Complete synthesis of the bicyclic ring of a mutacin analog with orthogonally protected lanthionine via solid-phase intracyclization.

Mutacin 1140 (MU1140) is a naturally occurring lantibiotic derived from posttranslational modifications of a ribosomally synthesized peptide during the fermentation of a bacterium called Streptococcus mutans, the etiological agent of dental cavities. A practical approach for chemically synthesizing lantibiotics would be a valuable tool to expand the MU1140 library with additional semisynthetic analogs. In turn, an expanded library may prove useful to explore additional therapeutic indications for this pipeline of novel compounds. In this work, orthogonally protected lanthionine analogs were synthesized via an aziridine ring opening strategy. This lanthionine was utilized to synthesize a cysteamine (Cya) instead of the (S)-aminovinyl-D-cysteine (AviCys) that is naturally found in MU1140. The Cya containing bicyclic C/D ring of MU1140 was synthesized by Fmoc solid-phase peptide synthesis (SPPS). The linear peptides were synthesized using OPfp ester derivatives and using various common coupling reagents such as COMU and TCTU. The linear peptide was intracyclized with DEPBT to construct the so-called bicyclic ring C/D. This is the first report on the complete chemical synthesis of the bicyclic C/D ring of a MU1140 analog using orthogonally protected lanthionines using SPPS.

Site-directed mutations in the lanthipeptide mutacin 1140.

The oral bacterium Streptococcus mutans, strain JH1140, produces the antibiotic mutacin 1140. Mutacin 1140 belongs to a group of antibiotics called lanthipeptides. More specifically, mutacin 1140 is related to the epidermin type A(I) lanthipeptides. Mutagenesis experiments of this group of lanthipeptides have been primarily restricted to the posttranslationally modified meso-lanthionine and 3-methyllanthionine residues. Site-directed mutagenesis of the core peptide of mutacin 1140 was performed using the suicide vector pVA891. Substitutions of the N-terminal residue, the charged residue in the hinge region, and residues in ring A and intertwined rings C and D were investigated. A truncation and insertion of residues in ring A and intertwined rings C and D were also performed to determine whether or not they would alter the antimicrobial activity of the producing strain. Bioassays revealed that five of 14 mutants studied had improved antimicrobial activity against the indicator strain Micrococcus luteus ATCC 10240. MICs against Streptococcus mutans UA159, Streptococcus pneumoniae ATCC 27336, Staphylococcus aureus ATCC 25923, Clostridium difficile UK1, and Micrococcus luteus ATCC 10240 were determined for three mutacin 1140 variants that had the most significant increases in bioactivity in the M. luteus bioassay. This mutagenesis study of the epidermin group of lanthipeptides shows that antimicrobial activity can be significantly improved.

Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B.

BACKGROUND: Shigatoxigenic E. coli are a global and emerging health concern. Shiga toxin, Stx, is encoded on the genome of temperate, lambdoid Stx phages. Genes essential for phage maintenance and replication are encoded on approximately 50% of the genome, while most of the remaining genes are of unknown function nor is it known if these annotated hypothetical genes are even expressed. It is hypothesized that many of the latter have been maintained due to positive selection pressure, and that some, expressed in the lysogen host, have a role in pathogenicity. This study used Change Mediated Antigen Technology (CMAT)™ and 2D-PAGE, in combination with RT-qPCR, to identify Stx phage genes that are expressed in E. coli during the lysogenic cycle. RESULTS: Lysogen cultures propagated for 5-6 hours produced a high cell density with a low proportion of spontaneous prophage induction events. The expression of 26 phage genes was detected in these cultures by differential 2D-PAGE of expressed proteins and CMAT. Detailed analyses of 10 of these genes revealed that three were unequivocally expressed in the lysogen, two expressed from a known lysogenic cycle promoter and one uncoupled from the phage regulatory network. CONCLUSION: Propagation of a lysogen culture in which no cells at all are undergoing spontaneous lysis is impossible. To overcome this, RT-qPCR was used to determine gene expression profiles associated with the growth phase of lysogens. This enabled the definitive identification of three lambdoid Stx phage genes that are expressed in the lysogen and seven that are expressed during lysis. Conservation of these genes in this phage genome, and other Stx phages where they have been identified as present, indicates their importance in the phage/lysogen life cycle, with possible implications for the biology and pathogenicity of the bacterial host.

