Global analysis of transcriptional regulators in Neisseria meningitidis with a focus on two regulators found only in pathogenic Neisseria species

Regulation of gene expression in the human pathogen Neisseria meningitidis remains poorly understood. The meningococcus is a good model for a global analysis of transcriptional regulation as it has fewer transcriptional regulators and proteins able to modulate gene expression compared to other species with genomes of similar size. The genes encoding proteins predicted to modulate transcription in the meningococcus were identified using a dedicated database for systematical functional analysis (NeMeSys) in this species and mutated by utilising an improved in vitro transposon mutagenesis system. The resulting mutants were subjected to phenotypic analysis for growth and functions linked to one of the major virulence factors of the meningococcus, Type-four pili (Tfp). Tfp are essential for adhesion, aggregation, twitching motility and DNA competence in pathogenic Neisseria species. However, not much is known about the expression of the 16 proteins essential for Tfp biogenesis, and the seven proteins playing important roles in Tfp biology. The mutants were assessed for the Tfp-dependent phenotypes: aggregation, adhesion to human cells and twitching motility. No mutants were found to be dramatically impaired for these properties. However, two transcriptional regulators, HexR (NMV_1005) and FarR (NMV_2033), were found to influence the aggregative abilities of the meningococcus, with mutations in these genes resulting in a slow aggregating, and consequently, slow adhering phenotype. In parallel, two transcriptional regulators were chosen for further characterisation because they are encoded on genomic islands absent in the nonpathogenic Neisseria strain, Neisseria lactamica and could thus contribute to virulence. One island encoded an AraC type regulator divergently transcribed from genes encoding a putative TonB-dependent iron uptake system, whilst the other island encoded a putative LysR type regulator divergently transcribed from a putative transporter. These regulators are likely to require specific inducer molecules that may be absent from experiments performed in vitro

Vaccinia virus protein complex F12/E2 interacts with kinesin light chain isoform 2 to engage the kinesin-1 motor complex.

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.This work was supported by grant G1000207 from the Medical Research Council, UK and grant 090315 from The Wellcome Trust. GLS is a Wellcome Trust Principal research Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final published version. It first appeared at http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004723

NeMeSys: a biological resource for narrowing the gap between sequence and function in the human pathogen Neisseria meningitidis

The genome of a clinical isolate of Neisseria meningitidis is described. This and other reannotated Neisseria genomes are compiled in a database

Acidic residues in the membrane-proximal stalk region of vaccinia virus protein B5 are required for glycosaminoglycan-mediated disruption of the extracellular enveloped virus outer membrane

The extracellular enveloped virus (EEV) form of vaccinia virus (VACV) is surrounded by two lipid envelopes. This presents a topological problem for virus entry into cells, because a classical fusion event would only release a virion surrounded by a single envelope into the cell. Recently, we described a mechanism in which the EEV outer membrane is disrupted following interaction with glycosaminoglycans (GAGs) on the cell surface and thus allowing fusion of the inner membrane with the plasma membrane and penetration of a naked core into the cytosol. Here we show that both the B5 and A34 viral glycoproteins are required for this process. A34 is required to recruit B5 into the EEV membrane and B5 acts as a molecular switch to control EEV membrane rupture upon exposure to GAGs. Analysis of VACV strains expressing mutated B5 proteins demonstrated that the acidic stalk region between the transmembrane anchor sequence and the fourth short consensus repeat of B5 are critical for GAG-induced membrane rupture. Furthermore, the interaction between B5 and A34 can be disrupted by the addition of polyanions (GAGs) and polycations, but only the former induce membrane rupture. Based on these data we propose a revised model for EEV entry

Global analysis of transcriptional regulators in Neisseria meningitidis with a focus on two regulators found only in pathogenic Neisseria species

Regulation of gene expression in the human pathogen Neisseria meningitidis remains poorly understood. The meningococcus is a good model for a global analysis of transcriptional regulation as it has fewer transcriptional regulators and proteins able to modulate gene expression compared to other species with genomes of similar size. The genes encoding proteins predicted to modulate transcription in the meningococcus were identified using a dedicated database for systematical functional analysis (NeMeSys) in this species and mutated by utilising an improved in vitro transposon mutagenesis system. The resulting mutants were subjected to phenotypic analysis for growth and functions linked to one of the major virulence factors of the meningococcus, Type-four pili (Tfp). Tfp are essential for adhesion, aggregation, twitching motility and DNA competence in pathogenic Neisseria species. However, not much is known about the expression of the 16 proteins essential for Tfp biogenesis, and the seven proteins playing important roles in Tfp biology. The mutants were assessed for the Tfp-dependent phenotypes: aggregation, adhesion to human cells and twitching motility. No mutants were found to be dramatically impaired for these properties. However, two transcriptional regulators, HexR (NMV_1005) and FarR (NMV_2033), were found to influence the aggregative abilities of the meningococcus, with mutations in these genes resulting in a slow aggregating, and consequently, slow adhering phenotype. In parallel, two transcriptional regulators were chosen for further characterisation because they are encoded on genomic islands absent in the nonpathogenic Neisseria strain, Neisseria lactamica and could thus contribute to virulence. One island encoded an AraC type regulator divergently transcribed from genes encoding a putative TonB-dependent iron uptake system, whilst the other island encoded a putative LysR type regulator divergently transcribed from a putative transporter. These regulators are likely to require specific inducer molecules that may be absent from experiments performed in vitro.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

