OppA Escherichia coli mutants have osmodependent resistance to aminoglycoside antibiotics

OppA Escherichia coli mutants have osmodependent resistance to aminoglycoside antibiotics

Maria H. Tsuhako¹, Luis Carlos S. Ferreira ² and Sérgio Olavo P. da Costa¹

¹ Departamento de Microbiologia, ICB II, Universidade de São Paulo, Av. Lineu Prestes, 1374, 05508-900 São Paulo, SP, Brasil. Fax: [phone]. E.mail: [e-mail]. Send correspondence to S.O.P.C.

² Instituto de Biofísica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, RJ, Brasil.

ABSTRACT

The oligopeptide permease (OppA) protein was found to be missing in the periplasmic fractions of Escherichia coli kanamycin-resistant mutants selected under high osmotic conditions. The growth behavior of one mutant in media containing kanamycin or the toxic peptide triornithine suggests that OppA and another cell envelope component contribute to the osmolarity-dependent aminoglycoside resistance of E. Coli.

INTRODUCTION

The uptake of aminoglycoside antibiotics is complex and involves several components of the bacterial cell envelope which are regulated by growth conditions as pH, osmolarity, concentration of divalent cations, carbon source and polyamines (Medeiros et al., 1971; Höltje, 1979; Taber et al., 1987; Rodriguez et al., 1990; Hancock et al., 1991; Xiong et al., 1996). We have previously isolated osmolarity-dependent amino-glycoside-resistant E. coli clones with reduced expression of a 61-kDa periplasmic protein, which could represent the OppA protein based on its electrophoretic behavior and subcellular location (Rodriguez, 1991; Rodriguez et al., 1995).

The OppA protein is a periplasmic component of the oligopeptide transport system with a broad substrate specificity, required for nutrient (peptides) captation and recycling of cell wall peptides in gram-negative bacteria as E. coli and Salmonella thyphimurium (Goodell and Higgins, 1987; Hiles and Higgins, 1986). Moreover, Kashiwagi et al. (1992) have reported that the aminoglycoside sensitivity of E. coli clones transformed with a plasmid carrying the OppA-coding gene is enhanced.

In the present study we have evaluated the involvement of the OppA protein in an osmolarity-dependent aminoglycoside-resistant E. coli mutant. The results further support the important role of the OppA protein in the uptake of aminoglycoside antibiotics but suggest that other cell envelope components might contribute for to osmolarity-dependent behavior of the analyzed mutant.

MATERIAL AND METHODS

Bacterial strains, media and growth conditions

The E. coli strain BB (kindly donated by Dr. R. Hausmann; Albert Ludwigs University, Freiburg, Germany) was used throughout this work. Bacteria growth was monitored by optical density at 540 nm. Minimal medium described by Davis and Mingioli (1950) was used as high osmolarity medium (158 mOsm). Minimal medium of low osmolarity (94 mOsm) was obtained after dilution (1:2) of the high osmolarity medium with distilled water but the glucose content was kept constant at 0.2%. One representative kanamycin-resistant mutant was tested with the toxic tripeptide triornithine (Sigma Chemical Co., St. Louis, MO) and kanamycin (Bristol Laboratories, UK) at final concentration of 500 mg/ml and 20 mg/ml, respectively. The composition of the double strength LB agar plates was 1% Bacto yeast extract, 2% Bacto tryptone, 1% sodium chloride and 1% agar (448.6 mOsm). The media osmolarities were measured in a model 3D II osmometer (Osmette Advanced, Needhan Heights, Massachusetts, USA).

Isolation of osmolarity-dependent kanamycin-resistant mutants

Kanamycin-resistant mutants were selected after plating overnight grown cells (approximately 2 x 10⁸ cells) on double strength LB agar plates added with the antibiotic (Rodriguez et al., 1995).

Isolation of periplasmic proteins

The periplasmic protein fractions were recovered after osmotic shock of cells grown overnight in minimal medium as described by Higgins and Hardie (1983). The protein content of each fraction was quantified by the Lowry method (Lowry et al., 1951), using bovine serum albumin (BSA) as protein standard.