Safety assessment of ProBiora3, a probiotic mouthwash: subchronic toxicity study in rats.

Streptococcus viridans are commensal bacteria that constitute a significant portion of the resident oral microflora. The objective of the present study is to investigate adverse effects, if any, of a blend of 3 natural strains, Streptococcus uberis KJ2, Streptococcus oralis KJ3, and Streptococcus rattus JH145 (probiotic mouthwash, ProBiora(3)). The blend is administered to rats orally once daily (5 days per week) at doses of 0, 10(6), or 10(9) colony-forming units of each strain for 14 weeks. No treatment-related adverse effects are observed in the physiological parameters during the study or in the evaluation of blood and tissue samples taken from the animals at the end. Results of an in vitro antibiotic susceptibility study demonstrate that all 3 ProBiora(3) strains are susceptible to commonly used therapeutic antibiotics. The results of these investigations reveal that the no-observed-adverse-effect level of the probiotic mouthwash is 2.16 x 10(9) colony-forming units per strain per kilogram of body weight per day, the highest dose used.

In vivo induced antigenic determinants of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a Gram-negative capnophilic rod and the etiological agent of localized aggressive periodontitis. The genome-wide survey of A. actinomycetemcomitans using in vivo induced antigen technology (IVIAT) has previously resulted in the discovery of antigenic determinants expressed specifically in diseased patients. The present study evaluated the potential of these antigens as putative disease markers, and investigating their contribution to the pathogenesis of the microorganism. Sera from patients had a significantly greater antibody titer than sera from healthy controls against six antigens, which supports the in vivo expression of these antigens, and suggests their usefulness as disease markers. A. actinomycetemcomitans invasion of epithelium-derived HeLa cells resulted in the induction of all three genes tested, as evidenced by real-time PCR. Isogenic mutants of these three genes were constructed and the adhesion and intracellular survival of the mutants was assayed in a competition assay with the wild-type strain. A significant defect in the intracellular survival of two of these mutant strains (orf1402 and orf859) was found. This defect could not be attributed to an adhesion defect. In contrast, a mutation in vapA, a homologue of a novel putative transcriptional regulator, out-competed the wild-type strain in the same assay. The virulent phenotype was restored for a mutant strain in orf859 upon complementation. This data provided new insight into the pathogenic personality of A. actinomycetemcomitans in vivo and supported the use of HeLa cells as a valid in vitro host-pathogen interactions model for that microorganism. IVIAT is applicable to most pathogens and will undoubtedly lead to the discovery of novel therapies, antibiotics and diagnostic tools.

Human serum binding and its effect on the pharmacodynamics of the lantibiotic MU1140.

The degree of MU1140 binding to human serum was measured and the effect of serum on MU1140 pharmacodynamics against Streptococcus pneumoniae and Staphylococcus aureus was investigated. 92.7% ± 2.0% of total MU1140 was bound to serum components as determined by ultrafiltration when tested in the concentration range 6.25-200 µg/ml. MIC and time-kill studies were used to study the effect of serum on the dynamics of MU1140. Serum inhibited MU1140 activity against S. pneumoniae but was found to enhance its activity against S. aureus. This phenomenon has not been reported for any other lantibiotic. Time-kill studies of MU1140 against S. aureus in various concentrations of serum revealed that the greatest bactericidal effect was observed at the lowest serum concentration. Mathematical modeling was used to quantify serum augmentation of MU1140 activity against S. aureus. Serum, at the lowest concentration, was shown to decrease MU1140 EC(50) against S. aureus by an order of magnitude. The data suggests that unbound MU1140 comprise the pharmacologically active fraction. Further, these findings suggest the possible existence of a complex dual inhibition and augmentation effect of serum on MU1140's activity against S. aureus. The molecular mechanism responsible for the synergistic action of human serum on MU1140's activity against S. aureus remains to be elucidated.

Pharmacokinetic and pharmacodynamic evaluation of the lantibiotic MU1140.