F12 co-immunoprecipitates with kinesin light chain isoform 2.

<p>(A) SDS-PAGE and immunoblot analysis of anti-Flag immunoprecipitations. HeLa cells were transfected with plasmids expressing Flag-tagged GFP, KLC1 or KLC2 and were infected 24 h later with vF12-HA (5 PFU/cell) for 14 h. Cell lysates were prepared and immunoprecipitated with anti-Flag antibody. (i) Clarified cell lysate (Input) and immunoprecipitated samples were immunoblotted with an anti-Flag antibody. (ii) As in (i) but immunoblotted with anti-KIF5B to show equal loading of cell lysate (Input) and the ability of Flag-KLC1 and Flag-KLC2 to associate with the endogenous kinesin-1 complex (αFlag IP). (iii) As in (i) but immunoblotted with an anti-HA antibody. (B) The experiment described in (A) was repeated in HeLa cells expressing a V5 epitope-tagged A36 protein. Samples were immunoblotted with anti-V5 antibody. (C) The experiment shown in (A) (iii) was repeated in triplicate and band intensities of co-immunoprecipitated F12 were quantified using a LiCor Odyssey Infrared Imager. Numbers represent the relative integrated intensities (with local background correction) normalised to the intensity of the band in the pFlag-GFP lane of 3 independent experiments ±sd. (D) SDS-PAGE and immunoblot analysis of a reciprocal anti-HA immunoprecipitation. HeLa cells were transfected with plasmids expressing either Flag-tagged KLC1 or KLC2 and were infected 24 h later with vF12-HA or vB14-HA. HA-tagged proteins were immunoprecipitated using anti-HA antibody-coated beads. Samples were immunoblotted with (i) anti-HA, (ii) anti-Flag and (iii) anti-KIF5B (input loading control) antibodies. The positions of molecular mass markers (kDa) are shown on the left for all immunoblots.</p

F12 and E2 form part of the IEV trafficking complex.

<p>(A) Schematic model (not to scale) of the IEV/kinesin-1 interaction complex showing the potential spacial arrangement of A36, F12 and E2 in relation to the KLC2 TPR structural model (shown as a surface rendering) published by Pernigo <i>et al</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004723#ppat.1004723.ref067" target="_blank">67</a>] (PDB # 3ZFW). The A36 WD/E motif interacts with the TPR groove in a similar manner to that shown for SifA-kinesin interacting protein (shown as an atomic space filling model) while E2 interacts with the C-terminal tail of KLC. F12 can interact with both A36 and E2. (B) (i) For fully wrapped IEVs to be transported efficiently from the site of wrapping to the cell surface the presence of A36, F12 and E2 is required. (ii) In the absence of either F12 or E2 IEV trafficking is almost entirely abrogated. (iii) In the absence of A36 some IEV egress can still take place. The E2/F12 complex might mediate the interaction between IEVs and kinesin-1 either directly or via another viral or cell protein present in IEVs. (iv) siRNA knockdown of KLC1 may result in an increased association of IEVs with KLC2 containing motor complexes, increasing the efficiency of trafficking.</p

The effect of KLC knock-down by siRNA on virus egress.

