Bacteriophages (phages) are obligate intracellular parasites of bacteria that usually kill the bacterial host. Bacteriophage therapy is a recently revived approach for treating bacterial infection that relies on the traits of the phage lytic cycle. A lot of attention has been given to phage therapy with new research being published weekly and international conferences organised every year, bringing together the academic and industrial phage communities. However, despite this huge effort and considerable scientific interest there is still a great lack of understanding on how to use phage effectively and overcome the many obstacles in the near future. One of the main triggers for such interest was the increasing evidence of antibiotic resistance among human bacterial pathogens, which were once efficiently eliminated by drugs but are now causing alarmingly high levels of morbidity and mortality. Also, bacteria when causing a disease are able to produce highly protective biofilm communities. Biofilms are major causes of impairment of wound healing and two of the most common and aggressive wound pathogens are Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative), both displaying a large repertoire of virulence factors and reduced susceptibility to antibiotics. This work reports and explores the use of phages to target both S. aureus and P. aeruginosa pathogen biofilm producers. Firstly, isolation of promising phage candidates was performed and cocktails were established. Two phages (DRA88 and phage K) formed the cocktail to target S. aureus and six phages (DL52, DL54, DL60, DL62, DL64 and DL68) formed a cocktail to target P. aeruginosa. A thorough characterisation of each of the selected phages was performed, including their range of host infectivity and their genome sequences were analysed. The phage’s ability to infect and kill planktonic cultures was successfully studied and afterwards such ability was assayed on biofilms using an in vitro static biofilm system (microtitre-plate), followed by an in vitro dynamic biofilm system (The Modified Robbins Device). Both cocktails were shown to be effective in reducing and dispersing biofilms formed by the clinical strains showing them to be promising not only to combat topical bacterial infections (related to biofilm production), but also to control biofilms produced on the surfaces of medical devices, such as catheters. Finally, the phage cocktail’s ability to treat systemic infections caused by the two pathogens was assessed in an in vivo G. mellonella infection model. In the case of the P. aeruginosa infection, although the phages were not able to fully treat the larvae, the cocktail allowed a delay of larval death, caused by the infection. For the S. aureus infection, the cocktail did not show the same trend, but most likely the high bacterial cell numbers involved in the experiment interfered with a successful study on the phage cocktail. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus and P. aeruginosa biofilms.

Thesis (M.A.)--Boston University / In view of the success of the Danish investigators Christensen and Kjems in inducing regressions of malignant tumors with streptococcus bacteriophage lysates, it was decided to test lysates of hemolytic Staphylococcus aureus upon malignancies in hamsters and mice. [TRUNCATED]

Staphylococcus aureus (S. aureus) is a significant pathogen that causes a wide variety of disease in humans and animals. Methicillin resistant S. aureus (MRSA) isolates carrying mecC, the gene that confers resistance to the antibiotic, have been isolated from humans but also from diverse animal species covering livestock, domestic and wild animals throughout Europe. Many of the known MRSA mecC isolates have been whole-genome sequenced by our group to gain insight into the evolution and epidemiology of these emerging lineages. For microbes and humans alike, iron is an essential cofactor in many biochemical reactions and S. aureus requires iron for colonisation and subsequent pathogenesis. The success of S. aureus is partly attributed to its ability to exploit the host iron pool. It does this through multiple iron uptake mechanisms, including at least two high-affinity iron scavenging siderophores (staphyloferrins A and B) and an iron-regulated surface determinant (Isd) pathway for haem-iron acquisition. Here I describe the identification of a novel locus encoding a siderophore-like non-ribosomal peptide synthetase (NRPS), directly downstream of the SCCmec insertion site in mecC S. aureus isolates. A homologous region was identified in Streptococcus equi 4047 (S. equi) which encodes a NRPS termed 'equibactin' that is involved in iron acquisition. I have therefore named the NRPS product 'staphylobactin' in MRSA, and the aim of this study was to determine the function of the staphylobactin biosynthesis cluster: is this region involved in iron acquisition and how might it be regulated? Analysis of the prevalence of isolates containing the staphylobactin locus showed it to be present in a large number of mecC strains in our collection but also identified homologues in other staphylococcus isolates. The region is highly conserved in all S. aureus isolates belonging to