Final Report Summary - RESTAVAC (Regulation of Expression of Staphylococcus aureus Vaccine Antigen Candidates)
Staphylococcus aureus is a remarkably versatile organism. S. aureus is a commensal of the human skin and nares, and a major cause of skin, and soft tissue as well as invasive infections. Bacterial pathogenicity results from a complex interplay of virulence factors with regulatory systems that respond to multiple external signals from the host environment and the bacterial population.
The large variety of disease and the increasing spread of antimicrobial resistance in the S. aureus population have resulted in multiple attempts to generate an effective vaccine formulation over the last decades. Two classes of methicillin-resistant S. aureus (MRSA) strains have emerged that are either associated with the hospital environment (HA-MRSA) or the community (CA-MRSA). These strains show profound differences within their virulence profiles and preliminary studies indicated that major changes in regulatory networks of virulence factor regulation could account for some, if not all, of the observed phenotypes.
Nevertheless, almost all projects thus far have failed and no vaccine has yet been licensed. From genome mining, a list of candidate antigen genes has been identified and an exploratory vaccine formulation from this list has recently been shown to have protective efficacy in mice (Bagnoli et al., 2015). In order to understand the potential protective efficacy of a vaccine, it is paramount to understand the expression of vaccine antigens within the host and disease environment. RESTAVAC aimed to elucidate the expression and regulation of the virulence factors and potential vaccine antigens of S. aureus using a combination of molecular and epidemiological approaches as well as their assessment during infection within animal models. RESTAVAC was structured into five main objectives.
O1: Generation of staphylococcal reporter strains and monitoring of gene expression of vaccine antigens and key virulence genes in vitro and in ex vivo virulence models.
We generated fluorescent and bioluminescent reporters of the promoters of candidate vaccine antigen genes and key reference virulence genes and screened their expression profiles under defined in vitro growth conditions either using standard growth media or inducing conditions that would be encountered by the S. aureus within the host environment during infections. We focused our attention on pH, iron and zinc starvation and cell envelope stresses and determined that several of the antigens altered their expression when exposed to these stimuli. This enabled us to identify specific signals for several of the vaccine antigens allowing us to predict potential regulatory networks involved in their control of expression. We further characterized expression of vaccine antigen in strains from diverse backgrounds such as HA- and CA-MRSA, which allowed us to either further corroborate or to exclude certain regulatory pathways from our investigation.
O2: Identification of molecular mechanisms involved in the regulation of the novel vaccine antigens.
Promoter regions for current and potential vaccine antigens were initially identified by bioinformatics. Expression of fhuD2, a key iron uptake gene and major vaccine candidate, was further characterized by using transcriptional promoter fusion and mutants in its predicted regulator, Fur by using qRT-PCR. This analysis showed that fhuD2 was indeed controlled by Fur in an iron-dependent manner in which iron acts as a cofactor to Fur resulting in the increase of its affinity to a specific direct inverted repeat region within the fhuD2 promoter. We investigated the regulatory architecture of the promoter of the LukED operon which encodes a bipartite leukocidin to explore further the molecular basis of the induction (approximately 2500-fold) in its expression during infection (see work performed for O3 and O4). We performed DNA pulldown experiments followed by non-targeted mass spectroscopy to identify direct interactors with its promoter region. Several regulatory proteins were identified from the pulldown experiment, and the most abundant of these appeared to be the SarA and SarS DNA-binding proteins as well as major virulence regulators MgrA, Rot, SarR and a regulator of the DeoR family suggesting that a multi-component regulatory complex is present on the lukED promoter and responsible for its specific in vivo induction.
O3: Monitoring of gene expression in infection models and in vivo (animal models).
We have developed a stable plasmid bioluminescent reporter for maintenance within S. aureus without the presence of antibiotic selection, through the exploitation of toxin/antitoxin and partitioning systems. We have infected mice with the stabilized bioluminescent promoter-reporter construct and monitored the in vivo expression of fhuD2, demonstrating stable maintenance of the plasmid until day 7 of the infection. This system has allowed us to follow the expression of fhuD2 via bioluminescent signal throughout a time course experiment in a temporal and spatial manner, using both 2D and 3D computer tomography as well as looking at specific promoter activity within different organs. This analysis revealed that fhuD2 was highly induced in all host organs yet the specific induction response differed slightly between host organs. The analysis also allowed us to identify progression of staphylococcal bacteria after intravenous infection.
O4: Investigation of global gene expression of staphylococcal genes in vitro, ex vivo and in vivo in the abscess models and air pouch models.
