The SloR metalloregulator is involved in the Streptococcus mutans oxidative stress response.

SloR, a 25-kDa metalloregulatory protein in Streptococcus mutans modulates the expression of multiple genes, including the sloABC operon that encodes essential Mn2+ transport and genes that promote cariogenesis. In this study, we report on SloC- and SloR-deficient strains of S. mutans (GMS284 and GMS584, respectively) that demonstrate compromised survivorship compared with their UA159 wild-type progenitor and their complemented strains (GMS285 and GMS585, respectively), when challenged with streptonigrin and/or in growth competition experiments. The results of streptonigrin assays revealed significantly larger zones of inhibition for GMS584 than for either UA159 or GMS585, indicating weakened S. mutans survivorship in the absence of SloR. Competition assays revealed a compromised ability for GMS284 and GMS584 to survive peroxide challenge compared with their SloC- and SloR-proficient counterparts. These findings are consistent with a role for SloC and SloR in S. mutans aerotolerance. We also predicted differential expression of oxidative stress tolerance genes in GMS584 versus UA159 and GMS585 when grown aerobically. The results of quantitative RT-PCR experiments revealed S. mutans sod, tpx, and sloC expression that was upregulated in GMS584 compared with UA159 and GMS585, indicating that the impact of oxidative stress on S. mutans is more severe in the absence of SloR than in its presence. The results of electrophoretic mobility shift assays indicate that SloR does not bind to the sod or tpx promoter regions directly, implicating intermediaries that may arbitrate the SloR response to oxidative stress.

A role for the DtxR family of metalloregulators in gram-positive pathogenesis.

Given the central role of transition metal ions in a variety of biochemical processes, the colonization, survival, and proliferation of a bacterium within a host hinges upon its ability to overcome the metal ion deprivation that characterizes nutritional immunity. Metalloregulatory, or 'metal-sensing' proteins have evolved in bacteria to mediate metal ion homeostasis by activating or repressing the expression of genes encoding metal ion transport systems upon binding their cognate metal ion. Yet increasing evidence in the literature supports an additional role for these metalloregulatory proteins in pathogenesis. Herein, we survey studies on the DtxR family of metalloregulators, namely DtxR (Cornyebacterium diphtheriae), SloR (Streptococcus mutans), MtsR (Streptococcus pyogenes), and MntR (Staphylococcus aureus) to describe how metalloregulation enables adaptive virulence gene expression within the mammalian host. This research has important implications for drug design, as the generation of hyper-repressive metalloregulatory proteins may represent a mechanism by which to attenuate bacterial pathogenicity. The fact that metalloregulators are unique to prokaryotes makes these proteins especially attractive therapeutic targets.

Gene regulation in S. mutans: complex control in a complex environment.

Dental caries is a chronic infectious disease of multifactorial etiology that derives from the interplay among cariogenic bacteria on the dentition, the host diet, and other environmental exposures. Streptococcus mutans proliferates as a biofilm on the tooth surface, where it obtains nutrients and metabolizes fermentable dietary carbohydrates. The accumulation of lactic acid as a by-product of fermentation results in acidification of the plaque biofilm and demineralization of tooth enamel, marking the onset of decay. The ability of S. mutans to respond to environmental stresses presented by salivary flow, acid pH, oxidative stress, and changes in carbohydrate source and availability is essential for its survival and predominance in caries lesions. Importantly, S. mutans has evolved a network of regulators to integrate its cellular response to environmental change. Herein we describe the latest insights into global gene regulation in S. mutans, including mechanisms of signal transduction, carbon catabolite repression, and quorum-sensing. An improved understanding of these regulatory networks can provide a basis for novel therapeutic applications aimed at treating and/or preventing caries.

Regulated expression of the Streptococcus mutans dlt genes correlates with intracellular polysaccharide accumulation.

Intracellular polysaccharides (IPS) are glycogen-like storage polymers which contribute significantly to Streptococcus mutans-induced cariogenesis. We previously identified and cloned a locus from the S. mutans chromosome which is required for the accumulation of IPS. Sequencing of this locus revealed at least four contiguous open reading frames, all of which are preceded by a common promoter region and are transcribed in the same direction. Analysis of the amino acid sequence deduced from the first of these open reading frames (ORF1) revealed domains which are highly conserved among D-alanine-activating enzymes (DltA) in Lactobacillus rhamnosus (formerly Lactobacillus casei) and Bacillus subtilis. The deduced amino acid sequences derived from ORF2, -3, and -4 also exhibit extensive similarity to DltB, -C, and -D, respectively, in these microorganisms. However, Southern hybridization experiments indicate that this operon maps to a locus on the S. mutans chromosome which is separate from the glgP, glgA, and glgD genes, whose products are known mediators of bacterial IPS accumulation. We therefore assigned a new dlt designation to the locus which we had formerly called glg. We maintain that the dlt genes are involved in S. mutans IPS accumulation, however, since they complement a mutation in trans which otherwise renders S. mutans IPS deficient. In this study, we found that expression of the S. mutans dlt genes is growth phase dependent and is modulated by carbohydrates internalized via the phosphoenolpyruvate phosphotransferase system (PTS). We demonstrated that the S. mutans dlt genes are expressed constitutively when non-PTS sugars are provided as the sole source of carbohydrate. Consistent with a role for the PTS in dlt expression is a similar constitutive expression of the dlt genes in an S. mutans PTS mutant grown in a chemically defined medium supplemented with glucose. In summary, these findings support a novel role for the dlt gene products in S. mutans IPS accumulation and suggest that dlt expression in this oral pathogen is subject to complex mechanisms of control imposed by growth phase, dietary carbohydrate, and other factors present in the plaque environment.

