Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane.

Bacterial cell growth necessitates synthesis of peptidoglycan. Assembly of this major constituent of the bacterial cell wall is a multistep process starting in the cytoplasm and ending in the exterior cell surface. The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II. After synthesis this lipid intermediate is translocated across the cell membrane. The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase). Here, we show that the integral membrane protein FtsW, an essential protein of the bacterial division machinery, is a transporter of the lipid-linked peptidoglycan precursors across the cytoplasmic membrane. Using Escherichia coli membrane vesicles we found that transport of Lipid II requires the presence of FtsW, and purified FtsW induced the transbilayer movement of Lipid II in model membranes. This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.

Lipidation of the autotransporter NalP of Neisseria meningitidis is required for its function in the release of cell-surface-exposed proteins.

Autotransporters of Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain and the secreted passenger domain in between. The autotransporter NalP of Neisseria meningitidis includes a protease domain that facilitates the release of several immunogenic proteins from the cell surface into the extracellular milieu. Rather exceptionally among autotransporters, NalP is a lipoprotein. We investigated the role of lipidation in the biogenesis and function of the protein. To this end, the N-terminal cysteine, which is lipidated in the wild-type protein, was substituted by alanine. Like the wild-type protein, the mutant protein was secreted into the medium, demonstrating that lipidation is not required for biogenesis of the protein. However, the non-lipidated NalP variant had a drastically reduced capacity to cleave its substrate proteins from the cell surface, suggesting that the lipid moiety is important for function. Kinetic experiments demonstrated that the autocatalytic processing of the non-lipidated protein at the cell surface was much faster than that of the wild-type protein. Thus, the lipid moiety delays the release of NalP from the cell surface, thereby allowing it to release other surface-exposed proteins into the milieu.

In vivo quantification of pulmonary inflammation in relation to emphysema severity via partial volume corrected (18)F-FDG-PET using computer-assisted analysis of diagnostic chest CT.

Our aim was to quantify the degree of pulmonary inflammation associated with centrilobular emphysema using fluoro-18-2-fluoro-2-deoxy-D-glucose positron emission tomography ((18)F-FDG-PET) and diagnostic unenhanced computed tomography (CT) based image segmentation and partial volume correction. Forty-nine subjects, with variable amounts of centrilobular emphysema, who had prior diagnostic unenhanced chest CT and either (18)F-FDG-PET or (18)F-FDG-PET/CT were selected. Lung parenchymal volume (L) (in cm³) excluding large and small pulmonary vessels, emphysema volume (E) (in cm³) based on a -910HU threshold, fraction of lung emphysema (F=E/L), and mean attenuation (HU) of non-emphysematous lung parenchyma (A) were calculated from CT images using the image analysis software 3DVIEWNIX. Lung uncorrected maximum SUV (USUVmax) was measured manually from PET images on a dedicated workstation. A first level of partial volume correction (PVC) of lung SUVmax to account for presence and degree of macroscopic emphysematous air space was calculated as CSUVmax=USUVmax/(1-F). A second level of PVC of non-emphysematous lung SUVmax to account for the mixture of air and lung parenchyma at the microscopic level was then estimated as CCSUVmax=CSUVmax/(A+1000/1040), assuming that air is -1000HU in attenuation and gasless lung parenchyma is 40HU in attenuation. The correlation of F with USUVmax, CSUVmax, CCSUVmax, % change between CSUVmax and USUVmax (%UC), and % change between CCSUVmax and USUVmax (%UCC) were then tested. The results showed that USUVmax was not significantly correlated with F (r=-.0973, P=0.34). CSUVmax (r=0.4660, P<0.0001) and CCSUVmax were significantly positively correlated with F (r=0.5479, P<0.0001), as were %UC (r=0.9383, P<0.0001) and %UCC (r=0.9369, P<0.0001). In conclusion, the degree of pulmonary inflammation increases with emphysema severity based on (18)F-FDG-PET or (18)F-FDG-PET/CT assessment, but is only detectable when (18)F-FDG uptake is corrected for the partial volume effect based on data provided from diagnostic chest CT images. These results support the notion that pulmonary inflammation plays an important role in the pathophysiology of emphysema. This novel image analysis approach has great potential for practical, accurate, and precise combined structural-functional PET quantification of pulmonary inflammation in patients with emphysema or other pulmonary conditions, although further validation and refinement will be required.

Specific labeling of peptidoglycan precursors as a tool for bacterial cell wall studies.

