Control of a Programmed Cell Death Pathway in Pseudomonas aeruginosa by an Antiterminator

In Pseudomonas aeruginosa the alp system encodes a programmed cell death pathway that is switched on in a subset of cells in response to DNA damage and is linked to the virulence of the organism. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified target locus, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. AlpA thus functions in a mechanistically unusual manner to control the expression of virulence genes in this opportunistic pathogen

A Self-Lysis Pathway that Enhances the Virulence of a Pathogenic Bacterium

In mammalian cells, programmed cell death (PCD) plays important roles in development, in the removal of damaged cells, and in fighting bacterial infections. Although widespread among multicellular organisms, there are relatively few documented instances of PCD in bacteria. Here we describe a potential PCD pathway in Pseudomonas aeruginosa that enhances the ability of the bacterium to cause disease in a lung infection model. Activation of the system can occur in a subset of cells in response to DNA damage through cleavage of an essential transcription regulator we call AlpR. Cleavage of AlpR triggers a cell lysis program through de-repression of the alpA gene, which encodes a positive regulator that activates expression of the alpBCDE lysis cassette. Although this is lethal to the individual cell in which it occurs, we find it benefits the population as a whole during infection of a mammalian host. Thus, host and pathogen each may use PCD as a survival-promoting strategy. We suggest that activation of the Alp cell lysis pathway is a disease-enhancing response to bacterial DNA damage inflicted by the host immune system

Safety, immunogenicity, and efficacy of a COVID-19 vaccine (NVX-CoV2373) co-administered with seasonal influenza vaccines: an exploratory substudy of a randomised, observer-blinded, placebo-controlled, phase 3 trial

Background: Safety and immunogenicity of COVID-19 vaccines when co-administered with influenza vaccines have not yet been reported. Methods: A sub-study on influenza vaccine co-administration was conducted as part of the phase 3 randomised trial of NVX-CoV2373’s safety and efficacy; ~400 participants meeting main study entry criteria, with no contraindications to influenza vaccination, were enroled. After randomisation to receive NVX-CoV2373 or placebo, sub-study participants received an open-label influenza vaccine at the same time as the first dose of NVX-CoV2373. Reactogenicity was evaluated for 7 days post-vaccination plus monitoring for unsolicited adverse events (AEs), medically-attended AEs (MAAEs), and serious AEs (SAEs). Vaccine efficacy against COVID-19 was assessed. Findings: Sub-study participants were younger (median age 39; 6.7 % ≥65 years), more racially diverse, and had fewer comorbid conditions than main study participants. Reactogenicity events more common in co-administration group included tenderness (70.1% vs 57.6%) or pain (39.7% vs 29.3%) at injection site, fatigue (27.7% vs 19.4%), and muscle pain (28.3% vs 21.4%). Rates of unsolicited AEs, MAAEs, and SAEs were low and balanced between the two groups. Co-administration resulted in no change to influenza vaccine immune response, while a reduction in antibody responses to the NVX-CoV2373 vaccine was noted. Vaccine efficacy against COVID-19 was 87.5% (95% CI: -0.2, 98.4) in those 18-<65 years in the sub-study while efficacy in the main study was 89.8% (95% CI: 79.7, 95.5).  Interpretation: This is the first study to demonstrate safety, immunogenicity, and efficacy of a COVID-19 vaccine when co-administered with influenza vaccines

Gene regulation and the leucine-responsive regulatory protein of Salmonella enterica serovar Typhimurium

THESIS 8421The leucine-responsive regulatory protein of Escherichia coli (E. coli) is a well characterised global regulator of transcription. Comparatively little is known about the Lrp regulon of Salmonella enterica serovar Typhimurium (S. Typhimurium ). In this study, the Irp gene was found to be regulated by nutrient availability, and negatively autoregulated, in a similar manner to that previously shown in E. coli. Autolysis o f S. Typhimurium in MOPS minimal medium was examined, and conditions that reduced autolysis were determined, which allowed for further experimentation. The regulon of Lrp was examined in S. Typhimurium , during growth in rich and nutrient poor media, using DNA microarray analysis. This revealed that Lrp regulates many genes that have previously been identified as Lrp-regulated in E. coli as well as Salmonella-specific virulence determinants, type 1 fimbriae of the fim gene cluster, and the genes of SPI-2. This also revealed genes that are affected by leucine in S. Typhimurium

The Leucine-Responsive Regulatory Protein, Lrp, Activates Transcription of the fim Operon in Salmonella enterica Serovar Typhimurium via the fimZ Regulatory Gene▿

