Differences in colonisation of five marine bacteria on two types of glass surfaces

The retention patterns of five taxonomically different marine bacteria after attachment on two types of glass surfaces, as-received and chemically etched, have been investigated. Contact angle measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), X-ray fluorescence spectroscopy (XRF) and X-ray photoelectron spectrometry (XPS) were employed to investigate the impact of nanometer scale surface roughness on bacterial attachment. Chemical modification of glass surfaces resulted in a ～1 nm decrease in the average surface roughness (R _a) and the root-mean-squared roughness (R_q ) and in a ～8 nm decrease in the surface height and the peak-to-peak (R _max) and the 10-point average roughness (R_z ). The study revealed amplified bacterial attachment on the chemically etched, nano-smoother glass surfaces. This was a consistent response, notwithstanding the taxonomic affiliation of the selected bacteria. Enhanced bacterial attachment was accompanied by elevated levels of secreted extracellular polymeric substances (EPS). An expected correlation between cell surface wettability and the density of the bacterial attachment on both types of glass surfaces was also reported, while no correlation could be established between cell surface charge and the bacterial retention pattern.     

Bacterial attachment on optical fibre surfaces

Optical fibres have received considerable attention as high-density sensor arrays suitable for both in vitro and in vivo measurements of biomolecules and biological processes in living organisms and/or nano-environments. The fibre surface was chemically modified by exposure to a selective etchant that preferentially erodes the fibre cores relative to the surrounding cladding material, thus producing a regular pattern of cylindrical wells of approximately 2.5 μm in diameter and 2.5 μm deep. The surface hydrophobicity of the etched and non-etched optical fibres was analysed using the sessile pico-drop method. The surface topography was characterised by atomic force microscopy (AFM), while the surface chemistry was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Six taxonomically different bacterial strains showed a consistent preference for attachment to the nano-scale smoother (R _q = 273 nm), non-etched fibre surfaces (water contact angle, θ = 106° ± 4°). In comparison, the surfaces of the etched optical fibres (water contact angle, θ = 96° ± 10°) were not found to be amenable to bacterial attachment. Bacterial attachment on the non-etched optical fibre substrata varied among different strains.     

Topographic cues guide the attachment of diatom cells and algal zoospores

Surface topography plays a key role in the colonization of substrata by the colonizing stages of marine fouling organisms. For the innovation of marine antifouling coatings, it is essential to understand how topographic cues affect the settlement of these organisms. In this study, tapered, spiked microstructures and discrete honeycombs of varying feature dimensions were designed and fabricated in order to examine the influence of topography on the attachment of zoospores of the green macroalga Ulva linza and cells of the diatom (microalga) Navicula incerta. Contrasting results were obtained with these two species of algae. Indeed, the preferred location of cells of N. incerta was dominated by attachment point theory, which suggested a positive correlation between the density of cells adhering and the amount of available attachment points, while the settlement of spores of U. linza was mainly regulated by both Wenzel roughness and local binding geometry.     

Engineered antifouling microtopographies: kinetic analysis of the attachment of zoospores of the green alga Ulva to silicone elastomers

Microtopography has been demonstrated as an effective deterrent to biofouling. The majority of published studies are fixed-time assays that raise questions regarding the kinetics of the attachment process. This study investigated the time-dependent attachment density of zoospores of Ulva, in a laboratory assay, on a micropatterned and smooth silicone elastomer. The attachment density of zoospores was reduced on average 70–80% by the microtopography relative to smooth surfaces over a 4 h exposure. Mapping the zoospore locations on the topography revealed that they settled preferentially in specific, recessed areas of the pattern. The kinetic data fit, with high correlation (r ² > 0.9), models commonly used to describe the adhesion of bacteria to surfaces. The grouping of spores on the microtopography indicated that the pattern inhibited the ability of attached spores to recruit neighbors. This study demonstrates that the antifouling mechanism of topographies may involve disruption of the cooperative effects exhibited by fouling organisms such as Ulva.     