Because of its importance for bacterial cell survival, the bacterial cell wall is an attractive target for new antibiotics in a time of great demand for new antibiotic compounds. Therefore, more knowledge about the diverse processes related to bacterial cell wall synthesis is needed. The cell wall is located on the exterior of the cell and consists mainly of peptidoglycan, a large macromolecule built up from a three-dimensional network of aminosugar strands interlinked with peptide bridges. The subunits of peptidoglycan are synthesized inside the cell before they are transported to the exterior in order to be incorporated into the growing peptidoglycan. The high flexibility of the cell wall synthesis machinery towards unnatural derivatives of these subunits enables research on the bacterial cell wall using labeled compounds. This review highlights the high potential of labeled cell wall precursors in various areas of cell wall research. Labeled precursors can be used in investigating direct cell wall-antibiotic interactions and in cell wall synthesis and localization studies. Moreover, these compounds can provide a powerful tool in the elucidation of the cell wall proteome, the "wallosome," and thus, might provide new targets for antibiotics.

Lipid II: a central component in bacterial cell wall synthesis and a target for antibiotics.

The bacterial cell wall is mainly composed of peptidoglycan, which is a three-dimensional network of long aminosugar strands located on the exterior of the cytoplasmic membrane. These strands consist of alternating MurNAc and GlcNAc units and are interlinked to each other via peptide moieties that are attached to the MurNAc residues. Peptidoglycan subunits are assembled on the cytoplasmic side of the bacterial membrane on a polyisoprenoid anchor and one of the key components in the synthesis of peptidoglycan is Lipid II. Being essential for bacterial cell survival, it forms an attractive target for antibacterial compounds such as vancomycin and several lantibiotics. Lipid II consists of one GlcNAc-MurNAc-pentapeptide subunit linked to a polyiosoprenoid anchor 11 subunits long via a pyrophosphate linker. This review focuses on this special molecule and addresses three questions. First, why are special lipid carriers as polyprenols used in the assembly of peptidoglycan? Secondly, how is Lipid II translocated across the bacterial cytoplasmic membrane? And finally, how is Lipid II used as a receptor for lantibiotics to kill bacteria?

Mutations in PIK3CA are infrequent in neuroblastoma.

BACKGROUND: Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported. METHODS: Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain "hot spots" where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. RESULTS: We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. CONCLUSION: These data suggest that activating mutations in the Ras/Raf-MAPK/PI3K signaling cascades occur infrequently in neuroblastoma. Further, despite compelling evidence for MYC and RAS cooperation in vitro and in vivo to promote tumourigenesis, activation of RAS signal transduction does not constitute a preferred secondary pathway in neuroblastomas with MYCN deregulation in either human tumors or murine models.

Generating knock-out and complementation strains of Neisseria meningitidis.

The human-restricted pathogens Neisseria meningitidis and Neisseria gonorrhoeae are naturally competent for DNA uptake. This trait has been exploited extensively for genetic manipulation of these bacteria in the laboratory. Most transformation protocols were developed for N. gonorrhoeae, but appear to work also for N. meningitidis. In this chapter, we describe a number of protocols for genetic manipulation of N. meningitidis. Specifically, we describe how to (1) obtain knock-out mutants containing antibiotic-resistance markers, (2) generate markerless knock-out mutants, and (3) construct complementation strains. The generation of such mutants provides a valuable resource for studies of bacterial pathogenesis and vaccine development.

Clinical, radiographic, and audiometric predictors in conservative management of vestibular schwannoma.

OBJECTIVE: Vestibular schwannomas (VS) can be managed by observation. The goals were to examine clinical, radiographic, and audiometric variables at presentation and during observation that may predict which patients fail conservative management. METHODS: A retrospective chart review was performed of 202 patients who elected observation primarily. Data collection included presenting symptoms, symptom progression, tumor size, audiologic measures, and global clinical outcomes. Univariate and multivariate analyses were performed. RESULTS: Follow-up ranged from 1 month to 16 years (mean, 2.48 yr). Nineteen patients (9.4%) in the study group failed. Disequilibrium as a presenting symptom appeared more often in patients who failed observation (58% versus 32%; p = 0.039), as did new-onset disequilibrium. Presenting tumor size differed for patients who failed conservative management, with a mean of 14.0 versus 8.4 mm (p = 0.0006). Neurotologic complications compared favorably to those treated with primary surgery or radiotherapy. CONCLUSION: Patients with subjective disequilibrium at presentation and subjective disequilibrium developed during observation may be more likely to fail conservative management. Increased tumor size at presentation also may indicate the same, although no threshold could be achieved.

Transmembrane transport of peptidoglycan precursors across model and bacterial membranes.

Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane.