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CONCISE REVIEW |
Institute of Dental Research, Westmead Centre for Oral Health, PO Box 533, Wentworthville, NSW 2145, Australia;
* corresponding author, njacques{at}dental.wsahs.nsw.gov.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODS OF PROTEOME ANALYSISPROTEOMICS OF ORAL PATHOGENSCURRENT LIMITATIONS OF AND...CONCLUSIONREFERENCES |
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KEY WORDS: proteomics • two-dimensional electrophoresis • MUDPIT • mass spectrometry • oral micro-organisms
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODS OF PROTEOME ANALYSISPROTEOMICS OF ORAL PATHOGENSCURRENT LIMITATIONS OF AND...CONCLUSIONREFERENCES |
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METHODS OF PROTEOME ANALYSIS |
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TOPABSTRACTINTRODUCTIONMETHODS OF PROTEOME ANALYSISPROTEOMICS OF ORAL PATHOGENSCURRENT LIMITATIONS OF AND...CONCLUSIONREFERENCES |
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Quantitative visualization of proteins on 2-DE gels is paramount for differential or comparative proteome analyses (Fig. 1
). Silver staining offers high sensitivity but poor linear dynamic range, while Coomassie Blue staining lacks sensitivity. Many researchers overcome this problem by first using a fluorescent stain, such as SYPRO Ruby, that has a linear dynamic range almost 700 times greater than that of silver stain and allows for accurate visualization and quantification of proteins with the use of a variety of analytical software (Lopez et al., 2000). A second stain, such as Coomassie Blue, is then used to reveal proteins for excision prior to analysis (Cordwell et al., 2002).
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Mass spectrometry is by far the most common technique used in proteome analysis for the identification of an unknown protein, although alternatives such as N-terminal amino acid analysis or Western blotting can also be used. Several different mass spectrometers are commercially available with various levels of sensitivity (Aebersold and Cravatt, 2002). Matrix-assisted laser desorption-ionization (MALDI)-time of flight (TOF) instruments measure peptide mass only. These instruments are robust and relatively cheap and, while not highly sensitive, can generally analyze excised proteins from Coomassie-Blue-stained 2-DE gels. In the MALDI-TOF process, proteins are first digested with proteinases such as trypsin and the peptides mixed with a large excess of UV-absorbing matrix (usually a cinnamic acid derivative) and allowed to dry to a small spot on a MALDI plate (Fig. 2B). A pulsed laser illuminates the spot in a vacuum, and the peptide ions are vaporized in the resulting gas plume and extracted by an electric field into a TOF mass analyser. More sophisticated tandem mass spectrometers allow peptide sequences to be determined. In these spectrometers, a peptide ion is selected by mass/charge and broken down further into fragments that allow the peptide to be sequenced. Sequence plus mass is more informative than mass alone, and for small amounts of sample, or a mixture of a few peptides, sequencing is essential is a protein is to be identified with confidence.
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). In practice, a comprehensive proteome analysis requires the PMF to be compared with an annotated protein database constructed from the genome of the organism under investigation. If the genome has not been sequenced, the PMF can be compared with very closely related species from the same genera. For example, PMFs from 2-DE protein spots obtained from Streptococcus oralis and S. mutans have been used to search the annotated genomic database of Streptococcus pneumoniae and Streptococcus pyogenes (Wilkins et al., 2001, 2002). Unfortunately, successful protein identification relies on a high degree of peptide sequence identity between species. Any alteration in peptide sequences and peptide masses will result in a failure of the protein to be identified by MALDI-TOF techniques. As noted, MALDI-TOF is not that sensitive, and hence many proteins detected on 2-DE gels with the use of fluorescent stains or radiolabeling are of insufficient concentration for analysis by this technique (Len et al., 2003). Where peptides already exist, such as those eluted by MUDPIT, they can be analyzed online by mass spectrometers equipped with electrospray ionization (ESI) injectors rather than MALDI plates. In these instruments, a voltage is applied to a fine needle containing a dilute solution of the peptides. This results in a spray of droplets typically containing just a few molecules. Repeated break-up of the droplets due to evaporation eventually leads to release of intact peptide ions into the gas phase, whence they are sampled into the mass spectrometer and analyzed. Since these instruments work online and at atmospheric pressure, continuous uninterrupted analysis of peptides is possible.
