Recent research has focussed on the capabilities of oral bacteria to withstand acid stress typical of conditions in the biofilms on teeth (dental plaque). The capacity to invoke an acid tolerance response (ATR) ensuring increased survival at very low pH (i.e., 4-3) is particularly important for those agents associated with dental caries since lesions are highly acidic (1).
Recent research (2) has tested the acid sensitivity of 21 strains in six acidogenic oral genera and shown that the strains can be categorized as non-, weak, or strong adapters to acid tolerance, with the more aciduric bacteria in the latter group consisting of strains of Streptococcus mutans, Lactobacillus casei, S. salivarius and S. gordonii.
A subsequent study (3) examining the proteins induced during the ATR by 1D PAGE revealed that S. mutans LT11 had the most complex 'log-phase ATR' (LATR) when the cells received an 'acid shock' from pH 7.5 to 5.5 for 2 h resulting in the enhanced and transient synthesis of 36 proteins, 25 appearing in the first 30 min. Comparisons between Lactobacillus casei and S. mutans LT11 indicated that acid tolerance involved a combination of constitutive and inducible proteins.
More recent 2DE analysis of the LATR has just been completed with S. mutans H7 involving multiple stresses (acid, salt, heat, oxidative and starvation) that revealed significant responses to acid, starvation and heat stress with the synthesis of 52, 36 and 54 proteins, respectively, enhanced in the first 30 min of stress initiation (4). The proteins could be classified as general stress proteins (9), stress-specific proteins (acid-10) and a variable number of shared proteins common to other stresses, but not all stresses.
Cross protection studies showed a link between starvation and acid tolerance. Future studies will be directed at the isolation and identification of the 'key' stress proteins in S. mutans H7 by employing mass spectrometric amino acid analysis and protein database searches. Selected stress proteins will be selected based on label intensity, novelty or location, and its gene located using the amino acid sequence to generate a nucleotide probe. Following gene sequencing and inspection of adjacent regions for regulatory elements, the gene will be inactivated in order to carry out functional studies in comparison to the wild-type. These will include: (a) growth characteristics, (b) survival to multiple stresses, (c) screening tests for the induction of log and stationary phase acid responses, and (d) 2DE analysis to 'map' the protein.
More recent studies (5) with strains of oral streptococci has focussed on the capacity of log-phase (L) and stationary-phase (S) cells to survive a three hour exposure at pH 3.5. When cells were tested during normal batch growth, they exhibited pH-dependent L responses, however, only some strains exhibited S responses and these could be confirmed with glucose-starved cells incubated in fresh complex medium. The capacity of cells to exhibit stationary-phase acid tolerance was associated with their capacity to maintain endogenous metabolism in the absence of an exogenous carbon source.
Associated with the physiological studies on acid tolerance were molecular biological studies on the genes associated with acid tolerance in S. mutans. In order to identify genes important in acid homeostasis, we have subjected S. mutans LT11 to transposon mutagenesis with the vector, pGh9:ISSI, selecting for the acid-sensitive phenotype. One mutant (IS1A) was isolated unable to grow at pH 5.0 and, following marker rescue and identification of the flanking DNA sequences, GenBank Database searches revealed that the ISSI had inserted into a gene having homology to the dltB gene of the dlt operon of Lactobacillus casei. The dlt operon is involved in the biosynthesis of D-alanine-lipoteichoic acid and essential for a variety of cellular processes.
Since the original mutant had a 6.3 kb deletion upstream of the dltB gene, and included four complete genes, we have selectively inactivated the dltC gene, encoding the Dcl carrier protein, in the wild-type strain to determine the effect of disruption of the dlt operon on acid sensitivity. This defect was shown to result in a low growth rate in complex medium, the loss of the ability to initite growth below pH 7.0, and a significant reduction in the log-phase ATR.
The above research is funded by a grant from the Medical Research Council of Canada.