clonal complex (CC) 130 (broad host range lineage), suggesting that staphylobactin might impact on S. aureus's ability to infect a broad range of host species. The staphylobactin gene cluster contains 14 coding sequences, stbB-F, F1, G-M and O. Bioinformatic analysis results in predictions of domain and gene functions associated with iron acquisition. I hypothesized that staphylobactin might have been acquired to compensate for the lack of another siderophore, such as staphyloferrin B, but the staphyloferrin B biosynthesis cluster and transport is present in nearly all S. aureus strains, ruling out this model. Unlike the equibactin locus, however, the staphylobactin locus lacks a homolog for the iron-dependent regulator eqbA. Instead, expression of this locus appears to be regulated by MntR, a DtxR-like regulator. The staplylobactin gene cluster is flanked by direct repeats which suggest staphylobactin could have been gained by horizontal gene transfer. In order to study the role of the staphylobactin gene cluster, deletion mutants of MntR, the staphylobactin locus and staphyloferrins A and B, were generated using the pIMAY two step gene deletion procedure in the previously un-manipulated mecC S. aureus CC130 strains - a challenging protocol that required significant optimization given the difficulties with manipulating this bacterium. Analysis of the MntR mutant suggests that the staphylobactin operon is regulated by MntR, acting as a positive regulator, in an iron-dependent manner. By RT-PCR, I found that expression of the staphylobactin NRPS genes is increased when cultures are grown in the absence of iron, suggesting an involvement with iron acquisition. Genomic inactivation of the staphyloferrins resulted in a mutant severely incapacitated for growth in serum and transferrin as the sole iron source, and addition of iron reversed this phenotype. However, deletion of staphylobactin alone or in addition to the staphyloferrins, lacked an iron-dependent growth defect, and numerous assays failed to identify a clear role for staphylobactin in iron metabolism. Therefore, further experiments are needed to elucidate the function of this siderophore like NRPS. Analysis of the same sequenced CC130 mecC isolates from our strain collection in which the staphylobactin locus was found, led to the identification of a novel Von Willebrand (vwb) gene. In order to investigate possible reasons for these isolates to infect a wide range of host species, wild-type and vwb deletion mutant strains, along with the novel vwb expressed in lactococcus, were tested using a coagulation assay and were able to clot plasma from a broad range of host species. Thus the specificity of vWbp proteins can be used to infer the host specificity and evolutionary history of the S. aureus strains that harbour them. Although I was unable to generate definitive evidence revealing the biological role for the staphylobactin locus this study has generated valuable tools for further studies and thoroughly tested a number of hypotheses concerning its role in cation metabolism.

As interações que ocorrem entre as bactérias e amebas podem dar-se através de relações mútuas, onde ambos os organismos se beneficiam da associação ou parasitárias, em que um organismo se beneficia em detrimento do outro. Quando esses organismos compartilham o mesmo ambiente, pode resultar em algumas alterações, seja no crescimento dos organismos, nos padrões de adaptação, na morfologia, no seu desenvolvimento ou até mesmo na sua capacidade para sintetizar proteínas e outras substâncias. No presente estudo, avaliou-se a interação entre Acanthamoeba polyphaga e Staphylococcus aureus (MRSA) através de um modelo de cocultivo em diferentes tempos de incubação. A partir deste, 89% das células amebianas permaneceram viáveis após contato com a bactéria. O isolado bacteriano foi visualizado no interior da ameba através de microscopia confocal e de fluorescência em até 216 horas de cocultivo, sendo considerado um microrganismo resistente à ameba. O contato de A. polyphaga com S. aureus (MRSA) não demonstrou alteração fenotípica da ameba através dos testes fisiológicos de osmo e termotolerância. O lisado da cultura amebiana aumentou o crescimento de S. aureus (MRSA) nos diferentes tempos de incubação, porém houve diferença significativa apenas entre o controle e 96 horas de cocultivo. O crescimento de S. aureus (MRSA) foi inibido ao longo dos tempos de incubação pelo efeito do sobrenadante da cultura amebiana apresentando diferença significativa entre o controle e 96 horas de cocultivo, sugerindo-se que A polyphaga tenha secretado algum tipo de metabólito, que inibiu o crescimento da bactéria. A interação dos microrganismos não apresentou alterações significativas no perfil de susceptibilidade aos antibióticos testados. S. aureus (MRSA) permaneceu viável em cistos de A. polyphaga, reforçando a hipótese de que Acanthamoeba pode desempenhar um papel crucial na propagação de S. aureus (MRSA) na comunidade e ambiente hospitalar. O maior percentual de amebas encistadas deu-se em 96 horas de incubação quando cocultivadas com o isolado de S. aureus (MRSA), apresentando um aumento progressivo deste percentual a cada período de