A Streptococcus mutans mutant that synthesizes elevated levels of intracellular polysaccharide is hypercariogenic in vivo.

We used the streptococcal transposon, Tn916 to identify and isolate mutants of Streptococcus mutans with altered intracellular polysaccharide (IPS) accumulation. We report on the isolation and characterization of S. mutans SMS202, a transposon mutant which accumulated the glycogen-like IPS in excess of wild-type levels. Southern blot analysis confirmed a single Tn916 insertion into the SMS202 chromosome. Moreover, quantitative ultrastructural analysis revealed significantly increased concentrations of IPS in SMS202 relative to those of the wild-type progenitor strain, UA130. The activities of ADPglucose pyrophosphorylase (GlgC) and glycogen synthase (GlgA), enzymes required for the biosynthesis of bacterial IPS, were also elevated in the IPS excess mutant. Furthermore, SMS202 was significantly more cariogenic on the molar surfaces of germ-free rats than the wild type (P < 0.01), thus confirming a central role for IPS in S. mutants-induced caries formation. We propose that the increased cariogenic potential of SMS202 is due to constitutive expression of genes which encode glycogen biosynthesis in this oral pathogen. The coordinate expression of GlgC and GlgA along with the results of ongoing nucleotide sequence analysis and Northern hybridization experiments support an operon-like arrangement for the glg genes of this oral pathogen.

The fimA locus of Streptococcus parasanguis encodes an ATP-binding membrane transport system.

The gene encoding fimA, a 36 kDa fimbrial adhesion of Streptococcus parasanguis FW213, is highly conserved in all four genetic groups of sanguis streptococci. FimA-like peptides were produced by all strains tested. The nucleotide sequence directly upstream of fimA contains two open reading frames, ORF5 and ORF1, whose deduced protein products are homologous to members of a superfamily of ATP-binding cassette membrane transport proteins, including both prokaryotic and eukaryotic uptake and export systems. The amino acid sequence of FimA contains the consensus prolipoprotein cleavage site (LxxC) common to the 'periplasmic' binding proteins of Gram-positive transport systems. The deduced product of ORF5 is a 28.6 kDa membrane-associated protein that has the consensus binding site for ATP (GxxGxGKS). It shares significant homology with AmiE of Streptococcus pneumoniae as well as with Escherichia coli proteins involved in iron(III) uptake. Allelic-replacement mutagenesis of ORF5 resulted in greatly increased resistance to aminopterin. These data demonstrate functionality with the amiE locus as well. The deduced product of ORF1 is an extremely hydrophobic integral membrane protein of 30.8 kDa with a pattern of six potential membrane-spanning regions, typical of a component of these types of transport system. The nucleotide sequence downstream of fimA, ORF3, encodes a 20 kDa protein having 78% identity with the 20 kDa protein encoded downstream of ssaB, a fimA homologue in S. sanguis 12. It also exhibits significant homology with bacterioferritin co-migratory protein (Bcp) of E. coli K-12. Allelic-replacement mutagenesis in the fimA locus of FW213 showed that (i) expression of fimA was initiated at a site far upstream of the fimA start codon, and (ii) expression of fimA was not linked to expression of ORF3. Northern blots probed with internal fragments of ORF5, ORF1, fimA or ORF3 hybridized to the same transcript of 3.3 kb, which suggested that these loci were transcribed as a polycistronic message. The ORF3 probe also hybridized to a 540 bp transcript consistent with the size of ORF3 alone and supportive of the mutagenesis data of non-linkage. Strains mutated in fimA continued to produce fimbriae, indicating that FimA was not the fimbrial structural subunit. Immunoelectron microscopy revealed FimA was localized at the tips of the fimbriae of FW213. This is the first study that demonstrates that an adhesin which binds a bacterial cell to a substrate is associated with an ATP-binding cassette.

Chemotherapeutic treatment of recurrent and/or metastatic nasopharyngeal carcinoma: a retrospective analysis of 40 cases.

