility and colonization of the CF lung remains poorly understood.</p> <p>Results</p> <p>A dual proteomics approach consisting of gel-based and gel-free comparisons were undertaken to analyse protein profiles in a transmissible, early (acute) isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1. Over 1700 <it>P. aeruginosa </it>proteins were confidently identified. AES-1R protein profiles revealed elevated abundance of proteins associated with virulence and siderophore biosynthesis and acquisition, antibiotic resistance and lipopolysaccharide and fatty acid biosynthesis. The most abundant protein in AES-1R was confirmed as a previously hypothetical protein with sequence similarity to carbohydrate-binding proteins and database search revealed this gene is only found in the CF-associated strain PA2192. The link with CF infection may suggest that transmissible strains have acquired an ability to rapidly interact with host mucosal glycoproteins.</p> <p>Conclusions</p> <p>Our data suggest that AES-1R expresses higher levels of proteins, such as those involved in antibiotic resistance, iron acquisition and virulence that may provide a competitive advantage during early infection in the CF lung. Identification of novel proteins associated with transmissibility and acute infection may aid in deciphering new strategies for intervention to limit <it>P. aeruginosa </it>infections in CF patients.</p

A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia

Comparative proteomics and glycoproteomics reveal increased n-linked glycosylation and relaxed sequon specificity in campylobacter jejuni nctc11168 o

Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the-2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA; Gln at the-2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.15 page(s

Comparative Proteomics and Glycoproteomics Reveal Increased N‑Linked Glycosylation and Relaxed Sequon Specificity in <i>Campylobacter jejuni</i> NCTC11168 O

Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC–MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC–MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the <i>C. jejuni </i>N-linked sequon D/E-X-<u>N</u>-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the −2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA; Gln at the −2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in <i>C. jejuni</i>