A model that predicts the attachment behavior of Ulva linza zoospores on surface topography

A predictive model for the attachment of spores of the green alga Ulva on patterned topographical surfaces was developed using a constant refinement approach. This ‘attachment model’ incorporated two historical data sets and a modified version of the previously-described Engineered Roughness Index. Two sets of newly-designed surfaces were used to evaluate the effect of two components of the model on spore settlement. Spores attached in fewer numbers when the area fraction of feature tops increased or when the number of distinct features in the design increased, as predicted by the model. The model correctly predicted the spore attachment density on three previously-untested surfaces relative to a smooth surface. The two historical data sets and two new data sets showed high correlation (R ² = 0.88) with the model. This model may be useful for designing new antifouling topographies.     

Barnacle settlement and the adhesion of protein and diatom microfouling to xerogel films with varying surface energy and water wettability

Previous work has shown that organosilica-based xerogels have the potential to control biofouling. In this study, modifications of chemistry were investigated with respect to their resistance to marine slimes and to settlement of barnacle cyprids. Adhesion force measurements of bovine serum albumin (BSA)-coated atomic force microscopy (AFM) tips to xerogel surfaces prepared from aminopropylsilyl-, fluorocarbonsilyl-, and hydrocarbonsilyl-containing precursors, indicated that adhesion was significantly less on the xerogel surfaces in comparison to a poly(dimethylsiloxane) elastomer (PDMSE) standard. The strength of adhesion of BSA on the xerogels was highest on surfaces with the highest and the lowest critical surface tensions, γ_C and surface energies, γ_S, and duplicated the ‘Baier curve’. The attachment to and removal of cells of the diatom Navicula perminuta from a similar series of xerogel surfaces were examined. Initial attachment of cells was comparable on all of the xerogel surfaces, but the percentage removal of attached cells by hydrodynamic shear stress increased with γ_C and increased wettability as measured by the static water contact angle, θ_Ws, of the xerogel surfaces. The percentage removal of cells of Navicula was linearly correlated with both properties (R ² = 0.74 for percentage removal as a function of θ_Ws and R ² = 0.69 for percentage removal as a function of γ_C). Several of the aminopropylsilyl-containing xerogels showed significantly greater removal of Navicula compared to a PDMSE standard. Cypris larvae of the barnacle B. amphitrite showed preferred settlement on hydrophilic/higher energy surfaces. Settlement was linearly correlated with θ_Ws (R ² = 0.84) and γ_C (R ² = 0.84). Hydrophilic xerogels should prove useful as coatings for boats in regions where fouling is dominated by microfouling (protein and diatom slimes).     

Dependence of the initial adhesion of biofilm forming Pseudomonas putida mt2 on physico-chemical material properties

Bacterial adhesion is strongly dependent on the physico-chemical properties of materials and plays a fundamental role in the development of a growing biofilm. Selected materials were characterized with respect to their physico-chemical surface properties. The different materials, glass and several polymer foils, showed a stepwise range of surface tensions (γ_s) between 10.3 and 44.7 mN m^?1. Measured zeta potential values were in the range between ?74.8 and ?28.3 mV. The initial bacterial adhesion parameter q _max was found to vary between 6.6 × 10⁶ and 28.1 × 10⁶ cm^?2. By correlation of the initial adhesions kinetic parameters with the surface tension data, the optimal conditions for the immobilization of Pseudomonas putida mt2 were found to be at a surface tension of 24.7 mN m^?1. Both higher and lower surface tensions lead to a smaller number of adherent cells per unit surface area. Higher energy surfaces, commonly termed hydrophilic, could constrain bacterial adhesion because of their more highly ordered water structure (exclusion zone) close to the surface. At low energy surfaces, commonly referred to as hydrophobic, cell adhesion is inhibited due to a thin, less dense zone (depletion layer or clathrate structure) close to the surface. Correlation of q _max with zeta potential results in a linear relationship. Since P. putida carries weak negative charges, a measurable repulsive effect can be assumed on negative surfaces.     

