Dental caries is the most common bacterial disease of humans and occurs when oral bacteria produce acids, following their fermentation of dietary carbohydrates. This acid can then cause a localised demineralisation of the tooth surface. A group of seven species of bacteria, collectively known as the mutans streptococci, have been predominantly implicated in the onset of dental caries. In particular, Streptococcus mutans and Streptococcus sobrinus have been shown to be the main aetiological agents of this disease in humans. Most attempts to control the microbial component of caries target these bacteria.
The past 50 years has provided considerable information about the pathogenesis of dental caries, the likely route and time of transmission of cariogenic bacteria to susceptible hosts and possible ways of either treating or controlling the onset of this disease. In regards to the latter, many techniques (such as the use of tooth brushes, mouth washes, dental floss and tooth paste) for the control of plaque build-up exist and the examples listed are generally part of a daily routine. However, these techniques need to be applied regularly, and as such only highly-motivated individuals generally experience improved oral health. Therefore, the search for more effective less labour-intensive approaches continues. One area of research is into the potential application of small ribosomally-synthesised antimicrobial peptides, known as bacteriocins. Bacteriocins generally inhibit closely-related species that occupy the same ecological niche. Their relatively-specific targeting, plus the fact that many are remarkably heat and chemically-stable molecules, makes them excellent candidates for possible anti-caries applications.
Numerous bacteriocins produced by the lactic acid bacteria have now been identified. Most can be broadly categorised into one of four main classes, of which Class I, the lantibiotics and Class II, the small (<10 kDa) non-modified peptides, contain the most examples. Many screens for anti-mutans streptococcal (MS) bacteriocins have been carried out and it appears that the best source of anti-MS bacteriocins are the mutans streptococci themselves. Research in this laboratory has identified examples of anti-mutans streptococcal bacteriocins produced by both mutans streptococci and non-mutans streptococci. The present study investigated the anti-MS inhibitors produced by two streptococcal strains, S. mutans N and Streptococcus sanguis K11. During the course of this study a third strain, S. mutans UA159, was also studied for its bacteriocinogenic properties.
Although S. sanguis K11 produces anti-mutans streptococcal inhibitory activity, this appears only effective against Streptococcus rattus. In addition however, the inhibitory activity of this strain is also directed against all tested strains of Streptococcus agalactiae and ca. 50% of Streptococcus pyogenes. In the present study a 5069 Da novel inhibitory agent (sanguicin K11) was characterised and shown responsible for this unusual inhibitory spectrum. Through reverse genetics the sanK11 locus was identified and shown to encode a Class II type bacteriocin, the first shown to be produced by S. sanguis. Following screens of additional S. sanguis, sanK11 was shown to be present only in strains producing the same type of inhibitory pattern (P-type) as strain K11. The cysteine residues at positions 7 and 38 of the sanguicin K11 propeptide were shown to form a disulphide bridge essential for sanguicin K11 inhibitory activity.
S. mutans N and eight other S. mutans strains have been found to have what appears to be the same inhibitory spectrum, which includes members of the mutans streptococci and several other oral streptococcal species. One strain (UA140) of the eight has previously been shown to produce the lantibiotic mutacin I and the non-lantibiotic mutacin IV. S. mutans N was known to produce the non-lantibiotic mutacin N. The current study set out to investigate how two strains, apparently producing completely different bacteriocins could have the same inhibitory spectrum. Reverse genetics identified the mutacin N structural gene (mutN) and mutagenesis studies showed that this bacteriocin was responsible only for the inhibitory activity against mutans streptococci. Further sequencing around the mutN locus identified a second bacteriocin-like locus (mutO) adjacent to mutN. mutO was also identified to have anti-mutans streptococcal inhibitory activity and because of the close proximity of mutO and mutN and given the homology they share with other known two-peptide bacteriocins it seemed probable that mutacins O and N are components of a new member of this special class of bacteriocins (Class IIb, the two peptide bacteriocins) in which the optimal inhibitory activity is dependent on the co-operative activity of the two peptides.
Further investigations of strain N examined the expression of mutacins O and N. During a search for a suitable heterologous non-mutacinogenic S. mutans strain to act as an expression host, the genome reference strain, S. mutans UA159 was given consideration. However, contrary to previous reports, this strain was found to exhibit bacteriocin-like inhibitory activity. During a follow-up investigation, strain UA159 was found to inhibit 84 strains representing 11 different species of bacteria, but no inhibition of mutans streptococci was detected. The locus (nlmAB) encoding the two-peptide bacteriocin mutacin IV was identified within the UA159 genome. Using genetic dissection of nlmA and nlmB, the contribution of each peptide was examined and it was found that only the NlmA* propeptide appears to be active, raising doubts as to whether mutacin IV is a bona fide two-peptide bacteriocin. Deletion of the entire nlmAB locus created a mutant strain that exhibited a loss of inhibitory activity against the same 64 strains as was found for the nlmA mutant. A BLASTP search for the consensus leader sequence that precedes the propeptide of Class II bacteriocins, identified ORFs encoding 9 more putative bacteriocin-like peptides. Further genetic dissection identified the SMU.1914c locus as being responsible for the inhibitory activity against a further 15 strains not already sensitive to mutacin IV. SMU.1914c was renamed mutacin V. However, it appears that another as yet unidentified mutacin(s) is also produced by strain UA159 given that three indicator strains still remained sensitive to a double mutant [UA[Delta](1914/NlmAB)] in which both the mutacin IV and putative mutacin V loci were inactivated.
Export of Class II bacteriocins has been found to occur by either a SEC-dependent system or via a dedicated peptide ATP Binding Cassette (ABC) transporter. Three potential ABC transporter ORFs were identified in S. mutans UA159. Two (comA and cslA) had the characteristic accessory factor ORF (comB and cslB respectively) located adjacent to the main ABC transporter ORF, while the third ORF763 appeared to lack this. Mutagenesis of each of these five ORFS was carried out and confirmed cslAB to be the ABC transporter involved in the export of the competence stimulating factor, while the function of ORF763 could not be established in this study. Mutagenesis of either comA or comB resulted in a complete cessation of bacteriocin production by the respective mutant strains. Historically, comA and comB is the nomenclature used for loci encoding the exporter of the competence inducing factors in streptococci. In light of this new information, comA and comB were renamed nlmT and nlmE respectively, to account for the newly defined role of this ABC transporter.
The present study investigated four bacteriocins two of which (sanguicin K11 and mutacin ON) appear to have some potential for application to anti-caries control, and the others (mutacins IV and V) being shown to be produced by the genome reference strain (UA159). All three mutacins were shown to be exported from their respective producer cells by the NlmTE ABC transporter, while sanguicin K11 is predicted to be exported by a peptide ABC transporter located adjacent to sanK11.
Bacteriocins may yet provide a novel alternative for the treatment and control of dental caries. In their favour is that fact that they have relatively narrow defined inhibitory spectra and thus are unlikely to produce widespread changes to plaque ecosystems. Potential uses include as topical agents where bacteriocin preparations could be incorporated into dentrifices such as toothpastes or mouthwashes. Alternatively, streptococci producing anti-mutans streptococcal bacteriocins could be implanted into the oral cavity in strain replacement therapy strategies. There are pros and cons to each technique and the most effective anti-caries control appears more likely to result from "cocktail therapy" where bacteriocins are combined with a number of other anti-mutans streptococcal agents to achieve long-lasting protection against mutans streptococcus proliferation.
| Identifer | oai:union.ndltd.org:ADTP/217398 |
| Date | January 2006 |
| Creators | Hale, John D. F., n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright John D. F. Hale |
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