In order to get a more holistic characterization of gene expression response in S. aureus, we generated global expression profiles by DNA microarray analysis of S. aureus strain Newman during in vitro growth as well as for targeted mutants under defined conditions. This allowed us to identify the general response of S. aureus during in vitro growth and to characterize the response of S. aureus regulatory networks. In particular, we investigated metal-responsive gene regulation and the regulons under the control of the metal-responsive transcriptional regulator, Fur, Zur and PerR. We furthermore used defined stimuli that mimicked environmental conditions that the bacterium could encounter in vivo. In particular, exposure to mouse and human serum revealed profound differences in the bacterial response to sera from these different sources highlighting that S. aureus might have a completely altered expression profile during human infection when compared to mouse models. We developed a high-throughput qRT-PCR assay using nanofluidic technology (Biomark HD, Fluidigm), to monitor a selected set of approximately 90 S. aureus genes involved in virulence from very limited quantities of RNA samples. This approach has permitted the global monitoring of expression profiles in individual mice rather than in pooled samples as would have been required for microarray analysis. This analysis has identified a subset of genes that are highly and specifically up-regulated during experimental infection, including lukED was upregulated 2500-fold. A parallel proteomics approach using targeted and non-targeted mass-spectrometry approaches confirmed that while the protein is undetectable in lysates of in vitro cultures the LukED leucocidin is one of the most abundant proteins detectable in bacteria isolated from infected mouse organs. The genes for this leucocidin, unlike the PVL which is only present in a subset of strains, are present in all clinical isolates but are expressed to low expression levels in vitro. The surprisingly high induction of these genes under experimental infection has highlighted their potential use as vaccine candidates.
O5: Identification of the diverse regulatory circuits and transcriptional landscape in community-associated (CA) and hospital-associated (HA) S. aureus strains.
The kinetic expression profile of S. aureus during in vitro growth mimics the temporal onset of virulence regulatory circuits and our hypothesis is that it can be used as a molecular probe for more in-depth epidemiological studies. We have performed comparative expression of the reporter fusions within different HA- and CA- strain backgrounds revealing profound regulatory differences in the expression of vaccine antigens and known regulators. We furthermore characterized the temporal expression profile of HA-MRSA strains relative to our lab reference strains using the high-throughput qRT-PCR array that we developed for this project. Transcriptional profiling for a more in-depth epidemiological study between recent USA100 and USA300 isolates from different geographic origins is still ongoing in which we will characterize selected subsets of isolates using our high-throughput qRT-PCR assay. These analyses suggest that through transcriptional profiling we can identify a molecular signature that may describe the epidemiology of emerging clones such as US100 and US300 and variants within these clonal lineages.
In summary, we expect our final results to be shared with the scientific community in several publications that are currently being written. A better understand of how S. aureus regulates expression of its virulence repertoire in diverse genetic S. aureus backgrounds is essential to understand if future vaccine formulations will show sufficient coverage in order to be implemented in public healthcare endeavors. The identification and elucidation of novel regulatory mechanisms will provide novel targets for vaccine and non-vaccine antimicrobial drugs and can help to better control the spread of the bacterium. Differences between HA- and CA-MRSA strains will give insight into the evolution of these two classes of S. aureus and understanding of the different virulence profiles will facilitate more targeted treatment regimes.
The large variety of disease and the increasing spread of antimicrobial resistance in the S. aureus population have resulted in multiple attempts to generate an effective vaccine formulation over the last decades. Two classes of methicillin-resistant S. aureus (MRSA) strains have emerged that are either associated with the hospital environment (HA-MRSA) or the community (CA-MRSA). These strains show profound differences within their virulence profiles and preliminary studies indicated that major changes in regulatory networks of virulence factor regulation could account for some, if not all, of the observed phenotypes.
Nevertheless, almost all projects thus far have failed and no vaccine has yet been licensed. From genome mining, a list of candidate antigen genes has been identified and an exploratory vaccine formulation from this list has recently been shown to have protective efficacy in mice (Bagnoli et al., 2015). In order to understand the potential protective efficacy of a vaccine, it is paramount to understand the expression of vaccine antigens within the host and disease environment. RESTAVAC aimed to elucidate the expression and regulation of the virulence factors and potential vaccine antigens of S. aureus using a combination of molecular and epidemiological approaches as well as their assessment during infection within animal models. RESTAVAC was structured into five main objectives.
O1: Generation of staphylococcal reporter strains and monitoring of gene expression of vaccine antigens and key virulence genes in vitro and in ex vivo virulence models.
We generated fluorescent and bioluminescent reporters of the promoters of candidate vaccine antigen genes and key reference virulence genes and screened their expression profiles under defined in vitro growth conditions either using standard growth media or inducing conditions that would be encountered by the S. aureus within the host environment during infections. We focused our attention on pH, iron and zinc starvation and cell envelope stresses and determined that several of the antigens altered their expression when exposed to these stimuli. This enabled us to identify specific signals for several of the vaccine antigens allowing us to predict potential regulatory networks involved in their control of expression. We further characterized expression of vaccine antigen in strains from diverse backgrounds such as HA- and CA-MRSA, which allowed us to either further corroborate or to exclude certain regulatory pathways from our investigation.
O2: Identification of molecular mechanisms involved in the regulation of the novel vaccine antigens.