Testing attachment point theory: diatom attachment on microtextured polyimide biomimics

Abstract

This paper explores diatom attachment to a range of laser etched polyimide surfaces to directly test ‘attachment point theory’. Static bioassays were conducted on microtextured polyimide surfaces using four diatom species, Fallacia carpentariae, Nitzschia cf. paleacea, Amphora sp. and Navicula jeffreyi with cell sizes ranging from 1 – 14 μm. The microtextured polyimides were modelled from natural fouling resistant bivalve surfaces and had wavelengths above, below and at the same scale as the diatom cell sizes. Diatoms attached in significantly higher numbers to treatments where the numbers of attachment points was highest. The lowest diatom attachment occurred where cells were slightly larger than the microtexture wavelength, resulting in only two theoretical points of attachment. The results support attachment point theory and highlight the need to address larval/cell size in relation to the number of attachment points on a surface. Further studies examining a range of microtexture scales are needed to apply attachment point theory to a suite of fouling organisms and to develop structured surfaces to control the attachment and development of fouling communities.     

The role of nano-roughness in antifouling

Nano-engineered superhydrophobic surfaces have been investigated for potential fouling resistance properties. Integrating hydrophobic materials with nanoscale roughness generates surfaces with superhydrophobicity that have water contact angles (θ) >150° and concomitant low hysteresis (<10°). Three superhydrophobic coatings (SHCs) differing in their chemical composition and architecture were tested against major fouling species (Amphora sp., Ulva rigida, Polysiphonia sphaerocarpa, Bugula neritina, Amphibalanus amphitrite) in settlement assays. The SHC which had nanoscale roughness alone (SHC 3) deterred the settlement of all the tested fouling organisms, compared to selective settlement on the SHCs with nano- and micro-scale architectures. The presence of air incursions or nanobubbles at the interface of the SHCs when immersed was characterized using small angle X-ray scattering, a technique sensitive to local changes in electron density contrast resulting from partial or complete wetting of a rough interface. The coating with broad spectrum antifouling properties (SHC 3) had a noticeably larger amount of unwetted interface when immersed, likely due to the comparatively high work of adhesion (60.77 mJ m^?2 for SHC 3 compared to 5.78 mJ m^?2 for the other two SHCs) required for creating solid/liquid interface from the solid/vapour interface. This is the first example of a non-toxic, fouling resistant surface against a broad spectrum of fouling organisms ranging from plant cells and non-motile spores, to complex invertebrate larvae with highly selective sensory mechanisms. The only physical property differentiating the immersed surfaces is the nano-architectured roughness which supports longer standing air incursions providing a novel non-toxic broad spectrum mechanism for the prevention of biofouling.     

Impact of diatomaceous biofilms on the frictional drag of fouling-release coatings

Skin-friction results are presented for fouling-release (FR) hull coatings in the unexposed, clean condition and after dynamic exposure to diatomaceous biofilms for 3 and 6 months. The experiments were conducted in a fully developed turbulent channel flow facility spanning a wide Reynolds number range. The results show that the clean FR coatings tested were hydraulically smooth over much of the Reynolds number range. Biofilms, however, resulted in an increase in skin-friction of up to 70%. The roughness functions for the biofilm-covered surfaces did not display universal behavior, but instead varied with the percentage coverage by the biofilm. The effect of the biofilm was observed to scale with its mean thickness and the square root of the percentage coverage. A new effective roughness length scale (k_eff) for biofilms based on these parameters is proposed. Boundary layer similarity-law scaling is used to predict the impact of these biofilms on the required shaft power for a mid-sized naval surface combatant at cruising speed. The increase in power is estimated to be between 1.5% and 10.1% depending on the biofilm thickness and percentage coverage.     

Roughness effects of diatomaceous slime fouling on turbulent boundary layer hydrodynamics

Abstract

Biofilm fouling significantly impacts ship performance. Here, the impact of biofilm on boundary layer structure at a ship-relevant, low Reynolds number was investigated. Boundary layer measurements were performed over slime-fouled plates using high resolution particle image velocimetry (PIV). The velocity profile over the biofilm showed a downward shift in the log-law region (ΔU⁺), resulting in an effective roughness height (k_s) of 8.8?mm, significantly larger than the physical thickness of the biofilm (1.7?±?0.5?mm) and generating more than three times as much frictional drag as the smooth-wall. The skin-friction coefficient, C_f, of the biofilm was 9.0?×?10^?3 compared with 2.9?×?10^?3 for the smooth wall. The biofilm also enhances turbulent kinetic energy (tke) and Reynolds shear stress, which are more heterogeneous in the streamwise direction than smooth-wall flows. This suggests that biofilms increase drag due to high levels of momentum transport, likely resulting from protruding streamers and surface compliance.     

