scholarly journals The Versatile Pseudomonas aeruginosa Biofilm Matrix Protein CdrA Promotes Aggregation through Different Extracellular Exopolysaccharide Interactions

2020 ◽  
Vol 202 (19) ◽  
Author(s):  
Courtney Reichhardt ◽  
Holly M. Jacobs ◽  
Michael Matwichuk ◽  
Cynthis Wong ◽  
Daniel J. Wozniak ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Matrix Protein ◽  
Specific Interaction ◽  
Aggregate Stability ◽  
Laboratory Strain ◽  
Chronic Infections ◽  
Content Type ◽  
Chemical Structures ◽  
Biofilm Matrix

ABSTRACT Pseudomonas aeruginosa is an important pathogen that causes chronic infections that involve multicellular aggregates called biofilms. Within biofilms, bacteria are surrounded in a protective extracellular matrix of proteins, exopolysaccharides (EPS), and DNA. A key P. aeruginosa matrix protein is an extracellular adhesin called CdrA, which promotes aggregation by binding to the EPS Psl and via CdrA-CdrA interactions. We hypothesized that because of its ability to bind Psl, CdrA would be important only for strains that use Psl as the primary EPS (e.g., the laboratory strain PAO1). Thus, we predicted that cdrA might be dispensable for biofilm formation by strains that do not utilize Psl (e.g., the laboratory strain PA14). Instead, we observed that cdrA deletion strains exhibited biofilm defects, regardless of their EPS dependencies. We screened a panel of clinical and environmental P. aeruginosa isolates for the presence of the cdrA allele and production of CdrA protein. All isolates that we tested contained the cdrA allele, and these alleles had minimal sequence variation compared to the reference PAO1 cdrA gene. Additionally, all isolates except one produced detectable CdrA protein. We investigated the possible mechanisms of CdrA-promoted biofilm formation in these strains where Psl is not dominant, and we discovered that CdrA binds to Pel. Although Psl and Pel chemical structures are distinct, this appears to be a specific interaction, since previous work has shown that CdrA binds discriminately to other EPS. Our findings provide new understanding of biofilm formation across P. aeruginosa isolates and emphasize the versatility of CdrA. IMPORTANCE Depending upon the strain, Pseudomonas aeruginosa can use different exopolysaccharides (e.g., Psl, Pel, and alginate) to build its biofilm matrix. Previously, we demonstrated that the biofilm matrix protein CdrA binds to Psl, promoting biofilm formation and aggregate stability. As such, it was thought that CdrA might be important for biofilm assembly only in strains that rely upon Psl. However, past studies indicated that CdrA can interact with monosaccharides not present in Psl, including N-acetylglucosamine, a constituent of another EPS called Pel. We discovered that CdrA also binds to Pel and promotes biofilm formation by strains in which Psl is not dominant. Thus, our findings suggest that CdrA plays a common role as a biofilm matrix cross-linker across P. aeruginosa isolates with different EPS.

scholarly journals Pseudomonas aeruginosa AlgU Contributes to Posttranscriptional Activity by IncreasingrsmAExpression in amucA22Strain

2016 ◽  
Vol 198 (13) ◽  
pp. 1812-1826 ◽  
Author(s):  
Sean D. Stacey ◽  
Christopher L. Pritchett
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Laboratory Strain ◽  
Immunocompromised Patients ◽  
Chronic Infections ◽  
Rnase Protection ◽  
Content Type ◽  
Mutant Strains ◽  
Life Threatening ◽  
Severe Infections

