Background to the project
Staphylococcus aureus is a major human pathogen capable of infecting most tissues. The organism causes a variety of diseases, ranging from localized skin and wound infections to severe sepsis, and is a leading cause of hospital-acquired infections. The incidence of S.aureus infections has increased steadily over the last 20 years, and infections due to multidrug-resistant strains has become an increasing problem worldwide, matters that have urged many investigators to search for new alternative ways to combat this organism.
Humans are a natural reservoir of S. aureus, and the bacteria can be found in the nose and on the skin in 25-30% of healthy people. Although most staphylococcal infections are caused by the patients own S. aureus , it is unclear whether all carrier strains are equally prone to cause an infection or if some strains are more virulent than the others. An enormous amount of research has been directed toward identifying the genes and regulatory mechanisms associated with S. aureus pathogenesis. This work has clearly demonstrated that the staphylococcal pathogenesis is multifactorial, and can be attributed the production of a large number of extracellular toxins (hemolysins, leukocidins, superantigens, etc.), secreted enzymes (proteases, lipases, coagulase, etc.), and cell wall associated proteins that bind extracellular matrix and plasma proteins such as fibronectin, collagen, fibrinogen, and immunoglobulins. The expression of most virulence genes is controlled in a growth-phase-dependent manner by more than a dozen global regulators, including agr, sarA, sarS, sarT, rot, and sigB. Generally, genes coding for cell wall associated proteins are expressed during early exponential phase of growth, while genes encoding secreted toxins and enzymes are expressed during the post-exponential phase. As many of the regulators are regulated by each other, the level of expression of a specific virulence gene is determined by the relative activities of several regulators.
The overall aim of this project was to further our understanding of the molecular basis of Staphylococcus aureus virulence in order to suggest new ways of preventing and treating infections caused by this organism. The specific objectives were to analyse virulence gene expression and regulation in strains of S. aureus isolated from human infections and from healthy carriers, and to apply a new light-emitting reporter gene system developed, to monitor virulence gene expression during bacterial growth under different environmental conditions in vitro, and later in animal models of infection and bacterial carriage.
Results
The overall aim for our group is to further understand the molecular basis of Staphylococcus aureus virulence by analysing the regulation of virulence gene expression. Two global regulators, agr and sar, seem to be involved in transition of S.aureus cells from the adhesive to the invasive phenotype. Factors that promote invasion are positively regulated by agr, while adhesive factors are negatively regulated. SarA , an the other hand, is a DNA binding protein that stimulates transcription of agr but acts also as a repressor of some virulence genes. The specific objectives of our projects are to characterise in detail the mechanisms of action of agr and sar among others (altogether up to 140 different), and to study their role of different target genes that are either positively or negatively regulated ( i.e. protein A, fibronectin binding protein, alpha-toxin, several proteases).
We have cloned several regulatory elements to control reporter gene expression in various S. aureus genetic backgrounds especially related to virulence associated mechanisms. Thus far we have shown that regulation of virulence can be monitored in real time using luciferase reporter gene systems. We have had technical problems mainly in two areas, namely there has existed severe genetic instability in single copy states. We would like to study virulence in as authentic settings as possible in order to see regulations such as it is in natural conditions to prevent for example repressor out-titration when using multicopy plasmid reporters. After several trials and errors, changing transducing phages and strains we were not able to stabilize the strains. Another problem has been the fact that bacterial luciferase seem to report regulation only in early stages of growth, i.e. light emission drops down regardless whatever regulatory element, constitutive, early or late, is inserted to control the luciferase operon. One assumption was that one of the substrates for luciferase reaction would be consumed in late stages and therefore we studied a coexpression of FMN reductase genes but this did not help. Addition of long-chain aldehyde increased significantly the light emission levels. Therefore a study is going on where Northern-blots and real-time quantitative PCR are used to estimate whether we have specific transcripts for all of the five genes in the luciferase reporter operon.
New methods for studies on Staphylococcus aureus virulence gene expression with real-time quantitative time-resolved RT- PCR were generated and compared to the bacterial luciferase reporter system in the final year of the project.