incubação. A partir disso, estudos devem intensificar-se para melhor compreender os mecanismos de virulência envolvidos na interação entre ambos os microrganismos. / The interactions that occur between bacteria and amoebae can give through mutual relations, where both organisms benefit from the association or parasitic in which one organism benefits at the expense of the other. When these organisms share the same environment, can result in some changes in the growth of organisms, in adaptation patterns, in morphology, development or even in their ability to synthesize proteins and other substances. In the present study, we evaluated the interaction between Acanthamoeba polyphaga and Staphylococcus aureus (MRSA) using a coculture model at different incubation times. From this, 89% of amoebic cells remain viable after contact with the bacteria. The bacterial isolate was visualized inside the amoeba through confocal microscopy and fluorescence for up to 216 hours of cocultivation, being considered a resistant microorganism to the amoeba. The contact of A. polyphaga with S. aureus (MRSA) showed no phenotypic changes of amoeba through physiologic osmo or thermotolerance tests. The lysate of amoebic culture increased the growth of S. aureus (MRSA) in the different incubation times, but there was a significant difference only between the control and 96 hours of cocultivation. The growth of S. aureus (MRSA) has been inhibited over the incubation times for the effect of amoebic culture supernatant showing a significant difference between the control and 96 hours of coculture, suggesting tha A. polyphaga has some kind of secreted metabolites inhibiting the growth of bacteria. The interaction of microorganisms showed no significant changes in the susceptibility profile of the tested antibiotics. S. aureus (MRSA) remained viable cysts of A. polyphaga, reinforcing the hypothesis that Acanthamoeba can play a crucial role in the spread of S. aureus (MRSA) in the community and hospital. The highest percentage of encysted amoebae occurred in 96 hours of incubation when cocultured with the isolate of S. aureus (MRSA), with a progressive increase in this percentage to each incubation period. From this, studies should be intensified to better understand the virulence mechanisms involved in the interaction between these two organisms.

Staphylococcus aureus, particularly the epidemic methicillin-resistant S. aureus (MRSA) USA300 strain, is a major cause of severe necrotizing lung, skin and systemic infection. Although these infections are generally attributed to the pathogen’s multiple toxins, exactly how S. aureus cause disease is not clearly defined. In this research, we sought to establish the role of necroptosis, a programmed form of necrosis, in the pathophysiology of S. aureus pneumonia, skin infection and sepsis. S. aureus, mainly through its multiple toxins, induced RIPK1/RIPK3/MLKL-mediated necroptosis in multiple host cells including human cell lines, freshly obtained alveolar macrophages, peripheral blood macrophages and epithelial cells. S. aureus toxin-associated pore-formation was essential for necroptosis, as cell death was blocked by exogenous K+ or dextran as well as by MLKL inhibition. To understand the role of necroptosis in S. aureus pneumonia, we used Ripk3-/- mice and mice treated with necrostatin-1s (Nec-1s), a potent inhibitor of RIPK1. Inhibition of necroptosis in a mouse model of pneumonia led to significantly improved outcome from S. aureus infection marked by increased bacterial clearance, preserved lung architecture, decreased inflammatory markers in the airway and retention of an anti-inflammatory macrophage population. In contrast, inhibiting necroptosis in vivo during skin infection led to worse outcome as determined by bacterial clearance and lesion sizes, which occurred in spite of the presence of neutrophils, macrophages and γδ T cells. Nec-1s-treated mice and Mlkl-/- mice had significantly larger lesions, increased cytokine response and more S. aureus recovered from the infected areas compared to control groups. We observed a similar outcome in Casp1/4-/- mice, which have limited ability to process IL-1β. Unlike Mlkl-/- mice, Ripk3-/- mice had improved outcome with increased bacterial clearance and decreased inflammation because of the effects of RIPK3 in activating the NLRP3 inflammasome and apoptosis during S. aureus skin infection. Casp1/4-/- immune cells showed a significant defect in their ability to kill S. aureus, whereas Mlkl-/- peritoneal exudate cells and bone marrow-derived macrophages did not. These results show that caspase-1 is essential for bacterial killing whereas necroptosis is necessary for regulating excessive inflammation. Similar to our findings in skin infection, inhibition of the executioner of necroptosis (using Mlkl-/- mice) or pyroptosis (using Casp1/4-/- mice) decreased survival in a mouse model of S. aureus sepsis. Ripk3-/- and wild type mice were equally resistant to S. aureus sepsis. Overall, these findings provide new insights into the complex roles of necrosome components in different tissues during S. aureus infection and may provide potential therapeutic targets to combat these infections.

