Departments of Biochemistry and Child Health, University of Missouri, Columbia, MO, USA1
Author for correspondence: Deborah L. Chance. Tel: +1 573 884 6078. Fax: +1 573 882 4287. e-mail: ChanceD{at}health.missouri.edu
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ABSTRACT |
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Keywords: Pseudomonas aeruginosa, growth, sulfate, mucin, cystic fibrosis
Abbreviations: CF, cystic fibrosis
a Present address: Department of Molecular Microbiology & Immunology, M616 Medical Sciences Building, University of Missouri, Columbia, MO 65212, USA.
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INTRODUCTION |
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With impaired mucociliary clearance in CF, respiratory mucins may serve not only to bind bacteria, but also to provide nourishment for entrapped organisms. Approximately 7080% of the mass of these normally protective structures is carbohydrate, consisting of heterologous oligosaccharide chains attached via O-glycosidic linkages of N-acetylgalactosamine to serine or threonine residues of the protein backbone (Boat et al., 1976 ; Roussel et al., 1975
). The principle sugars of mucin oligosaccharide chains are galactose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid and fucose. In the absence of disease, a small percentage of the oligosaccharide chains possess sulfate esters, whereas in CF there is generally a significantly higher level of oligosaccharide sulfation (Boat et al., 1976
; Chace et al., 1983
; Cheng et al., 1989
; Scharfman et al., 1996
). Structural analysis of these sulfated mucins has revealed terminal galactose 3-sulfate and galactose 6-sulfate, and internal galactose 6-sulfate and N-acetylglucosamine 6-sulfate residues (Chance & Mawhinney, 1996
; Mawhinney et al., 1987
, 1992a
, b
; Mawhinney & Chance, 1994
). Whilst mucinases have been found in a number of pathogenic bacteria, (Schneider & Parker, 1982
; Prizont & Reed, 1991
; Smith et al., 1994
), implying that mucins may be a potential source of nutrients for these organisms, it is suggested that sulfation may confer a protection to mucins against degradative enzymes of bacteria (Amerongen et al., 1998
). In studies of growth of Pseudomonas fluorescens with glucose 6-sulfate as the sole carbon source in a minimal salts solution, Fitzgerald & Dodgson (1971a
, b
) demonstrated that over time this organism was able to grow on this sugar sulfate by an undefined mechanism. The identification of mucin sulfatase activity from Helicobacter pylori (Slomiany et al., 1992
), Porphyromonas gingivalis (Slomiany et al., 1993
) and recently Burkholderia cepacia and P. aeruginosa (Jansen et al., 1999
) suggests that these adaptive organisms may in fact also be capable of utilization of sulfated sugars, including sulfomucins.
This study was designed to test whether carbohydrate sulfation affects the in vitro growth of clinical isolates and laboratory strains of P. aeruginosa in an environment of limited nutrients, as might be experienced in the CF airway.
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METHODS |
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For initial growth experiments, laboratory standard ATCC 39018 was incubated for 1 week in minimal medium containing 56 mM glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, mannose, fucose or no sugar at all. Growth was measured as described below. The effect of carbohydrate concentration on P. aeruginosa growth in minimal medium was assessed for a panel of eight strains (see Fig. 2) by culture as described above, for up to 5 d with 56, 5·6 and 0·56 mM glucose-supplemented minimal medium.
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To assess whether sulfation of glucose affected its utilization across a wide variety of strains of P. aeruginosa, a panel of 12 clinical isolates and 2 laboratory standard strains of P. aeruginosa were cultured with glucose 6-sulfate or glucose as the sole carbon source. Similarly, to further investigate the growth of P. aeruginosa on (or adaptation to growth on) sugars commonly found at the termini of tracheobronchial mucin oligosaccharides, this panel of 14 strains was screened for growth in minimal medium with galactose, galactose 3-sulfate or galactose 6-sulfate as the sole carbon source. To minimize any negative effects of manipulation of cells prior to assay for growth on galactose and galactose sulfates, test media was inoculated directly with 50 µl aliquots of overnight cultures. Growth contributed by residual glucose in the inocula was determined by parallel inoculation and incubation in medium with no other carbon source.
Assessment of growth.
