Summary analysis of Effects of Oxygen on Virulence Traits of Streptococcus Mutans

I wrote summary of this publication for my microbial genetics class. Probably pretty bland to most, but might as well post it.

Chalmer Wren

March 20, 2008

Summary analysis of Effects of Oxygen on Virulence Traits of Streptococcus Mutans

This is a summary of Effects of Oxygen on Virulence Traits of Streptococcus Mutans, by Sang-Joon Ahn, Zezhang T. Wen, and Robert A. Burne. The researchers sought to investigate the effects of aerobic and anaerobic conditions on the virulence traits of S. mutans. S. mutans, a gram-positive lactic acid bacterial species, is the primary contributor to tooth decay (1, p.266). This species of bacteria is localized in the crevices and pits of the teeth, which are a normal part of tooth topology. Tooth decay occurs as the result the acidic byproducts of the bacteria’s metabolic processes. Differential gene expression was first investigated by means of DNA microarray analysis, and the results validated by real-time quantitative RT-PCR on a subset of genes. Included among the genes which were down-regulated under aerobic conditions is gtfB, which encodes an enzyme critical to the process of biofilm production. The biofilm is an extracellular polysaccharide matrix which contributes to the virulence of S. mutans by protecting it from adverse environmental conditions and allowing it to adhere strongly to its substrate. Because the GtfB and GtfC proteins both play a significant role in biofilm production, the regulation of the genes encoding them were further investigated under aerobic and anaerobic conditions using a CAT assay and a western blot. Furthermore, the role of the VicK sensor kinase of a CovRS-like two component system (TCS) and the autolysin AtlA in S. mutans response to oxygen were also studied.

DNA microarray analysis is a powerful tool for studying genetics because it allows researchers to potentially monitor thousands of genes. Microarray analysis measures the relative amounts of RNA being produced via transcription by a cell (5, p.517). First, the desired RNA is isolated from the cells and purified. This RNA is then treated with reverse transcriptase and appropriate primers, resulting in the formation of a DNA RNA hybrid. The original RNA is then nicked by an enzyme so that DNA polymerase can utilize the RNA as a primer and then replace the RNA with DNA. The result of this process is called complementary DNA, or cDNA (5, p.769). The cDNA can then be cloned into a vector and labeled with a reporter gene. The cDNA is then allowed to hybridize with the immobilized DNA fragments of the microarray (5). This type of analysis indicates whether genes transcription is taking place, but is limited in its analysis because transcribed mRNA is not always necessarily translated. While useful for studying gene regulation at the transcriptional level, DNA microarray analysis reveals less about gene regulation at the level of translation.

DNA microarray analysis revealed that about 5% of the S. mutans genome displayed differences in genes expression, the majority of which were up-regulated under aerobic conditions. Table 3 lists the differentially expressed genes and the magnitude of difference in expression of each gene between aerobic and anaerobic conditions. Also listed in Table 3 are the P-values, which is an indication of the reasonableness of acquired measurements. Under aerobic conditions 83 genes displayed up-regulation while only 23 genes displayed down-regulation. Figure 1 shows the distribution of genes whose expression was altered by the availability of oxygen. Up-regulated genes were placed in five categories. 28 of the up-regulated genes were classified as hypothetical, unassigned, or unknown, 23 were classified as encoding energy metabolism proteins, 9 encoding signal transduction proteins, 8 encoding transport and binding proteins, and 4 encoding cellular process-proteins.

Of the 16 genes displaying a 10 to 42 fold difference in expression, many are either established or predicted to encode mutacins, which are the bacteriocins of S. mutans. Mutacins are exported by the cell with the intention of limiting competition from closely related species by killing them or interfering with their growth. The researchers postulate that the increased production of mutacins is an effort to limit competitive pressures prior to biofilm maturation. The fact that biofilm formation is a significant contributor to the virulence of S. mutans, and that biofilm formation is inhibited by aeration, is consistent with the observed increase in mutacin production. Lack of a biofilm exposes the S. mutans to a significant number of environmental pressures, including competition from other organisms in the environment, thus dedicating cell resources to creating mutacins would be advantageous.

bip, which encodes a reputed bacteriocin immunity protein, was up-regulated 18-fold. The Bip protein has been implicated in providing resistance not only to bacteriocins, but to a variety of other environmental pressures. These include, but are not limited to, antibiotics, metals, and attacks from the host organisms’ immune system. As mentioned earlier, exposure to inhibitory components of the environment increases when the formation of the protective biofilm is compromised. Therefore the necessity to produce Bip and related proteins becomes increasingly important.

comD, a gene which codes for the histidine kinase of a TCS and functions to increase the expression of com genes via the process of induction, was up-regulated 2.3 fold under aerobic conditions. The comD gene also functions in the development of competence, which is the ability of a cell to import naked DNA from the extracellular space (1, p. 278). The product of the comC gene is a competence stimulating peptide (CSP), the receptor of which is the integral membrane protein called ComD with histidine kinase activity. When ComD is bound to extracellular CSP, the histidine kinase activity of ComD is activated. This leads to altered expression of gene associated with competence (8). This particular instance of regulating gene expression is part of the comC quorum-sensing system (ComCDE), which describes an intracellular communication system of bacteria which alters gene expression in response to population density (9). Previous studies have also shown ComCDE to play a role in appropriate biofilm formation for S. mutans (9).

Previous studies have established that aerobic conditions prompt lactic acid bacteria to resort to a heterofermentative metabolism. The same should also be true for S. mutans. As expected, differential gene expression associated with metabolism was observed in the genes associated with the partial tricarboxylic acid cycle, which will hereafter be referred to as the Szent-Györgyi-Krebs cycle to honor its principle contributors, of S. mutans. The genes associated with the Szent-Györgyi-Krebs cycle were up-regulated. As well, up-regulation of enzymes such as NADH oxidases and pyruvate formate lyase were also observed. NADH oxidase enzymes allow the organism to metabolize oxygen, a function that is important for survival in aerobic environments. Pyruvate dehydrogenase oxidizes pyruvate, which is a product of glycolysis, to form acetyl-CoA, an important precursor of the Szent-Györgyi-Krebs cycle (4). .

Increased production of CcpA, a DNA binding protein, was also indicated by microarray analysis. One aspect of metabolism affected by cultivation in the presence of oxygen is glycolytic rates, the variation of which would lead to a change in the concentration glycolytic intermediates. CcpA is part of a global regulatory system which, in response to the variation of in the concentration of glycolytic intermediates, alters the transcription of genes encoding enzymes of the glycolytic pathway, the Szent-Györgyi-Krebs cycle, and enzymes involved in carbohydrate acquisition and catabolism. Additionally, CcpA has been shown to play an important role in regulating expression of genes whose products are used in biofilm formation. These genes are gtfB, gtfC and ftf, which encode enzymes needed for the production of a major component of the biofilm, called exopolysaccharides.

As mentioned previously, genes affected by the regulatory role of CcpA include those corresponding to the importation carbohydrates from the extracellular environment. So, the observation of up-regulation in genes associated with the phosphophenopyruvate:sugar transerase system (PTF) and the ATP-binding cassette is consistent with what is known about the function of CcpA. The affect aeration has on the production of CcpA and its regulatory role in the expression genes associated with the acquisition of carbohydrates and their subsequent metabolism is an important observation because it indicates a response by S. mutans at the level of transcription.

Of the down regulated genes, 8 are classified as hypothetical or unknown, 4 are involved in transport and binding, 2 in energy metabolism, 2 cellular processes, and 2 in protein fate. One important observation was the up-regulation of relP in conjunction with the down-regulation of genes involved in amino acid metabolism. The product relP catalyzes the synthesis of (p)ppGpp, whose suggested function is to alter metabolism. (p)ppGpp compromises expression of genes needed for cell growth in order to produce an increased expression of genes which aid in stress tolerance and amino acid synthesis.

