A new regulator of pathogenicity (bvlR) is required for full virulence and tight microcolony formation in Pseudomonas aeruginosa

LysR-type transcriptional regulators (LTTRs) are the most common family of transcriptional regulators found in the opportunistic pathogen Pseudomonas aeruginosa. They are known to regulate a wide variety of virulence determinants and have emerged recently as positive global regulators of pathogenicity in a broad spectrum of important bacterial pathogens. However, in spite of their key role in modulating expression of key virulence determinants underpinning pathogenic traits associated with the process of infection, surprisingly few are found to be transcriptionally altered by contact with host cells. BvlR (PA14_26880) an LTTR of previously unknown function, has been shown to be induced in response to host cell contact, and was therefore investigated for its potential role in virulence. BvlR expression was found to play a pivotal role in the regulation of acute virulence determinants such as type III secretion system and exotoxin A production. BvlR also played a key role in P. aeruginosa pathogenicity within the Caenorhabditis elegans acute model of infection. Loss of BvlR led to an inability to form tight microcolonies, a key step in biofilm formation in the cystic fibrosis lung, although surface attachment was increased. Unusually for LTTRs, BvlR was shown to exert its influence through the transcriptional repression of many genes, including the virulence-associated cupA and alg genes. This highlights the importance of BvlR as a new virulence regulator in P. aeruginosa with a central role in modulating key events in the pathogen–host interactome.

Download Full-text

Local and Global Regulators Linking Anaerobiosis to cupA Fimbrial Gene Expression in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.01344-07 ◽

2007 ◽

Vol 189 (23) ◽

pp. 8667-8676 ◽

Cited By ~ 29

Author(s):

Isabelle Vallet-Gely ◽

Josh S. Sharp ◽

Simon L. Dove

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Opportunistic Pathogen ◽

Phase Variable ◽

Global Regulators ◽

Variable Expression ◽

Regulatory Cascade ◽

Abiotic Surfaces ◽

Positive Regulators ◽

Anaerobic Environment

ABSTRACT The cupA gene cluster of Pseudomonas aeruginosa encodes components and assembly factors of a putative fimbrial structure that enable this opportunistic pathogen to form biofilms on abiotic surfaces. In P. aeruginosa the control of cupA gene expression is complex, with the H-NS-like MvaT protein functioning to repress phase-variable (on/off) expression of the operon. Here we identify four positive regulators of cupA gene expression, including three unusual regulators encoded by the cgrABC genes and Anr, a global regulator of anaerobic gene expression. We show that the cupA genes are expressed in a phase-variable manner under anaerobic conditions and that the cgr genes are essential for this expression. We show further that cgr gene expression is negatively controlled by MvaT and positively controlled by Anr and anaerobiosis. Expression of the cupA genes therefore appears to involve a regulatory cascade in which anaerobiosis, signaled through Anr, stimulates expression of the cgr genes, resulting in a concomitant increase in cupA gene expression. Our findings thus provide mechanistic insight into the regulation of cupA gene expression and identify anaerobiosis as an inducer of phase-variable cupA gene expression, raising the possibility that phase-variable expression of fimbrial genes important for biofilm formation may occur in P. aeruginosa persisting in the largely anaerobic environment of the cystic fibrosis host lung.

Download Full-text

cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers

Journal of Bacteriology ◽

10.1128/jb.00135-18 ◽

2018 ◽

Vol 200 (12) ◽

Cited By ~ 9

Author(s):

Alice Berry ◽

Kook Han ◽

Julian Trouillon ◽

Mylène Robert-Genthon ◽

Michel Ragno ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Transcriptional Activation ◽

Opportunistic Pathogen ◽

Secretion System ◽

Intracellular Camp ◽

Virulence Determinants ◽

Cell Infection ◽

Content Type

ABSTRACT The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [ K eq ] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ , resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers. IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

Download Full-text

PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

Infection and Immunity ◽

10.1128/iai.01388-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1638-1647 ◽

Cited By ~ 28

Author(s):

