scholarly journals Modulation of Pseudomonas aeruginosa Biofilm Dispersal by a Cyclic-Di-GMP Phosphodiesterase with a Putative Hypoxia-Sensing Domain

2010 ◽  
Vol 76 (24) ◽  
pp. 8160-8173 ◽  
Author(s):  
Shuwen An ◽  
Ji'en Wu ◽  
Lian-Hui Zhang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Regulatory Protein ◽  
Phosphodiesterase Activity ◽  
Low Oxygen ◽  
Ggdef Domain ◽  
Biofilm Dispersal ◽  
Biofilm Development

ABSTRACT Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.

scholarly journals Rhamnose Binding Protein as an Anti-Bacterial Agent—Targeting Biofilm of Pseudomonas aeruginosa

Marine Drugs ◽  
2019 ◽  
Vol 17 (6) ◽  
pp. 355 ◽  
Author(s):  
Tse-Kai Fu ◽  
Sim-Kun Ng ◽  
Yi-En Chen ◽  
Yuan-Chuan Lee ◽  
Fruzsina Demeter ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Lung Epithelial Cells ◽  
Bacterial Agent ◽  
Lung Epithelial ◽  
Biofilm Development

More than 80% of infectious bacteria form biofilm, which is a bacterial cell community surrounded by secreted polysaccharides, proteins and glycolipids. Such bacterial superstructure increases resistance to antimicrobials and host defenses. Thus, to control these biofilm-forming pathogenic bacteria requires antimicrobial agents with novel mechanisms or properties. Pseudomonas aeruginosa, a Gram-negative opportunistic nosocomial pathogen, is a model strain to study biofilm development and correlation between biofilm formation and infection. In this study, a recombinant hemolymph plasma lectin (rHPLOE) cloned from Taiwanese Tachypleus tridentatus was expressed in an Escherichia coli system. This rHPLOE was shown to have the following properties: (1) Binding to P. aeruginosa PA14 biofilm through a unique molecular interaction with rhamnose-containing moieties on bacteria, leading to reduction of extracellular di-rhamnolipid (a biofilm regulator); (2) decreasing downstream quorum sensing factors, and inhibiting biofilm formation; (3) dispersing the mature biofilm of P. aeruginosa PA14 to improve the efficacies of antibiotics; (4) reducing P. aeruginosa PA14 cytotoxicity to human lung epithelial cells in vitro and (5) inhibiting P. aeruginosa PA14 infection of zebrafish embryos in vivo. Taken together, rHPLOE serves as an anti-biofilm agent with a novel mechanism of recognizing rhamnose moieties in lipopolysaccharides, di-rhamnolipid and structural polysaccharides (Psl) in biofilms. Thus rHPLOE links glycan-recognition to novel anti-biofilm strategies against pathogenic bacteria.

scholarly journals Use of In-Biofilm Expression Technology To Identify Genes Involved in Pseudomonas aeruginosa Biofilm Development

2003 ◽  
Vol 185 (9) ◽  
pp. 2700-2710 ◽  
Author(s):  
Antonio Finelli ◽  
Claude V. Gallant ◽  
Keith Jarvi ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Three Dimensional ◽  
Streptomyces Griseus ◽  
Form Complex ◽  
Technology System ◽  
Planktonic Growth ◽  
Biofilm Development

ABSTRACT Mature Pseudomonas aeruginosa biofilms form complex three-dimensional architecture and are tolerant of antibiotics and other antimicrobial compounds. In this work, an in vivo expression technology system, originally designed to study virulence-associated genes in complex mammalian environments, was used to identify genes up-regulated in P. aeruginosa grown to a mature (5-day) biofilm. Five unique cloned promoters unable to promote in vitro growth in the absence of purines after recovery from the biofilm environment were identified. The open reading frames downstream of the cloned promoter regions were identified, and knockout mutants were generated. Insertional mutation of PA5065, a homologue of Escherichia coli ubiB, was lethal, while inactivation of PA0240 (a porin homologue), PA3710 (a putative alcohol dehydrogenase), and PA3782 (a homologue of the Streptomyces griseus developmental regulator adpA) had no effect on planktonic growth but caused defects in biofilm formation in static and flowing systems. In competition experiments, mutants demonstrated reduced fitness compared with the parent strain, comprising less than 0.0001% of total biofilm cells after 5 days. Therefore, using in-biofilm expression technology, we have identified novel genes that do not affect planktonic growth but are important for biofilm formation, development, and fitness.