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PROTEOMICS OF ORAL PATHOGENS |
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TOPABSTRACTINTRODUCTIONMETHODS OF PROTEOME ANALYSISPROTEOMICS OF ORAL PATHOGENSCURRENT LIMITATIONS OF AND...CONCLUSIONREFERENCES |
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Dental Caries
The mutans streptococci, including S. mutans and Streptococcus sobrinus, are generally associated with the initial phase of human dental caries, since their acidogenic and aciduric properties allow them to create a low-pH environment in dental plaque following the ingestion of sugars. It is not surprising, therefore, that several 2-DE proteome studies have concentrated on the physiological adaptations associated with S. mutans survival in the oral cavity. For example, 2-DE protein analyses of 14C-labeled cellular proteins of S. mutans have been used to characterize changes in protein expression following the imposition of pH, temperature, salt, and oxidative and starvation stresses (Svensäter et al., 2000). S. mutans responded to these adverse environmental conditions by a complex and diverse alteration in protein synthesis. For instance, the protein profile of cells shocked from pH 7.5 to pH 5.0 revealed 64 proteins that were up-regulated (25 of them acid-specific) and 49 that were down-regulated. In a similar study, 78 14C-labeled cellular proteins were diminished and 57 enhanced out of a total of 694 analyzed, when S. mutans underwent a transition from the planktonic to the biofilm state (Svensäter et al., 2001). S. mutans also expressed 13 unique proteins in the biofilm state, while 9 others present in planktonic cells could not be detected. Mass spectral analysis resulted in the identification of 41 proteins, 21 of which were enhanced in biofilm cells and the remainder reduced. In general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while proteins associated with protein synthesis, protein folding, and replication were enhanced.
In a third 2-DE study, 18 proteins were up-regulated and 12 down-regulated when S. mutans was grown at pH 5.2 compared with cells grown at pH 7.0. These proteins were involved in energy metabolism, cell division, translation, and transport (Wilkins et al., 2002). Although the general conclusions of previous studies were confirmed, anomalies were observed. For instance, the protein DnaK was down-regulated when S. mutans was grown at low pH, while transcriptional studies had shown that dnaK gene expression was up-regulated under similar conditions (Jayaraman et al., 1997). The failure of other stress-related proteins to be detected emphasizes an important aspect of current proteomics involving incomplete 2-DE displays, since any undetected changes may be of equal importance in an understanding of the true nature of phenotypic change. The role of stress-related proteins, however, has been examined in S. mutans lacking the Clp ATPase, ClpP. S. mutans lacking ClpP was impaired in its ability to grow at low pH and had a reduced capacity to form biofilms (Lemos and Burne, 2002). Comparison of silver-stained 2-DE gels revealed at least 28 proteins with altered levels of expression in the clpP mutant compared with the parent. In particular, evidence was presented that the molecular chaperones DnaK, GroEL, and GroES were elevated in the mutant. The loss of ClpP appeared to induce a stress response in the cells, possibly due to the accumulation of denatured proteins that are normally targeted by the ClpP proteinase.
Following a similar mutagenic approach, a two-component regulator system, defined by the genes hk11 and rr11, was also shown to be involved in biofilm formation and acid resistance in S. mutans (Li et al., 2002). Deletion of the hk11 gene resulted in a mutant that formed biofilms with reduced biomass and possessed greatly reduced resistance to low pH. Autoradiograms of 2-DE gels revealed 594 cellular proteins, 19 of which were acid-regulated in the parent, but only 15 in the mutant. Two of the 4 missing proteins were putatively identified as an exopolyphosphatase previously linked to biofilm formation, and the histidine kinase HK11 itself.