Polyacrylamine gel electrophoresis (SDS-PAGE) and immunoblotting

Proteins (5 mg/ml) were sorted in 9% acrylamine gels (acrylamide to bisacrylamide ratio of 19:1) at 100 V for 2 h following the conditions published by Ames (1974). Silver staining of gels was performed according to Blum et al. (1987). Electroblotting of proteins to nitrocellulose sheets was carried out in a model EPS 500/400 transfer electro-blotter unit (Pharmacia, Upsalla, Sweden) following conditions suggested by the manufacturer. The nitrocellulose sheets were blocked with phosphate buffered saline (PBS) containing 1% BSA and 0.05% Tween 20. Rabbit anti-OppA serum (kindly supplied by Dr. D.S. Santos, Centro de Biotecnologia do Estado do Rio Grande do Sul) was used at final dilution of 1:1,000 in PBS-Tween 20 and incubated for 1 h. Membranes were washed with PBS-Tween 20 and then incubated with goat anti-rabbit IgG horseradish peroxidase conjugate (Sigma) for 1 h, followed by development with 3,3-diaminobenzidine.

RESULTS

Plating of the E.coli BB strain on high osmolarity LB (double strength) agar plates containing 20mg/ml kanamycin resulted in the selection of resistant clones in elevated frequencies of approximately 10^-6 (Rodriguez, 1991). Similar isolation frequencies of kanamycin-resistant (Km^r) mutants were found in other high osmolarity media such as Müeller-Hinton agar (307.2 mOsm), but not in the low osmolarity NA medium (63.5 mOsm). These mutants were resistant to the inhibitory action of kanamycin as well as to other aminoglycosides due to reduced uptake of the drugs (Rodriguez, 1991). One Km^r clone named BBKR was selected for further analysis.

The OppA content proteins in periplasmic fraction of the wild type strain and the aminoglycoside-resistant mutant were evaluated by silver stained polyacrylamine gels and immunoblots developed with OppA-specific rabbit anti-serum. There was no significant difference in OppA levels in the periplasmic fractions of BB cells grown in high or low osmolarity minimal media (Figure 1). On the other hand, no OppA protein was detected in the periplasmic fraction of BBKR either at low or high osmotic condition (Figure 1). Other 24-kanamycin resistant independent E. coli mutants selected in high osmolarity medium showed reduced expression of OppA and 6 had undetectable levels of the protein (data not shown). These results indicate that the osmolarity-dependent amino-glycoside-resistant E. coli mutants have altered expression of the OppA protein.

Figure 1
- Analysis of periplasmic enriched fractions of the E. coli BB strain and the BBKR mutant. Samples (5 mg/ml) were separated by SDS-PAGE and silver stained (left panel) or subjected to Western blot analysis employing antibodies raised against the OppA protein (right panel). The strains were cultivated in low osmolarity (94 mOsm) (a,b,c), or high osmolarity (158 mOsm) (d,e,f) minimal medium. Periplasmic fractions were harvested from E. coli BB (lanes a,d) and two aminoglycoside-resistant mutants (lanes b,c,e,f). Molecular weight markers (M) are indicated on the right side of the left panel.

Reduced expression of OppA can affect the peptide uptake system mediated by this protein (Barak and Gilvarg, 1974). Therefore, resistance to the toxic effects of the peptide triornithine was evaluated in the osmolarity-dependent aminoglycoside-resistant E. coli BBKR in the presence of triornithine or kanamycin in low or high osmolarity media. As shown in Figure 2, the BBKR mutant was able to grow in the presence of both inhibitors, but only in the high osmolarity medium. There was an initial reduction in the growth rate of the mutant strain in relation to cultures grown in plain medium, but similar densities were reached after 24 h (data not shown). Cultures kept in low osmolarity medium were not able to grow in the presence of either kanamycin or triornithine. These data further support the involvement of the OppA protein in the uptake of aminoglycoside antibiotics. Moreover, resistance to kanamycin and triornithine of the BBKR mutant was clearly affected by the osmolarity of the growth medium.