Cultivation in aerobic conditions yielded 3.3 fold down-regulation of gtfB. Gtfb is an essential enzyme needed for the production of the glucan polymers, a component of the biofilm. These glucan polymers are water insoluble and adhesive, properties which contribute significantly to the functions of biofilms. According to the researchers, the down regulation of gtfB may be a factor in reducing S. mutans’ ability to form biofilms. gtbC, a gene which is cotranscribed with gtfB, was neither up-regulated or down regulated according to DNA microanalysis. Because of their significance in establishing a biofilm, the expression of these genes was further investigated.

Figure 2 shows a CAT assay whose results establish the same trend as the microarray analysis, which is the up-regulation gtfB and a lack of differential expression for gtfC. The CAT assay was produced by fusing the gtfB and gtfC genes with a reporter gene in a process described in more detail in the subsequent paragraph, and then inserted as a single copy into strain UA159, which is wild type, to produce two new strains. The resulting strains, indicated in table 1, were TW54 and TW55. TW54 contained gtfB promoter fusion, while TW55 contained the gtfC promoter fusion. The reporter gene for this analysis is the chloramphenicol acetyltransferase (cat) gene. Chloramphenicol is an antibiotic that functions by binding the ribosome and inhibiting the process of translation. The cat gene gives a bacterium a resistance chloramphenicol (1). Following cultivation of cells, the relative amount of protein produced by cat can be measured. The CAT assay essentially measures effectiveness of a genes promoter, within a given environmental context. The original gene is replaced with the reporter, and the reporters production measured.

In order to construct the reporter gene fusion, PCR was performed on a fragment of DNA containing the promoter regions and ribosome binding sites (RBS) for each gene. The primers used for this process are listed in Table 2. In order to produce the expression of the reporter gene, the products of PCR were cloned into plasmid pU1. Cloning a gene into a plasmid first requires that the plasmid is digested to a linear DNA molecule by a restriction endonuclease. The restriction endonucleases used in this part of the experiment were BamHI and PstI, which create a staggered or sticky end (5). They are capable of creating a staggered break because they recognize sequences with twofold rotational symmetry. The resulting staggered unpaired ends read the same in the 5’ to 3’ direction and this can pair with and be ligated to any other piece of DNA digested by the same restriction endonuclease (5,). The transcriptional fusions were then released by digestion with SmaI and HindIII, also restriction endonucleases, and inserted into pBluescript KS(+) which had been digested by the restriction. endonucleases Eco-RV and HindIII. The resulting plasmid was then digested by restriction endonucleases SmaI and HincIII. HincIII and SmaI differ from the restriction nucleases previously described because they create blunt ends instead of sticky ends. Blunt ends lack the unpaired bases of staggered ends and this have a much lower affinity for the ends of other DNA strands (6,7). The DNA fragments were then purified and ligated into the integration vector, which was then inserted into the gtfA locus of strain UA159 to create strains TW54 and TW55. The results of the CAT assay displays the same trend observed in the DNA microarray analysis, which was that gtfB was up-regulated and gtfC was unaffected.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and a Western blot was performed of protein fractions from S. mutans in order to compare the localization of GtfB and GtfC proteins of cells cultured in aerobic to those cultured in anaerobic conditions. Sodium dodecyl sulfate (SDS) is a detergent which binds to protein in an amount approximately proportional to the molecular weight of that protein and gives it a net negative charge. Electrophoresis in the presences of SDS separates proteins on the basis of mass(4, p.92). The results of the SDS-PAGE and Western blot, to be discussed later, are displayed in Figure 3. GtfB and GtfC were detected using a polyclonal rabbit antibody. The protein fractions used for SDS-PAGE and Western blot were obtained in a series of steps, the first of which was centrifugation and two washes with Tris-buffered saline. Supernatant proteins were obtained by passing the supernatant fluid through a filter and precipitating the proteins with trichloroacetic acid. Whole cell lysates were obtained by homogenization in an SDS boiling buffer with glass beads in a Bead Beater. Surface associated proteins were obtained by incubating cells in a 4% SDS solution for 30 minutes. The soluble fraction, containing soluble proteins of the cytosol, was obtained by centrifugation of the homogenized cells, which separates soluble and insoluble proteins. Soluble proteins remain in the supernatant, and the pellet is resuspended in SDS boiling buffer to create the insoluble fraction.