The understanding of Staphylococcus aureus virulence associated gene expression is important in order to find new means for preventing hospital-acquired, potentially lethal infections. We designed a real-time quantitative time-resolved reverse transcription PCR (qTR RT-PCR) detection method which was validated for S. aureus virulence expression studies by comparing it to bacterial luciferase reporter gene measurements. The expression of lux genes controlled by hla promoter (alpha toxin gene) in S. aureus strain RN4220 was monitored both by bioluminescence measurements and qTR RT-PCR using novel europium labelled probes and a quencher probes specific to luxB and luxE genes. The bioluminescence measurements allowed real-time detection of gene expression on protein synthesis level. The time-resolved fluorescence based method measured gene expression on mRNA level. As internal standards for the qTR RT-PCR measurements we used probes specific for S. aureus 16S rRNA. The methods developed are highly sensitive. The results show, that the expression of luxB and luxE mRNA exhibit similar features despite of slight differences in the shape of the qTR RT-PCR curves. The amount of luxB mRNA increases slightly earlier than the bioluminescence signal, but those two methods give highly correlating results.
Selected publications:
Lorenzo Galluzzi and Matti Karp (2006) Intracellular redox equilibrium and growth phase affect the performance of luciferase based biosensors. Submitted to Journal of Biotechnology.
Lorenzo Galluzzi and Matti Karp (2006) Whole cell strategies based on LUX genes for high throughput applications toward new antimicrobials. Submitted to Combinatorional Chemistry and High Throughput Screening.
Johanna Mäkeläinen, Hanna Polari, Jussi Nurmi, Staffan Arvidson, Matti Karp (2006). New methods for studies on Staphylococcus aureus virulence gene expression with real-time quantitative time-resolved RT- PCR and bacterial luciferase reporter system. Submitted to Journal of Microbiological Methods.
Results of the ARVIDSON'S GROUP:
Key words: Staphylococcus aureus, virulence factors, gene regulation, luciferase reporter system
Backgroud
Staphylococcus aureus is a major human pathogen capable of infecting most tissues. The organism causes a variety of diseases, ranging from localized skin and wound infections to severe sepsis, and is a leading cause of hospital-acquired infections. The incidence of S.aureus infections has increased steadily over the last 20 years, and infections due to multidrug-resistant strains has become an increasing problem worldwide, matters that have urged many investigators to search for new alternative ways to combat this organism
Humans are a natural reservoir of S. aureus, and the bacteria can be found in the nose and on the skin in 25-30% of healthy people. Although most staphylococcal infections are caused by the patients own S. aureus , it is unclear whether all carrier strains are equally prone to cause an infection or if some strains are more virulent than the others.It seems clear that the S. aureus virulence is multifactorial, and can be attributed the production of a large number of extracellular toxins (hemolysins, leukocidins, superantigens, etc.), secreted enzymes (proteases, lipases, coagulase, etc.), and cell wall associated proteins that bind extracellular matrix and plasma proteins such as fibronectin, collagen, fibrinogen, and immunoglobulins.The expression of most virulence genes is controlled in a growth-phase-dependent manner by more than a dozen global regulators, including agr, sarA, sarS, sarT, rot, and sigB. Generally, genes coding for cell wall associated proteins are expressed during early exponential phase of growth, while genes encoding secreted toxins and enzymes are expressed during the post-exponential phase. As many of the regulators are regulated by each other, the level of expression of a specific virulence gene is determined by the relative activities of several regulators.
The overall aim of this project is to further our understanding of the molecular basis of Staphylococcus aureus virulence in order to suggest new ways of preventing and treating infections caused by this organism. The specific objectives are to analyze virulence gene expression and regulation in strains of S. aureus isolated from human infections and from healthy carriers, and to apply a new light-emitting reporter gene system developed by our Finnish partners, to monitor virulence gene expression during bacterial growth under different environmental conditions in vitro, and later in animal models of infection and bacterial carriage.