Ziyu Sun ◽

Jing Shi ◽

Chang Liu ◽

Yongxin Jin ◽

Kewei Li ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Bacterial Colonization ◽

Mrna Level ◽

Sos Response ◽

Opportunistic Pathogen ◽

Host Cells ◽

Chronic Infections ◽

Mobility Shift ◽

Content Type

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced byP. aeruginosathat are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component ofP. aeruginosa. In this study, we demonstrate that mutation inprtRresults in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by atacpromoter repressed the endogenousprtRpromoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

Download Full-text

Regulation of Quorum Sensing by RpoS inPseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.182.15.4356-4360.2000 ◽

2000 ◽

Vol 182 (15) ◽

pp. 4356-4360 ◽

Cited By ~ 179

Author(s):

Marvin Whiteley ◽

Matthew R. Parsek ◽

E. P. Greenberg

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Gene Transcription ◽

Opportunistic Pathogen ◽

Null Mutant ◽

Global Regulators ◽

Acylhomoserine Lactone ◽

Sensing Systems

ABSTRACT The LasR-LasI and RhlR-RhlI quorum-sensing systems are global regulators of gene expression in the opportunistic pathogenPseudomonas aeruginosa. Previous studies suggest that the RhlR-RhlI system activates expression of rpoS. We constructed merodiploid strains of P. aeruginosa containing the native rpoS gene and an rpoS-lacZ fusion. Studies of lacZ transcription in these strains indicated that rpoS was not regulated by RhlR-RhlI. We also generated an rpoS null mutant. This rpoS mutant showed elevated levels of rhlI (but not rhlR) transcription, elevated levels of the RhlI-generated acylhomoserine lactone quorum-sensing signal, and elevated levels of RhlR-RhlI-regulated gene transcription. These findings indicate that there is a relationship between RpoS and quorum sensing, but rather than the RhlR-RhlI system influencing the expression ofrpoS, it appears that RpoS regulates rhlI.

Download Full-text

Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients

Microbiology ◽

10.1099/mic.0.26822-0 ◽

2004 ◽

Vol 150 (5) ◽

pp. 1315-1326 ◽

Cited By ~ 101

Author(s):

Julianne V. Kus ◽

Elizabeth Tullis ◽

Dennis G. Cvitkovitch ◽

Lori L. Burrows

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Opportunistic Pathogen ◽

Host Cells ◽

Phylogenetic Groups ◽

Allele Distribution ◽

Specific Pcr ◽

Type Iv ◽

Accessory Genes ◽

Group I

Type IV pili (TFP) are important colonization factors of the opportunistic pathogen Pseudomonas aeruginosa, involved in biofilm formation and attachment to host cells. This study undertook a comprehensive analysis of TFP alleles in more than 290 environmental, clinical, rectal and cystic fibrosis (CF) isolates of P. aeruginosa. Based on the results, a new system of nomenclature is proposed, in which P. aeruginosa TFP are divided into five distinct phylogenetic groups. Each pilin allele is stringently associated with characteristic, distinct accessory genes that allow the identification of the allele by specific PCR. The invariant association of the pilin and accessory genes implies horizontal transfer of the entire locus. Analysis of pilin allele distribution among isolates from various sources revealed a striking bias in the prevalence of isolates with group I pilin genes from CF compared with non-CF human sources (P<0·0001), suggesting this particular pilin type, which can be post-translationally modified by glycosylation via the action of TfpO (PilO), may confer a colonization or persistence advantage in the CF host. This allele was also predominant in paediatric CF isolates (29 of 43; 67·4 %), showing that this bias is apparent early in colonization. Group I pilins were also the most common type found in environmental isolates tested. To the authors' knowledge, this is the first example of a P. aeruginosa virulence factor allele that is strongly associated with CF isolates.

