Histatins are a group of small cationic peptides in human saliva which are well known for their antibacterial and antifungal activities. In a previous study we demonstrated that histatin 5 kills both blastoconidia and germ tubes of Candida albicans in a time-and concentration-dependent manner at 37°C, whereas no killing was detected at 4°C. This indicated that killing activity depends on cellular energy. To test histatin 5 killing activity at lower cellular ATP levels at 37°C, respiratory mutants, or so-called petite mutants, of C. albicans were prepared. These mutants are deficient in respiration due to mutations in mitochondrial DNA. Mutants were initially identified by their small colony size and were further characterized with respect to colony morphology, growth characteristics, respiratory activity, and cytochrome spectra. The killing activity of histatin 5 at the highest concentration was only 28 to 30% against respiratory mutants, whereas 98% of the wild-type cells were killed. Furthermore, histatin 5 killing activity was also tested on wild-type cells in the presence of the respiratory inhibitor sodium azide or, alternatively, the uncoupler carbonyl cyanide m-chlorophenylhydrazone. In both cases histatin 5 killing activity was significantly reduced. Additionally, supernatants and pellets of cells incubated with histatin 5 in the presence or absence of inhibitors of mitochondrial ATP synthesis were analyzed by sodium dodecyl sulfate gel electrophoresis. It was observed that wild-type cells accumulated large amounts of histatin 5, while wild-type cells treated with inhibitors or petite mutants did not accumulate significant amounts of the peptide. These data showed first that cellular accumulation of histatin 5 is necessary for killing activity and second that accumulation of histatin 5 depends on the availability of cellular energy. Therefore, mitochondrial ATP synthesis is required for effective killing activity of histatin 5.

Histatins, a group of histidine-rich proteins in human saliva, exhibit antimicrobial activity and are therefore considered to be important in the prevention of infections in the oral cavity. Although killing of C. albicans by histatins has been extensively studied, little is known about the processes responsible for this antifungal activity. Recent studies show the requirement of metabolic activity and ATP production for histatin 5 killing activity. Therefore, the goal of this study was to investigate the kinetics of histatin 5 interaction at different temperatures with C. albicans wild type cells and with respiratory deficient mutants of C. albicans. Synthetic histatin 5 was labeled with fluorescein-5-isothiocyanate (FITC) and its association with C. albicans cells was followed by epi-fluorescence microscopy and fluorescence confocal microscopy. At 37 degrees C, histatin 5 accumulates intracellularly, and both killing activity and uptake of unlabeled and FITC-labeled histatin 5 are time- and concentration-dependent. At 4 degrees C, no killing is observed and FITC-histatin 5 is only associated with the cytoplasmic membrane. Internalization and killing activity only occurs after cells are transferred to 37 degrees C. In addition, cellular accumulation of histatin 5 is concomitant with a moderate alteration of membrane integrity leading to the release of UV-absorbing cell components into the medium. The uptake of histatin 5, the release of UV-absorbing materials and killing of C. albicans are markedly decreased by the respiratory inhibitor sodium azide. Concomitantly, respiratory deficient mutants of C. albicans are also less susceptible to histatin 5. These results indicated that histatin 5 killing activity could be directly correlated to histatin 5 internalization. Both of these processes are prevented by modulators of cellular metabolic activity.

Streptococcus gordonii, a primary colonizer, is part of the pioneer biofilm consortium that initiates dental plaque development on tooth surfaces. An insertion of Tn917-lac transposon into the adcR gene produced a biofilm-defective phenotype. S. gordonii adcR is a regulatory gene and is part of an operon (adc) that includes three other genes, adcCBA. AdcC contains a putative consensus-binding site for adenosine triphosphate, AdcB is a putative hydrophobic membrane protein, and AdcA is a putative lipoprotein permease. Mutants were constructed by insertional inactivation in each of the three adcCBA genes and their effects on biofilm formation examined. The adcC::spec(R) and adcB::spec(R) mutations displayed a biofilm-defective phenotype, whereas the adcA::spec(R) mutant was biofilm-positive in a static polystyrene microtiter plate biofilm assay. All three mutants formed poor biofilms in a flow-cell system and were competence-defective, suggesting the adc operon plays an important role in S. gordonii biofilm formation and competence.

Oral streptococci such as Streptococcus gordonii are facultative anaerobes that initiate biofilm formation on tooth surfaces. An isolated S. gordonii::Tn917-lac biofilm-defective mutant contained a transposon insertion in an open reading frame (ORF) encoding a homolog of NosX of Ralstonia eutropha, a putative maturation factor of nitrous oxide reductase. Located downstream are two genes, qor1 and qor2, predicted to encode two putative NADPH quinone oxidoreductases. These three genes are cotranscribed, forming a putative oxidative stress response (osr) operon in S. gordonii. Inactivation of nosX, qor1, or qor2 resulted in biofilm-defective phenotypes. Expression of nosX, measured by the ␤-galactosidase activity of the nosX::Tn917-lac mutant, was growth-phase dependent and enhanced when grown under aerobic conditions or in the presence of paraquat. Real-time reverse transcription-PCR revealed that nosX-specific mRNA levels were increased approximately 8.4 and 3.5 fold in biofilm-derived cells grown on plastic and glass, respectively, when compared to planktonic cells. Expression of nosX increased 19.9 fold in cells grown under aerated aerobic conditions and 4.7 fold in cells grown under static aerobic conditions. Two ORFs immediately adjacent to the osr operon encode a putative NADH oxidase (Nox) and a putative thiol-specific antioxidant enzyme (AhpC, for alkyl hydroperoxide peroxidase C). Expression of nox and ahpC was also significantly increased in cells grown under aerated and static aerobic conditions when compared to anaerobic conditions. In addition, nox expression was increased in biofilm cells compared to planktonic cells. These genes may be part of an island that deals with oxidoreductive response, some of which may be important in S. gordonii biofilm formation.

MG2 (the MUC7 gene product) is a low-molecular-mass mucin found in human submandibular/sublingual secretions. This mucin is believed to agglutinate a variety of microbes and thus is considered an important component of the non-immune host defence system in the oral cavity. We have shown that MUC7 can bind to cariogenic strains of Streptococcus mutans and that this binding requires a structural determinant in the N-terminal region. In the present study an expression construct, pNMuc7, encoding the N-terminal 144 amino acids of MUC7 was generated, and the recombinant protein rNMUC7 was expressed in Escherichia coli. Purified rNMUC7 was characterized and the binding of this protein to oral bacteria was investigated in an established assay. The results showed that the recombinant protein bound to S. mutans ATCC 25175 and ATCC 33402, and that alkylation of the two cysteine residues (Cys(45) and Cys(50)) resulted in the complete loss of bacterial binding. This suggests that binding of MUC7 to S. mutans occurs between the N-terminal region of the mucin molecule and the bacterial surface, and that this interaction is dependent on a cysteine-containing domain within this region of MUC7. In addition, the killing activity of rNMUC7 was compared with that of the candidacidal salivary protein histatin 5 in an established Candida albicans (ATCC 44505) blastoconidia killing assay. It was found that the LD(50) values of rNMUC7 and histatin 5 were comparable, and that the recombinant protein displayed significant killing activity at the physiological concentration range of MUC7 in whole saliva. This study is the first to show that the N-terminal region of MUC7 contains a structural determinant for bacterial binding and that this region exhibits candidacidal activity.