No description available.

As doenças infecciosas estão entre as principais causas de morte no mundo. Nas infecções bacterianas, os componentes das paredes destes organismos são descritos como os principais antígenos. Os macrófagos estão diretamente envolvidos no reconhecimento e ataque de agentes patogênicos, além de atuarem como moduladores da resposta imunológica. Os macrófagos participam do combate às infecções bacterianas através da fagocitose dos agentes patogênicos e da produção de diversos mediadores inflamatórios como citocinas, metaloproteinases (MMP), espécies reativas de oxigênio (ROS) e óxido nítrico(NO). Estes mediadores são importantes na resposta inflamatória, contribuindo para o controle da infecção. Entretanto, a produção exacerbada destas moléculas contribui na patogênese das complicações associadas à inflamação, como a sepse e a falência múltipla de órgão. A maior parte dos estudos tem focado a resposta das células imunes a infecções por bactérias gram-negativas ou ao seu principal antígeno, o lipopolisacarídeo (LPS). No entanto, as infecções por bactérias gram-positivas são freqüentes e resultam em grande taxa de mortalidade. As doenças infecciosas estão entre as principais causas de morte no mundo. Nas infecções bacterianas, os componentes das paredes destes organismos são descritos como os principais antígenos. Os macrófagos estão diretamente envolvidos no reconhecimento e ataque de agentes patogênicos, além de atuarem como moduladores da resposta imunológica. Os macrófagos participam do combate às infecções bacterianas através da fagocitose dos agentes patogênicos e da produção de diversos mediadores inflamatórios como citocinas, metaloproteinases (MMP), espécies reativas de oxigênio (ROS) e óxido nítrico(NO). Estes mediadores são importantes na resposta inflamatória, contribuindo para o controle da infecção. Entretanto, a produção exacerbada destas moléculas contribui na patogênese das complicações associadas à inflamação, como a sepse e a falência múltipla de órgão. A maior parte dos estudos tem focado a resposta das células imunes a infecções por bactérias gram-negativas ou ao seu principal antígeno, o lipopolisacarídeo (LPS). No entanto, as infecções por bactérias gram-positivas são freqüentes e resultam em grande taxa de mortalidade. Os receptores de adenosina, em macrófagos, possuem função antiinflamatória em modelos de infecções por bactérias gram-negativas, no entanto, pouco se investigou a ação destes receptores em infecções por bactérias gram-positivas. O diabetes é uma doença metabólica comum que apresenta diversas complicações secundárias, muitas delas associadas aos níveis elevados de glicose. Os pacientes diabéticos apresentam uma ocorrência aumentada de infecções bacterianas, além de o diabetes tipo 2 estar associado com uma inflamação crônica, caracterizada por níveis plasmáticos aumentados de citocinas pró-inflamatórias. Adicionalmente, estes pacientes possuem maior risco de desenvolver doenças cardiovasculares e aterosclerose, o que tem sido relacionado à hiperglicemia e à inflamação crônica. A regulação, pela glicose, da resposta inflamatória em macrófagos expostos a bactérias gram-negativas ou ao seu antígeno principal, o LPS, é descrita, entretanto, a ação da glicose em modelos de infecções por bactérias gram-positivas não é conhecida. O tratamento dos macrófagos RAW 264.7 com concentrações elevadas de glicose aumentaram a resposta inflamatória ao LTA, com o aumento da produção de NO, TNF-a e MMP-9. Estes resultados mostram que tanto os receptores de adenosina como concentrações elevadas de glicose modulam a resposta inflamatória a S. aureus. Os receptores de adenosina parecem atuar num mecanismo autócrino de modulação da resposta inflamatória, apontando