Promoter regions for current and potential vaccine antigens were initially identified by bioinformatics. Expression of fhuD2, a key iron uptake gene and major vaccine candidate, was further characterized by using transcriptional promoter fusion and mutants in its predicted regulator, Fur by using qRT-PCR. This analysis showed that fhuD2 was indeed controlled by Fur in an iron-dependent manner in which iron acts as a cofactor to Fur resulting in the increase of its affinity to a specific direct inverted repeat region within the fhuD2 promoter. We investigated the regulatory architecture of the promoter of the LukED operon which encodes a bipartite leukocidin to explore further the molecular basis of the induction (approximately 2500-fold) in its expression during infection (see work performed for O3 and O4). We performed DNA pulldown experiments followed by non-targeted mass spectroscopy to identify direct interactors with its promoter region. Several regulatory proteins were identified from the pulldown experiment, and the most abundant of these appeared to be the SarA and SarS DNA-binding proteins as well as major virulence regulators MgrA, Rot, SarR and a regulator of the DeoR family suggesting that a multi-component regulatory complex is present on the lukED promoter and responsible for its specific in vivo induction.
O3: Monitoring of gene expression in infection models and in vivo (animal models).
We have developed a stable plasmid bioluminescent reporter for maintenance within S. aureus without the presence of antibiotic selection, through the exploitation of toxin/antitoxin and partitioning systems. We have infected mice with the stabilized bioluminescent promoter-reporter construct and monitored the in vivo expression of fhuD2, demonstrating stable maintenance of the plasmid until day 7 of the infection. This system has allowed us to follow the expression of fhuD2 via bioluminescent signal throughout a time course experiment in a temporal and spatial manner, using both 2D and 3D computer tomography as well as looking at specific promoter activity within different organs. This analysis revealed that fhuD2 was highly induced in all host organs yet the specific induction response differed slightly between host organs. The analysis also allowed us to identify progression of staphylococcal bacteria after intravenous infection.
O4: Investigation of global gene expression of staphylococcal genes in vitro, ex vivo and in vivo in the abscess models and air pouch models.
In order to get a more holistic characterization of gene expression response in S. aureus, we generated global expression profiles by DNA microarray analysis of S. aureus strain Newman during in vitro growth as well as for targeted mutants under defined conditions. This allowed us to identify the general response of S. aureus during in vitro growth and to characterize the response of S. aureus regulatory networks. In particular, we investigated metal-responsive gene regulation and the regulons under the control of the metal-responsive transcriptional regulator, Fur, Zur and PerR. We furthermore used defined stimuli that mimicked environmental conditions that the bacterium could encounter in vivo. In particular, exposure to mouse and human serum revealed profound differences in the bacterial response to sera from these different sources highlighting that S. aureus might have a completely altered expression profile during human infection when compared to mouse models. We developed a high-throughput qRT-PCR assay using nanofluidic technology (Biomark HD, Fluidigm), to monitor a selected set of approximately 90 S. aureus genes involved in virulence from very limited quantities of RNA samples. This approach has permitted the global monitoring of expression profiles in individual mice rather than in pooled samples as would have been required for microarray analysis. This analysis has identified a subset of genes that are highly and specifically up-regulated during experimental infection, including lukED was upregulated 2500-fold. A parallel proteomics approach using targeted and non-targeted mass-spectrometry approaches confirmed that while the protein is undetectable in lysates of in vitro cultures the LukED leucocidin is one of the most abundant proteins detectable in bacteria isolated from infected mouse organs. The genes for this leucocidin, unlike the PVL which is only present in a subset of strains, are present in all clinical isolates but are expressed to low expression levels in vitro. The surprisingly high induction of these genes under experimental infection has highlighted their potential use as vaccine candidates.
O5: Identification of the diverse regulatory circuits and transcriptional landscape in community-associated (CA) and hospital-associated (HA) S. aureus strains.
The kinetic expression profile of S. aureus during in vitro growth mimics the temporal onset of virulence regulatory circuits and our hypothesis is that it can be used as a molecular probe for more in-depth epidemiological studies. We have performed comparative expression of the reporter fusions within different HA- and CA- strain backgrounds revealing profound regulatory differences in the expression of vaccine antigens and known regulators. We furthermore characterized the temporal expression profile of HA-MRSA strains relative to our lab reference strains using the high-throughput qRT-PCR array that we developed for this project. Transcriptional profiling for a more in-depth epidemiological study between recent USA100 and USA300 isolates from different geographic origins is still ongoing in which we will characterize selected subsets of isolates using our high-throughput qRT-PCR assay. These analyses suggest that through transcriptional profiling we can identify a molecular signature that may describe the epidemiology of emerging clones such as US100 and US300 and variants within these clonal lineages.
In summary, we expect our final results to be shared with the scientific community in several publications that are currently being written. A better understand of how S. aureus regulates expression of its virulence repertoire in diverse genetic S. aureus backgrounds is essential to understand if future vaccine formulations will show sufficient coverage in order to be implemented in public healthcare endeavors. The identification and elucidation of novel regulatory mechanisms will provide novel targets for vaccine and non-vaccine antimicrobial drugs and can help to better control the spread of the bacterium. Differences between HA- and CA-MRSA strains will give insight into the evolution of these two classes of S. aureus and understanding of the different virulence profiles will facilitate more targeted treatment regimes.