[Show abstract][Hide abstract] ABSTRACT: 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
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Dec 2010 · European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Nov 2009 · Journal of Pharmaceutical Sciences
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Oct 2009 · International Journal of Toxicology
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jun 2009 · Journal of pharmaceutical and biomedical analysis
[Show abstract][Hide abstract] ABSTRACT: To conduct a pilot human clinical trial to assess the safety and to test the ability of a probiotic mouthwash, ProBiora(3), to affect the levels of Streptococcus mutans and certain known periodontal pathogens in the mouth when administered twice daily over a period of 4 weeks.
The mouthwash contained three specific strains of naturally occurring oral bacteria and was tested at two dose levels: 10(6) and 10(8) colony forming units each of Strep. oralis strain KJ3sm, Strep. uberis strain KJ2sm, and the spontaneous lactic acid-deficient variant of Strep. rattus, strain JH145. Substantial decreases in the levels of the marker bacteria were observed. No safety issues were noted with the twice daily application of this mouthwash.
Despite the small number of subjects and the use of young, orally healthy adults, along with the inherent variability in the microbiological measurements, the probiotic mouthwash was able to substantially affect the levels of dental pathogens in saliva and periodontal pathogens in subgingival plaque.
The results of this pilot human study suggest that the probiotic mouthwash product may be safe for daily use as an aid in maintaining both dental and periodontal health.
No preview · Article · Apr 2009 · Journal of Applied Microbiology
[Show abstract][Hide abstract] ABSTRACT: To study the ability of daily applications of Streptococcus rattus strain JH145 to affect the numbers of an implanted Streptococcus mutans strain in a rat model.
A spontaneous L(+)-lactate dehydrogenase (LDH)-deficient mutant of Streptococcus rattus, JH146, was isolated by screening on selective medium and compared with a previously isolated spontaneous LDH deficient strain, JH145. Both strains were shown to have single base pair deletion mutations in the structural gene (ldh) for LDH, and reversion frequencies were approximately the same. Animals treated once daily with >or=10(6) CFU (colony forming units) of JH145 showed a statistically significant decrease in the proportion of implanted S. mutans to total cultivable bacteria in oral swab samples. The rate of decrease in S. mutans levels was dose-dependent. No adverse effects were observed by in-life observation of treated animals, and histopathological, haematological and blood chemistry analyses were unremarkable.
The results presented indicate that daily application of JH145, a naturally occurring LDH-deficient variant of S. rattus, can compete with S. mutans for its habitat on the tooth surface.
S. rattus JH145 has potential as a probiotic for use in the prevention of dental caries.
[Show abstract][Hide abstract] ABSTRACT: Mutacin 1140 and nisin A are peptide antibiotics that belong to the lantibiotic family. N-Terminal rings A and B of nisin A and mutacin 1140 (lipid II-binding domain) share many structural and sequence similarities. Nisin A binds lipid II and thus disrupts cell wall synthesis and also forms transmembrane pores. Very little is known about mutacin 1140 in this regard. We performed fluorescence-based studies using a bacteria-mimetic membrane system. The results indicated that lipid II monomers are arranged differently in the mutacin 1140 complex than in the nisin A complex. These differences in complex formation may be attributed to the fact that nisin A uses lipid II to form a distinct pore complex, while mutacin 1140 does not form pores in this membrane system. Further experiments demonstrated that the mutacin 1140-lipid II and nisin A-lipid II complexes are very stable and capable of withstanding competition from each other. Transmembrane electrical potential experiments using a Streptococcus rattus strain, which is sensitive to mutacin 1140, demonstrated that mutacin 1140 does not form pores in this strain even at a concentration 8 times higher than the minimum inhibitory concentration (MIC). Circular complexes of mutacin 1140 and nisin A were observed by electron microscopy, providing direct evidence for a lateral assembly mechanism for these antibiotics. Mutacin 1140 did exhibit a membrane disruptive function in another commonly used artificial bacterial membrane system, and its disruptive activity was enhanced by increasing amounts of anionic phospholipids.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Molecular pathogenesis of Tannerella forsythia, a putative periodontal pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here, identification of in vivo expressed antigens of T. forsythia is reported using in vivo-induced antigen technology (IVIAT). Among 13 000 recombinant clones screened, 16 positive clones were identified that reacted reproducibly with sera obtained from patients with periodontal disease. DNA sequences from 12 of these in vivo-induced genes were determined. IVIAT-identified protein antigens of T. forsythia include: BspA, a well-defined virulence factor of T. forsythia; enzymes involved in housekeeping functions (tRNA synthetases, glycine hydroxymethyltransferase, and glucoside glucohydrolase); enzymes implicated in tissue destruction (dipeptidyl peptidase IV); a DNA mismatch repair protein; and putative outer membrane proteins of unknown function. The in vivo gene expression of these IVIAT-identified antigens was confirmed by a quantitative real-time PCR analysis. This is, to the best of the authors' knowledge, the first report using IVIAT in T. forsythia. It is anticipated that detailed analysis of the in vivo-induced genes identified by IVIAT in this study will lead to a better understanding of the molecular mechanisms mediating periodontal infection by T. forsythia.
Full-text · Article · Nov 2007 · FEMS Microbiology Letters
[Show abstract][Hide abstract] ABSTRACT: To construct a genetically modified strain of Streptococcus mutans for dental caries prevention. The strain has significantly reduced cariogenicity owing to a deletion of the entire open reading frame for lactate dehydrogenase, and has excellent colonization potential through the production of a natural antibiotic called mutacin 1140. For use in human clinical trials, additional mutations were introduced to enable rapid elimination of the strain in case of adverse side effects and to increase genetic stability.
Deletion mutations were introduced into the dal gene for d-alanine biosynthesis and the comE gene for genetic transformation. The resulting strain, A2JM, was tested for dependence on exogenous d-alanine and its ability to be eradicated from colonized rats. The strain was also tested for its ability to exchange DNA with another strain of S. mutans in in vitro and in vivo models.
A2JM was completely dependent on exogenous d-alanine, but could colonize the oral cavity of rats in low numbers in the absence of dietary d-alanine. Results indicated that A2JM can scavenge d-alanine from other plaque bacteria. Lowering of the total oral bacterial load through daily application of chlorhexidine enabled virtually complete eradication of A2JM. The introduction of the comE gene did not significantly decrease the transformability of A2JM in in vitro or in vivo models. The addition of a deletion in the comE gene does, nonetheless, provide additional safety as it has a very low reversion frequency.
Based on the safety and efficacy profiles established in vitro and in animal models, A2JM appears suitable for safe use in human clinical trials.
Full-text · Article · Jun 2007 · Journal of Applied Microbiology
[Show abstract][Hide abstract] ABSTRACT: Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II-targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Oct 2006 · Infectious Disorders - Drug Targets(Formerly Current Drug Targets - Infectious Disorders)
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Oct 2005 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: 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°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.
Full-text · Article · Aug 2005 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jun 2005 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Feb 2005 · Cellular Microbiology