Elucidation of the DNA-interacting properties and anticancer activity of a Ni(II)-coordinated mithramycin dimer complex

Mithramycin (Mith) forms a drug-metal complex with a 2:1 stoichiometry by chelation with a Ni(II) ion, which was determined using circular dichroism spectroscopy. Mith exhibits an increased affinity (~55 fold) for Ni(II) in the presence of DNA compared to the absence of DNA, suggesting that DNA acts as an effective template to facilitate chelation. Also, we characterized the DNA-acting properties of a Ni(II) derivative of Mith. Kinetic analysis using surface plasmon resonance and UV melting studies revealed that Ni^II(Mith)₂ binds to duplex DNA with a higher affinity compared to Mg^II(Mith)₂. The thermodynamic parameters revealed a higher free energy of formation for duplex DNA in the presence of Ni^II(Mith)₂ compared to duplex DNA in the presence of Mg^II(Mith)₂. The results of a DNA-break assay indicated that Ni^II(Mith)₂ is capable of promoting one-strand cleavage of plasmid DNA in the presence of hydrogen peroxide; the DNA cleavage rate of Ni^II(Mith)₂ was calculated to be 4.1 × 10^?4 s^?1. In cell-based experiments, Ni^II(Mith)₂ exhibited a more efficient reduction of c-myc and increased cytotoxicity compared to Mith alone because of its increased DNA-binding and cleavage activity. The evidence obtained in this study suggests that the biological effects of Ni^II(Mith)₂ require further investigation in the future.     

Effect of ship hull form on the resistance penalty from biofouling

Hull biofouling is a well-known problem for the shipping industry, leading to increased resistance and fuel consumption. Considering that the effects of hull form on resistance are known to be higher for a less slender hull, it is hypothesised in this paper that the effect of biofouling roughness on resistance is also dependent on the hull form. To test this hypothesis, previously reported full-scale numerical results on a containership are re-analysed. Form effects on roughness penalties, corresponding to K_ΔCT = 0.058 ± 0.025, are observed at a low speed (19 knots, Re_s = 2.29 × 10⁹), which are however cancelled out by traditionally neglected roughness effects on wave-making resistance at a higher speed (24 knots, Re_s = 2.89 × 10⁹). It is concluded that hull form effects on biofouling penalties can be significant at low speeds, though not generalisable for higher speeds, namely when wave-making resistance corresponds to ≥ 29% of total resistance.     

Interaction of gangliosides with plasma low density lipoproteins

The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous G_M1- and G_D1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (G_M3, G_D1a, G_T1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (G_M1, G_M2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 M concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.Abbreviations MAH mouse ascites hepatoma 22a - LDL low density lipoprotein - ASM anthrylvinyl-labeled sphingomyelin [N-12-(9-anthryl-trans-dodecanoyl-sphingosine-1-phosphocholine] - RITC rhodamine isothiocyanate. The designation of gangliosides follows the IUPAC-IUB recommendation [1]: G_M3, II³NeuAc-LacCer, II³-N-acetylneuraminosyllactosylceramide - G_M2 II³-NeuAc-GgOse₃Cer, II³-N-acetylneuraminosylgangliotriaosylceramide - G_M1 II³-NeuAc-GgOse₄Cer, II³-N-acetylneuraminosylgangliotetraosylceramide - G_D1a, II³ IV³(NeuAc)₂-GgOse₄Cer, II³, IV³-di(N-acetylneuraminosyl)gangliotetraosylceramide - G_T1b II³(NeuAc)₂, IV³ NeuAc-GgOse₄Cer, II³-di-N-acetylneuraminosyl, IV³-N-acetylneuraminosylgangliotetraosylceramide     

The effect of butenolide on behavioral and morphological changes in two marine fouling species,the barnacle Balanus amphitrite and the bryozoan Bugula neritina

Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC₅₀ = 0.82 μg ml^?1) and B. neritina (EC₅₀ = 3 μg ml^?1). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 μg ml^?1 and 10 μg ml^?1, butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.     