DISCUSSION

In this work we have evaluated the involvement of the OppA protein in the previously described osmolarity-dependent aminoglycoside resistance of E. coli (Rodriguez et al., 1995). This protein has already been shown to be involved in the uptake of aminoglycoside antibiotics in E. coli (Kashiwagi, et al., 1992) and, therefore, represents the most probable candidate for the altered uptake to aminoglycosides displayed by mutants selected under high osmolarity media. The uptake of aminoglycoside antibiotics by aerobically growing gram-negative bacterial cells occurs in three consecutive steps. An initial rapid electrostatic binding to the outer surface is followed by two energy dependent phases (EDP). The EDP I represents a slow rate of energized accumulation which involves the passage of the antibiotic through the cell envelope; EDP II requires active protein synthesis and involves an enhanced rate of uptake and the binding of the drug to ribosomes (Taber et al., 1987; Davis, 1987). Based on the available evidence OppA would probably participate in aminoglycoside uptake during the EDP I as a periplasmic carrier of the antibiotic molecules which passed through the outer membrane. Evidence for the direct binding of OppA to aminoglycoside antibiotics has already been obtained (Kashiwagi et al., 1992). Nonetheless, the role of osmolarity on the aminoglycoside resistance of the BBKR mutant probably involves other cell envelope components.

The sensitivity of gram-negative bacteria to several aminoglycoside antibiotics is known to be affected by the osmotic condition of the growth media (Rodriguez et al., 1990). An osmolarity-dependent behavior has also been observed in the aminoglycoside resistance observed in the OppA-deficient BBKR mutant. Some evidence indicates that osmolarity of the growth medium might affect the uptake of aminoglycoside antibiotics in a step different from that performed by the OppA protein. First, as seen with the parental strain, there was no significant difference in the amounts of OppA expressed by cells cultivated under high or low osmolarity medium. This finding was supported by previous data which demonstrated the lack of significant growth medium osmolarity effect on the transcription of the OppA-coding gene (Hiles et al., 1987). Second, no OppA protein has been detected in the BBKR mutant but different resistant levels to kanamycin and triornithine were observed in high or low osmotic environments (Tsuhako, M.H. and Costa, S.O.P., unpublished observations). Changes in the osmolarity of the growth medium are known to affect the expression of the porin-coding genes and alter the composition of periplasmic polysaccharides, two components of the bacterial cell envelope which may interfere with the initial step of the aminoglycoside uptake (Lugtenberg et al., 1976; Kennedy, 1982). Therefore, the osmolarity-dependent aminoglycoside resistance of the BBKR mutant might reflect the combined effects of the OppA-deficient expression and physiological adaptation of the cell envelope component induced by the high osmolarity of the growth medium.

Resistance to aminoglycoside antibiotics in clinically relevant bacteria has been usually associated with the widespread occurrence of plasmids encoding modifying enzymes (Davies, 1994). As far as we know no attempt to evaluate aminoglycoside resistance mediated by mechanisms not related to modifying enzymes has been carried out. The isolation of aminoglycoside-resistant clones affected in antibiotic uptake only in high osmolarity media suggests that clinically relevant isolates could arise in specific environments as those found in the gastrointestinal or urinary tracts. The finding of osmolarity-dependent aminoglycoside-resistant isolates in clinical settings may reveal unsuspected relevance of this new strategy of aminoglycosides resistance in E. coli.

ACKNOWLEDGMENTS

The authors wish to thank the financial support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We are grateful to Dr. Gabriel M. Padilla for critically reading the manuscript. Publication supported by FAPESP.

RESUMO

Mutantes de Escherichia coli resistentes aos aminoglicosídios por seleção in vitro em meio de cultivo de alta osmolaridade contendo canamicina podem apresentar ausência da proteína periplásmica oligopeptídio permease (OppA). O comportamento desse mutante em presença de canamicina ou do peptídio tóxico triornitina sugere que a proteína OppA e um outro componente do envoltório bacteriano contribuem para a resistência osmodependente em E. coli.

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(Received April 16, 1997)

Figure 2 - Effect of kanamycin and triornithine on the growth curves of the E. coli BBKR mutant at low and high osmotic condition. A - Growth curves of the BBKR mutant in the presence (open symbols) or not (closed symbols) of kanamycin (20 mg/ml). B - Growth curves of the BBKR mutant in the presence (open symbols) or not (closed symbols) of triornithine (500 mg/ml). The strains were grown in high osmolarity (158 mOsm) minimal medium (circles) or low osmolarity (94 mOsm) minimal medium (squares).

Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  Mar 1998

History

• Received
  16 Apr 1997