A novel antibacterial agent derived from the C-terminal domain of Streptococcus mutans GTP-binding protein

Seung-Ho Ohk and Howard K. Kuramitsu*

Department of Oral Biology, State University of New York at Buffalo, 3435 Main Street, Buffalo, NY 14214, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A decapeptide, P1, homologous to part of the Streptococcus mutans G-protein (SGP) and the Escherichia coli Era protein, was synthesized and examined for growth-inhibitory effects. When P1 10 mg/L was added to E. coli DH5, the viability of the cells was reduced by 13%. Addition of lauric acid enhanced the bactericidal effects of P1 (68% killing in the presence of P1 plus lauric acid). Similar enhancements were observed for mono lauroyl-rac-glycerol (MLG) and sodium dodecyl sulphate (SDS). In cultures treated with both P1 and MLG, there were more elongated cells than in cultures treated with detergent or peptide alone. As with E. coli, the bactericidal effects of P1 on S. mutans were significantly enhanced in the presence of the detergent lauric acid. The combination of the two effectors produced >90% killing of S. mutans. Likewise, the combined action of P1 plus lauric acid reduced the viability of Listeria monocytogenes. P1 did not appear to be toxic to human gingival epithelial cells when added at concentrations <= 1000 mg/L. Therefore, P1 has properties which could allow it to be used as an antibacterial agent.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
GTP-binding proteins (G-proteins) are ubiquitous in eukaryotic and prokaryotic cells and many of them are involved in important signal transduction events.1 A family of G-proteins homologous to the essential Escherichia coli Era protein have now been identified in all those bacterial genomes that have been sequenced.2 The Streptococcus mutans G-protein (SGP) complements an era mutation in E. coli.3 The sgp gene appears to be essential for cell growth since attempts to mutate this gene have been unsuccessful.4 In addition, evidence has been presented that SGP plays a role in the stress response of S. mutans.5 Although a number of bacterial cell functions, including cell cycle regulation, are influenced by G-proteins,6 the precise role of these proteins in cellular physiology has yet to be determined.

This investigation was initiated to determine whether inhibitors of bacterial G-protein function would act as antimicrobial agents against a variety of Gram-positive and Gram-negative bacteria. Potential analogues of SGP were synthesized and examined for antibacterial activity. This report indicates that one of these, the P1 peptide, which is identical to the C-terminal region of SGP, is bactericidal for both Gram-positive and Gram-negative bacteria.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and growth

E. coli DH5 cells (Gibco, Gaithersburg, MD, USA) were grown in Luria broth (Gibco). An overnight culture was inoculated (0.1%) into medium containing peptides and/or detergents and incubated at 37°C with shaking. S. mutans GS-5 (serotype c human isolate)4 was grown in Todd– Hewitt broth (THB, Gibco) and Listeria monocytogenes L83 in brain–heart infusion (BHI, Gibco). L. monocytogenes L83 was kindly provided by M. Wiedmann of Cornell University (Ithaca, NY, USA). The growth of bacterial cells was monitored with a spectrophotometer (Shimatsu UV-1201) at 550 nm.

Detergents and peptides

The detergents used in this study, mono lauroyl-rac- glycerol (MLG), sodium dodecyl sulphate (SDS) and lauric acid, were obtained from Sigma (St Louis, MO, USA). The peptides used in this investigation, P1 (VYLETWVKVK), P2 (RFLVSEMIREK) and P3 (TIMVERDSQKG), were synthesized by Afiniti Research Products (Exeter, UK).

Assays of bactericidal activity

Bactericidal activity was assayed in microdilution plates in the presence or absence of peptides. Early stationary-phase cells were harvested, washed twice with 10 mM Tris–HCl buffer (pH 8.0) and resuspended to a concentration of 1.0 x 105 cells/mL. Twenty microlitres of each cell suspension was then mixed with 20 µL of 10 mM Tris–HCl buffer (pH 8.0) containing the peptides or detergents. The mixtures were incubated at 37°C for 2 h with or without shaking. After incubation, 360 µL of growth medium was added; 10 µL of the suspensions was then spread on to agar plates and incubated at 37°C for 24 h for E. coli and L. monocytogenes or anaerobically (Gibco BRL GasPak Plus system) for 48 h for S. mutans.

