scholarly journals Role of Hibernation Promoting Factor in Ribosomal Protein Stability during Pseudomonas aeruginosa Dormancy

2020 ◽  
Vol 21 (24) ◽  
pp. 9494
Author(s):  
Sokuntheary Theng ◽  
Kerry S. Williamson ◽  
Michael J. Franklin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ribosomal Protein ◽  
Stringent Response ◽  
Opportunistic Pathogen ◽  
Growth Conditions ◽  
Physiological Status ◽  
Metabolic Remodeling ◽  
Its Rrna ◽  
Mutant Cells

Pseudomonas aeruginosa is an opportunistic pathogen that causes biofilm-associated infections. P. aeruginosa can survive in a dormant state with reduced metabolic activity in nutrient-limited environments, including the interiors of biofilms. When entering dormancy, the bacteria undergo metabolic remodeling, which includes reduced translation and degradation of cellular proteins. However, a supply of essential macromolecules, such as ribosomes, are protected from degradation during dormancy. The small ribosome-binding proteins, hibernation promoting factor (HPF) and ribosome modulation factor (RMF), inhibit translation by inducing formation of inactive 70S and 100S ribosome monomers and dimers. The inactivated ribosomes are protected from the initial steps in ribosome degradation, including endonuclease cleavage of the ribosomal RNA (rRNA). Here, we characterized the role of HPF in ribosomal protein (rProtein) stability and degradation during P. aeruginosa nutrient limitation. We determined the effect of the physiological status of P. aeruginosa prior to starvation on its ability to recover from starvation, and on its rRNA and rProtein stability during cell starvation. The results show that the wild-type strain and a stringent response mutant (∆relA∆spoT strain) maintain high cellular abundances of the rProteins L5 and S13 over the course of eight days of starvation. In contrast, the abundances of L5 and S13 reduce in the ∆hpf mutant cells. The loss of rProteins in the ∆hpf strain is dependent on the physiology of the cells prior to starvation. The greatest rProtein loss occurs when cells are first cultured to stationary phase prior to starvation, with less rProtein loss in the ∆hpf cells that are first cultured to exponential phase or in balanced minimal medium. Regardless of the pre-growth conditions, P. aeruginosa recovery from starvation and the integrity of its rRNA are impaired in the absence of HPF. The results indicate that protein remodeling during P. aeruginosa starvation includes the degradation of rProteins, and that HPF is essential to prevent rProtein loss in starved P. aeruginosa. The results also indicate that HPF is produced throughout cell growth, and that regardless of the cellular physiological status, HPF is required to protect against ribosome loss when the cells subsequently enter starvation phase.

scholarly journals Role of an Alginate Lyase for Alginate Transport in Mucoid Pseudomonas aeruginosa

2005 ◽  
Vol 73 (10) ◽  
pp. 6429-6436 ◽  
Author(s):  
Sumita Jain ◽  
Dennis E. Ohman
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Alginate Lyase ◽  
Opportunistic Pathogen ◽  
Chronic Pulmonary Disease ◽  
Host Defenses ◽  
Alginate Production ◽  
Mutant Cells ◽  
Alginate Biosynthesis

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa secretes a capsule-like polysaccharide called alginate that is important for evasion of host defenses, especially during chronic pulmonary disease of patients with cystic fibrosis (CF). Most proteins for alginate biosynthesis are encoded by the 12-gene algD operon. Interestingly, this operon also encodes AlgL, a lyase that degrades alginate. Mutants lacking AlgG, AlgK, or AlgX, also encoded by the operon, synthesize alginate polymers that are digested by the coregulated protein AlgL. We examined the phenotype of an ΔalgL mutation in the highly mucoid CF isolate FRD1. Generating a true ΔalgL mutant was possible only when the algD operon was under the control of a LacIq-repressed trc promoter. Upon induction of alginate production with isopropyl-β-d-thiogalactopyranoside, the ΔalgL mutant cells were lysed within a few hours. Electron micrographs of the ΔalgL mutant showed that alginate polymers accumulated in the periplasm, which ultimately burst the bacterial cell wall. The requirement of AlgL in an alginate-overproducing strain led to a new model for alginate secretion in which a multiprotein secretion complex (or scaffold, that includes AlgG, AlgK, AlgX, and AlgL) guides new polymers through the periplasm for secretion across the outer membrane. In this model, AlgL is bifunctional with a structural role in the scaffold and a role in degrading free alginate polymers in the periplasm.

Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells

1980 ◽  
Vol 29 (3) ◽  
pp. 1146-1151 ◽  
Author(s):  
D E Woods ◽  
D C Straus ◽  
W G Johanson ◽  
V K Berry ◽  
J A Bass
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Opportunistic Pathogen ◽  
In Vitro System ◽  
Heterologous Antiserum ◽  
Buccal Epithelial Cells ◽  
Serological Studies ◽  
Seriously Ill Patients

Adherence of Pseudomonas aeruginosa organisms to the upper respiratory epithelium of seriously ill patients in vitro is correlated with subsequent colonization of the respiratory tract by this opportunistic pathogen. The role of pili in the attachment to epithelial cells of P. aeruginosa was studied in an in vitro system employing human buccal epithelial cells and P. aeruginosa pretreated by various means. Pretreatment of the bacteria with proteases, heat, or Formalin caused a significant decrease in adherence. A decrease when compared with controls was also noted in the adherence of P. aeruginosa organisms to buccal epithelial cells preincubated with purified pili prepared from the strain used for adherence testing; however, pili prepared from a heterologous strain failed to block adherence. Similar results were obtained in serological studies when antisera to purified pili prepared from the strain used for adherence testing decreased adherence, whereas heterologous antiserum to pili did not decrease adherence. From these results it appears that pili mediate the adherence of P. aeruginosa organisms to human buccal epithelial cells.

scholarly journals Pseudomonas aeruginosa-Mediated Damage Requires Distinct Receptors at the Apical and Basolateral Surfaces of the Polarized Epithelium

2009 ◽  
Vol 78 (3) ◽  
pp. 939-953 ◽  
Author(s):  
Iwona Bucior ◽  
Keith Mostov ◽  
Joanne N. Engel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mdck Cells ◽  
Three Dimensional ◽  
Opportunistic Pathogen ◽  
Necessary And Sufficient ◽  
Plastic Surfaces ◽  
Pharmacologic Inhibition ◽  
Increased Susceptibility ◽  
Polarized Epithelium

ABSTRACT Pseudomonas aeruginosa, an important opportunistic pathogen of humans, exploits epithelial damage to establish infection. We have rigorously explored the role of N-glycoproteins and heparan sulfate proteoglycans (HSPGs) in P. aeruginosa-mediated attachment and subsequent downstream events at the apical (AP) and basolateral (BL) surfaces of polarized epithelium. We demonstrate that the N-glycan chains at the AP surface are necessary and sufficient for binding, invasion, and cytotoxicity to kidney (MDCK) and airway (Calu-3) cells grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation enhanced binding, whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at the AP surface, explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK cells grown as three-dimensional cysts as a model for epithelial organs, we show that P. aeruginosa specifically colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P. aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity toward isolated HS chains. Together, these findings demonstrate that P. aeruginosa recognizes distinct receptors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains and/or in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa.

scholarly journals PmtA Regulates Pyocyanin Expression and Biofilm Formation in Pseudomonas aeruginosa

2021 ◽  
Vol 12 ◽  
Author(s):  
Amy V. Thees ◽  
Kathryn M. Pietrosimone ◽  
Clare K. Melchiorre ◽  
Jeremiah N. Marden ◽  
Joerg Graf ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pseudomonas Aeruginosa ◽  
Metal Binding ◽  
Biofilm Formation ◽  
Binding Capacity ◽  
Opportunistic Pathogen ◽  
Small Molecular Weight ◽  
Heavy Metal Binding ◽  
The Impact