ABSTRACTPseudomonas aeruginosathrives in multiple environments and is capable of causing life-threatening infections in immunocompromised patients. RsmA is a posttranscriptional regulator that controls virulence factor production and biofilm formation. In this study, we investigated the expression and activity ofrsmAand the protein that it encodes, RsmA, inP. aeruginosamucAmutant strains, which are common in chronic infections. We determined that AlgU regulates a previously unknownrsmApromoter inP. aeruginosa. Western blot analysis confirmed that AlgU controlsrsmAexpression in both a laboratory strain and a clinical isolate. RNase protection assays confirmed the presence of tworsmAtranscripts and suggest that RpoS and AlgU regulatersmAexpression. Due to the increased amounts of RsmA inmucAmutant strains, a translational leader fusion of the RsmA target,tssA1, was constructed and tested inmucA,algU,retS,gacA, andrsmAmutant backgrounds to examine posttranscriptional activity. From these studies, we determined that RsmA is active inmucA22mutants, suggesting a role for RsmA inmucAmutant strains. Taken together, we have demonstrated that AlgU controlsrsmAtranscription and is responsible for RsmA activity inmucAmutant strains. We propose that RsmA is active inP. aeruginosamucAmutant strains and that RsmA also plays a role in chronic infections.IMPORTANCEP. aeruginosacauses severe infections in immunocompromised patients. The posttranscriptional regulator RsmA is known to control virulence and biofilm formation. We identify a newrsmApromoter and determine that AlgU is important in the control ofrsmAexpression. MutantmucAstrains that are considered mucoid were used to confirm increasedrsmAexpression from the AlgU promoter. We demonstrate, for the first time, that there is RsmA activity in mucoidP. aeruginosastrains. Our work suggests that RsmA may play a role during chronic infections as well as acute infections.

scholarly journals Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

2012 ◽  
Vol 78 (15) ◽  
pp. 5060-5069 ◽  
Author(s):  
Morten T. Rybtke ◽  
Bradley R. Borlee ◽  
Keiji Murakami ◽  
Yasuhiko Irie ◽  
Morten Hentzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Subsequent Development ◽  
Cellular Level ◽  
Host Immune System ◽  
Chronic Infections ◽  
Content Type ◽  
Genes Encoding ◽  
Green Fluorescent

ABSTRACTThe increased tolerance toward the host immune system and antibiotics displayed by biofilm-formingPseudomonas aeruginosaand other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsivecdrApromoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP inP. aeruginosastrains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment ofP. aeruginosawith the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology ofP. aeruginosa.

scholarly journals Pseudomonas aeruginosa Requires the DNA-Specific Endonuclease EndA To Degrade Extracellular Genomic DNA To Disperse from the Biofilm

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Kathryn E. Cherny ◽  
Karin Sauer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Genomic Dna ◽  
Early Stage ◽  
Content Type ◽  
Biofilm Dispersion ◽  
Biofilm Structure ◽  
First Time ◽  
Biofilm Matrix ◽  
Dispersed Cells

ABSTRACT The dispersion of biofilms is an active process resulting in the release of planktonic cells from the biofilm structure. While much is known about the process of dispersion cue perception and the subsequent modulation of the c-di-GMP pool, little is known about subsequent events resulting in the release of cells from the biofilm. Given that dispersion coincides with void formation and an overall erosion of the biofilm structure, we asked whether dispersion involves degradation of the biofilm matrix. Here, we focused on extracellular genomic DNA (eDNA) due to its almost universal presence in the matrix of biofilm-forming species. We identified two probable nucleases, endA and eddB, and eddA encoding a phosphatase that were significantly increased in transcript abundance in dispersed cells. However, only inactivation of endA but not eddA or eddB impaired dispersion by Pseudomonas aeruginosa biofilms in response to glutamate and nitric oxide (NO). Heterologously produced EndA was found to be secreted and active in degrading genomic DNA. While endA inactivation had little effect on biofilm formation and the presence of eDNA in biofilms, eDNA degradation upon induction of dispersion was impaired. In contrast, induction of endA expression coincided with eDNA degradation and resulted in biofilm dispersion. Thus, released cells demonstrated a hyperattaching phenotype but remained as resistant to tobramycin as biofilm cells from which they egress, indicating EndA-dispersed cells adopted some but not all of the phenotypes associated with dispersed cells. Our findings indicate for the first time a role of DNase EndA in dispersion and suggest weakening of the biofilm matrix is a requisite for biofilm dispersion. IMPORTANCE The finding that exposure to DNase I impairs biofilm formation or leads to the dispersal of early stage biofilms has led to the realization of extracellular genomic DNA (eDNA) as a structural component of the biofilm matrix. However, little is known about the contribution of intrinsic DNases to the weakening of the biofilm matrix and dispersion of established biofilms. Here, we demonstrate for the first time that nucleases are induced in dispersed Pseudomonas aeruginosa cells and are essential to the dispersion response and that degradation of matrix eDNA by endogenously produced/secreted EndA is required for P. aeruginosa biofilm dispersion. Our findings suggest that dispersing cells mediate their active release from the biofilm matrix via the induction of nucleases.