Download Full-text

Syndecan 1 Shedding Contributes to Pseudomonas aeruginosa Sepsis

Infection and Immunity ◽

10.1128/iai.73.12.7914-7921.2005 ◽

2005 ◽

Vol 73 (12) ◽

pp. 7914-7921 ◽

Cited By ~ 45

Author(s):

Allan Haynes ◽

Frank Ruda ◽

Jeffrey Oliver ◽

Abdul N. Hamood ◽

John A. Griswold ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Thermal Injury ◽

Opportunistic Pathogen ◽

Multiple Organ ◽

Innate Immune ◽

Host Cells ◽

Ectodomain Shedding ◽

Systemic Spread ◽

Immunocompromised State ◽

First Time

ABSTRACT The innate immune system is comprised of many components that function coordinately to prevent bacterial sepsis. However, thermal injury suppresses many of these factors, and the opportunistic pathogen Pseudomonas aeruginosa takes advantage of this condition, making it one of the leading causes of morbidity and mortality in the setting of thermal injury. P. aeruginosa is extremely efficient at colonizing burn wounds, spreading systemically, and causing sepsis, which often results in a systemic inflammatory response, multiple-organ failure, and death. The pathogenicity of P. aeruginosa is due to the arsenal of virulence factors produced by the pathogen and the immunocompromised state of the host. Syndecan 1 is a major heparan sulfate proteoglycan present on many host cells involved in thermal injury. Syndecan 1 anchored to the cell surface can be cleaved in a process termed ectodomain shedding. Syndecan 1 shedding results in the release of intact, soluble proteoglycan ectodomains that have diverse roles in innate immunity. Here we show for the first time that thermal injury results in shedding of syndecan 1 from host tissue. Our data show that syndecan 1 null mice are significantly less susceptible to P. aeruginosa infection than their wild-type counterparts, as demonstrated by (i) significantly lower mortality; (ii) absence of systemic spread of P. aeruginosa; and (iii) significant reductions in some proinflammatory cytokines. These results suggest that shed syndecan 1 plays an important role in the pathogenesis of P. aeruginosa infection of thermal injury and that syndecan 1-neutralizing agents may be effective supplements to current P. aeruginosa treatments.

Download Full-text

Exoenzyme Y Contributes to End-Organ Dysfunction Caused by Pseudomonas aeruginosa Pneumonia in Critically Ill Patients: An Exploratory Study

Toxins ◽

10.3390/toxins12060369 ◽

2020 ◽

Vol 12 (6) ◽

pp. 369

Author(s):

Brant M. Wagener ◽

Naseem Anjum ◽

Sarah C. Christiaans ◽

Morgan E. Banks ◽

Jordan C. Parker ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Organ Dysfunction ◽

Virulence Factors ◽

Opportunistic Pathogen ◽

Lavage Fluid ◽

Host Cells ◽

Clinical Isolates ◽

Icu Patients ◽

Edema Factor ◽

Host Infection

Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia in immunocompromised and intensive care unit (ICU) patients. During host infection, P. aeruginosa upregulates the type III secretion system (T3SS), which is used to intoxicate host cells with exoenzyme (Exo) virulence factors. Of the four known Exo virulence factors (U, S, T and Y), ExoU has been shown in prior studies to associate with high mortality rates. Preclinical studies have shown that ExoY is an important edema factor in lung infection caused by P. aeruginosa, although its importance in clinical isolates of P. aeruginosa is unknown. We hypothesized that expression of ExoY would be highly prevalent in clinical isolates and would significantly contribute to patient morbidity secondary to P. aeruginosa pneumonia. A single-center, prospective observational study was conducted at the University of Alabama at Birmingham Hospital. Mechanically ventilated ICU patients with a bronchoalveolar lavage fluid culture positive for P. aeruginosa were included. Enrolled patients were followed from ICU admission to discharge and clinical P. aeruginosa isolates were genotyped for the presence of exoenzyme genes. Ninety-nine patients were enrolled in the study. ExoY was present in 93% of P. aeruginosa clinical isolates. Moreover, ExoY alone (ExoY+/ExoU−) was present in 75% of P. aeruginosa isolates, compared to 2% ExoU alone (ExoY−/ExoU+). We found that bacteria isolated from human samples expressed active ExoY and ExoU, and the presence of ExoY in clinical isolates was associated with end-organ dysfunction. This is the first study we are aware of that demonstrates that ExoY is important in clinical outcomes secondary to nosocomial pneumonia.