The opportunistic pathogen Pseudomonas aeruginosa expresses a small molecular weight, cysteine-rich protein (PmtA), identified as a metallothionein (MT) protein family member. The MT family proteins have been well-characterized in eukaryotes as essential for zinc and copper homeostasis, protection against oxidative stress, and the ability to modify a variety of immune activities. Bacterial MTs share sequence homology, antioxidant chemistry, and heavy metal-binding capacity with eukaryotic MTs, however, the impact of bacterial MTs on virulence and infection have not been well-studied. In the present study, we investigated the role of PmtA in P. aeruginosa PAO1 using a PmtA-deficient strain (ΔpmtA). Here we demonstrated the virulence factor, pyocyanin, relies on the expression of PmtA. We showed that PmtA may be protective against oxidative stress, as an alternative antioxidant, glutathione, can rescue pyocyanin expression. Furthermore, the expression of phzM, which encodes a pyocyanin precursor enzyme, was decreased in the ΔpmtA mutant during early stationary phase. Upregulated pmtA expression was previously detected in confluent biofilms, which are essential for chronic infection, and we observed that the ΔpmtA mutant was disrupted for biofilm formation. As biofilms also modulate antibiotic susceptibility, we examined the ΔpmtA mutant susceptibility to antibiotics and found that the ΔpmtA mutant is more susceptible to cefepime and ciprofloxacin than the wild-type strain. Finally, we observed that the deletion of pmtA results in decreased virulence in a waxworm model. Taken together, our results support the conclusion that PmtA is necessary for the full virulence of P. aeruginosa and may represent a potential target for therapeutic intervention.

scholarly journals The Role of Pseudomonas aeruginosa Lipopolysaccharide in Bacterial Pathogenesis and Physiology

Pathogens ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 6 ◽  
Author(s):  
Steven M. Huszczynski ◽  
Joseph S. Lam ◽  
Cezar M. Khursigara
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Opportunistic Pathogen ◽  
Major Constituent ◽  
Bacterial Survival ◽  
Core Oligosaccharide ◽  
Persistent Infections ◽  
The Relationship ◽  
Extracellular Environment

The major constituent of the outer membrane of Gram-negative bacteria is lipopolysaccharide (LPS), which is comprised of lipid A, core oligosaccharide, and O antigen, which is a long polysaccharide chain extending into the extracellular environment. Due to the localization of LPS, it is a key molecule on the bacterial cell wall that is recognized by the host to deploy an immune defence in order to neutralize invading pathogens. However, LPS also promotes bacterial survival in a host environment by protecting the bacteria from these threats. This review explores the relationship between the different LPS glycoforms of the opportunistic pathogen Pseudomonas aeruginosa and the ability of this organism to cause persistent infections, especially in the genetic disease cystic fibrosis. We also discuss the role of LPS in facilitating biofilm formation, antibiotic resistance, and how LPS may be targeted by new antimicrobial therapies.

scholarly journals Budding Yeast CTDK-I Is Required for DNA Damage-Induced Transcription

2003 ◽  
Vol 2 (2) ◽  
pp. 274-283 ◽  
Author(s):  
Denis Ostapenko ◽  
Mark J. Solomon
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Scale ◽  
Large Subunit ◽  
Growth Conditions ◽  
Dna Damaging Agents ◽  
Irradiation Treatment ◽  
Mutant Cells

ABSTRACT CTDK-I phosphorylates the C-terminal domain (CTD) of the large subunit of yeast RNA polymerase II in a reaction that stimulates transcription elongation. Mutations in CTDK-I subunits—Ctk1p, Ctk2p, and Ctk3p—confer conditional phenotypes. In this study, we examined the role of CTDK-I in the DNA damage response. We found that mutation of individual CTDK-I subunits rendered yeast sensitive to hydroxyurea (HU) and UV irradiation. Treatment with DNA-damaging agents increased phosphorylation of Ser2 within the CTD repeats in wild-type but not in ctk1Δ mutant cells. Using microarray hybridization, we identified genes whose transcription following DNA damage is Ctk1p dependent, including several DNA repair and stress response genes. Following HU treatment, the level of Ser2-phosphorylated RNA polymerase II increased both globally and on the CTDK-I-regulated genes. The pleiotropic phenotypes of ctk mutants suggest that CTDK-I activity is essential during large-scale transcriptional repatterning under stress and unfavorable growth conditions.

scholarly journals Recent advances in understanding Pseudomonas aeruginosa as a pathogen

