Genetic Regulation of Mucoidy in Pseudomonas aeruginosa

Cystic fibrosis is a genetic disorder that results from mutations in the CF transmembrane conductance regulator gene. These mutations cause a disruption in the chloride transport in mucosal tissues causing the accumulation of dehydrated mucus, and a decrease in the mucocilliary removal of environmental pathogens within the lungs. Additionally, the accumulation of dehydrated mucus within the lungs provides a hospitable environment for various bacteria, including the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa uses the overproduction of a surface polysaccharide called alginate to form a biofilm to evade the host’s immunological defenses. The overproduction of alginate, often referred to as mucoidy, is a virulence factor that is responsible for chronic P. aeruginosa infections, as well as an increased resistance to antibiotics and phagocytosis by the host defense cells. Chronic P. aeruginosa infections are the leading cause of morbidity and mortality in CF patients, and the detection of mucoid isolates is a proven predictor of a decline in the patient’s health. The transition from the non-mucoid phenotype, found in environmental isolates, to the mucoid phenotype found within the CF lung is typically due to “loss-of-function” mutations in the transmembrane anti-sigma factor MucA. However, P. aeruginosa can overproduce alginate independent of mutations in mucA, through the regulated proteolysis of MucA. A series of proteases, beginning with AlgW, can degrade MucA, and release the alternative sigma factor AlgU to drive transcription of the alginate biosynthetic operon. It is generally accepted that the regulated proteolysis of MucA is a mechanism used by early colonizing strains prior to the selection for MucA mutations. Therefore, understanding this mechanism employed by those early colonizing strains may prove beneficial in preventing the establishment of chronic P. aeruginosa respiratory infection. In this dissertation, I identify and characterize two novel regulators of alginate overproduction in P. aeruginosa strains possessing a wildtype MucA. Using the model strain PAO579, I determined that mutations that result in the truncation of the type-IV pilin precursor protein, PilA, can induce alginate overproduction through activation of the AlgW resulting in an increased rate of proteolysis of MucA. Additionally, I identify that expression of the genetic locus PA1494, referred to as mucoid inhibitor A (muiA), can suppress mucoidy in P. aeruginosa strains with a wild-type MucA. Collectively, these findings provide needed insight into the regulation of mucoidy in those early colonizing strains, as well as identifies potential therapeutic targets for the prevention of chronic P. aeruginosa infections in the CF lung

Draft Genome Sequence for Pseudomonas aeruginosa Strain PAO579, a Mucoid Derivative of PAO381

Pseudomonas aeruginosa is an opportunistic pathogen that establishes a chronic lung infection in individuals afflicted with cystic fibrosis. Here, we announce the draft genome of P. aeruginosa strain PAO579, an alginate-overproducing derivative of strain PAO381

Evidence for Sigma Factor Competition in the Regulation of Alginate Production by Pseudomonas aeruginosa

Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cystic fibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, the mucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon library using CF149, a non-mucoid clinical isolate with a misssense mutation in algU(AlgUA61V). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified with transposon insertions that caused 1) an overexpression of AlgUA61V, 2) an overexpression of the stringent starvation protein A (SspA), and 3) a reduced expression of the major sigma factor RpoD (σ70). Induction of AlgUA61V in trans caused conversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgUA61V is functional in activating alginate production. Furthermore, the level of AlgUA61V was increased in all three mutants relative to CF149. However, compared to the wild type AlgU, AlgUA61V had a reduced activity in promoting alginate production in PAO1ΔalgU. SspA and three other anti-σ70 orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149. Conversely, RpoD overexpression resulted in suppression of mucoidy in all mucoid strains tested, indicating that sigma factor competition can regulate mucoidy. Additionally, an RpoD-dependent promoter (PssrA) was more active in non-mucoid strains than in isogenic mucoid variants. Altogether, our results indicate that the anti-σ70 factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD

Draft Genome Sequence of a Mucoid Isolate of Pseudomonas aeruginosa Strain C7447m from a Patient with Cystic Fibrosis

Alginate overproduction by Pseudomonas aeruginosa, or mucoidy, plays an important role in the pathogenesis of chronic lung infections in cystic fibrosis (CF) patients. Here we report the draft genome sequence of a clinical isolate of mucoid P. aeruginosa strain C7447m from a CF patient with chronic lung infection

Truncation of type IV pilin induces mucoidy in Pseudomonas aeruginosa strain PAO579

Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host\u27s defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ22). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP causing the conversion to mucoidy. Pseudomonas aeruginosastrain PAO579, a derivative of the nonmucoid strain PAO1, is mucoid due to an unidentified mutation (muc-23). Using whole genome sequencing, we identified 16 nonsynonymous and 15 synonymous single nucleotide polymorphisms (SNP). We then identified three tandem single point mutations in the pilA gene (PA4525), as the cause of mucoidy in PAO579. These tandem mutations generate a premature stop codon resulting in a truncated version of PilA (PilA108), with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF). Inactivation of pilA108 confirmed it was required for mucoidy. Additionally, algW and algU were also required for mucoidy of PAO579. Western blot analysis indicated that MucA was less stable in PAO579 than nonmucoid PAO1 or PAO381. The mucoid phenotype and high PalgU and PalgD promoter activities of PAO579 require pilA108, algW, algU, and rpoN encoding the alternative sigma factor σ54. We also observed that RpoN regulates expression of algW and pilA in PAO579. Together, these results suggest that truncation in type IV pilin in P. aeruginosa strain PAO579 can induce mucoidy through an AlgW/AlgU-dependent pathway