The presence of fluoride in the environment represents another stress factor for many cariogenic bacteria. Fluoride not only protects tooth enamel from bacterial acids by forming fluorapatite in the outer layers of enamel, it also directly alters the bacterial phenotype. For example, 2-DE analysis of S. sobrinus grown in the presence of fluoride revealed that several proteins were influenced by the presence of fluoride, including the loss of a glucan-binding lectin activity (Cox et al., 1999).
Infective Endocarditis
Streptococcus gordonii and S. oralis are among the earliest colonizers of the primary dentition and are part of the healthy microbial flora in dental plaque (Whiley and Beighton, 1998). Both S. gordonii and S. oralis, however, are associated with community-acquired infective endocarditis, should they gain access to the vascular system (Douglas et al., 1993). Oral bacteria in a healthy mouth are exposed to slightly acidic conditions (pH 6.0 to 6.5), but when they gain access to the bloodstream, there is an immediate rise to pH 7.3. This may be a stimulus for a change in protein expression and the subsequent ability of the bacteria to colonize a damaged heart valve (Vriesema et al., 2000). Glyceraldehyde-3-phosphate dehydrogenase is implicated as a virulence determinant aiding S. pyogenes invasion of tissues and is considered to act as a possible defense against the immune system in S. gordonii (Nelson et al., 2001). Interestingly, when S. oralis grown at either pH 5.2 or pH 7.0 was analyzed by 2-DE, 28 cellular proteins, including glyceraldehyde-3-phosphate dehydrogenase, were down-regulated at pH 7.0 (Wilkins et al., 2001). At first sight, this observation seems incompatible with a role for the protein as a virulence determinant in infective endocarditis. However, in S. gordonii, the glyceraldehyde-3-phosphate dehydrogenase becomes the major extracellular protein as the pH rises to pH 7.5 (Nelson et al., 2001). Since the amount of extracellular glyceraldehyde-3-phosphate dehydrogenase was not determined when S. oralis was grown at different pHs, it is not clear whether the reduction in the amount of cellular protein is due to down-regulation of expression or simply secretion into the extracellular milieu as the pH rises. Clearly, further analysis of the extracellular proteins of S. oralis is warranted to determine the true nature of the change in expression of this and other proteins.
Antibiotic resistance is a major problem for the control of infective endocarditis, requiring an understanding of the changes in the proteome of the resistant phenotype. To this end, the mechanism of penicillin tolerance has been examined in S. gordonii by 2-DE (Caldelari et al., 2000). A tolerant mutant contained two proteins with increased intensity. Comparison of the sequences of their N-terminal amino acids with known proteins showed that they were homologous to the N-termini of arginine deiminase and ornithine carbamoyl transferase of the arginine deiminase (arc) operon. Although the penicillin-tolerance mutation mapped at a physically distinct location on the chromosome from the arc operon, genetic transformation of tolerance always conferred arc deregulation. It was concluded that the tolerance mutant affected a global regulatory mechanism that was important for survival in the presence of penicillin. The correlation between tolerance and arc deregulation has allowed a reporter system to be developed that will aid in determining the central mechanism of penicillin tolerance underlying this clinically important phenotype.
Periodontal Disease
Porphyromonas gingivalis is associated with chronic periodontitis and co-adheres to primary colonizers such as S. gordonii and Actinomyces spp. present in dental plaque. Since surface proteins are associated with adhesion and other virulence traits, the outer membrane sub-proteome of P. gingivalis was studied by 2-DE (Veith et al., 2001). Of the 39 outer membrane proteins identified, several displayed pI heterogeneity that was observed as a train of horizontal spots on 2-DE gels. For the proteins Omp40 and Omp41, conformational equilibria resulting from incomplete denaturation were shown to account for this phenomenon (Veith et al., 2001). Other charged isoforms were not investigated, so it is not known whether this phenomenon can account for all of these observations or whether the isoforms are due to post-translational modifications or some other artefact. However, analysis of the multiple Mr forms of the cysteine proteinases, RgpA and Kgp, and the putative hemagglutinin, HagA, indicated that they resulted from a series of specific C-terminal truncations due to the RgpA and Kgp proteinases themselves. The observed vertical streaking of RgpA appeared to be due to the covalent binding of lipopolysaccharide to the C-terminus of the proteinase, suggesting a possible mode of attachment of the enzyme to the outer membrane of P. gingivalis. These results are a clear example of how proteomics in combination with other techniques can lead to a better understanding of the biochemistry and physiology of a cell—in this case, the nature of expression of key virulence traits.