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1261 ◽  
Author(s):  
Jens Klockgether ◽  
Burkhard Tümmler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Population Biology ◽  
Recent Progress ◽  
Human Subjects ◽  
Opportunistic Pathogen ◽  
Mammalian Host ◽  
Secretion Systems ◽  
Chronic Infections ◽  
Type Vi Secretion

The versatile and ubiquitousPseudomonas aeruginosais an opportunistic pathogen causing acute and chronic infections in predisposed human subjects. Here we review recent progress in understandingP. aeruginosapopulation biology and virulence, its cyclic di-GMP-mediated switches of lifestyle, and its interaction with the mammalian host as well as the role of the type III and type VI secretion systems inP. aeruginosainfection.

scholarly journals The stringent stress response controls proteases and global regulators under optimal growth conditions in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Daniel Pletzer ◽  
Travis M. Blimkie ◽  
Heidi Wolfmeier ◽  
Yicong Li ◽  
Arjun Baghela ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stress Response ◽  
Stringent Response ◽  
Optimal Growth ◽  
Transcriptional Regulators ◽  
Growth Conditions ◽  
Exponential Growth Phase ◽  
Rna Seq ◽  
Signaling Molecule ◽  
Response Mutant

AbstractThe bacterial stringent stress response, mediated by the signaling molecule guanosine tetra-phosphate, ppGpp, has recently gained attention as being important during normal cellular growth and as potential new therapeutic target, which warrants detailed mechanistic understanding. Here, we used intracellular protein tracking in Pseudomonas aeruginosa PAO1, which indicated that RelA was bound to the ribosome, while SpoT localized at the cell poles. RNA-Seq was used to investigate the transcriptome of a ppGpp-deficient strain under non-stressful nutrient-rich broth conditions where the mutant grew at the same rate as the parent strain. In exponential growth phase, the lack of ppGpp led to >1,600 transcriptional changes (fold-change cut-off ±1.5), providing further novel insights into the normal physiological role of ppGpp. The stringent response was linked to gene expression of various proteases and secretion systems including aprA, PA0277, impA, and clpP2. The previously observed reduction in cytotoxicity towards red blood cells, in a stringent response mutant, appeared to be due to aprA. Investigation of an aprA mutant in a murine skin infection model, showed increased survival rates of the aprA mutant consistent with previous observations that stringent-response mutants have reduced virulence. In addition, the overexpression of relA, but not induction of ppGpp with serine hydroxamate, dysregulated global transcriptional regulators as well as >30% of the regulatory networks controlled by AlgR, OxyR, LasR, and AmrZ. Together these data expand our knowledge about ppGpp and its regulatory network and role in environmental adaptation. It also confirms its important role throughout the normal growth cycle of bacteria.Significance StatementMicroorganisms need to adapt rapidly to survive harsh environmental changes. Here, we showed the broad influence of the highly studied bacterial stringent stress response under non-stressful conditions that indicate its general physiological importance and might reflect the readiness of bacteria to respond to and activate acute stress responses. Using RNA-Seq to investigate the transcriptional network of Pseudomonas aeruginosa cells revealed that >30% of all genes changed expression in a stringent-response mutant under optimal growth conditions. This included genes regulated by global transcriptional regulators and novel downstream effectors. Our results help to understand the importance of this stress regulator in bacterial lifestyle under relatively unstressed conditions. As such it draws attention to the consequences of targeting this ubiquitous bacterial signaling molecule.

scholarly journals Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Ian T. Hill ◽  
Thomas Tallo ◽  
Matthew J. Dorman ◽  
Simon L. Dove
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Toxic Effects ◽  
Growth Defect ◽  
Wild Type ◽  
Rna Chaperone ◽  
Small Protein ◽  
Content Type ◽  
Transcription Regulator ◽  
Mutant Cells

ABSTRACT Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR. We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT. IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa. We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

scholarly journals Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

2016 ◽  
Vol 12 ◽  
pp. 1428-1433 ◽  
Author(s):  
Bernardas Morkunas ◽  
Balint Gal ◽  
Warren R J D Galloway ◽  
James T Hodgkinson ◽  
Brett M Ibbeson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Cell Signalling ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Wide Range ◽  
Potential Applications ◽  
Therapeutic Context ◽  
Pyocyanin Production

Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable.

Sign in / Sign up

Export Citation Format

Share Document