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa

Background Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ22) to initiate transcription of the alginate biosynthetic operon. Results In the current study, we mapped the mucE transcriptional start site, and determined that PmucE activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in PmucE activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. Conclusions Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa

The effect on the small bowel of 5-FU and oxaliplatin in combination with radiation using a microcolony survival assay

<p>Abstract</p> <p>Background</p> <p>In locally advanced rectal cancer, 5-Fluorouracil (5-FU)-based chemoradiation is the standard treatment. The main acute toxicity of this treatment is enteritis. Due to its potential radiosensitizing properties, oxaliplatin has recently been incorporated in many clinical chemoradiation protocols. The aim of this study was to investigate to what extent 5-FU and oxaliplatin influence the radiation (RT) induced small bowel mucosal damage when given in conjunction with single or split dose RT.</p> <p>Methods</p> <p>Immune competent balb-c mice were treated with varying doses of 5-FU, oxaliplatin (given intraperitoneally) and total body RT, alone or in different combinations in a series of experiments. The small bowel damage was studied by a microcolony survival assay. The treatment effect was evaluated using the inverse of the slope (D₀) of the exponential part of the dose-response curve.</p> <p>Results</p> <p>In two separate experiments the dose-response relations were determined for single doses of RT alone, yielding D₀values of 2.79 Gy (95% CI: 2.65 - 2.95) and 2.98 Gy (2.66 - 3.39), for doses in the intervals of 5-17 Gy and 5-10 Gy, respectively. Equitoxic low doses (IC5) of the two drugs in combination with RT caused a decrease in jejunal crypt count with significantly lower D₀: 2.30 Gy (2.10 - 2.56) for RT+5-FU and 2.27 Gy (2.08 - 2.49) for RT+oxaliplatin. Adding both drugs to RT did not further decrease D₀: 2.28 Gy (1.97 - 2.71) for RT+5-FU+oxaliplatin. A clearly higher crypt survival was noted for split course radiation (3 × 2.5 Gy) compared to a single fraction of 7.5 Gy. The same difference was seen when 5-FU and/or oxaliplatin were added.</p> <p>Conclusion</p> <p>Combining 5-FU or oxaliplatin with RT lead to an increase in mucosal damage as compared to RT alone in our experimental setting. No additional reduction of jejunal crypt counts was noted when both drugs were combined with single dose RT. The higher crypt survival with split dose radiation indicates a substantial recovery between radiation fractions. This mucosal-sparing effect achieved by fractionation was maintained also when chemotherapy was added.</p

SPARC 2022 book of abstracts

Welcome to the Book of Abstracts for the 2022 SPARC conference. Our conference is called “Moving Forwards” reflecting our re-emergence from the pandemic and our desire to reconnect our PGR community, in celebration of their research. PGRs have continued with their research endeavours despite many challenges, and their ongoing successes are underpinned by the support and guidance of dedicated supervisors and the Doctoral School Team. To recognise supervision excellence we will be awarding our annual Supervisor of the Year prizes, based on the wonderful nominations received from their PGR students.Once again, we have received a tremendous contribution from our postgraduate research community; with over 60 presenters, 12 Three-Minute Thesis finalists, and 20 poster presentations, the conference showcases our extraordinarily vibrant, inclusive, and resilient PGR community at Salford. This year there will be prizes to be won for ‘best in conference’ presentations, in addition to the winners from each parallel session. Audience members too could be in for a treat, with judges handing out spot prizes for the best questions asked, so don’t miss the opportunity to put your hand up. These abstracts provide a taster of the diverse and impactful research in progress and provide delegates with a reference point for networking and initiating critical debate. Take advantage of the hybrid format: in online sessions by posting a comment or by messaging an author to say “Hello”, or by initiating break time discussions about the amazing research you’ve seen if you are with us in person. Who knows what might result from your conversation? With such wide-ranging topics being showcased, we encourage you to take up this great opportunity to engage with researchers working in different subject areas from your own. As recent events have shown, researchers need to collaborate to meet global challenges. Interdisciplinary and international working is increasingly recognised and rewarded by all major research funders. We do hope, therefore, that you will take this opportunity to initiate interdisciplinary conversations with other researchers. A question or comment from a different perspective can shed new light on a project and could lead to exciting collaborations, and that is what SPARC is all about. SPARC is part of a programme of personal and professional development opportunities offered to all postgraduate researchers at Salford. More information about this programme is available on our website: Doctoral School | University of Salford. Registered Salford students can access full details on the Doctoral School hub: Doctoral School Hub - Home (sharepoint.com) You can follow us on Twitter @SalfordPGRs and please use the #SPARC2022 to share your conference experience.We particularly welcome taught students from our undergraduate and master’s programmes as audience members. We hope you enjoy the presentations on offer and that they inspire you to pursue your own research career. If you would like more information about studying for a PhD here at the University of Salford, your lecturers can advise, or you can contact the relevant PGR Support Officer; their details can be found at Doctoral School | University of Salford. We wish you a rich and rewarding conference experience