scholarly journals BifA, a Cyclic-Di-GMP Phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility by Pseudomonas aeruginosa PA14

2007 ◽  
Vol 189 (22) ◽  
pp. 8165-8178 ◽  
Author(s):  
Sherry L. Kuchma ◽  
Kimberly M. Brothers ◽  
Judith H. Merritt ◽  
Nicole T. Liberati ◽  
Frederick M. Ausubel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Intracellular Signaling ◽  
Biofilm Formation ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Phosphodiesterase Activity ◽  
Swarming Motility ◽  
Ggdef Domain

ABSTRACT The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifA mutant but does not contribute to the observed swarming defect. The ΔbifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.

scholarly journals Staphylococcus aureus Serves as an Iron Source for Pseudomonas aeruginosa during In Vivo Coculture

2005 ◽  
Vol 187 (2) ◽  
pp. 554-566 ◽  
Author(s):  
Lauren M. Mashburn ◽  
Amy M. Jett ◽  
Darrin R. Akins ◽  
Marvin Whiteley
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Iron Acquisition ◽  
Low Oxygen ◽  
Dialysis Membrane ◽  
Opportunistic Human Pathogen ◽  
The Impact

ABSTRACT Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo compared to in vitro controls. Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in this environment. We propose a model where P. aeruginosa lyses S. aureus and uses released iron for growth in low-iron environments.

scholarly journals Early Biofilm Formation on UV Light Activated Nanoporous TiO2SurfacesIn Vivo

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Nagat Areid ◽  
Eva Söderling ◽  
Johanna Tanner ◽  
Ilkka Kangasniemi ◽  
Timo O. Närhi
Keyword(s):  
Titanium Alloy ◽  
Biofilm Formation ◽  
Uv Light ◽  
Antibacterial Properties ◽  
Mutans Streptococci ◽  
Human Gingival Fibroblast ◽  
Gingival Fibroblast ◽  
Biofilm Development

Purpose. To explore earlyS. mutansbiofilm formation on hydrothermally induced nanoporous TiO2surfacesin vivoand to examine the effect of UV light activation on the biofilm development.Materials and Methods. Ti-6Al-4V titanium alloy discs (n = 40) were divided into four groups with different surface treatments: noncoated titanium alloy (NC); UV treated noncoated titanium alloy (UVNC); hydrothermally induced TiO2coating (HT); and UV treated titanium alloy with hydrothermally induced TiO2coating (UVHT).In vivoplaque formation was studied in 10 healthy, nonsmoking adult volunteers. Titanium discs were randomly distributed among the maxillary first and second molars. UV treatment was administered for 60 min immediately before attaching the discs in subjects’ molars. Plaque samples were collected 24h after the attachment of the specimens. Mutans streptococci (MS), non-mutans streptococci, and total facultative bacteria were cultured, and colonies were counted.Results. The plaque samples of NC (NC + UVNC) surfaces showed over 2 times more oftenS. mutanswhen compared to TiO2surfaces (HT + UVHT), with the number of colonized surfaces equal to 7 and 3, respectively.Conclusion. Thisin vivostudy suggested that HT TiO2surfaces, which we earlier showed to improve blood coagulation and encourage human gingival fibroblast attachmentin vitro, do not enhance salivary microbial (mostly mutans streptococci) adhesion and initial biofilm formation when compared with noncoated titanium alloy. UV light treatment provided Ti-6Al-4V surfaces with antibacterial properties and showed a trend towards less biofilm formation when compared with non-UV treated titanium surfaces.

Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions

2009 ◽  
Vol 58 (6) ◽  
pp. 765-773 ◽  
Author(s):  
Che Y. O'May ◽  
Kevin Sanderson ◽  
Louise F. Roddam ◽  
Sylvia M. Kirov ◽  
David W. Reid
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Low Oxygen ◽  
Anaerobic Conditions ◽  
Chronic Infections ◽  
Strain Pao1 ◽  
Biofilm Inhibition ◽  
Oxygen Conditions ◽  
Biofilm Development ◽  
Chelating Compounds

