Högskolan i Skövde

Correlation between antibody response and clinical outcome in Staphylococcus aureus bacteremia has yielded conflicting results. Immunization schedules have failed in clinical trials. Is the humoral response toward S. aureus of protective nature? A prospective study was performed in patients with invasive S. aureus (ISA) infections during the period 2003–2005. The antibody levels were determined at the beginning and at the end of treatment and one month later (n = 96, n = 71, and n = 51, respectively). As controls, 115 healthy individuals were used. A quantitative enzyme-linked immunosorbent assay (ELISA) against eight purified antigens was performed. Bacterial isolates were grouped as to the production of alpha-toxin, agr type, and pulsed-field gel electrophoresis (PFGE) type. Large variations were seen in the antibody levels. The levels in the second sample were the highest. A correlation between agr group, PFGE group, alpha-toxin production, and initial antibody levels was observed. Patients with fatal outcome displayed lower initial antibody levels to all antigens and significantly so in regard to teichoic acid, lipase, enterotoxin A, and scalded skin syndrome toxin. In episodes with complicated bacteremia, initial significantly low levels to teichoic acid and lipase were registered. Low initial antibody levels against several antigens were associated with increased mortality and complicated bacteremia in invasive S. aureus infections. Bacterial properties, strain, and toxin production affected the antibody response.

In the present study, we have investigated 37 invasive Staphylococcus aureus strains (collected between 1997 and 2005) from 33 human episodes of septicaemia causing either endocarditis or vertebral osteomyelitis. All S. aureus strains were typed using pulsed field gel electrophoresis (PFGE), and most strains belonged to any of 4 different PFGE clusters. There was no correlation between any of the PFGE clusters with site of infection. All strains showed highly different expression patterns of extracellular proteins, i.e. we found a vast variation in the number of proteins and amount of individual proteins expressed by the different strains. There was no correlation between any cluster of exoprotein patterns with endocarditis or with vertebral osteomyelitis. We did not find any correlation between agr group and endocarditis, as previously reported. On the other hand, a correlation between some of the PFGE clusters with a certain agr group was found. Known risk factors for S. aureus infections were observed in a majority of the patients.

In the present work a general systems biology approach has been used to study the complex regulatory network controlling the transcription of the spa gene, encoding protein A, a major surface protein and an important virulence factor of Staphylococcus aureus. A valid mathematical model could be formulated using parameter values, which were fitted to quantitative Northern blot data from various S. aureus regulatory mutants using a gradient search method. The model could correctly predict spa expression levels in 4 different regulatory mutants not included in the parameter value search, and in 2 other S. aureus strains, SH 1000 and UAMS-1. The mathematical model revealed that sarA and sarS seem to balance each other in a way that when the activating impact of sarS is small, e.g. in the wild-type, the repressive impact of sarA is small, while in an agr-deficient background, when the impact of sarS is maximal, the repressive impact of sarA is close to its maximum. Furthermore, the model revealed that Rot and SarS act synergistically to stimulate spa expression, something that was not obvious from experimental data. We believe that this mathematical model can be used to evaluate the significance of other putative interactions in the regulatory network governing spa transcription. (C) 2008 Elsevier GmbH. All rights reserved.

Staphylococcus aureus pathogenicity is dependent on the coordinated action of a number of virulence factors and the expression of these virulence factors is determined by several global regulators. The main regulator seems to be agr but there are several additional regulators (mostly sarA homologues) involved that mainly act downstream of agr. Some of these regulators control virulence gene expression directly but they also regulate each other forming complex regulatory networks. The work described in this thesis aims at better understanding of the function of the agr system and how different regulators act together in controlling transcription of virulence genes.

Most virulence factors in S. aureus are expressed in a growth phase-dependent manner governed by the auto-inducible quorum sensing system agr. Activation of agr results in rapid increase of the regulator RNAIII and occurs in response to accumulation of the auto-inducing peptide (AIP). In order to activate the agr system a low basal transcription of the agr operon must be assumed. This basal activity of the operon is stimulated by sarA. To be able to study how SarA would affect activation of agr, a mathematical model of the agr system was set up. The model predicted that the agr system is hysteretic, meaning that activation of agr occurs in a switch-like manner at a specific concentration of AIP, whereas it is inactivated at a specific lower concentration of AIP. According to the model, SarA does not seem essential for the function of the agr switch but alters the concentration of AIP (cell density) at which agr is activated. This was supported by Northern blot analysis of RNAIII in S. aureus mutants with different levels of sarA expression.

To determine how agr and the other regulators act together in controlling transcription of virulence genes, we studied the regulation of one gene (spa) that is negatively regulated by agr and the genes encoding extracellular proteases (aur and sspA), which are positively regulated by agr. To analyze the general principles of how each component in a regulatory system contributes to expression of a virulence gene, a mathematical model of the regulation of spa (protein A) transcription was developed.