scholarly journals Pushing beyond the Envelope: the Potential Roles of OprF inPseudomonas aeruginosaBiofilm Formation and Pathogenicity

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Erin K. Cassin ◽  
Boo Shan Tseng
Keyword(s):  
Extracellular Matrix ◽  
Outer Membrane ◽  
Biofilm Formation ◽  
Matrix Protein ◽  
Cell Envelope ◽  
Outer Membrane Vesicles ◽  
Extracellular Dna ◽  
Future Research ◽  
Content Type ◽  
Biofilm Matrix

ABSTRACTThe ability ofPseudomonas aeruginosato form biofilms, which are communities of cells encased in a self-produced extracellular matrix, protects the cells from antibiotics and the host immune response. While some biofilm matrix components, such as exopolysaccharides and extracellular DNA, are relatively well characterized, the extracellular matrix proteins remain understudied. Multiple proteomic analyses of theP. aeruginosasoluble biofilm matrix and outer membrane vesicles, which are a component of the matrix, have identified OprF as an abundant matrix protein. To date, the few reports on the effects ofoprFmutations on biofilm formation are conflicting, and little is known about the potential role of OprF in the biofilm matrix. The majority of OprF studies focus on the protein as a cell-associated porin. As a component of the outer membrane, OprF assumes dual conformations and is involved in solute transport, as well as cell envelope integrity. Here, we review the current literature on OprF inP. aeruginosa, discussing how the structure and function of the cell-associated and matrix-associated protein may affect biofilm formation and pathogenesis in order to inform future research on this understudied matrix protein.

scholarly journals Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Keehoon Lee ◽  
Kang-Mu Lee ◽  
Donggeun Kim ◽  
Sang Sun Yoon
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enterococcus Faecalis ◽  
Synergistic Effects ◽  
Bacterial Species ◽  
Extracellular Dna ◽  
Infection Model ◽  
Surgical Removal ◽  
Chronic Infections ◽  
Content Type ◽  
Biofilm Matrix

ABSTRACT Biofilms are microbial communities that inhabit various surfaces and are surrounded by extracellular matrices (ECMs). Clinical microbiologists have shown that the majority of chronic infections are caused by biofilms, following the introduction of the first biofilm infection model by J. W. Costerton and colleagues (J. Lam, R. Chan, K. Lam, and J. W. Costerton, Infect Immun 28:546–556, 1980). However, treatments for chronic biofilm infections are still limited to surgical removal of the infected sites. Pseudomonas aeruginosa and Enterococcus faecalis are two frequently identified bacterial species in biofilm infections; nevertheless, the interactions between these two species, especially during biofilm growth, are not clearly understood. In this study, we observed phenotypic changes in a dual-species biofilm of P. aeruginosa and E. faecalis, including a dramatic increase in biofilm matrix thickness. For clear elucidation of the spatial distribution of the dual-species biofilm, P. aeruginosa and E. faecalis were labeled with red and green fluorescence, respectively. E. faecalis was located at the lower part of the dual-species biofilm, while P. aeruginosa developed a structured biofilm on the upper part. Mutants with altered exopolysaccharide (EPS) productions were constructed in order to determine the molecular basis for the synergistic effect of the dual-species biofilm. Increased biofilm matrix thickness was associated with EPSs, not extracellular DNA. In particular, Pel and Psl contributed to interspecies and intraspecies interactions, respectively, in the dual-species P. aeruginosa and E. faecalis biofilm. Accordingly, targeting Pel and Psl might be an effective part of eradicating P. aeruginosa polymicrobial biofilms. IMPORTANCE Chronic infection is a serious problem in the medical field. Scientists have observed that chronic infections are closely associated with biofilms, and the vast majority of infection-causing biofilms are polymicrobial. Many studies have reported that microbes in polymicrobial biofilms interact with each other and that the bacterial interactions result in elevated virulence, in terms of factors, such as infectivity and antibiotic resistance. Pseudomonas aeruginosa and Enterococcus faecalis are frequently isolated pathogens in chronic biofilm infections. Nevertheless, while both bacteria are known to be agents of numerous nosocomial infections and can cause serious diseases, interactions between the bacteria in biofilms have rarely been examined. In this investigation, we aimed to characterize P. aeruginosa and E. faecalis dual-species biofilms and to determine the molecular factors that cause synergistic effects, especially on the matrix thickening of the biofilm. We suspect that our findings will contribute to the development of more efficient methods for eradicating polymicrobial biofilm infections.

