My Lien Dao

Identification and analysis of a collagenolytic activity in Streptococcus mutans.

Abstract.Streptococcus mutans is an important pathogen in coronal caries and is implicated in dental root decay by its ability to bind collagen from various sources. In the present study, electron microscopic analysis demonstrated the ability of S. mutans to bind and to disrupt collagen fibrils of the amniotic membrane. The synthetic peptide FALGPA, which is similar in structure to collagen, was degraded by S. mutans, with a lower level of FALGPA hydrolytic activity observed in sucrose-grown cells compared with cells grown in the absence of sucrose. Inhibition studies of FALGPA hydrolytic activity showed a pattern characteristic of collagenase activity, with inhibition by 1,10-phenanthroline and EDTA, but not by phenylmethylsulfonyl fluoride (PMSF). Additionally, immunological cross-reactivity was observed between proteins from disrupted cells of S. mutans and antiserum to collagenase from Clostridium histolyticum. Gelatinolytic activity was demonstrated by gelatin zymogram analysis. These findings suggest that collagenolytic activity by S. mutans may be an important virulence factor in dental root decay.

Identification, molecular cloning, and sequence analysis of a deoxyribose aldolase in Streptococcus mutans GS-5

Bacterial fitness in the environment, where nutrients are limited and competition is intense, plays a central role in survival and virulence of the organisms. Deoxyribose aldolase, found in several species of bacteria, is known to be involved in the catabolism of deoxynucleosides arising from dead cells, thereby giving a selective advantage to the microorganisms with a capability to consume DNA as an alternative carbon and energy source. A gene encoding a deoxyribose aldolase gene (deoC) was identified in the cariogenic Streptococcus mutans strain GS-5 by comparative sequence analysis and gene cloning. The gene encodes a protein of 220 amino acids, having a predicted molecular weight of 23.3 kDa with a pI of 5.44. The gene was cloned into the expression vector pFLAG-1™, and the biological function of the gene product was analyzed by a complementation assay with a deoC−Escherichia coli mutant SΦ063. Transformation of the E. coli SΦ063 with the plasmid construct allowed this organism to grow on glucose minimal medium supplemented with 2 mM deoxyadenosine or deoxythymidine. These results showed activity of deoxyribose aldolase, confirming the identity of the gene. Utilization of exogenous deoxynucleotides as a carbon and energy source may confer a survival and growth advantage to S. mutans over other bacteria in dental plaque, suggesting that deoxyribose aldolase may be a contributing factor to virulence.

Dietary Manipulation of Mammary Tumor Development in Adult C3H/Bi Mice

Abstract Chronic energy intake restriction (CEIR) in virgin female mice is one of the most effective ways of reducing significantly mammary adenocarcinoma in C3H/Bi mice, a strain which develops mammary adenocarcinoma associated with the murine mammary tumor virus spontaneously and at high incidence. In this study, the influence of chronic energy intake restriction imposed on fully mature (4- to 5-month-old), breeding female C3H/Bi mice was addressed, and the influence of energy intake where energy was derived largely from fat versus diets in which energy was derived largely from carbohydrates on tumor development and survival rate was investigated. The results show that chronic energy intake restriction can be delayed until full maturation and successful reproduction and still reduce significantly the incidence of mammary tumor development in this relatively short-lived strain of mice. Our findings demonstrate that the overriding dietary factor controlling mammary tumor development in these experiments in C3H/Bi mice was the level of energy intake, regardless of the primary source of energy (fat or carbohydrates).

High-Level Expression of a Truncated Wall-Associated Protein A from the Dental Cariogenic Streptococcus mutans