Each fraction, the sum of which comprise the whole cell lysate, indicates the amount of GtfB or GtfC within different parts of the cell, this providing data about protein localization. For each fraction, the 4% SDS extract isolates the proteins associated with the cell surface, the soluble fraction contains proteins of the cytosol, and the supernatant fraction contain proteins exported by the cell. The results in Figure 3 indicate that for the 4% SDS extracts (B), the soluble fraction (C), and the insoluble fraction (D), the amount of GtfC as indicated by the intensity of the band was greater in magnitude for the aerobically cultivated cells than for the cells grown in anaerobic conditions. In the supernatant fraction (C), the relative amounts of GtfC were equivalent in magnitude. While Western blot results of GtfB are consistent with the DNA microarray analysis and the CAT assay, it indicates an overall increase in the amount of produced GtfC, which neither of the previous two methods indicated.

Unlike the CAT assay, SDS-PAGE and the subsequent Western blot used a polyclonal antibody to detect GtfC and GtfB directly. The variation in methodology might play a role is the variation observed results.

This study also showed that VicK sensor kinase of the CovRS-like TCS plays a role in the response of S. mutans to aerobic environments. The sensor kinase of a TCS detects changes in the environment and when the appropriate conditions are satisfied it, as its name would imply, phosphorylates protein of the cytosol which plays a role in regulating gene expression (10). The CovRS TCS of S. mutans is a regulatory system which regulates the expression of glucosyltransferase genes, ftf, and a gene encoding a glucan-binding protein (11). Analysis, discussed in further detail in the subsequent paragraph, revealed inactivation of the genes for VicK restored the ability of S. mutans to form biofilms in the presence of oxygen.

In a recent study performed by the investigators it was observed that the VicK-deficient strain, indicated as vicK-NP in Table 1, produced elevated levels of GtfC. VicK is a component of the VicRKX TCS. A PAS domain, which was recently reported to be present in the VicRKX TCS, is a potential sensor for redox potential. This is important because redox potential of mature biofilms differs from that of early biofilms. When subjected to SDS-PAGE analysis, both GtfB and GtfC on the vicK-NP strain an compared to UA159, the amount of both proteins had increased in the soluble and insoluble fractions from vicK-NP strain, while, in the same strain, a reduction in the levels of both proteins was observed for the supernatant fractions. The investigators also measured the levels of mRNA for vicK-NP, the results of which are consistent with the SDS-PAGE analysis in that they display and increase in transcription of gtfC. Figure 4A displays the results of the SDS-PAGE comparison of UA159 and vicK-NP, and Figure 4B displays the results of real-time PCR, used to measure amount of transcribed mRNA.

In addition to the VicK sensor kinase, this study also investigated the role of the AtlA autolysin pathway and its role in meadiating S. mutans’ response to oxygen. Like the VicK sensor kinase, inactivation of the genes for AtlA restored the bacteria’s ability to form biofilms in aerobic conditions. The AtlA autolysins pathway plays an essential role in the formation and maturation of the biofilm, and the regulation of its associated genes by the ComCDE regulatory system has been implicated in other studies (11).

This study established that exposure to oxygen has a profound effect on the virulence of S. mutans, especially in regards to biofilm formation. As well, it was demonstrated that VicK sensor kinase and the AtlA autolysin pathway both play an important regulatory role with gene expression associated with biofilm was established.

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