The success of Pseudomonas aeruginosa in cystic fibrosis (CF) and other chronic infections is largely attributed to its ability to grow in antibiotic-resistant biofilm communities. This study investigated the effects of limiting iron levels as a strategy for preventing/disrupting P. aeruginosa biofilms. A range of synthetic and naturally occurring iron-chelating agents were examined. Biofilm development by P. aeruginosa strain PAO1 and CF sputum isolates from chronically infected individuals was significantly decreased by iron removal under aerobic atmospheres. CF strains formed poor biofilms under anaerobic conditions. Strain PAO1 was also tested under anaerobic conditions. Biofilm formation by this model strain was almost totally prevented by several of the chelators tested. The ability of synthetic chelators to impair biofilm formation could be reversed by iron addition to cultures, providing evidence that these effective chelating compounds functioned by directly reducing availability of iron to P. aeruginosa. In contrast, the biological chelator lactoferrin demonstrated enhanced anti-biofilm effects as iron supplementation increased. Hence biofilm inhibition by lactoferrin appeared to occur through more complex mechanisms to those of the synthetic chelators. Overall, our results demonstrate the importance of iron availability to biofilms and that iron chelators have potential as adjunct therapies for preventing biofilm development, especially under low oxygen conditions such as encountered in the chronically infected CF lung.

scholarly journals Function of BriC Peptide in the Pneumococcal Competence and Virulence Portfolio

2018 ◽  
Author(s):  
Surya D. Aggarwal ◽  
Rory Eutsey ◽  
Jacob West-Roberts ◽  
Arnau Domenech ◽  
Wenjie Xu ◽  
...  
Keyword(s):  
Murine Model ◽  
Biofilm Formation ◽  
Opportunistic Pathogen ◽  
Nasopharyngeal Colonization ◽  
Point Of Entry ◽  
Abiotic Surfaces ◽  
Biofilm Development

AbstractStreptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

scholarly journals Differential Gene Expression Patterns ofYersinia pestisandYersinia pseudotuberculosisduring Infection and Biofilm Formation in the Flea Digestive Tract

mSystems ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Iman Chouikha ◽  
Daniel E. Sturdevant ◽  
Clayton Jarrett ◽  
Yi-Cheng Sun ◽  
B. Joseph Hinnebusch
Keyword(s):  
Digestive Tract ◽  
Biofilm Formation ◽  
Yersinia Pseudotuberculosis ◽  
Expression Patterns ◽  
Type Vi Secretion System ◽  
Genetic Changes ◽  
Content Type ◽  
Biofilm Development

ABSTRACTYersinia pestis, the etiologic agent of plague, emerged as a fleaborne pathogen only within the last 6,000 years. Just five simple genetic changes in theYersinia pseudotuberculosisprogenitor, which served to eliminate toxicity to fleas and to enhance survival and biofilm formation in the flea digestive tract, were key to the transition to the arthropodborne transmission route. To gain a deeper understanding of the genetic basis for the development of a transmissible biofilm infection in the flea foregut, we evaluated additional gene differences and performedin vivotranscriptional profiling ofY. pestis, aY. pseudotuberculosiswild-type strain (unable to form biofilm in the flea foregut), and aY. pseudotuberculosismutant strain (able to produce foregut-blocking biofilm in fleas) recovered from fleas 1 day and 14 days after an infectious blood meal. Surprisingly, theY. pseudotuberculosismutations that increased c-di-GMP levels and enabled biofilm development in the flea did not change the expression levels of thehmsgenes responsible for the synthesis and export of the extracellular polysaccharide matrix required for mature biofilm formation. TheY. pseudotuberculosismutant uniquely expressed much higher levels ofYersiniatype VI secretion system 4 (T6SS-4) in the flea, and this locus was required for flea blockage byY. pseudotuberculosisbut not for blockage byY. pestis. Significant differences between the two species in expression of several metabolism genes, the Psa fimbrial genes, quorum sensing-related genes, transcription regulation genes, and stress response genes were evident during flea infection.IMPORTANCEY. pestisemerged as a highly virulent, arthropod-transmitted pathogen on the basis of relatively few and discrete genetic changes fromY. pseudotuberculosis. Parallel comparisons of thein vitroandin vivotranscriptomes ofY. pestisand twoY. pseudotuberculosisvariants that produce a nontransmissible infection and a transmissible infection of the flea vector, respectively, provided insights into howY. pestishas adapted to life in its flea vector and point to evolutionary changes in the regulation of metabolic and biofilm development pathways in these two closely related species.

scholarly journals Emergent Bacteria in Cystic Fibrosis:In VitroBiofilm Formation and Resilience under Variable Oxygen Conditions

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Susana P. Lopes ◽  
Nuno F. Azevedo ◽  
Maria O. Pereira
Keyword(s):  
Bacterial Species ◽  
Low Oxygen ◽  
Antibiotic Susceptibilities ◽  
Oxygen Conditions ◽  
Mode Of Life ◽  
Lung Infections ◽  
Biofilm Development ◽  
The Impact