Streptococcus mutans plays a primary role in the formation of dental caries. Previously, in our laboratory, an S. mutans genomic library was prepared, and the wapA gene was cloned into the shuttle vector, pSA4/4B2. To generate overexpression of wapA and to facilitate efficient purification of the WapA protein for use as an immunogen, an expression vector with the strong tac promoter was used. In order to answer questions regarding the optimization of solubility and expression based on gene size or the hydrophobicity of the protein product, 12 truncated constructs of the wapA gene were prepared using PCR. The truncated products were subcloned into the pGEX-6P-1 glutathione S-transferase (GST) fusion vector and expressed in E. coli BL21. The fusion proteins were analyzed by SDS-PAGE and confirmed by analysis with anti-GST and anti-WapA antibodies. Our study suggests that abrogation of the wapA promoter is necessary for expression of this gene in this expression system. Deletion of the signal peptide and the hydrophobic C terminus of WapA increased expression compared with the full-length construct, and truncation at the protease cleavage site of the C-terminal region greatly increased the stability of the protein without a loss in reactivity with the anti-WapA antibody. Western immunoblot analysis with anti-WapA antiserum clearly showed that the majority of the epitopes of the GST-WapA fusions are located in the N-terminal region of WapA. The immunogenicity of the various WapA fusion products is being examined in mice and rats to further map the immunologically dominant regions of the protein. This method effectively increased the expression of WapA and should contribute to the further understanding of gene expression of E. coli, as well as aid in the characterization of this protein for future immunologic evaluation.

The impact of fatty acids on the antibacterial properties of N-thiolated β-lactams.

Bacterial fatty acid synthesis (FAS) is a potentially important, albeit controversial, target for antimicrobial therapy. Recent studies have suggested that the addition of exogenous fatty acids (FAs) to growth media can circumvent the effects of FAS-targeting compounds on bacterial growth. Consequently, such agents may have limited in vivo applicability for the treatment of human disease, as free FAs are abundant within the body. Our group has previously developed N-thiolated β-lactams and found they function by interfering with FAS in select pathogenic bacteria, including MRSA. To determine if the FAS targeting activity of N-thiolated β-lactams can be abrogated by exogenous fatty acids, we performed MIC determinations for MRSA strains cultured with the fatty acids oleic acid and Tween 80. We find that, whilst the activity of the known FAS inhibitor triclosan is severely compromised by the addition of both oleic acid and Tween 80, exogenous FAs do not mitigate the antibacterial activity of N-thiolated β-lactams towards MRSA. Consequently, we propose that N-thiolated β-lactams are unique amongst FAS-inhibiting antimicrobials, as their effects are unimpeded by exogenous FAs.

Effects of brevetoxins on murine myeloma SP2/O cells: aberrant cellular division.

Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-k Da protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclindependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.

Modified FALGPA assay for cell-associated collagenolytic activity

A continuous spectrophotometric assay monitoring the hydrolysis of the synthetic peptide 2-furanacryloyl-l-leucylglycyl-l-prolyl-l-alanine (FALGPA) is used to measure collagenase activity of both bacterial and vertebral collagenases. In the present study, a protocol was developed to adapt this assay to the measurement of cell-associated FALGPA hydrolytic activity in bacteria. The bacteria tested included Bacillus cereus, Streptococcus agalactiae, Streptococcus mutans, Enterococcus faecalis, and Escherichia coli, and various levels of activity were identified. The method presented here allows the detection of FALGPA hydrolysis using a small quantity of cells without the need for prior purification of the collagenolytic enzyme or collection and concentration of a large volume of culture supernatant fluid.

Expression of Streptococcus mutans Wall-Associated Protein A Gene in Chinese Hamster Ovary Cells: Prospect for a Dental Caries DNA Vaccine

The Streptococcus mutans strain GS-5 wall-associated protein A (Wap-A) is a precursor to the extracellular antigen A (AgA), a recognized candidate dental caries vaccine. The full-length wapA gene (wapA-E) and a C-terminal truncated version (wapA-G) encoding the AgA were cloned into the mammalian expression vector pcDNA 3.1/V5/His-TOPO. The resulting constructs were propagated in the Escherichia coli Top10. To investigate the expression of the S. mutans genes in mammalian cells, the above constructs were used to transfect Chinese hamster ovary (CHO) cells in the presence of the cationic lipid pfx-8. Transient expression of the wapA-E and wapA-G genes was observed at 24 h post-transfection, as shown by Western immunoblot analysis using a rabbit antiserum to S. mutans cell wall. Immunochemical staining of the transfected CHO cells showed expression of WapA mainly in the cells and budding vesicles, whereas AgA was found mainly in the transfected cells and extracellular medium. The expression of S. mutans proteins in CHO cells, in either vesicles or soluble form, suggested an antibody response to the above DNA constructs. Work is under way to test the efficacy of these as DNA vaccines against S. mutans.