Parameter values in this mathematical model were determined by fitting the output of the model to quantitative Northern blot data from various S. aureus regulatory mutants using a gradient search method. The model was validated by correctly predicted spa expression levels in different regulatory mutants not included in the parameter value search. The mathematical model revealed that Rot and SarS act synergistically to stimulate spa expression and that sarA and sarS seem to balance each other in a way that when the activating impact of sarS is small, e.g. in the wild type, the repressive impact of sarA is small, while in a agr-deficient background, when the impact of sarS is maximal, the repressive effect of sarA is close to its maximum.

Previous studies have shown that SarR down-regulates transcription of sarA, which is a repressor of the aur and sspA transcription. This means that inactivation of sarR would result in decreased transcription of aur and sspA, which was confirmed by mRNA analysis using quantitative real-time PCR. However, we also found that sarR acted as a direct stimulator of aur and sspA transcription and that sarR is required for maximal transcription of aur and sspA.

Previous studies have shown that expression of aur (metalloprotease; aureolysin) and sspA (V8 protease; serine protease) in Staphylococcus aureus strain 8325-4 is maximal in the postexponential phase of growth, when the agr (RNAIII) system is activated. Transcription of aur and sspA is mainly regulated through repression by sarA and rot, and RNAIII stimulates protease production by inhibiting translation of rot mRNA. As SarR is a repressor of sarA, inactivation of sarR would result in downregulation of aur and sspA transcription. This was confirmed by mRNA analysis using quantitative real-time PCR. However, we found that sarR acted as a direct stimulator, i.e. its positive effect on aur and sspA transcription did not require sarA (or rot) per se. In addition, aur and sspA were dependent on sarR for maximal transcription. This stimulating role of sarR was not restricted to the rsbU-deficient laboratory strain 8325-4 but was also demonstrated in S. aureus strain SH1000 (rsbU-complemented derivative of 8325-4) and in one clinical isolate.

We report a survey of invasive Staphylococcus aureus (ISA) infections concerning outcome variables such as mortality, recurrence and residual symptoms. A prospective, population-based study of all cases of ISA was conducted in the catchment area of Skaraborg Hospital (population 255,109) in western Sweden during the period from 1st March 2003 to 28th February 2005. One hundred and fifty-seven patients were included. Recurrences were seen in 13 cases (9.3%). Thirty patients (19.1%) died during the first 28 days. Mortality rates for complicated bacteraemia and severe sepsis were 32% and 54%, respectively. Older patients (> 65 years of age), patients with concomitant heart disease and patients with endovascular infections all suffered higher mortality. Line-associated infections had a higher recurrence rate. Residual symptoms were common, with 34% of the living patients reporting incomplete recovery. Accessory gene regulator (agr) type within the bacteria did not affect disease presentation. We conclude that ISA infections are of major medical importance, with high rates of mortality (19.1%), recurrence (9.3%) and residual functional impairment (34%).

Competence for genetic transformation seems to play a fundamental role in the biology of Streptococcus pneumoniae and is believed to account for serotype switching, evolution of virulence factors, and rapid emergence of antibiotic resistance. The initiation of competence is regulated by the quorum sensing system referred as the ComABCDE pathway. Experimental studies reveal that competence is down-regulated a short time after its induction and several hypotheses about the mechanism(s) responsible for this shut-down have been presented. Possibly, a ComX-dependent gene product, such as a repressor or a phosphatase, is involved. To better understand the down-regulation of the competence-evoking system in S. pneumoniae, a mathematical model was set up. By analyzing the model, we suggest that shut-down of competence possibly occurs at the transcriptional level on the comCDE operon. As a result of introducing a putative comX-dependent repressor, which inhibits expression of comCDE and comX, in the mathematical model, competence is demonstrated to appear in waves. This is supported by experimental studies showing the appearance of successive competence cycles in pneumococcal batch cultures.

The virulence determinants of Staphylococcus aureus are expressed in a growth phase-dependent manner governed by the autoinducible quorum-sensing system agr. Activation of the agr system results in a rapid increase in the regulator RNAIII and occurs in response to accumulation of AIP. In order to activate the agr system, a basal transcription of agr components must be assumed. This basal transcription of agr components seems to be stimulated by sarA. To better understand how SarA would affect activation of the agr system by modulating the basal agr activity, a mathematical model for autoactivation of the agr system was set up. The model predicted that the agr system is hysteretic, meaning that the agr system is activated at a specific concentration of autoinducing peptide (AIP), whereas it is inactivated at a specific lower concentration of AIP. According to the model, changing the basal agr activity only had a marginal effect on steady-state levels of RNAIII but changed the sensitivity of the cells to AIP. This was supported by Northern blot analysis of RNAIII in S. aureus mutants with different levels of SarA expression. Since natural antagonistic AIPs have been demonstrated, the effect of adding inhibitors to the system was analyzed.