um possível alvo terapêutico. Já a modulação da resposta inflamatória dos macrófagos por concentrações aumentadas de glicose pode contribuir para complicações associadas ao diabetes, como a aterosclerose e a inflamação crônica. / Infectious diseases are among the majors causes of mortality around the world. In bacterial infections, the bacterial cell wall components are described as the main antigens. The macrophages are key mediators of inflammatory response, acting in the attack to the pathogens and in the regulation of immune response. These cells act in the phagocytosis of pathogenic agents and in the production of inflammatory mediators, like cytokines, matrix-metalloproteinases (MMP), reactive oxygen species (ROS) and nitric oxide (NO). These mediators participate of inflammatory response, acting in infection control, notwithstanding, exacerbated production of these molecules could contribute to inflammatory complications, like sepsis and multiple organ failure. Several studies has addressed the inflammatory response to gram-negative bacterial infections or lipopolysaccharide (LPS), the main antigen of these microorganisms, however grampositive bacterial infections are prevalent and associated to high mortality. The adenosine receptors are described to possess anti-inflammatory properties in macrophages in gram-negative bacterial infections models but the role of these receptors in gram-positive bacterial infections is unknown. Diabetes is a prevalent metabolic disorder which presents several associated complications; much of them associated to increased glucose levels. Diabetic patients shown increased occurrence of bacterial infections and type 2 diabetes is associated to a chronic inflammatory state, with increased circulatory levels of pro-inflammatory cytokines. Type 2 diabetic patients possess increased risk of cardiovascular diseases and atherogenesis, which has been associated to hyperglycemia and chronic inflammation. The regulation of macrophages inflammatory response to gram-negative bacterial infections or to its antigen LPS by increased levels of glucose is described, however, the role of increased glucose in gram-positive bacterial infections is unknown. For this reasons, we studied the role of adenosine receptors and increased glucose in macrophages activation by Staphylococcus aureus antigens, considering that this bacteria is the major organism in hospital infections. The treatment of RAW 264.7 macrophages with S. aureus antigens lipoteichoic acid (LTA) and peptidoglycan (PEG) resulted in the production of proinflammatory mediators. Diabetes is a prevalent metabolic disorder which presents several associated complications; much of them associated to increased glucose levels. Diabetic patients shown increased occurrence of bacterial infections and type 2 diabetes is associated to a chronic inflammatory state, with increased circulatory levels of pro-inflammatory cytokines. Type 2 diabetic patients possess increased risk of cardiovascular diseases and atherogenesis, which has been associated to hyperglycemia and chronic inflammation. The regulation of macrophages inflammatory response to gram-negative bacterial infections or to its antigen LPS by increased levels of glucose is described, however, the role of increased glucose in gram-positive bacterial infections is unknown. For this reasons, we studied the role of adenosine receptors and increased glucose in macrophages activation by Staphylococcus aureus antigens, considering that this bacteria is the major organism in hospital infections. The treatment of RAW 264.7 macrophages with S. aureus antigens lipoteichoic acid (LTA) and peptidoglycan (PEG) resulted in the production of proinflammatory mediators.