Oral Candidiasis
Candida infections are the most frequently encountered fungal diseases of the oral cavity. As well as being associated with antibiotic or prolonged steroid use and with denture stomatitis, oral candidiasis presents as one of the initial manifestations of acquired immune deficiency syndrome. Virulence factors of Candida albicans have been widely studied and include its ability to convert from a yeast-like to a mycelial form capable of penetrating tissue (Niimi et al., 1999). Differential 2-DE analysis of this morphological transition failed to identify any proteins uniquely associated with either morphology, though many were preferentially synthesized in germ-tube-forming cells (Niimi et al., 1996). While these proteins may subsequently prove to be regulators of morphogenic change, none was identified, and so their biological significance could not be evaluated.
Several other studies of C. albicans have combined 2-DE with Western blotting to identify antigens that react to antibodies present in human sera. This method has successfully identified several new antigens that may be useful in developing diagnostic strategies aimed at treating systemic candidiasis (Barea et al., 1999; Pardo et al., 2000). Current treatment of Candida infections is impeded by the limited number of antifungal drugs and is complicated by the emergence of azole-resistant strains associated with changes in azole efflux (White et al., 1998). Azole-susceptible and -resistant isolates of Candida glabrata have been compared by 2-DE. Twenty-five proteins were up-regulated and 76 down-regulated in the resistant isolate (Marichal et al., 1997). Since these proteins were not identified, further research is needed to determine their involvement in drug resistance.
Other Pathogens
Micro-organisms other than bacteria and fungi are involved in oral diseases. These include protozoans and viruses. For example, the protozoan Entamoeba gingivalis is found in patients with destructive periodontal disease, where it attacks and destroys both erythrocytes and leukocytes (Lyons et al., 1983). Its presence has also been associated with human immunodeficiency virus diagnosis (Lucht et al., 1998). Unfortunately, research on protozoan proteomes is being hindered by the current lack of protozoan proteins in public databases (Rabilloud et al., 1999; Cohen et al., 2002). However, this situation should improve in the future as more genomes are sequenced.
Unlike protozoan proteomes, viral proteomes are relatively small, and many have already been predicted from their genomic sequence (Blattner et al., 1997). Viral proteome studies tend to center on the effects of a virus on its host. For instance, a recent study based on the 2-DE differential display of HeLa cell proteins has shown that the 2A proteinase of human Rhinovirus and Coxsachie virus cleaves cytokeratin 8 in the early stages of infection (Seipelt et al., 2000). In a similar study, a combination of 2-DE and Western blotting allowed the receptors on HeLa and human lung carcinoma cells that bind Parechovirus 1 and Echovirus 1 to be identified as integrins 
vß3 and 2ß1, respectively (Triantafilou and Triantafilou, 2001).
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CURRENT LIMITATIONS OF AND FUTURE DEVELOPMENTS FOR PROTEOME ANALYSIS |
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TOPABSTRACTINTRODUCTIONMETHODS OF PROTEOME ANALYSISPROTEOMICS OF ORAL PATHOGENSCURRENT LIMITATIONS OF AND...CONCLUSIONREFERENCES |
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMETHODS OF PROTEOME ANALYSISPROTEOMICS OF ORAL PATHOGENSCURRENT LIMITATIONS OF AND...CONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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Received January 3, 2003; Last revision June 2, 2003; Accepted August 6, 2003
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TOPABSTRACTINTRODUCTIONMETHODS OF PROTEOME ANALYSISPROTEOMICS OF ORAL PATHOGENSCURRENT LIMITATIONS OF AND...CONCLUSIONREFERENCES |
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