Presented are the pharmacokinetics (PK), exposure-response relationship, and the PK/pharmacodynamic (PD) index predictive of maximum therapeutic efficacy for the lantibiotic MU1140. MU1140, at a dose of 12.5 or 25 mg/kg, was administered intravenously, to characterize its PK parameters in rat. The recently developed in vitro PD model of MU1140 activity was enhanced by incorporation of the PK of MU1140 in rat. The linked PK/PD model was used in a simulation study to determine the PK/PD index predictive of in vivo efficacy. MU1140 total plasma concentration-time profiles declined biexponentially with elimination terminal half-life of 1.6 +/- 0.1 h. Rapid injection of MU1140 was associated with a hypersensitivity reaction that can be blocked by premedication with diphenhydramine. The simulation study revealed that Staphylococcus aureus concentrations correlated with T > MIC making it the PK/PD index best predictive of efficacy. Collectively, these findings suggest that the best route of administration of MU1140 is slow infusion which will increase the time its concentration remains above the MIC, thus maximizing the therapeutic effect and minimizing the observed toxicity.

Development and validation of a LC-MS quantification method for the lantibiotic MU1140 in rat plasma.

This study reports the first ever development and validation of a quantification method for a lantibiotic in plasma. This method was developed for the quantification of total MU1140 in Sprague Dawley rat plasma. The procedure involved acidification of plasma samples with formic acid followed by precipitation of plasma proteins using isopropanol, filtration, and analysis by RPLC-MS. The lantibiotic gallidermin was used as an internal standard (ISTD). The analyte and ISTD were eluted using a gradient of isopropanol and water, both acidified with 0.3% formic acid (v/v), at a flow rate of 250 microl/min. Positive electrospray ionization was utilized at the ion source and the analyte and ISTD were both detected by selected-ion monitoring (SIM). Total run time was 15 min. This method was validated for selectivity, sensitivity, linearity, recovery, accuracy, and precision. The method was shown to be selective, with a quantitative linear range of 0.39-100 microg/ml using 25 microl samples. The bias, intra- and inter-day percent relative standard deviation at all concentrations tested was lower than 15%. MU1140 mean extraction recovery was 96.1%. The analyte was shown to be stable to freeze/thaw and for short- and long-term storage. Extracted MU1140 was stable at 4 degrees C for over 5 days. This method was successfully applied to a preliminary pharmacokinetic study of intravenously administered MU1140 in Sprague Dawley rats. Overall, this method was shown to be applicable for quantification of MU1140 in plasma samples for the purpose of further MU1140 ADME or bioequivalence studies.

Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis.

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.

In vivo induced antigen technology (IVIAT) and change mediated antigen technology (CMAT).

In this chapter, an overview of in vivo induced antigen technology (IVIAT) and change mediated antigen technology (CMAT) will be presented, including a discussion of the advantages and limitations of these methods. Over fifteen different microbial pathogens have been or are known to be currently studied with these methods. Salient data obtained from the application of IVIAT and/or CMAT to a selection of human and plant pathogens will be summarized. This includes recent reports on Streptococcus pyogenes (Group A) in neurological disorders and invasive diseases, Xylella fastidiosa in Pierce's disease, Xanthomonas campestris in bean blight, Salmonella enterica serovar typhi in typhoid fever and Leishmania spp. related infections. Special emphasis will be given to those targets that have been further investigated for the development of novel vaccine, diagnostic and/or antibiotherapy strategies. This encompasses a new point-of-care serological diagnostic test for chronic periodontal diseases. Finally, Mycobacterium tuberculosis in vivo induced products will be described as providing a rational basis for differentiating subjects with primary, dormant or secondary tuberculosis infections, from control subjects who have or did not have prior vaccination with BCG.

Microarray analysis of quorum-sensing-regulated genes in Porphyromonas gingivalis.

Quorum sensing is a phenomenon defined as gene regulation in response to cell density that regulates various functions in bacteria. The periodontopathogen Porphyromonas gingivalis possesses a luxS gene homologue that may encode a quorum-sensing system. In order to identify genes of P. gingivalis that are regulated by luxS, gene expression analysis was done using microarrays and RNA samples from the W83 wild-type strain and an isogenic luxS mutant, LY2001. The results indicated that 17 open reading frames (ORFs) in LY2001 are upregulated and two are downregulated. Real-time PCR was done to confirm the microarray results. Among the upregulated ORFs is a group of stress-related genes, including htrA, clpB, groEL, dnaK, and the F subunit of alkyl hydroperoxide reductase. This suggested that luxS is involved in stress gene regulation in P. gingivalis. Stress response experiments, including high-temperature survival, resistance to hydrogen peroxide (H2O2), and survival during exposure to low and high pH, were performed on the P. gingivalis wild-type and LY2001 strains. LY2001 had a significantly higher survival rate than did W83 when stressed at 50 degrees C. No difference was found at pH 5, but LY2001 had increased survival compared to W83 at pH 9. LY2001 also survived better than W83 when stressed with 0.35 mM H2O2. These results suggest that luxS might be involved in promoting survival of P. gingivalis in the host by regulating its response to host-induced stresses such as temperature, H2O2, and pH.