Results
S. aureus strains with single copies of selected virulence gene promoters fused to the luxABCDE cassette, inserted in the chromosomal lipase gene (geh), did not generate enough light to be useful for further expression studies in vitro. We are therefore testing if plasmid constructs can be used instead. The problem with such an approach might be that the presence of multiple promoter copies might titrate regulating proteins and perturb regulatory circuits. Plasmid constructs with different promoters Phla (alpha toxin), Pspa (proteinA), P3agr (RNAIII) fused to the luxABCDE cassette, which were received from our Finnish partners, have been transferred to several S. aureus regulatory mutants, and light production in broth cultures and in bacterial colonies has been measured by the luminometer (Victor, Wallac) and the hyper-sensitive CCD camera (Xenogene) respectively. Although expression of the hla-lux fusion was regulated as expected by agr, sigB, sarA and sarS the ranges of activation or repression were not as wide as for the original chromosomal alleles. For example, the difference in light production by the hla-lux between wild type and agr mutant strains was only 3- to 4-fold in both cultures as compared to 10- to 20-fold for the chromosomal hla allele when measured by Northern blotting. Furthermore, light production ceased at the end of exponential phase of growth, several hours before alpha-toxin production normally stops.
Our collaborators in Finland have shown that light production stops because of lack of substrates required for light production. Adding aldehyde substrate to the culture prolonged light production in to the early stationary phase but did also increase the light signal indicating a deficiency in aldehyde production in the S. aureus strains tested so far. However, a good correlation between light production and the luxB, luxE mRNA levels were found when luciferase genes expression was under control of the hla promoter in an agr deficient S. aureus strain (Mäkeläinen et al . manuscript submitted), indicating that the system may be used for quantitative studies of genes that are expressed at a relatively low level.
To further test whether the plasmid-borne reporter system can be used to monitor gene expression regulation of spa (protein A) has been studied using both the luxABCDE reporter system and Northern blotting. The level of repression of spa by agr was almost the same as for the chromosomal spa allele and the spa-lux reporter, i.e. a ten-fold higher level in the agr mutant than in the corresponding wild type strain. Using both conventional methods and the lux reporter system we have elucidated the regulatory networks coverning the expression of spa, hla and the protease genes sspA and aur. Regulation of spa by agr seems to be mediated via rot, sarT, and sarS. DNA-binding experiments and transcriptional analysis of single, double, triple and quadruple mutants indicated that SarA represses sarS by direct binding to the promoter, and that stimulation of sarS by sarT is rot-dependent. We also found that MgrA bound to the sarS promoter and was required for expression of both sarS and spa in an agr mutant. However, mgrA and rot were not required for stimulation of sarS in the absence of SarA, suggesting that these regulators, together with SarT, counteract the repressive activity of SarA. Essentially the same regulatory network governs the expression of hla. However, in the case of hla, sarS acts as a repressor contrary to in the case of spa regulation where it is an activator. In the case of sspA and aur, these genes are more sensitive to direct repression by rot and sarA, while sarS seems to be less important.
The regulatory network controlling spa expression was described mathematically by six ordinary differential equations, for which the parameter values were generated by a gradient search method to give the best correlation with our Northern blot data. The quantitative predictions of spa expression in different regulatory knock-out mutants made in silico correlated extremely well with our experimental in vitro data. Simulations revealed that the regulatory net-work is very robust and tolerates substantial variations in regulator concentrations without collapsing (manuscript in preparation).
We also found that very high production of alpha-hemolysin and proteases in several natural isolates of S. aureus was associated with low level of sarA expression during post-exponential phase of growth as a result of mutations in the sigB operon leading to sigma factor B deficiency.
Selected publications:
Oscarsson J, Harlos C, Arvidson S. Regulatory role of proteins binding to the spa (protein A) and sarS (staphylococcal accessory regulator) promoter regions in Staphylococcus aureus NTCC 8325-4.Int J Med Microbiol. 2005 Aug;295(4):253-66.
Karlsson-Kanth A, Tegmark-Wisell K, Arvidson S, Oscarsson J. Natural human isolates of Staphylococcus aureus selected for high production of proteases and alpha-hemolysin are sigma(B) deficient. Int J Med Microbiol. 2006 Mar 8; [Epub ahead of print]
Oscarssoon J, Tegmark-Wisell, and Arvidson S. Coordinated and differential control of aureolysin (aur) and serine protease (sspA) expression in Staphylococcus aureus by sarA, rot and agr (RNAIII). Int. J. Med Microbiol. 2006 Apr. in press
An abstract of the research plan (January 2003)