Concurrent to conventional bacterial pathogens, unusual microbes are emerging from cystic fibrosis (CF) airways. Nonetheless, little is known about the contribution of these newly microbes to the resilience of CF-associated biofilms, particularly under variable-oxygen concentrations that are known to occurin vivoin the mucus of CF patients. Two CF-emergent bacterial species,Inquilinus limosusandDolosigranulum pigrum, and the major pathogenPseudomonas aeruginosawere studied in terms of biofilm development and antibiotic susceptibilities underin vitroatmospheres with different oxygen availabilities. All species were able to developin vitrobiofilms under different oxygen-available environments, withD. pigrumaccumulating high amounts of biomass and respiratory activities. When established, biofilms were of difficult eradication, with antibiotics losing their effectiveness in comparison with the corresponding planktonic populations. Surprisingly, biofilms of each emergent organism displayed multidrug resistance under aerobic environments, enduring even in low-oxygen atmospheres. This study suggests a potential prospect on the impact of nonconventional organismsI. limosusandD. pigrumon CF lung infections, demonstrating capacity to adapt to biofilm mode of life under restricted-oxygen atmospheres resembling CF airways, which may ultimately endanger the efficacy of currently used antibiotic regimens.

Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Anna Scherhag ◽  
Martina Rüger ◽  
Katrin Gerbracht ◽  
Jaqueline Rehner ◽  
Susanne Zehner ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Model Organism ◽  
Functional Complementation ◽  
Full Length ◽  
Protein Activity ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Ggdef Domain

<p>The molecule c-di-GMP is a bacterial second messenger that controls various processes such as motility or biofilm formation in bacteria [1]. To synthesize and degrade c-di-GMP, enzymes called diguanylate cyclases (DGC) containing a GGDEF-domain and phosphodiesterases (PDE) containing an EAL-domain or HD-GYP-domain are important [1, 2].<em> Pseudomonas aeruginosa</em>, a model organism for biofilm formation and dispersion, encodes for 18 GGDEF, 5 EAL, 16 GGDEF / EAL, and 3 HD-GYP-domain-containing proteins [3].<br />One of the GGDEF / EAL-containing proteins is NbdA. This protein also harbors an N-terminal membrane anchored MHYT-domain, that is predicted to be a sensor for NO, CO or O<sub>2</sub> [4]. In this work, recombinant and affinity purified NbdA was tested for its PDE activity. Three different methods were used to measure the PDE activity of NbdA: a bis-pNPP-assay in which the conversion of the pseudosubstrate bis-pNPP into p-nitrophenol was detected spectroscopically, an HPLC-analysis of an enzymatic assay with the native substrate c-di-GMP, and a MANT-c-di-GMP-assay in which a fluorescently labeled form of the presumed substrate c-di-GMP was utilized.<br />To establish these methods, the two known phosphodiesterases, PdeH from <em>Escherichia coli</em> [5] and RocR from <em>P. aeruginosa</em> [6], were also produced and tested. Subsequently, three variants of NbdA were investigated: the full-length version and two truncated versions of the protein. Activity was further assessed using functional complementation of an <em>E. coli</em> phosphodiesterase deficient strain with full-length and truncated NbdA variants confirming PDE activity <em>in vivo</em>.</p> <p> </p> <p> </p> <p>[1] Hengge, R. (2009) Nature Rev. Microbiol. 7: 263-273.</p> <p>[2] Römling, U., Gomelsky, M., Galperin, M.Y. (2005). Mol. Microbiol. 57: 629–639.</p> <p>[3] Valentini, M., Filloux, A. (2016). J. Biol. Chem. 291: 12547–12555.</p> <p>[4] Galperin, M.Y., Gaidenko, T.A., Mulkidjanian, A.Y., Nakano, M., und Price, C.W. (2001). FEMS Microbiol. Lett. 205, 17–23.</p> <p>[5] Pesavento, C., Becker, G., Sommerfeldt, N., Possling, A., Tschowri, N., Mehlis, A., Hengge, R. (2008). Genes Dev. 22: 2434–2446.</p> <p>[6] Chen et al. (2012) Chen, M.W., Kotaka, M., Vonrhein, C., Bricogne, G., Rao, F., Chuah, M.L.C., Svergun, D., Schneider, G., Liang, Z.-X., Lescar, J.  (2012). Signaling. J. Bacteriol. 194: 4837–4846</p> <p> </p>

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