Investigation on the interaction of newly designed potential antibacterial Zn(II) complexes with CT-DNA and HSA

Two Zn(II) complexes of formula [Zn(bpy)(Gly)]NO₃ (I) and [Zn(phen)(Gly)]NO₃ (II) (where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline and Gly = glycine) were synthesized and characterized by elemental analysis, molar conductance measurements, UV–vis, FT-IR, and ¹H NMR spectra. The interaction ability of these complexes with calf thymus DNA was monitored using spectroscopic methods, including UV–vis absorption spectroscopy, ethidium bromide displacement, Fourier transform infrared, and electrophoretic mobility assay. Further, the human serum albumin interactions of complexes I and II were investigated using UV–vis absorption spectroscopy, fluorescence quenching, circular dichroism, and Fourier transform infrared. The results obtained from these analyses indicated that both complexes interact effectively with CT-DNA and HSA. The binding constant (K_b), the Stern–Volmer constant (K_sv), and the number of binding sites (n) at different temperatures were determined for CT-DNA and HSA. Also, the negative ΔH° and ΔS° values showed that both hydrogen bonds and van der Waals forces played major roles in the association of CT-DNA-Zn(II) and HSA-Zn(II) complex formation. The displacement experiments suggested that Zn(II)-complexes primarily bound to Sudlow’s site II of HSA. The distance between the donor (HSA) and the acceptor (Zn(II) complexes) was estimated on the basis of the Forster resonance energy transfer (FRET) and the alteration of HSA secondary structure induced by the compounds were confirmed by FT-IR spectroscopy. The complexes follow the binding affinity order of I > II with DNA and II > I with HSA. Finally, Antibacterial activity of complexes I and II have been screened against gram positive and gram negative bacteria.     

Antibacterial combination therapy using Co3+, Cu2+, Zn2+ and Pd2+ complexes: Their calf thymus DNA binding studies

Four Co(III)-, Cu(II)-, Zn(II)-, and Pd(II)-based potent antibacterial complexes of formula K₃[Co(ox)₃].3H₂O (I), [Cu(bpy)₂Cl]Cl.5H₂O (II), [Zn(bpy)₃]Cl₂ (III), and [Pd(bpy)₂](NO₃)₂ (IV) (where ox is oxalate and bpy is 2,2′-bipyridine) were synthesized. They were characterized by elemental analyses, molar conductance measurements, UV–Vis, FTIR, ¹H NMR, and ¹³C NMR spectra. These metal complexes were ordered in three combination series of I + II, I + II + III, and I + II + III + IV. Antibacterial activity was tested for each of these four metal complexes and their combinations against Gram-positive and Gram-negative bacteria. All compounds were more potent antibacterial agents against the Gram-negative than those of the Gram-positive bacteria. The four metal complexes showed antibacterial activity in the order I > II > III > IV and the activity of their combinations followed the order of I + II + III + IV > I + II + III > I + II. CT-DNA binding studies of complex I and its three combinations were carried out using UV–vis spectral titration, displacement of ethidium bromide (EB), and electrophoretic mobility assay. The results obtained from UV–vis studies indicated that all series interact effectively with CT-DNA. Fluorescence titration revealed that the complexes quench DNA-EB strongly through the static quenching procedures. The binding constant (K_b), the Stern–Volmer constant (K_sv), and the number of binding sites (n) were determined at different temperatures of 293, 300, and 310 K, respectively. The calculated thermodynamic parameters supported that hydrogen binding and Van der Waals forces play a major role in association of each series of metal complexes with CT-DNA and follow the above-binding affinity order for the series.     

