scholarly journals Role of Alginate and Its O Acetylation in Formation of Pseudomonas aeruginosa Microcolonies and Biofilms

2001 ◽  
Vol 183 (3) ◽  
pp. 1047-1057 ◽  
Author(s):  
David E. Nivens ◽  
Dennis E. Ohman ◽  
Jessica Williams ◽  
Michael J. Franklin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Three Dimensional ◽  
Attenuated Total Reflection ◽  
Confocal Laser ◽  
Infrared Spectrometry ◽  
Ir Radiation ◽  
Mutant Strains ◽  
Ft Ir ◽  
Biofilm Development

ABSTRACT Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of Pseudomonas aeruginosa on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm−1. All strains produced biofilms that absorbed IR radiation near 1,650 cm−1 (amide I), 1,550 cm−1 (amide II), 1,240 cm−1 (PO stretching, C—O—C stretching, and/or amide III vibrations), 1,100 to 1,000 cm−1 (C—OH and P—O stretching) 1,450 cm−1, and 1,400 cm−1. The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm−1 (C—OH stretching of alginate) and 1,250 cm−1 (C—O stretching of the O-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 μm, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 μm in depth. In contrast, FRD1 produced microcolonies that were approximately 40 μm in depth. An algJ mutant strain that produced alginate lacking O-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the algJ mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for P. aeruginosa biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in P. aeruginosabiofilm architecture.

A Comparison of Pseudomonas Aeruginosa Biofilm Development on ZnSe and TiO2 Using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy

2009 ◽  
Vol 63 (9) ◽  
pp. 1000-1007 ◽  
Author(s):  
Jonathan W. D. Comeau ◽  
Judith Pink ◽  
Evan Bezanson ◽  
Colin D. Douglas ◽  
David Pink ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fourier Transform ◽  
Fourier Transform Infrared ◽  
Total Reflection ◽  
Attenuated Total Reflection ◽  
Lag Phase ◽  
Ft Ir ◽  
Coated Surface ◽  
Coated Surfaces ◽  
Biofilm Development

The growth of Pseudomonas aeruginosa PAO1 biofilms on ZnSe internal reflection elements (IREs) was compared with their growth on TiO2-coated ZnSe over several days using attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy. The effect of the TiO2 coating on the IR spectra of reference compounds and cell suspensions was determined to aid in the interpretation of the data. The presence of TiO2 on the surface of a ZnSe IRE tripled the size of the amide II peak and facilitated the detection of pyoverdin production due to its increased adsorption on the coated surface. A 50% increase in the length of the lag phase was observed for PAO1 growth on TiO2-coated surfaces as compared to growth on ZnSe. Biofilms on both surfaces exhibited a growth maximum for all components, followed by restructuring at the surface characterized by a decrease in the signal. The composition of biofilms grown on TiO2 was relatively constant after the restructuring phase, while the extracellular polymeric substance (EPS) component of the biofilms grown on ZnSe gradually increased. The peak due to the carbohydrate component of EPS was much larger in the spectra of biofilms than in those of planktonic cells. The increase of the pyoverdin signal over time in the spectra of the biofilms on TiO2 closely followed the overall increase in biomass. However, no signal from pyoverdin was detected in the presence of ferric ions.

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 Mucin inhibitsPseudomonas aeruginosabiofilm formation by significantly enhancing twitching motility

2014 ◽  
Vol 60 (3) ◽  
pp. 155-166 ◽  
Author(s):  
Cecily L. Haley ◽  
Cassandra Kruczek ◽  
Uzma Qaisar ◽  
Jane A. Colmer-Hamood ◽  
Abdul N. Hamood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iv Pili ◽  
Dependent Manner ◽  
Twitching Motility ◽  
Strain Pao1 ◽  
Concentration Dependent Manner ◽  
Dependent Effect ◽  
Type Iv ◽  
Biofilm Development

In Pseudomonas aeruginosa, type IV pili (TFP)-dependent twitching motility is required for development of surface-attached biofilm (SABF), yet excessive twitching motility is detrimental once SABF is established. In this study, we show that mucin significantly enhanced twitching motility and decreased SABF formation in strain PAO1 and other P. aeruginosa strains in a concentration-dependent manner. Mucin also disrupted partially established SABF. Our analyses revealed that mucin increased the amount of surface pilin and enhanced transcription of the pilin structural gene pilA. Mucin failed to enhance twitching motility in P. aeruginosa mutants defective in genes within the pilin biogenesis operons pilGHI/pilJK-chpA-E. Furthermore, mucin did not enhance twitching motility nor reduce biofilm development by chelating iron. We also examined the role of the virulence factor regulator Vfr in the effect of mucin. In the presence or absence of mucin, PAOΔvfr produced a significantly reduced SABF. However, mucin partially complemented the twitching motility defect of PAOΔvfr. These results suggest that mucin interferes with SABF formation at specific concentrations by enhancing TFP synthesis and twitching motility, that this effect, which is iron-independent, requires functional Vfr, and only part of the Vfr-dependent effect of mucin on SABF development occurs through twitching motility.

Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation

2004 ◽  
Vol 53 (7) ◽  
pp. 679-690 ◽  
Author(s):  
Andres Plata Stapper ◽  
Giri Narasimhan ◽  
Dennis E. Ohman ◽  
Johnny Barakat ◽  
Morten Hentzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Cell System ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Alginate Production ◽  
Green Fluorescent ◽  
Biofilm Development

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by Pseudomonas aeruginosa strain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg+ PAOmucA22 and Alg− PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (bip) and Community Statistics (comstat) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.

scholarly journals Cell Death in Pseudomonas aeruginosa Biofilm Development

2003 ◽  
Vol 185 (15) ◽  
pp. 4585-4592 ◽  
Author(s):  
Jeremy S. Webb ◽  
Lyndal S. Thompson ◽  
Sally James ◽  
Tim Charlton ◽  
Tim Tolker-Nielsen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Spatial Organization ◽  
Normal Component ◽  
Three Dimensional ◽  
Multicellular Development ◽  
Flow Cells ◽  
Viable Cells ◽  
Temporal And Spatial ◽  
Biofilm Development

ABSTRACT Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa. We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells.

scholarly journals Multiple Roles of Biosurfactants in Structural Biofilm Development by Pseudomonas aeruginosa

2007 ◽  
Vol 189 (6) ◽  
pp. 2531-2539 ◽  
Author(s):  
Sünje Johanna Pamp ◽  
Tim Tolker-Nielsen
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Initial Phase ◽  
Laser Scanning ◽  
Multiple Roles ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Structural Development ◽  
Biofilm Development ◽  
Later Phase

ABSTRACT Recent studies have indicated that biosurfactants produced by Pseudomonas aeruginosa play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. Through the use of flow cell technology and enhanced confocal laser scanning microscopy, we have obtained results which suggest that the biosurfactants produced by P. aeruginosa play additional roles in structural biofilm development. We present genetic evidence that during biofilm development by P. aeruginosa, biosurfactants promote microcolony formation in the initial phase and facilitate migration-dependent structural development in the later phase. P. aeruginosa rhlA mutants, deficient in synthesis of biosurfactants, were not capable of forming microcolonies in the initial phase of biofilm formation. Experiments involving two-color-coded mixed-strain biofilms showed that P. aeruginosa rhlA mutants were defective in migration-dependent development of mushroom-shaped multicellular structures in the later phase of biofilm formation. Experiments involving three-color-coded mixed-strain P. aeruginosa biofilms demonstrated that the wild-type and rhlA and pilA mutant strains formed distinct subpopulations on top of each other dependent on their ability to migrate and produce biosurfactants.

scholarly journals Cell Autoaggregation, Biofilm Formation, and Plant Attachment in a Sinorhizobium meliloti lpsB Mutant

2018 ◽  
Vol 31 (10) ◽  
pp. 1075-1082 ◽  
Author(s):  
Fernando Sorroche ◽  
Pablo Bogino ◽  
Daniela M. Russo ◽  
Angeles Zorreguieta ◽  
Fiorela Nievas ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Sinorhizobium Meliloti ◽  
Planktonic Cell ◽  
Confocal Laser ◽  
Bacterial Surface ◽  
Abiotic Surfaces ◽  
Defective Mutant ◽  
Mutant Strains ◽  
Adhesive Interactions ◽  
Biofilm Development

Bacterial surface molecules are crucial for the establishment of a successful rhizobia-legume symbiosis, and, in most bacteria, are also critical for adherence properties, surface colonization, and as a barrier for defense. Rhizobial mutants defective in the production of exopolysaccharides (EPSs), lipopolysaccharides (LPSs), or capsular polysaccharides are usually affected in symbiosis with their plant hosts. In the present study, we evaluated the role of the combined effects of LPS and EPS II in cell-to-cell and cell-to-surface interactions in Sinorhizobium meliloti by studying planktonic cell autoaggregation, biofilm formation, and symbiosis with the host plant Medicago sativa. The lpsB mutant, which has a defective core portion of LPS, exhibited a reduction in biofilm formation on abiotic surfaces as well as altered biofilm architecture compared with the wild-type Rm8530 strain. Atomic force microscopy and confocal laser microscopy revealed an increase in polar cell-to-cell interactions in the lpsB mutant, which might account for the biofilm deficiency. However, a certain level of biofilm development was observed in the lpsB strain compared with the EPS II-defective mutant strains. Autoaggregation experiments carried out with LPS and EPS mutant strains showed that both polysaccharides have an impact on the cell-to-cell adhesive interactions of planktonic bacteria. Although the lpsB mutation and the loss of EPS II production strongly stimulated early attachment to alfalfa roots, the number of nodules induced in M. sativa was not increased. Taken together, this work demonstrates that S. meliloti interactions with biotic and abiotic surfaces depend on the interplay between LPS and EPS II.

