The surface of
is decorated with over 20 proteins that are covalently anchored to peptidoglycan by the action of sortase A. These cell wall-anchored (CWA) proteins can be classified into several ...structural and functional groups. The largest is the MSCRAMM family, which is characterized by tandemly repeated IgG-like folded domains that bind peptide ligands by the dock lock latch mechanism or the collagen triple helix by the collagen hug. Several CWA proteins comprise modules that have different functions, and some individual domains can bind different ligands, sometimes by different mechanisms. For example, the N-terminus of the fibronectin binding proteins comprises an MSCRAMM domain which binds several ligands, while the C-terminus is composed of tandem fibronectin binding repeats. Surface proteins promote adhesion to host cells and tissue, including components of the extracellular matrix, contribute to biofilm formation by stimulating attachment to the host or indwelling medical devices followed by cell-cell accumulation via homophilic interactions between proteins on neighboring cells, help bacteria evade host innate immune responses, participate in iron acquisition from host hemoglobin, and trigger invasion of bacteria into cells that are not normally phagocytic. The study of genetically manipulated strains using animal infection models has shown that many CWA proteins contribute to pathogenesis. Fragments of CWA proteins have the potential to be used in multicomponent vaccines to prevent
infections.
Staphylococcus aureus is the most frequent causative organism of osteomyelitis. It is characterised by widespread bone loss and bone destruction. Previously we demonstrated that S. aureus protein A ...(SpA) is capable of binding to tumour necrosis factor receptor-1 expressed on pre-osteoblastic cells, which results in signal generation that leads to cell apoptosis resulting in bone loss. In the current report we demonstrate that upon S. aureus binding to osteoblasts it also inhibits de novo bone formation by preventing expression of key markers of osteoblast growth and division such as alkaline phosphatase, collagen type I, osteocalcin, osteopontin and osteocalcin. In addition, S. aureus induces secretion of soluble RANKL from osteoblasts which in turn recruits and activates the bone resorbing cells, osteoclasts. A strain of S. aureus defective in SpA failed to affect osteoblast growth or proliferation and most importantly failed to recruit or activate osteoclasts. These results suggest that S. aureus SpA binding to osteoblasts provides multiple coordinated signals that accounts for bone loss and bone destruction seen in osteomyelitis cases. A better understanding of the mechanisms through which S. aureus leads to bone infection may improve treatment or lead to the development of better therapeutic agents to treat this notoriously difficult disease.
Nonrandom collecting practices may bias conclusions drawn from analyses of herbarium records. Recent efforts to fully digitize and mobilize regional floras online offer a timely opportunity to assess ...commonalities and differences in herbarium sampling biases.
We determined spatial, temporal, trait, phylogenetic, and collector biases in c. 5 million herbarium records, representing three of the most complete digitized floras of the world: Australia (AU), South Africa (SA), and New England, USA (NE).
We identified numerous shared and unique biases among these regions. Shared biases included specimens collected close to roads and herbaria; specimens collected more frequently during biological spring and summer; specimens of threatened species collected less frequently; and specimens of close relatives collected in similar numbers. Regional differences included overrepresentation of graminoids in SA and AU and of annuals in AU; and peak collection during the 1910s in NE, 1980s in SA, and 1990s in AU. Finally, in all regions, a disproportionately large percentage of specimens were collected by very few individuals. We hypothesize that these mega-collectors, with their associated preferences and idiosyncrasies, shaped patterns of collection bias via ‘founder effects’.
Studies using herbarium collections should account for sampling biases, and future collecting efforts should avoid compounding these biases to the extent possible.
Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not ...been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans.
Protein-based biofilm matrices in Staphylococci Speziale, Pietro; Pietrocola, Giampiero; Foster, Timothy J ...
Frontiers in cellular and infection microbiology,
12/2014, Letnik:
4
Journal Article
Recenzirano
Odprti dostop
Staphylococcus aureus and Staphylococcus epidermidis are the most important etiological agents of biofilm associated-infections on indwelling medical devices. Biofilm infections may also develop ...independently of indwelling devices, e.g., in native valve endocarditis, bone tissue, and open wounds. After attachment to tissue or indwelling medical devices that have been conditioned with host plasma proteins, staphylococcal biofilms grow, and produce a specific environment which provides the conditions for cell-cell interaction and formation of multicellular communities. Bacteria living in biofilms express a variety of macromolecules, including exopolysaccharides, proteins, extracellular eDNA, and other polymers. The S. aureus surface protein C and G (SasC and SasG), clumping factor B (ClfB), serine aspartate repeat protein (SdrC), the biofilm-associated protein (Bap), and the fibronectin/fibrinogen-binding proteins (FnBPA and FnBPB) are individually implicated in biofilm matrix formation. In S. epidermidis, a protein named accumulation-associated protein (Aap) contributes to both the primary attachment phase and the establishment of intercellular connections by forming fibrils on the cell surface. In S. epidermidis, proteinaceous biofilm formation can also be mediated by the extracellular matrix binding protein (Embp) and S. epidermidis surface protein C (SesC). Additionally, multifunctional proteins such as extracellular adherence protein (Eap) and extracellular matrix protein binding protein (Emp) of S. aureus and the iron-regulated surface determinant protein C (IsdC) of S. lugdunensis can promote biofilm formation in iron-depleted conditions. This multitude of proteins intervene at different stages of biofilm formation with certain proteins contributing to biofilm accumulation and others mediating primary attachment to surfaces. This review examines the contribution of proteins to biofilm formation in Staphylococci. The potential to develop vaccines to prevent protein-dependent biofilm formation during staphylococcal infection is discussed.
Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into ...clinical isolates of S. aureus. Here, we describe the construction and characterization of the IMXXB series of Escherichia coli strains that mimic the type I adenine methylation profiles of S. aureus clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages.
The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus. The IMXXB series of E. coli strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus.
Macrocyclic ligands have been explored extensively as scaffolds for transition metal catalysts for oxygen and hydrogen atom transfer reactions. C–C reactions facilitated using earth abundant metals ...bound to macrocyclic ligands have not been well-understood but could be a green alternative to replacing the current expensive and toxic precious metal systems most commonly used for these processes. Therefore, the yields from direct Suzuki–Miyaura C–C coupling of phenylboronic acid and pyrrole to produce 2-phenylpyrrole facilitated by eight high-spin iron complexes (Fe 3+ L1(Cl) 2 + , Fe 3+ L4(Cl) 2 + , Fe 2+ L5(Cl) + , Fe 2+ L6(Cl) 2 , Fe 3+ L7(Cl) 2 + , Fe 3+ L8(Cl) 2 + , Fe 2+ L9(Cl) + , and Fe 2+ L10(Cl) + ) were compared to identify the effect of structural and electronic properties on catalytic efficiency. Specifically, catalyst complexes were compared to evaluate the effect of five properties on catalyst reaction yields: (1) the coordination requirements of the catalyst, (2) redox half-potential of each complex, (3) topological constraint/rigidity, (4) N atom modification(s) increasing oxidative stability of the complex, and (5) geometric parameters. The need for two labile cis-coordination sites was confirmed based on a 42% decrease in catalytic reaction yield observed when complexes containing pentadentate ligands were used in place of complexes with tetradentate ligands. A strong correlation between iron(III/II) redox potential and catalytic reaction yields was also observed, with Fe 2+ L6(Cl) 2 providing the highest yield (81%, −405 mV). A Lorentzian fitting of redox potential versus yields predicts that these catalysts can undergo more fine-tuning to further increase yields. Interestingly, the remaining properties explored did not show a direct, strong relationship to catalytic reaction yields. Altogether, these results show that modifications to the ligand scaffold using fundamental concepts of inorganic coordination chemistry can be used to control the catalytic activity of macrocyclic iron complexes by controlling redox chemistry of the iron center. Furthermore, the data provide direction for the design of improved catalysts for this reaction and strategies to understand the impact of a ligand scaffold on catalytic activity of other reactions.
Background Colonization of the skin by Staphylococcus aureus in individuals with atopic dermatitis exacerbates inflammation. Atopic dermatitis is associated with loss-of-function mutations in the ...filaggrin (FLG) gene, accompanied by reduced levels of filaggrin breakdown products on the skin. Objective To assess the affect of growth in the presence of the filaggrin breakdown products urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA) on fitness of and protein expression by S aureus. Methods S aureus was grown for 24 hours in the presence of UCA and PCA, and the density of the cultures was monitored by recording OD600 values. Cell wall extracts and secreted proteins of S aureus were isolated and analyzed by SDS-PAGE. Cell wall–associated proteins known to be involved in colonization and immune evasion including clumping factor B, fibronectin binding proteins, protein A, iron-regulated surface determinant A, and the serine-aspartate repeat proteins were examined by Western immunoblotting. Results Acidification of growth media caused by the presence of UCA and PCA resulted in reduced growth rates and reduced final cell density of S aureus. At the lower pH, reduced expression of secreted and cell wall–associated proteins, including proteins involved in colonization (clumping factor B, fibronectin binding protein A) and immune evasion (protein A), was observed. Decreased expression of iron-regulated surface determinant A due to growth with filaggrin breakdown products appeared to be independent of the decreased pH. Conclusion S aureus grown under mildly acidic conditions such as those observed on healthy skin expresses reduced levels of proteins that are known to be involved in immune evasion.
The strong restriction barrier present in Staphylococcus aureus and Staphylococcus epidermidis has limited functional genomic analysis to a small subset of strains that are amenable to genetic ...manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain of S. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiency Escherichia coli cloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates of S. aureus and S. epidermidis. We also show that the loss of restriction (both type I and IV) in an S. aureus USA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens.
Staphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding of Staphylococcus aureus and Staphylococcus epidermidis infections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.
Staphylococcus aureus surface protein SasG promotes cell–cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, ...we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn²⁺ strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell–cell adhesion via specific Zn²⁺-dependent homophilic bonds between β-sheet–rich G5–E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell–cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.
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