Toxicity

KB human gingival epithelial cells, grown in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum and gentamicin 50 mg/L, were used to test the toxicity of P1. Cells were incubated at 37°C with 5% CO2 in a 24-well plate. P1 was added when the cells became confluent (5 x 104–1 x 105 cells/well) and cultures were then incubated for another 24 h. The cells were collected by adding 100 µL of cell dissociation buffer. Five microlitres of trypan blue (Gibco) was added and viable cells were counted under a microscope.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of P1 and detergents on E. coli

Peptides P1, P2 and P3, corresponding to regions of SGP homologous with the E. coli Era protein, were examined for their ability to inhibit the growth of E. coli DH5. P1 showed a slight growth-inhibitory effect when added at <100 mg/L (data not shown). Since peptides as large as P1 may have difficulty penetrating bacterial membranes, it was of interest to determine whether the addition of low concentrations of detergents might increase the growth-inhibitory properties of P1. Addition of lauric acid (which reduced viability by 19%) enhanced the bactericidal effects of P1 (68% killing in the presence of P1 plus lauric acid; Figure 1Go). Similar enhancements were observed for MLG and SDS (data not shown). In the absence of detergents, P1 concentrations of c. 100 mg/L were required to produce rates of killing similar to those obtained with P1 10 mg/L plus detergents.



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Figure 1. Effects of P1 10 mg/L and lauric acid 0.1 mg/L on the viability of E. coli DH5.

 
Since recent results have suggested that Era plays a role in regulating cell division in E. coli,6 it was of interest to determine the effects of P1 on this process. The addition of P1 together with the detergent MLG resulted in elongation of some cells, unlike cultures treated with detergent or peptide alone (Figure 2Go). Not all of the cells were elongated in the presence of the inhibitors, which may indicate that the inhibitor was acting only at certain periods in the cell division cycle. Propidium iodide staining of the cells indicated that the elongated cells contained multiple chromosomes (data not shown). This suggested that the combination of P1 plus MLG inhibited cell septation but not chromosome replication.



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Figure 2. Effects of P1 and MLG on the division of E. coli DH5 cells. (a) Untreated control; (b), P1 10 mg/L; (c) MLG 1 mg/L; (d) combination of P1 10 mg/L and MLG 1 mg/L. Magnification x1000.

 
Effect of P1 on Gram-positive bacteria

In order to gauge the spectrum of activity of P1, the effects of this peptide on the growth of S. mutans were examined. As with E. coli, the growth-inhibitory effects of P1 were enhanced in the presence of the detergents (Figure 3aGo). The bactericidal effects of P1 on S. mutans were significantly enhanced in the presence of the detergent lauric acid (Figure 3bGo). P1 (1.0 mg/L) alone produced no significant loss in viability whereas lauric acid alone produced a slight inhibitory effect. However, the combination of the two effectors produced >90% killing of S. mutans under these conditions.



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Figure 3. Effects of P1 and lauric acid on the growth and viability of S. mutans GS-5. (a) Growth of S. mutans GS-5. {circ}, Untreated control; •, P1 10 mg/L; {square}, lauric acid 1 mg/L; {blacksquare}, combination of P1 10 mg/L and lauric acid 1 mg/L. (b) Viability of S. mutans GS-5 treated with P1 1 mg/L and/or lauric acid 1 mg/L.

 
Since expression of sgp antisense RNA decreases the growth of S. mutans under stress conditions,5 we tested the growth-inhibitory effects of P1 plus detergents at elevated temperature (42°C), and acidic pH (pH 5.0). The viability of S. mutans was not further reduced in the presence of P1 and lauric acid in either of these conditions (data not shown).