scholarly journals Pseudomonas aeruginosa Exopolysaccharide Psl Promotes Resistance to the Biofilm Inhibitor Polysorbate 80

2012 ◽  
Vol 56 (8) ◽  
pp. 4112-4122 ◽  
Author(s):  
Michael E. Zegans ◽  
Daniel Wozniak ◽  
Edward Griffin ◽  
Christine M. Toutain-Kidd ◽  
John H. Hammond ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nonionic Surfactant ◽  
Biofilm Formation ◽  
Laboratory Strain ◽  
Polysorbate 80 ◽  
Biosynthetic Pathways ◽  
Human Tissues ◽  
Biofilm Inhibition ◽  
Content Type ◽  
Biofilm Inhibitor

ABSTRACTPolysorbate 80 (PS80) is a nonionic surfactant and detergent that inhibits biofilm formation byPseudomonas aeruginosaat concentrations as low as 0.001% and is well tolerated in human tissues. However, certain clinical and laboratory strains (PAO1) ofP. aeruginosaare able to form biofilms in the presence of PS80. To better understand this resistance, we performed transposon mutagenesis with a PS80-resistant clinical isolate, PA738. This revealed that mutation ofalgCrendered PA738 sensitive to PS80 biofilm inhibition. AlgC contributes to the biosynthesis of the exopolysaccharides Psl and alginate, as well as lipopolysaccharide and rhamnolipid. Analysis of mutations downstream of AlgC in these biosynthetic pathways established that disruption of thepsloperon was sufficient to render the PA738 and PAO1 strains sensitive to PS80-mediated biofilm inhibition. Increased levels of Psl production in the presence of arabinose in a strain with an arabinose-induciblepslpromoter were correlated with increased biofilm formation in PS80. InP. aeruginosastrains MJK8 and ZK2870, known to produce both Pel and Psl, disruption of genes in thepslbut not thepeloperon conferred susceptibility to PS80-mediated biofilm inhibition. The laboratory strain PA14 does not produce Psl and does not form biofilms in PS80. However, when PA14 was transformed with a cosmid containing thepsloperon, it formed biofilms in the presence of PS80. Taken together, these data suggest that production of the exopolysaccharide Psl byP. aeruginosapromotes resistance to the biofilm inhibitor PS80.

scholarly journals Genome Sequences of Two Pseudomonas aeruginosa Isolates with Defects in Type III Secretion System Gene Expression from a Chronic Ankle Wound Infection

2021 ◽  
Author(s):  
Sardar Karash ◽  
Robert Nordell ◽  
Egon A. Ozer ◽  
Timothy L. Yahr
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Biofilm Formation ◽  
Secretion System ◽  
Chronic Infections ◽  
Genome Sequences ◽  
Content Type ◽  
Chronic Colonization ◽  
Host Tolerance

A common feature of microorganisms that cause chronic infections is a stealthy lifestyle that promotes immune avoidance and host tolerance. During chronic colonization of cystic fibrosis (CF) patients, Pseudomonas aeruginosa acquires numerous adaptations that include reduced expression of some factors, such as motility, O antigen, and the T3SS, and increased expression of other traits, such as biofilm formation.

scholarly journals 2,3-Dihydroxybenzoic Acid-Containing Nanofiber Wound Dressings Inhibit Biofilm Formation by Pseudomonas aeruginosa

2014 ◽  
Vol 58 (4) ◽  
pp. 2098-2104 ◽  
Author(s):  
Jayesh J. Ahire ◽  
Leon M. T. Dicks
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Iron Chelator ◽  
Wound Dressings ◽  
Tissue Destruction ◽  
Dihydroxybenzoic Acid ◽  
Chronic Infections ◽  
Content Type ◽  
Cell Numbers ◽  
Time Required