In vivo induced antigen technology (IVIAT).

In vivo induced antigen technology (IVIAT) is a technique that identifies pathogen antigens that are immunogenic and expressed in vivo during human infection. IVIAT is complementary to other techniques that identify genes and their products expressed in vivo. Genes and gene pathways identified by IVIAT may play a role in virulence or pathogenesis during human infection, and may be appropriate for inclusion in therapeutic, vaccine or diagnostic applications.

Identification of group A Streptococcus antigenic determinants upregulated in vivo.

Group A Streptococcus (GAS) causes a range of diseases in humans, from mild noninvasive infections to severe invasive infections. The molecular basis for the varying severity of disease remains unclear. We identified genes expressed during invasive disease using in vivo-induced antigen technology (IVIAT), applied for the first time in a gram-positive organism. Convalescent-phase sera from patients with invasive disease were pooled, adsorbed against antigens derived from in vitro-grown GAS, and used to screen a GAS genomic expression library. A murine model of invasive GAS disease was included as an additional source of sera for screening. Sequencing DNA inserts from clones reactive with both human and mouse sera indicated 16 open reading frames with homology to genes involved in metabolic activity to genes of unknown function. Of these, seven genes were assessed for their differential expression by quantitative real-time PCR both in vivo, utilizing a murine model of invasive GAS disease, and in vitro at different time points of growth. Three gene products-a putative penicillin-binding protein 1A, a putative lipoprotein, and a conserved hypothetical protein homologous to a putative translation initiation inhibitor in Vibrio vulnificus-were upregulated in vivo, suggesting that these genes play a role during invasive disease.

Use of in vivo-induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection.

Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.

Genetically modified Streptococcus mutans for the prevention of dental caries.

There are many examples of positive and negative interactions between different species of bacteria inhabiting the same ecosystem. This observation provides the basis for a novel approach to preventing microbial diseases called replacement therapy. In this approach, a harmless effector strain is permanently implanted in the host's microflora. Once established, the presence of the effector strain prevents the colonization or outgrowth of a particular pathogen. In the case of dental caries, replacement therapy has involved construction of an effector strain called BCS3-L1, which was derived from a clinical Streptococcus mutans isolate. Recombinant DNA technology was used to delete the gene encoding lactate dehydrogenase in BCS3-L1 making it entirely deficient in lactic acid production. This effector strain was also designed to produce elevated amounts of a novel peptide antibiotic called mutacin 1140 that gives it a strong selective advantage over most other strains of S. mutans. In laboratory and rodent model studies, BCS3-L1 was found to be genetically stable and to produce no apparent deleterious side effects during prolonged colonization. BCS3-L1 was significantly less cariogenic than wild-type S. mutans in gnotobiotic rats, and it did not contribute at all to the cariogenic potential of the indigenous flora of conventional Sprague-Dawley rats. And, its strong colonization properties indicated that a single application of the BCS3-L1 effector strain to human subjects should result in its permanent implantation and displacement over time of indigenous, disease-causing S. mutans strains. Thus, BCS3-L1 replacement therapy for the prevention of dental caries is an example of biofilm engineering that offers the potential for a highly efficient, cost effective augmentation of conventional prevention strategies. It is hoped that the eventual success of replacement therapy for the prevention of dental caries will stimulate the use of this approach in the prevention of other bacterial diseases.

Identification and testing of Porphyromonas gingivalis virulence genes with a pPGIVET system.