The bactericidal activity of P1 in the presence of detergents was not limited to S. mutans since similar effects were also observed with Listeria monocytogenes.7 Again, the killing effect of P1 plus lauric acid together was greater than the additive effects of either agent alone (Figure 4Go). However, L. monocytogenes did not appear to be as sensitive to P1 as S. mutans.



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Figure 4. Effects of P1 1 mg/L and lauric acid 1 mg/L on the viability of L. monocytogenes L83.

 
Toxicity of P1

Since the long-range goal of these experiments was to identify potential novel antibacterial agents which could be used medically or in food processing, it was important to determine if P1 was toxic to eukaryotic cells. When 1000 mg/L of P1 was added to KB human epithelial cells, the death rate was 4.08%, as compared with 3.63% in control cells. Therefore, P1 did not appear to be toxic to human gingival epithelial cells when added at concentrations of <=1000 mg/L. In addition, P1 did not appear to affect the growth of the fungus Candida albicans (data not shown). Thus, the peptide inhibitor appears to be relatively innocuous for eukaryotic cells.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Era, the signal transducing GTPase of E. coli, is a peripheral membrane protein which binds to the cytoplasmic surface of the inner membrane.8 A comparison of the amino acid sequences of SGP and other G-proteins indicated several regions of homology between the proteins.2 Besides extensive homology in the GTP-binding domain near the N terminus, at least three regions of significant homology were identified in the C-terminal region. Such regions could be important for binding to membranes or other downstream regulatory molecules.

Of the three decapeptides tested, only P1 inhibited the growth of E. coli DH5 when added up to a concentration of 100 mg/L; inhibition was slight. Since Era is probably involved in a GTPase-receptor-coupled membrane-signalling pathway, P1 could competitively inhibit the binding of Era to the membrane or a downstream GTPase-receptor coupled regulatory step.

In order to act as an inhibitor, P1 would probably have to penetrate the cellular membrane. Since large peptides like P1 might have difficulty penetrating the bacterial membrane, an increase in the lipophilicity of the inhibitor might increase the absorption of the P1 peptide. Our results suggested that detergents containing a lauroyl group (MLG, SDS and lauric acid) increased the permeability of cells to P1, although this has not been demonstrated directly. The precise role of the detergent in enhancing the activity of P1 remains to be determined. E. coli cells treated with P1 and MLG also appeared to be elongated, so P1 may inhibit the function of Era during cell division. Pogliano et al.9 demonstrated that cpxA mutants of E. coli, which exhibit impaired membrane functions, produce irregular septation. This could result from alteration of the signal transduction system required for normal cell division. It was also of interest that S. mutans cells treated with P1 plus lauric acid did not demonstrate a similar alteration in cell septation (data not shown). This apparent paradox may reflect fundamental differences in cell division mechanisms between rod-shaped E. coli and coccal S. mutans cells.10

SGP and Era share some amino acid motifs which facilitate GTP binding and hydrolysis; SGP can replace Era in E. coli3 and expression of SGP antisense RNA inhibits the growth of not only S. mutans but also E. coli under stress conditions.5 Our study showed that S. mutans was more sensitive to P1 in the presence of detergents relative to E. coli. This may be because P1 is identical to the SGP domain but not to the equivalent Era domain. However, cell division in S. mutans (assessed by microscopic examination and chromosomal DNA staining with propidium iodide) did not not appear to be affected, unlike that in E. coli. Since S. mutans normally has a chain-like morphology, it would be difficult to observe elongation if it had occurred. The sensitivity of bacteria to P1 also varies between species. L. monocytogenes was more sensitive than E. coli but less so than S. mutans. The sensitivity might be affected by structural differences in cell walls or cellular membranes in addition to the actual sequences of the G-proteins.