ABSTRACTPseudomonas aeruginosaforms biofilms in wounds, which often leads to chronic infections that are difficult to treat with antibiotics. Free iron enhances biofilm formation, delays wound healing, and may even be responsible for persistent inflammation, increased connective tissue destruction, and lipid peroxidation. Exposure ofP. aeruginosaXen 5 to the iron chelator 2,3-dihydroxybenzoic acid (DHBA), electrospun into a nanofiber blend of poly(d,l-lactide) (PDLLA) and poly(ethylene oxide) (PEO), referred to as DF, for 8 h decreased biofilm formation by approximately 75%. This was shown by a drastic decline in cell numbers, from 7.1 log10CFU/ml to 4.8 log10CFU/ml when biofilms were exposed to DF in the presence of 2.0 mM FeCl36H2O. A similar decline in cell numbers was recorded in the presence of 3.0 mM FeCl36H2O and DF. The cells were more mobile in the presence of DHBA, supporting the observation of less biofilm formation at lower iron concentrations. DHBA at MIC levels (1.5 mg/ml) inhibited the growth of strain Xen 5 for at least 24 h. Our findings indicate that DHBA electrospun into nanofibers inhibits cell growth for at least 4 h, which is equivalent to the time required for all DHBA to diffuse from DF. This is the first indication that DF can be developed into a wound dressing to treat topical infections caused byP. aeruginosa.

scholarly journals Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

2015 ◽  
Vol 197 (17) ◽  
pp. 2810-2820 ◽  
Author(s):  
John H. Hammond ◽  
Emily F. Dolben ◽  
T. Jarrod Smith ◽  
Sabin Bhuju ◽  
Deborah A. Hogan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Quorum Sensing ◽  
Cellular Response ◽  
Iron Acquisition ◽  
Laboratory Strain ◽  
Interspecies Interactions ◽  
Low Oxygen ◽  
Chronic Infections ◽  
Content Type

ABSTRACTInPseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanrderivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanrstrains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections.IMPORTANCEPseudomonas aeruginosacauses acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients.P. aeruginosauses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS,lasR, are frequently observed in clinical isolates. We demonstrated that the regulon attributed to Anr, an oxygen-sensitive transcription factor, was more highly expressed inlasRmutants. Furthermore, we show that Anr regulates the production of several different secreted factors inlasRmutants. These data demonstrate the importance of Anr in naturally occurring quorum sensing mutants in the context of chronic infections.

scholarly journals Involvement of Stress-Related GenespolBandPA14_46880in Biofilm Formation of Pseudomonas aeruginosa

2014 ◽  
Vol 82 (11) ◽  
pp. 4746-4757 ◽  
Author(s):  
Sahar A. Alshalchi ◽  
Gregory G. Anderson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Acute Infection ◽  
Regulatory Gene ◽  
Chronic Infections ◽  
Content Type ◽  
Abiotic Surfaces ◽  
Mutant Strains ◽  
In The Wild ◽  
Airway Cells

ABSTRACTChronic infections ofPseudomonas aeruginosaare generally established through production of biofilm. During biofilm formation, production of an extracellular matrix and establishment of a distinct bacterial phenotype make these infections difficult to eradicate. However, biofilm studies have been hampered by the fact that most assays utilize nonliving surfaces as biofilm attachment substrates. In an attempt to better understand the mechanisms behindP. aeruginosabiofilm formation, we performed a genetic screen to identify novel factors involved in biofilm formation on biotic and abiotic surfaces. We found that deletion of genespolBandPA14_46880reduced biofilm formation significantly compared to that in the wild-type strain PA14 in an abiotic biofilm system. In a biotic biofilm model, wherein biofilms form on cultured airway cells, the ΔpolBand ΔPA14_46880strains showed increased cytotoxic killing of the airway cells independent of the total number of bacteria bound. Notably, deletion mutant strains were more resistant to ciprofloxacin treatment. This phenotype was linked to decreased expression ofalgR, an alginate transcriptional regulatory gene, under ciprofloxacin pressure. Moreover, we found that pyocyanin production was increased in planktonic cells of mutant strains. These results indicate that inactivation ofpolBandPA14_46880may inhibit transition ofP. aeruginosafrom a more acute infection lifestyle to the biofilm phenotype. Future investigation of these genes may lead to a better understanding ofP. aeruginosabiofilm formation and chronic biofilm infections.

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