scholarly journals FpvA-Mediated Ferric Pyoverdine Uptake in Pseudomonas aeruginosa: Identification of Aromatic Residues in FpvA Implicated in Ferric Pyoverdine Binding and Transport

2005 ◽  
Vol 187 (24) ◽  
pp. 8511-8515 ◽  
Author(s):  
Jiang-Sheng Shen ◽  
Valérie Geoffroy ◽  
Shadi Neshat ◽  
Zongchao Jia ◽  
Allison Meldrum ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Aromatic Residues ◽  
Three Dimensional Structure

ABSTRACT A number of aromatic residues were seen to cluster in the upper portion of the three-dimensional structure of the FpvA ferric pyoverdine receptor of Pseudomonas aeruginosa, reminiscent of the aromatic binding pocket for ferrichrome in the FhuA receptor of Escherichia coli. Alanine substitutions in three of these, W362, W391, and F795, markedly compromised ferric pyoverdine binding and transport, consistent with a role of FpvA in ferric pyoverdine recognition.

scholarly journals A Nonfimbrial Adhesin ofAggregatibacter actinomycetemcomitansMediates Biofilm Biogenesis

2018 ◽  
Vol 87 (1) ◽  
Author(s):  
David R. Danforth ◽  
Gaoyan Tang-Siegel ◽  
Teresa Ruiz ◽  
Keith P. Mintz
Keyword(s):  
Biofilm Formation ◽  
Protein A ◽  
Three Dimensional ◽  
Binding Activity ◽  
Periodontal Pathogen ◽  
Collagen Binding ◽  
Content Type ◽  
Mutant Strains ◽  
Different Strains ◽  
Biofilm Development

ABSTRACTPeriodontitis is an inflammatory disease caused by polymicrobial biofilms. The periodontal pathogenAggregatibacter actinomycetemcomitansdisplays two proteinaceous surface structures, the fimbriae and the nonfimbrial extracellular matrix binding protein A (EmaA), as observed by electron microscopy. Fimbriae participate in biofilm biogenesis and the EmaA adhesins mediate collagen binding. However, in the absence of fimbriae,A. actinomycetemcomitansstill retains the potential to form robust biofilms, suggesting that other surface macromolecules participate in biofilm development. Here, isogenic mutant strains lacking EmaA structures, but still expressing fimbriae, were observed to have reduced biofilm potential. In strains lacking both EmaA and fimbriae, biofilm mass was reduced by 80%. EmaA enhanced biofilm formation in different strains, independent of the fimbriation state or serotype. Confocal microscopy revealed differences in cell density within microcolonies between the EmaA positive and mutant strains. EmaA-mediated biofilm formation was found to be independent of the glycosylation state and the precise three-dimensional conformation of the protein, and thus this function is uncorrelated with collagen binding activity. The data suggest that EmaA is a multifunctional adhesin that utilizes different mechanisms to enhance bacterial binding to collagen and to enhance biofilm formation, both of which are important forA. actinomycetemcomitanscolonization and subsequent infection.

scholarly journals Different Approaches to FT-IR Microspectroscopy on X-ray Exposed Human Cells

Proceedings ◽  
2019 ◽  
Vol 42 (1) ◽  
pp. 18
Author(s):  
Marianna Portaccio ◽  
Federico Manganello ◽  
Roberta Meschini ◽  
Ines Delfino ◽  
Valerio Ricciardi ◽  
...  
Keyword(s):  
Neuroblastoma Cells ◽  
Cell Interaction ◽  
Attenuated Total Reflection ◽  
X Ray ◽  
Infrared Microspectroscopy ◽  
Fourier Transform Infrared Microspectroscopy ◽  
Experimental Approaches ◽  
Ft Ir ◽  
Ir Microspectroscopy

Fourier-Transform Infrared microspectroscopy (μFT-IR) has been usefully applied in the analysis of the complex biological processes occurring during X-ray radiation-cell interaction. Different experimental approaches are available for FT-IR spectra collection (transmission, attenuated total reflection (ATR), and transflection modes) from cells samples. Recently, some problems have been raised about the role of transmitted and reflected components of the infrared beam in transflection mode. For this reason, we investigated two different transflection approaches for collecting spectra from cells exposed to X-ray. In the former approach, cells were grown on MirrIR slides, and for the second approach, cell pellets were prepared. In both cases, SH-SY5Y neuroblastoma cells were used. X-ray exposure was performed at doses of 2 and 4 Gy. Spectra were obtained by using both the approaches in the 600–4000 cm−1 spectral range from exposed and not-exposed samples. The main contributions from proteins, lipids, carbohydrates, and DNA were clearly evidenced in spectra obtained with the two different acquisition approaches. A comparison among them has been also reported.

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