Many G-proteins have been discovered in eukaryotic cells1 and there is cross-species complementation between some bacteria,3 so it is important to assess the potential toxicity of P1 if it is to be used in medicine or food processing. P1 was not toxic to fungal or human epithelial cells. However, additional testing will be necessary for further assessment of peptides similar to P1 as potential antibacterial agents and it will be of interest to enhance the susceptibility of the peptide agents in medical and industrial settings.

In summary, we have described the antibacterial effects of a peptide, P1, targeted against the Era-like G-proteins of bacteria. In the presence of permeability-enhancing detergents, the peptide showed antibacterial activity for several Gram-positive and Gram-negative bacteria. These results suggest that antimicrobial agents directed against these G-proteins may represent a novel class of antibacterial agents. Additional testing of P1 and related peptides with a broad spectrum of antibacterial activity both in vitro and in vivo will be necessary to assess adequately the applicability of such an approach.


    Acknowledgments
 
We gratefully acknowledge the assistance of Yiping Han in carrying out the toxicity assays. This work was supported by Unilever Research, Vlaardingen, The Netherlands.


    Notes
 
* Corresponding author. Tel: +1-716-829-2068; Fax: +1-716-829-3942; E-mail: kuramits{at}acsu.buffalo.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Bourne, H. R., Sanders, D. A. & McCormick, F. (1990). The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125–32.[ISI][Medline]

2 . Zuber, M., Hoover, T. A., Dertzbaugh, M. T. & Court, D. I. (1997). A Francisella tularensis DNA clone complements Escherichia coli defective for the production of Era, an essential Ras-like GTP-binding protein. Gene 189, 31–4.[ISI][Medline]

3 . Pillutla, R. C., Sharer, J. D., Gulati, P. S., Wu, E., Yamashita, Y., Lerner, C. G. et al. (1995). Cross-species complementation of the indispensable Escherichia coli era gene highlights amino acid regions essential for activity. Journal of Bacteriology 177, 2194–6.[Abstract]

4 . Yamashita, Y., Takehara, T. & Kuramitsu, H. K. (1993). Molecular characterization of a Streptococcus mutans mutant altered in environmental stress responses. Journal of Bacteriology 175, 6220–8.[Abstract]

5 . Sato, T., Wu, J. & Kuramitsu, H. (1998). The sgp gene modulates stress responses of Streptococcus mutans: utilization of an antisense RNA strategy to investigate essential gene functions. FEMS Microbiology Letters 159, 241–5.[ISI][Medline]

6 . Britton, R. A., Powell, B. S., Dasgupta, S., Sun, Q., Margolin, W., Lupski, J. R. et al. (1998). Cell cycle arrest in Era GTPase mutants: a potential growth rate-regulated checkpoint in Escherichia coli. Molecular Microbiology 27, 739–50.[ISI][Medline]

7 . Chakraborty, T., Ebel, F., Wehland, J., Dufrenne, J. & Notermans, S. (1994). Naturally occurring virulence-attenuated isolates of Listeria monocytogenes capable of inducing long term protection against infection by virulent strains of homologous and heterologous serotypes. FEMS Immunology and Medical Microbiology 10, 1–10.[ISI][Medline]

8 . Lin, Y. P., Sharer, J. D. & March, P. E. (1994). GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for Era in Escherichia coli. Journal of Bacteriology 176, 44–9.[Abstract]

9 . Pogliano, J., Dong, J. M., Wulf, P. D., Furlong, D., Boyd, D., Losick, R. et al. (1998). Aberrant cell division and random FtsZ ring positioning in Escherichia coli cpxA mutants. Journal of Bacteriology 180, 3486–90.[Abstract/Free Full Text]

10 . Ayala, J. A., Garrido, T., De Pedro, M. A. & Vicente, M. (1994). Molecular biology of bacterial septation. In Bacterial Cell Wall, (Ghuysen, J. M. & Hakenbeck, R., Eds), pp. 73–101. Elsevier, Amsterdam.

Received 16 September 1999; returned 28 November 1999; revised 4 January 2000; accepted 23 February 2000





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