O Staphylococcus aureus está freqüentemente envolvido em surtos de intoxicação alimentar. Geralmente esses surtos têm início abrupto, causando náusea e vômito nos acometidos. Na elucidação deste tipo de surtos é necessário que seja investigada a presença da enterotoxina no alimento e/ou a capacidade enterotoxigênica da bactéria isolada. A partir disso, o objetivo do presente estudo foi implantar uma técnica de detecção de enterotoxinas e avaliar a capacidade enterotoxigênica de linhagens de Staphylococcus aureus isolados de surtos. Trinta linhagens isoladas, no período de 2002 a 2003, de alimentos envolvidos em surtos no Rio Grande do Sul foram identificadas e avaliadas quanto à capacidade de produzir enterotoxinas SEA, SEB, SEC e SED, através da técnica de sensibilidade ótima em placa-OSP.Ao lado disso, os dados epidemiológicos relativos aos surtos de origem foram coletados. Os resultados obtidos demonstraram que todas as linhagens foram positivas no teste da catalase, coagulase em tubo, prova de termonuclease, teste de hemólise e fermentaram a maltose e o manitol, sendo confirmadas como Staphylococcus aureus. Todas as linhagens produziram enterotoxina SEA, 24 produziram SEB, 12 SEC e 6 SED, isoladamente ou em associação.A maioria das linhagens foi isolada de alimentos submetidos à grande manipulação durante seu preparo e apresentaram contagens superiores a 106 UFC/g de Staphylococcus aureus, demonstrando que condições favoráveis à produção de enterotoxinas devem ter ocorrido durante o seu preparo e armazenamento. / Staphylococcus aureus often occurs in food poisoning outbreaks. These outbreaks usually start abruptly, causing nausea and vomit on affected people. In order to investigate these outbreaks, it is necessary to check the presence of enterotoxin in food and/or enterotoxigenic capacity of isolate. So the purpose of this study was to propose a method to the enterotoxin detection and evaluate the enterotoxigenic capacity of coagulase positive Staphylococci strains which were isolated from foods involved with outbreaks. On the period of 2002 to 2003, thirty strains detected in foods poisoning outbreaks in RS were identified and evaluated on their SEA, SEB, SEC and SED enterotoxins production capacity checking their sensitivity on OSP - plates. Moreover, epidemiological information related to the origin outbreaks was collected. The results showed that all the strains were positive according to following tests: catalase, coagulase in vitro/tube, termonuclease and hemolysis. Besides the strains have fermented maltose and manitol as to confirm as being Staphylococcus aureus. All the strains produced SEA enterotoxin; 24 of them produced SEB, 12 SEC and 6 SED, isolated or in association. Most strains were isolated in food that had been under intense manipulation during their preparation and which had presented levels higher than 106 CFU/g to Staphylococcus aureus, indicating that favorable conditions to the production of enterotoxins should have occured during their preparation and storage.

Staphylococcus aureus encodes two extracellular nuclease enzymes, Nuc and Nuc2. Nuc is a secreted enzyme that is cut by signal peptidase (SpsB) at the cell membrane and is further processed into two active forms, NucA and NucB, by an unknown protease. Nuc2 is predicted to be a second extracellular nuclease based on sequence homology to the staphylococcal nuclease (SNase) and is tethered to the membrane with a N-terminal anchor. At the beginning of these studies, little was understood about the biological and physiological roles of Nuc and Nuc2 in S. aureus. The goal of this dissertation was to characterize the extracellular nuclease activity of S. aureus in order to better understand the contributions of Nuc and Nuc2 to the S. aureus life cycle. The studies presented in Chapter II focus on the role of Nuc in regulating S. aureus biofilm growth. The secreted forms of Nuc, called NucA and NucB, were first identified as anti-biofilm agents present in spent media from a S. aureus alternative sigma factor B (sigB) mutant. Regulation studies identified the major repressors and activators of nuc expression and showed that nuc is repressed under biofilm-forming conditions. By bypassing the native regulatory mechanisms using a nuc inducible plasmid, biofilm growth could be inhibited in a dose-dependent manner. Biofilm testing of nuc mutant strains across genetic backgrounds led to the observation that biofilm thickness increased two-fold in the absence of Nuc. More high molecular weight extracellular DNA (eDNA) accumulated in the nuc mutant compared to wild-type cells, indicating a direct link between Nuc and the availability of eDNA to contribute to the biofilm matrix. These studies showed that nuc expression is tightly regulated in S. aureus biofilms, and Nuc activity can greatly impact biofilm formation and maturation. In Chapter III, studies were performed to determine whether Nuc2 is an active nuclease in S. aureus and where the protein is localized in the cell. Upon initial comparison to Nuc, Nuc2 has 42% amino acid identity in the proposed SNase domain, and 7 of 9 residues known to be required for Nuc activity are conserved in Nuc2. Fluorescence microscopy of a Nuc2-sGFP translational fusion demonstrated the protein is localized to the cell membrane, and alkaline phosphatase fusion studies showed that the C-terminus of Nuc2 faces out of the cell. Fluorescence resonance energy transfer (FRET) assays facilitated the detection of low levels of Nuc2 activity on the S. aureus cell surface, demonstrating for the first time the enzyme is a functional nuclease, and mutations in the nuc2 gene eliminated this activity. Purification of recombinant Nuc2 also showed that enzyme has DNase activity that is calcium-dependent. Through the construction of Nuc/Nuc2 chimeric proteins, it was determined that localization to the cell membrane does not impair nuclease activity, and the low levels measured for Nuc2 on S. aureus cells is likely due instead to weak expression. The knowledge that Nuc2 is an active nuclease, localized to the cell surface, provides insight into the potential roles Nuc2 may play in a biofilm environment and during S. aureus infection.