The fim operon of Salmonella enterica serovar Typhimurium encodes type 1 fimbriae. The expression of fim is controlled in response to environmental signals through a complex regulatory cascade involving the proteins FimW, FimY, and FimZ and a genetic locus, fimU, that encodes a rare arginine tRNA. We discovered that a knockout mutation in lrp, the gene that codes for the leucine-responsive regulatory protein (Lrp), inhibited fim transcription. The loss of fim gene expression was accompanied by a corresponding loss of the mannose-sensitive hemagglutination that is a characteristic of type 1 fimbriae. Normal type 1 fimbrial expression was restored following the introduction into the knockout mutant of a plasmid carrying a functional copy of the lrp gene. Electrophoretic mobility shift analysis revealed no interactions between purified Lrp protein and the regulatory region of the fimA, fimU, or fimW gene. Instead, Lrp produced protein-DNA complexes with the regulatory region of the fimZ gene, and the nature of these complexes was leucine sensitive. DNase I footprinting showed that Lrp binds within a region between −65 and −170 with respect to the fimZ transcription start site, consistent with the binding and wrapping of the DNA in this upstream region. Ectopic expression of the fimZ gene from an inducible promoter caused Lrp-independent type 1 fimbriation in serovar Typhimurium. These data show that Lrp makes a positive contribution to fim gene expression through direct interaction with the fimZ promoter region, possibly by antagonizing the binding of the H-NS global repressor protein

A Novel AT-Rich DNA Recognition Mechanism for Bacterial Xenogeneic Silencer MvaT

Bacterial xenogeneic silencing proteins selectively bind to and silence expression from many AT rich regions of the chromosome. They serve as master regulators of horizontally acquired DNA, including a large number of virulence genes. To date, three distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, and MvaT of Pseudomonas sp. Although H-NS and Lsr2 family proteins are structurally different, they all recognize the AT-rich DNA minor groove through a common AT-hook-like motif, which is absent in the MvaT family. Thus, the DNA binding mechanism of MvaT has not been determined. Here, we report the characteristics of DNA sequences targeted by MvaT with protein binding microarrays, which indicates that MvaT prefers binding flexible DNA sequences with multiple TpA steps. We demonstrate that there are clear differences in sequence preferences between MvaT and the other two xenogeneic silencer families. We also determined the structure of the DNA-binding domain of MvaT in complex with a high affinity DNA dodecamer using solution NMR. This is the first experimental structure of a xenogeneic silencer in complex with DNA, which reveals that MvaT recognizes the AT-rich DNA both through base readout by an “AT-pincer” motif inserted into the minor groove and through shape readout by multiple lysine side chains interacting with the DNA sugar-phosphate backbone. Mutations of key MvaT residues for DNA binding confirm their importance with both in vitro and in vivo assays. This novel DNA binding mode enables MvaT to better tolerate GC-base pair interruptions in the binding site and less prefer A tract DNA when compared to H-NS and Lsr2. Comparison of MvaT with other bacterial xenogeneic silencers provides a clear picture that nature has evolved unique solutions for different bacterial genera to distinguish foreign from self DNA

Analysis of MvaT_ctd-bound DNA conformation.

<p>(A) Selected helix parameters of the solution structures of MvaT_ctd-bound 3AT DNA (blue), a free DNA with ATATAT sequence (PDB 2LWG) (magenta) and an A-tract DNA with AAAAAA sequence (PDB 1FZX [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004967#ppat.1004967.ref052" target="_blank">52</a>]) (gold). Average minor groove width, roll and inclination angles including standard deviations are shown. Base steps of 3AT are indicated. (B) Mean structures of MvaT_ctd-bound 3AT (blue), 2LWG (magenta) and 1FZX (gold). Axes are shown by sticks.</p

Restraints and structural statistics for MvaT_ctd and its complex with DNA.

<p>*Pairwise RMSD was calculated among 20 refined structures over residues A79-G124 (MvaT_ctd) and/or C1-G24 (DNA).</p><p>Restraints and structural statistics for MvaT_ctd and its complex with DNA.</p

Sequence alignments and structural comparisons of MvaT_ctd with AT-hook-like motif containing DNA-binding domains.

<p>(A) Alignment of <i>Pseudomonas</i> MvaT with members of the H-NS family reveals regions of similarity outside of the canonical H-NS motif. Clustal Omega alignment of C-terminal domains of MvaT and MvaU from <i>P</i>. <i>aeruginosa</i> PAO1 with Bv3F from <i>Burkholderia vietnamiensis</i> G4 (Buv), H-NS from <i>S</i>. <i>typhimurium</i> (Sal), <i>Xanthomonas albilineans</i> (Xan), and <i>X</i>. <i>fastidiosa</i> (Xyl), and Lsr2 from <i>M</i>. <i>tuberculosis</i>. The canonical H-NS motif is boxed. In bold are the residues corresponding to the AT-hook-like motif in Lsr2 and H-NS. (B) Superimposing structures of MvaT_ctd (cyan) and <i>S</i>. <i>typhimurium</i> H-NS_ctd (magenta). Side chain conformations of K97 and N100 of MvaT_ctd are nearly identical to those of T110 and R114 of H-NS_ctd, respectively. MvaT_ctd lacks the corresponding residue of Q112, functioning as the first critical residue of the “Q/RGR” AT-hook-like structure in H-NS_ctd. When MvaT_ctd is complexed with DNA, side chain of R80 occupies similar position as the side chain of Q112 from H-NS_ctd. A blue circle is drawn to indicate the gap between the R80 side chain and the backbone of loop2. (C) MvaT_ctd/DNA complex structure viewed from one end of the double helix. The cavity above A6-T19 base pair is shown by red spheres.</p