Download Full-text

Neutron crystallography reveals novel mechanisms used by Pseudomonas aeruginosa for host-cell binding

10.1101/2021.09.24.461693 ◽

2021 ◽

Author(s):

Lukas Gajdos ◽

Matthew P Blakeley ◽

Michael Haertlein ◽

V Trevor Forsyth ◽

Juliette M Devos ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Calcium Ions ◽

Bacterial Infections ◽

Opportunistic Pathogen ◽

Host Cells ◽

Carbohydrate Binding ◽

Hydrogen Atoms ◽

High Affinity ◽

Structural Details ◽

Neutron Crystallography

The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.

Download Full-text

Molecular Cloning, Expression and FPLC Purification of Lectin A from Pseudomonas aeruginosa

International Journal of Applied Sciences and Biotechnology ◽

10.3126/ijasbt.v2i4.11559 ◽

2014 ◽

Vol 2 (4) ◽

pp. 529-536

Author(s):

Peyman Ghoraishizadeh ◽

Shraddha Raikar ◽

Mahsa Takhtechian

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Molecular Cloning ◽

Opportunistic Pathogen ◽

Host Cells ◽

Related Protein ◽

Burn Victims ◽

Gene Coding ◽

Lectin Protein

Pseudomonas aeruginosa (PA) as an opportunistic pathogen infects the pulmonary tract, bladder, cystic fibrosis patients and burn victims. PA infections treatment is challenging because of its ability to rapidly develop resistance to multiple classes of antibiotics. Lectin is protein that isexpressed in cell of PA and cause of infection by attaching to the host cells. Lectin A gene coding lectin protein so we cloned and expressedthis gene then purified of related protein, that can be used in preparation of vaccine to treat PA infections.DOI: http://dx.doi.org/10.3126/ijasbt.v2i4.11559 Int J Appl Sci Biotechnol, Vol. 2(4): 529-536

Download Full-text

Pseudomonas aeruginosa at the dawn of a post-antibiotic era: clinical significance, resistance mechanisms, novel antibiotics and alternative treatments

Studia Universitatis Babeş-Bolyai Biologia ◽

10.24193/subbbiol.2020.2.03 ◽

2020 ◽

Vol 65 (2) ◽

pp. 31-67

Author(s):

Orsolya Kovács ◽

Endre Jakab

Keyword(s):

Pseudomonas Aeruginosa ◽

Treatment Options ◽

Opportunistic Pathogen ◽

Resistance Mechanisms ◽

Modern Medicine ◽

Host Cells ◽

Bacteriophage Therapy ◽

Priority List ◽

Resistant Strains ◽

Novel Antibiotics

Since their discovery, antibiotics have helped treat diseases prior to which many were untreatable, saving millions of lives. However, due to the overuse of antibiotics in medicine and agriculture, the advent of resistant strains of bacteria followed shortly after. The current antibiotic resistance crisis is bringing humanity closer to a post-antibiotic era, when all the advancements made by modern medicine could easily be reversed. Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium, ubiquitous owing to its minimal nutritional and growth requirements. P. aeruginosa is one of the pathogens included in the priority list of the WHO, being assessed as critical due to its high antimicrobial resistance, leaving only a few effective treatment options to combat it. As an opportunistic pathogen, P. aeruginosa establishes infection in immunocompromised patients, primarily in hospital settings. In order to initiate infection, it requires several virulence factors that mediate the invasion of the pathogen into host cells. Owing to the multiple resistance mechanisms of P. aeruginosa, it has developed resistance to most classes of antibiotics. Due to its increased resistance, treating P. aeruginosa infections is a great challenge for clinicians. Several β-lactam/β-lactamase combinations have been approved and are available as treatment options, which overall show high efficacy against P. aeruginosa. Moreover, novel antibiotics are currently in development as possible antipseudomonal agents, including a Pseudomonas-specific formulation. In addition, new strategies such as bacteriophage therapy, pyocins or the inhibition of the quorum sensing system are being investigated for the treatment of P. aeruginosa infections.

Download Full-text