Staphylococcus aureus (SA) causes nosocomial infections including life threatening sepsis by multi-resistant strains (MRSA). It has the ability to form biofilms to protect it from the host immune system and from anti staphylococcal drugs. Biofilm and planctonic life style is regulated by a complex Quorum-Sensing (QS) system with agr as a central regulator. To study biofilm formation and QS mechanisms in SA a Boolean network was build (94 nodes, 184 edges) including two different component systems such as agr, sae and arl. Important proteins such as Sar, Rot and SigB were included as further nodes in the model. System analysis showed there are only two stable states biofilm forming versus planctonic with clearly different subnetworks turned on. Validation according to gene expression data confirmed this. Network consistency was tested first according to previous knowledge and literature. Furthermore, the predicted node activity of different in silico knock-out strains agreed well with corresponding micro array experiments and data sets. Additional validation included the expression of further nodes (Northern blots) and biofilm production compared in different knock-out strains in biofilm adherence assays. The model faithfully reproduces the behaviour of QS signalling mutants. The integrated model allows also prediction of various other network mutations and is supported by experimental data from different strains. Furthermore, the well connected hub proteins elucidate how integration of different inputs is achieved by the QS network. For in silico as well as in vitro experiments it was found that the sae-locus is also a central modulator of biofilm production. Sae knock-out strains showed stronger biofilms. Wild type phenotype was rescued by sae complementation. To elucidate the way in which sae takes influence on biofilm formation the network was used and Venn-diagrams were made, revealing nodes regulated by sae and changed in biofilms. In these Venn-diagrams nucleases and extracellular proteins were found to be promising nodes. The network revealed DNAse to be of great importance. Therefore qualitatively the DNAse amount, produced by different SA mutants was measured, it was tried to dissolve biofilms with according amounts of DNAse and the concentration of nucleic acids, proteins and polysaccharides were measured in biofilms of different SA mutants. With its thorough validation the network model provides a powerful tool to study QS and biofilm formation in SA, including successful predictions for different knock-out mutant behaviour, QS signalling and biofilm formation. This includes implications for the behaviour of MRSA strains and mutants. Key regulatory mutation combinations (agr–, sae–, sae–/agr–, sigB+, sigB+/sae–) were directly tested in the model but also in experiments. High connectivity was a good guide to identify master regulators, whose detailed behaviour was studied both in vitro and in the model. Together, both lines of evidence support in particular a refined regulatory role for sae and agr with involvement in biofilm repression and/or SA dissemination. With examination of the composition of different mutant biofilms as well as with the examination of the reaction cascade that connects sae to the biofilm forming ability of SA and also by postulating that nucleases might play an important role in that, first steps were taken in proving and explaining regulatory links leading from sae to biofilms. Furthermore differences in biofilms of different mutant SA strains were found leading us in perspective towards a new understanding of biofilms including knowledge how to better regulate, fight and use its different properties. / Staphylococcus aureus (SA) ist Auslöser nosocomialer Infektionen, darunter auch die, durch multiresistente Stämme (MRSA) verursachte, lebensbedrohliche Sepsis. Er hat die Fähigkeit Biofilme zu bilden, um sich vor dem Immunsystem des Wirtes und vor Antibiotika zu