Biodegradation of Isopropanol by a Solvent-Tolerant Paracoccus denitrificans Strain

The biodegradation of high concentration isopropanol (2-propanol, IPA) at 16 g/L was investigated by a solvent-tolerant strain of bacteria identified as Paracoccus denitrificans for the first time by 16S rDNA gene sequencing. The strain P. denitrificans GH3 was able to utilize the high concentration of IPA as the sole carbon source within a minimal salts medium with a cell density of 1.5 × 10⁸ cells/mL. The optimal conditions were found as follows: initial pH 7.0, incubation temperature 30°C, with IPA concentration 8 g/L. Under the optimal conditions, strain GH3 utilized 90.3% of IPA in 7 days. Acetone, the major intermediate of aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Both IPA and acetone were completely removed from the medium following 216 hr and 240 hr, respectively. The growth of strain GH3 on IPA as a sole carbon and energy source was well described by the Andrews model with a maximum growth rate (μ _max ) = 0.0277/hr, a saturation constant (K _S ) = 0.7333 g/L, and an inhibition concentration (Ki) = 8.9887 g/L. Paracoccus denitrificans GH3 is considered to be well used in degrading IPA in wastewater.     

Optimization of ultraviolet ozone treatment process for improvement of polycaprolactone (PCL) microcarrier performance

Growing cells on microcarriers may have overcome the limitation of conventional cell culture system. However, the surface functionality of certain polymeric microcarriers for effective cell attachment and growth remains a challenge. Polycaprolactone (PCL), a biodegradable polymer has received considerable attention due to its good mechanical properties and degradation rate. The drawback is the non-polar hydrocarbon moiety which makes it not readily suitable for cell attachment. This report concerns the modification of PCL microcarrier surface (introduction of functional oxygen groups) using ultraviolet irradiation and ozone (UV/O₃) system and investigation of the effects of ozone concentration, the amount of PCL and exposure time; where the optimum conditions were found to be at 60,110.52 ppm, 5.5 g PCL and 60 min, respectively. The optimum concentration of carboxyl group (COOH) absorbed on the surface was 1495.92 nmol/g and the amount of gelatin immobilized was 320 ± 0.9 µg/g on UV/O₃ treated microcarriers as compared to the untreated (26.83 ± 3 µg/g) microcarriers. The absorption of functional oxygen groups on the surface and the immobilized gelatin was confirmed with the attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) and the enhancement of hydrophilicity of the surface was confirmed using water contact angle measurement which decreased (86.93°–49.34°) after UV/O₃ treatment and subsequently after immobilization of gelatin. The attachment and growth kinetics for HaCaT skin keratinocyte cells showed that adhesion occurred much more rapidly for oxidized surfaces and gelatin immobilized surface as compared to untreated PCL.     

Attachment of Pneumocystis carinii to Primary Cultures of Rat Alveolar Epithelial Cells

Pneumocystis carinii (PC) is an exclusively extracellular pathogen which causes pneumonia in immunocompromised individuals. Histologic studies have demonstrated that PC organisms attach preferentially to type I alveolar epithelial cells and rarely bind to type II cells. Previous reports have demonstrated that cultured type II cells develop a type I cell-like phenotype and express type I cell surface antigens. The current study examines the attachment of PC organisms to isolated rat type H alveolar epithelial cells as a function of time in culture. PC attachment to isolated type II cells increased as the type II cells differentiated in culture from 2.3 ± 1.2% on Day 2 to 18.4 ± 2.7% by Day 8. Previous studies have indicated a role for fibronectin (Fn) and Fn receptors as mediators of PC attachment. Addition of anti-Fn antibodies decreased attachment of PC to Day 8 type II cells from 19.4 ± 2.5% to 9.4 ± 1.9% (P < 0.01). Addition of antibodies to the α_v and α₅ integrin subunits resulted in significant decreases in PC attachment to Day 8 type II cells. Examination of expression of α_v and α₅ integrins on Day 2 and Day 8 type II cells demonstrated increased expression of both α_v and α₅ integrin subunits on Day 8 type II cells. Overall these data indicate that attachment of PC to isolated rat type II cells increases as the cells differentiate into a type I cell-like phenotype in vitro and correlates with increased expression of Fn-binding integrins on the cell surface of the cultured type II cells.