An in vivo expression technology (IVET) system was designed to identify previously unknown virulence genes of Porphyromonas gingivalis. Fourteen ivi (for in vivo induced) genes that are induced during infection in a mouse abscess model were identified in our study. Of these, seven had homology to genes in the NCBI database, and the rest had no homology to reported DNA sequences. In order to determine virulence-related properties of these genes, three mutant strains, deleted of ivi8 (no homology to genes in the database), ivi10 (homologous to a putative TonB-dependent outer membrane receptor protein), and ivi11 (an immunoreactive 33-kDa antigen PG125 in P. gingivalis), were created. The mutants were tested in a mouse abscess model for alterations in virulence relative to the wild type by a competition assay in BALB/c mice. After 5 days we observed the enrichment of the wild-type strain over mutant strains Deltaivi10 and Deltaivi11, which indicated that mutant strains Deltaivi10 and Deltaivi11 are less able to survive in this model than the wild-type strain, while Deltaivi8 survives as well as the wild-type strain. We propose that knockout of these ivi genes reduced the ability of the mutated P. gingivalis to survive and cause infection compared to the wild-type strain at the site of injection. Also, in separate experiments, groups of mice were challenged with subcutaneous injections of each individual mutant strain (Deltaivi8, Deltaivi10, and Deltaivi11) or with the wild-type strain alone and were then examined to assess their general health status. The results showed that knockout of these ivi genes conferred a reduction in virulence. The ability of the mutants to invade KB cells compared to the wild type was also determined. Interestingly, the CFU counts of the mutant strain Deltaivi10 recovered from KB cells were eight times lower than those of the wild type, indicating that this mutant has a lower capacity for invasion. These results demonstrate that IVET is a powerful tool in discovering virulence genes and the significant role that ivi genes play in the pathogenesis of this species.

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Identification of Candida albicans genes induced during thrush offers insight into pathogenesis.

Candida albicans causes a wide spectrum of diseases, ranging from mucocutaneous infections like oral thrush to disseminated candidiasis. Screening for C. albicans genes expressed within infected hosts might advance understanding of candidal pathogenesis, but is impractical using existing techniques. In this study, we used an antibody-based strategy to identify C. albicans genes expressed during thrush. We adsorbed sera from HIV-infected patients with thrush against candidal cells grown in vitro and screened a C. albicans genomic expression library. We identified 10 genes encoding immunogenic antigens and used reverse transcription-polymerase chain reaction to verify that they were induced within thrush pseudomembranes recovered from a patient. The in vivo induced genes are involved in diverse functions, including regulation of yeast-hyphal morphogenesis, adhesion to host cells, nutrient uptake, phospholipid biosynthesis and amino acid catabolism. Four genes encode known virulence determinants (HWP1, CST20, CPP1 and RBF1). Another gene, LPD1, for which a role in candidal pathogenesis is unknown, encodes a protein homologous to a bacterial virulence determinant. Most importantly, disruption of CaNOT5, a newly identified gene, conferred defects in morphogenesis, decreased adherence to human buccal epithelial cells and attenuated mortality during murine disseminated candidiasis, proving that our strategy can identify genes encoding novel virulence determinants.

Genes of periodontopathogens expressed during human disease.

BACKGROUND: Since many bacterial genes are environmentally regulated, the screening for virulence-associated factors using classical genetic and molecular biology approaches can be biased under laboratory growth conditions of a given pathogen, because the required conditions for expression of many virulence factors may not occur during in vitro growth. Thus, technologies have been developed during the past several years to identify genes that are expressed during disease using animal models of human disease. However, animal models are not always truly representative of human disease, and with many pathogens, there is no appropriate animal model. METHODS: A new technology, in vivo-induced antigen technology (IVIAT) was thus engineered and tested in our laboratory to screen for genes of pathogenic organisms induced specifically in humans, without the use of animal or artificial models of infection. This technology uses pooled sera from patients to probe for genes expressed exclusively in vivo (or ivi, in vivo-induced genes). IVIAT was originally designed for the study of Actinobacillus actinomycetemcomitans pathogenesis, but we have now extended it to other oral pathogens including Porphyromonas gingivalis. RESULTS: One hundred seventy-one thousand (171,000) clones from P. gingivalis strain W83 were screened and 144 were confirmed positive. Over 300,000 A. actinomycetemcomitans clones were probed, and 116 were confirmed positive using a quantitative blot assay. CONCLUSION: MAT has proven useful in identifying previously unknown in vivo-induced genes that are likely involved in virulence and are thus excellent candidates for use in diagnostic : and therapeutic strategies, including vaccine design.