OD600 measurements were made throughout the culture period of every original, independent, test culture tube and all control tubes, employing a Milton Roy Genesys II spectrophotometer (1 cm path length). Specific growth rates were not determined as growth on the test substrates was generally not exponential (linear with semilog analysis of opitical density vs time) in this minimal media. Optical density maxima were used to compare growth on various substrates and with various strains of bacteria, and were assumed to reflect maximum growth as preliminary studies yielded the highest viable cell counts at the optical density maxima. Data are expressed as mean OD600±SEM for each culture condition and for each bacterial strain. Cell counts (c.f.u. ml-1) and phenotypes were determined at the time of inoculation and approximately at the culture maximum optical density by plating serial dilutions of test culture aliquots onto trypticase soy agar plates. Culture phenotypes were recorded from these plates after 48 h incubation at 35 °C.
Statistical analysis.
Statistical significance was assessed by one-tailed t-test at a confidence interval of 0·05 (Voelker & Orton, 1993 ). A two-sample t-test for comparing two means was employed to compare mean optical density maxima for bacteria in media with sulfated sugar as the sole carbon source with parallel cultures in media containing the analogous nonsulfated sugar or glucose.
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RESULTS |
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Growth of P. aeruginosa with various monosaccharides as the sole carbon source
Growth of P. aeruginosa was first assessed with several neutral sugars or sialic acid as the sole carbon source in a minimal medium, intended here to parallel the conditions in the airway of patients with CF where nutrient availability may be quite limited. As shown in Fig. 1, P. aeruginosa strain ATCC 39018 readily grew on glucose in minimal medium, and, given time, achieved equally significant growth on N-acetylglucosamine. Other sugars potentially available in the airway, such as galactose, N-acetylgalactosamine, mannose, sialic acid and fucose, did not support marked growth under these conditions.
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Effects of carbohydrate sulfation on the growth of P. aeruginosa
The influence of carbohydrate sulfation on the growth of P. aeruginosa was initially evaluated for three strains: ATCC 39018 as a nonmucoid, motile, laboratory standard, and CF 8314-1 and CF 8314-2 as representative nonmucoid and mucoid CF sputum isolates, respectively. Growth curves of these strains in minimal medium with 56 mM glucose, glucose 3-sulfate, N-acetylglucosamine, N-acetylglucosamine 6-sulfate, or with no sugar are presented in Fig. 3. Growth on glucose was similar for these three strains. N-acetylglucosamine growth curves were comparable for the two nonmucoid strains, whilst the mucoid isolate showed a steeper slope, quite similar to its glucose curve after about a 12 h lag. Glucose 3-sulfate and N-acetylglucosamine 6-sulfate supported markedly reduced growth relative to the nonsulfated analogues, with only limited immediate growth of these strains and then maintenance of the characteristic optical density for the remaining duration of the experiment. Mean optical densities of cultures and cell counts (not shown) for cultures with no carbon source indicated only slow decreases in population over the experimental period. Of note, in preliminary tests, the addition of free potassium sulfate to media supplemented with sulfated monosaccharides had no effect on growth curves.
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Survey of effects of glucose and galactose sulfation on growth of a variety of P. aeruginosa strains
A panel of 14 strains of P. aeruginosa, subjected to culture with glucose 6-sulfate, galactose 3-sulfate, galactose 6-sulfate, their nonsulfated analogues, or no sugar at all, generally demonstrated comparable substrate utilization profiles to those reported above. Growth on glucose and glucose 6-sulfate paralleled previous results, with glucose 6-sulfate supporting little growth for all strains (maximum optical densities ranged between 0·046 and 0·092). As presented in Table 3, galactose supported intermediate growth with the exceptions of two clinical isolates, CF 8981-1, which showed no growth on galactose or glucose, and CF 6935, which showed significant growth on galactose, reaching an optical density of 0·845 by day 8. As seen previously, galactose 3-sulfate and galactose 6-sulfate supported minimal to no growth of clinical isolates or laboratory strains of P. aeruginosa. Removing the cell washing step, with its inherent cell manipulation, prior to inoculation did not improve utilization of galactose and galactose sulfates. For this panel of bacteria, the maximum optical densities of galactose 6-sulfate cultures, with the exception of one isolate, CF 21165, although not statistically different from cultures with no test sugar, were consistently slightly higher than cultures with galactose 3-sulfate. Interestingly, isolate CF 8981-1, which showed no growth on glucose or galactose, showed a gradual small increase in optical density and cell number when cultured with galactose 3-sulfate and galactose 6-sulfate. Comparisons of nonmucoid and mucoid cultures as groups yielded no significant differences in growth on sulfated or on nonsulfated sugars (P>0·2) based on maximum optical densities of the P. aeruginosa strains in this survey.