<p>(A) SDS-PAGE and immunoblotting analysis of the efficiency of siRNA knockdown of KLC1 and KLC2 in the human osteosarcoma cell line U-2 OS. Cells were treated with siRNA targeting KLC1 (siKLC1), or KLC2 (siKLC2) or a mix of both (siKLC1 & 2) and compared to cells treated with two independent non-targeting siRNAs (nsA and nsB). Cells were harvested 72 hpi and protein levels were analysed by SDS-PAGE. Tubulin levels were measured to confirm equivalent protein loading levels using an antibody specific to α-tubulin. Levels of KLC1 and KLC2 were measured by staining both with the pan-KLC 63–90 antibody, detecting both KLC1 (lower band) and KLC2 (upper band), and antibodies specific for KLC1 and KLC2. (B) Plaque size determination of vA5GFP on siRNA treated U-2 OS cells. Cells were treated with siRNA to KLC1, KLC2, KLC1&2 or two independent non-silencing RNAs (nsA and nsB). Monolayers of siRNA-treated cells were infected with vA5GFP to generate well separated plaques by 3 dpi. Cells were fixed and plaques positive for GFP expression were imaged using an inverted fluorescence microscope with a mounted digital camera and plaque surface area was measured using Axiovision (Zeiss) software. The average size of 20–35 plaques per sample and 3 replicate samples per condition were calculated and compared by student t-test (**** p<0.0001). (C) Estimation of virus egress from siRNA-treated cells by flow cytometry. Cells infected with vA5GFP at 5 PFU/cell and stained at various times pi prior to fixation for the CEV-associated B5 protein. Levels of staining were quantified by flow cytometry. (i) To validate this method of measuring egress an initial experiment compared three viruses known to display different levels of virion egress; vA5GFP (WT), vA5GFP-ΔA36 (vΔA36) and vA5GFP-ΔF12 (vΔF12). Background staining levels were monitored by including a sample stained with an isotype control antibody (iso). The three viruses showed levels of surface staining similar to their known relative levels of virion egress. (ii) To measure the effect of siRNA treatment on egress, cells were treated with siRNAs for 48 h and then infected with vA5GFP and stained for surface B5 at the indicated times. (D) Single step growth curve of released and cell-associated virus from siRNA-treated cells. U-2 OS cells were treated with siRNA targeting either KLC1 or KLC2 or a non-silencing (ns) control RNA and infected with vA5GFP at 10 PFU/cell 48 h after siRNA treatment. The supernatant (i) and cells (ii) were harvested separately at 1 hpi and 16 hpi. The infectious virus titre of triplicate samples was determined by plaque assay and numbers were analysed by student’s T-test.</p

The F12/E2 interaction with KLC maps to the KLC2 C-terminal tail.

<p>(A) Schematic representation of KLC and chimeric alleles used. (i) Conservation score plot of murine KLC1 and KLC2 protein sequence alignment (shown in supplemental information <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004723#ppat.1004723.s002" target="_blank">S2 Fig.</a>). The physiochemical conservation was calculated for each residue using the PET91 matrix (1 = complete conservation, 0 = no conservation, see key for colour values, positions within KLC2 that have no corresponding KLC1 residue are coloured black) and plotted onto the KLC domain organisation diagram to scale (note the alignment of the KLC1 C-terminal tail to a region in the centre of the KLC2 C-terminal tail as denoted by hatched grey lines). (ii) KLC domain organisation. All KLC molecules possess an N-terminal coiled-coil region that mediates interaction with KHC, a TPR region consisting of 6 copies of the tetratricopeptide repeat (TPR) motif, that affects interaction with certain cargo proteins containing a tryptophan acidic (WD) motif, and a highly variable C-terminal tail. (iii) Schematic of KLC1/KLC2 chimeric proteins generated. The regions used in each chimera are colour coded; red for KLC1 and green for KLC2. The amino acid positions of KLC1 and KLC2 included in each chimera are listed alongside. A summary of the results described in results below is also shown. (B) and (C) Co-immunoprecipitation analysis of the interaction of KLC chimeras with the F12/E2 complex. (i) Cells transfected with FLAG-KLC chimeras were infected with vF12-HA and clarified cell lysates produced 16 hpi. Chimeric Flag-KLC proteins were immunoprecipitated and co-precipitating F12-HA and KHC were analysed by immunoblot as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004723#ppat.1004723.g001" target="_blank">Fig. 1A</a>. (ii) The experiment was repeated using vHA-E2 (a virus expressing HA-tagged E2) to analyse the interaction of E2 with the various chimeras. (iii) The experiment was repeated in a HeLa cell line expressing V5-tagged A36 to analyse the ability of A36 to bind to the different chimeras. (B) shows results for the chimeras described in (a iii) and (C) shows the more detailed mapping using the chimeras described in (a iv).</p

KLC1 and KLC2 associate with peripheral virions.

<p>Confocal laser scanning microscopy of HeLa cells grown on glass coverslips. (A) Cells were infected with vA5GFP at 5 PFU/cell and fixed at 1 or 8 hpi. Cells were immunostained with anti-KLC2 antibody (red) and mounted in Mowiol-containing DAPI to stain DNA (blue). (B) HeLa cells were transfected with Flag-KLC1 or Flag-KLC2 and infected with vA5GFP at 5 PFU/cell, fixed 8 hpi, immunostained with an anti-Flag antibody (red) and mounted in Mowiol containing DAPI (blue). Images shown are maximum intensity projections of z-stack data sets acquired of the complete cell volume. The boxed regions are expanded in the inserts. Scale bars represent 20 μm, or when indicated, 5 μm (inserts).</p