schützen. Biofilm und planktonische Lebensweise werden durch ein komplexes Quorum-Sensing (QS) System mit agr als zentralem Regulator gesteuert. Um die Biofilm Bildung und QS Mechanismen in SA zu untersuchen, wurde ein Boole´sches Netzwerk erstellt (94 Knoten, 184 Kanten) das verschiedene Zwei-Komponenten-Systeme wie agr, sae und arl mit einschließt. Wichtige Proteine wie Sar, Rot und SigB wurden als weitere Knoten im Modell eingefügt. Die Systemanalyse zeigte, dass es nur zwei stabile Zustände gibt, Biofilm bildend versus planktonisch, in denen deutlich unterschiedliche Subnetzwerke angeschaltet sind. Überprüfungen anhand von Gen-Expressions-Daten bestätigten dies. Die Netzwerkstabilität wurde zuerst an Hand von bestehendem Wissen und Literatur getestet. Zudem stimmte die vorhergesagte Aktivität der Knoten in verschiedenen in silico Knock-out Stämmen sehr gut mit den zugehörigen Micro-array Experimenten und Daten überein. Zusätzliche Validierungen schlossen die Expression weiterer Knoten (Northern Blots) und die Biofilm Produktion, verglichen durch Biofilm adherence assays, in verschiedenen Knock-out Stämmen mit ein. Das Modell spiegelt zuverlässig das Verhalten von QS-Signal Mutanten wieder. Das integrierte Modell erlaubt auch Vorhersagen von diversen anderen Netzwerk Mutationen und wird durch experimentelle Daten unterschiedlicher Stämme gestützt. Außerdem zeigen die gut vernetzten Hubproteine im Detail auf, wie die Verarbeitung unterschiedlicher Eingangssignale durch das QS-Netzwerk erreicht wird. Sowohl für in silico als auch für in vitro Experimente konnte gezeigt werden, dass der sae-Locus auch einen zentralen Modulator der Biofilm Produktion darstellt, sae Knock-out Stämme zeigten stärkere Biofilme. Der Wildtyp Phänotyp wurde durch sae Komplementierung wiederhergestellt. Um die Art und Weise, mit der sae Einfluss auf die Biofilm Bildung nimmt, aufzuklären wurde das Netzwerk genutzt und Venn-Diagramme angefertigt, welche Knoten aufzeigten, die durch sae reguliert- und in Biofilmen verändert sind. In den Venn-Diagrammen wurden Nucleasen und extrazelluläre Proteine als vielversprechende Knoten gefunden. Das Netzwerk zeigte, dass DNAse von großer Bedeutung ist. Deswegen wurde qualitativ die, durch unterschiedliche SA Mutanten produzierte, DNAse-Menge gemessen, es wurde versucht den Biofilm mit vergleichbaren DNAse-Mengen aufzulösen und die Konzentration von Nukleinsäuren, Proteinen und Polysacchariden wurde in Biofilmen unterschiedlicher SA Mutanten gemessen. Aufgrund seiner sorgfältigen Überprüfung stellt das Netzwerk-Modell ein mächtiges Werkzeug zur Untersuchung von QS und Biofilm Bildung in SA dar, erfolgreiche Vorhersagen über das Verhalten unterschiedlicher Knock-out Mutanten, QS Signale und Biofilm Bildung eingeschlossen. Dies beinhaltet Prognosen für das Verhalten von MRSA Stämmen und Mutanten. Zentrale regulatorische Mutationskombinationen (agr–, sae–, sae–/agr–, sigB+, sigB+/sae–) wurden direkt im Model aber auch in Experimenten getestet. Hohe Konektivität war ein guter Anhaltspunkt, um Hauptregulatoren zu identifizieren, deren Verhalten in vitro und im Modell untersucht wurde. Zusammen unterstützen beide Beweisführungen im Besonderen eine präzise regulatorische Rolle von sae und agr in Bezug auf Biofilm Unterdrückung und/oder SA Ausbreitung. Mit der Untersuchung der Zusammensetzung von Biofilmen unterschiedlicher Mutanten, ebenso wie mit der Untersuchung der Reaktionskaskade die sae mit der Biofilm Bildungsfähigkeit von SA verbindet und auch dem Überprüfen der Annahme, dass Nukleasen eine bedeutende Rolle hierin spielen könnten, wurden erste Schritte unternommen, um regulatoische Interaktionen zwischen sae und Biofilmen zu belegen und zu untersuchen. Des Weiteren wurden Unterschiede in Biofilmen verschiedener mutierter SA Stämme gefunden, die uns voraussichtlich zu einem neuem Verständnis von Biofilmen und damit zu Wissen führen, wie ihre Eigenschaften reguliert, bekämpft und genutzt werden können.