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DISCUSSION |
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This study indicates that many of the sugars composing mucins are not readily used to support growth of laboratory or clinical strains of P. aeruginosa, supporting the hypothesis that mucins by their structure are protected from bacterial degradation and therefore more able to perform their clearance role under normal conditions. Sulfation of these sugars appears to add another level of protection against degradation. Given time though, as expected in a stagnant airway such as is seen in CF, P. aeruginosa may utilize mucin-derived sugars. As observed here, with time, N-acetylglucosamine supports as much growth as glucose, possibly reflecting amidase/acetylase activity like that responsible for the hydrolysis of and growth on acetamide by this organism (Clarke & Slater, 1986 ). O-Sulfatase has recently been described by Jansen et al. (1999)
for several clinical P. aeruginosa isolates. Evidence of such activity was not observed in the current study, suggesting that under these minimal media conditions, the organisms tested here were incapable of removal of the sulfate ester or of internalization and usage of the intact sugar sulfates. Additional experiments would be necessary to ascertain whether the strains surveyed here express detectable sulfatase activity in vivo or under different in vitro conditions.
The capability of P. aeruginosa to survive in suboptimal conditions until a better means of survival becomes available was evidenced in these studies. Galactose and other sugars, which did not support rapid growth in these minimal medium-based experiments, did supply enough nutritive value for a low level of growth and maintenance of viability. The apparent improvement in utilization of available substrates noted for some strains on galactose and galactose derivatives suggests that P. aeruginosa has additional mechanisms which are inducible in times of need or through which a mutation proves beneficial. Unlike P. fluorescens in previous studies (Fitzgerald & Dodgson, 1971a , b
), P. aeruginosa, under similar conditions, did not adapt to utilize sulfated sugars. Data revealed no statistically significant patterns of carbohydrate utilization among nonmucoid versus mucoid isolates, suggesting that improved carbohydrate metabolism may not necessarily be a general feature related to the prevalent mucoid phenotype in CF. Likewise, the growth of these strains of P. aeruginosa on sulfated and nonsulfated sugars common to mucin, as the sole carbon source, did not appear to promote conversion to the mucoid phenotype of nonmucoid strains or enhance the mucoid character of mucoid strains.
Whilst not directly part of this study, it should be noted that liberation of mucin carbohydrates for use as an energy source for bacteria presumably requires glycosidase activity. Although there are no reports of mucinase activity from P. aeruginosa, as there are for other pathogens, (Prizont & Reed, 1991 ; Schneider & Parker, 1982
; Smith et al., 1994
), there have been reports of ß-galactosidase activity (Cybulski et al., 1993
; Vieu et al., 1987
), which could liberate terminal galactose residues from mucins as well as make accessible N-acetylglucosamine, which is often the penultimate sugar on mucin oligosaccharide chains. The inability of the methyl glycosides or sulfated methyl glycosides tested here to support significant growth of P. aeruginosa suggests that under these conditions of limited nutrients, glycosidases are not elaborated.
Conclusions
These data suggest that the increased tracheobronchial mucin oligosaccharide sulfation seen in CF may in fact serve to protect the mucins from degradation during prolonged contact with P. aeruginosa, and limit the bacteriums growth on the basis of substrate availability, regardless of its mucoid status. Additional detailed in vitro research with clinical isolates and defined laboratory strains is required to assess whether other culture conditions may affect P. aeruginosa growth in the presence of sulfated mucins, before proceeding to the more complex analysis of in vivo P. aeruginosa responses to CF airway mucin sulfation.
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ACKNOWLEDGEMENTS |
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Received 16 March 2000;
accepted 24 March 2000.