Authors carried out a review of 40 cases of recurrent and/or metastatic nasopharyngeal carcinoma (NPC) treated with cisplatin-based chemotherapy at the Division of Othorhinolaryngology and the Service of Chemotherapy of the University of Palermo between July 1984 and July 1992. All patients were treated with regimens comprising high dose cisplatin (80-100 mg m-2). Histologically there were 29 squamous cell and 11 undifferentiated NPC. Thirty-nine patients were evaluable for response and toxicity. The overall response rate was 64%, with a 20.5% complete response rate and a 43.5% partial response rate. The mean duration of complete responses was 10.2+months, while that of partial responses was 8.6+months. The mean survival of the whole group was 11.4+months, with four patients alive after 2 years of follow-up. No statistically significant difference in response rate and survival was found between patients with metastatic disease and those with locoregional recurrency, and between patients with squamous cell NPC and those with undifferentiated histology. The employed regimens have been generally well tolerated. These data confirm that NPC is a neoplasm highly responsive to chemotherapy. However, duration of objective response and survival are still largely unsatisfactory.

Head and neck carcinoma with distant metastases: a retrospective analysis of 44 cases treated with cisplatin-based chemotherapeutic regimens.

The rarity of medical reports on the chemotherapeutic management of head and neck cancer metastatic to distant organs prompted us to review the effect of cisplatin-based regimens in this clinical setting. Out of 44 eligible patients, 10 patients (23%) achieved a CR, 16 patients (36%) has a PR, 7 (16%) no change, and 11 (25%) progressed. Patients with rhinopharyngeal carcinoma showed a 69% overall response rate, while those with other head and neck carcinomas had a 54% overall response rate. No preferential site of response was detected. The difference in mean survival of responding patients between the rhinopharyngeal group and the non-rhinopharyngeal group was statistically significant (P < 0.05). Responding patients survived longer than non responders (P < 0.05 in both groups). Interestingly, 3 patients in the rhinopharyngeal cancer group survived more than 2 years from the start of chemotherapy for metastatic disease. These data strengthen the observation that rhinopharyngeal carcinoma, even with distant metastases, responds to chemotherapy better than other carcinomas arising in the head and neck region. Moreover, although survival is still dismal, cisplatin-based systemic chemotherapy seems an effective palliative treatment for metastatic head and neck cancer.

High-dose folinic acid and 5-fluorouracil plus cisplatin on a weekly schedule in the treatment of advanced cancer of the head and neck.

A group of 60 patients with advanced head/neck cancer were treated with high-dose folinic acid (500 mg/m-2/week-1) plus 5-fluorouracil (400 mg/m-2/week-1 on day 1, and cisplatin (20 mg/m-2/week-1) 24 h after folinic acid infusion was completed. Out of 55 evaluable patients, 10 patients (18%) experienced a complete response with a mean duration of 11.4+ months, 25 patients had a partial response (45%) of 6.7+ months, 6 patients (11%) showed a stabilization of 4.8+ months, and 14 (25%) progressed. The overall response rate was 63.6% (95% confidence limits 56.5%-69.5%). Patients pretreated with radiotherapy had a 67% overall response rate, while those pretreated with chemotherapy showed a 54% overall response rate. All patients with cancer of the oropharynx had a major response, while patients with cancer of the oral cavity had the lowest response rate. The mean survival of patients who attained a complete response was 14.5+ months. Partial responders had a mean survival of 10.6+ months, while patients who progresses survived a mean of 3.6+ months. The treatment has been very well tolerated with few cases of grade 3 gastrointestinal toxicity. Grade 1-2 leukopenia was recorded in 64% of cases, grade 1-2 nausea/vomiting in 85%. In one case therapy was stopped because of persistent diarrhoea.

Bactericidal activity and toxicity of iodine-containing solutions in wounds.

Complexing iodine with povidone (polyvinylpyrrolidone) or surfactants significantly limits the quantity of free iodine. Reduction of the free iodine level eliminates the adverse properties of staining, instability, and irritation and also alters bactericidal activity. Addition of detergents to create surgical scrub solutions further reduces the activity of iodine. In vitro testing indicated that the bactericidal activity of iodophors was inferior to that of uncomplexed aqueous iodine. In vivo tests proved that aqueous iodine significantly potentiated the development of infection. Although the povidone iodophor did not enhance the rate of wound or infection, it offered no therapeutic benefit when compared with control wounds treated with saline solution. Addition of detergents to the povidone iodophor was deleterious, with the wounds exposed to this combination displaying significantly higher infection rates than untreated control wounds. Based on these results, aqueous iodine solutions and iodophor surgical scrub solutions should not be used on broken skin. Aqueous iodophors can be used in wounds, but no therapeutic benefit from such use was found in this study.