Structural relationships among chloramphenicol-resistance plasmids of Staphylococcus aureus

Abstract Twelve different chloramphenicol-resistance (Cm^r) plasmids detected in Staphylococcus aureus strains isolated between 1952 and 1981 were characterized by restriction endonuclease, DNA hybridization and heteroduplex analyses. These studies revealed three families of Cm^r plasmids which were distinguished by their chloramphenicol acetyltransferase sequences; the prototype plasmids of the families were pC221, pC223 and pC194. The cat and replication ( rep ) genes of the plasmid pC221 were highly conserved in other pC221 family members and were related to their homologs in the pC223 family plasmids; however, the cat and rep genes of the pC194 family plasmids were distinct.     

Structural and functional characterization of Staphylococcus aureus dihydrodipicolinate synthase

Lysine biosynthesis is crucial for cell-wall formation in bacteria. Enzymes involved in lysine biosynthesis are thus potential targets for anti-microbial therapeutics. Dihydrodipicolinate synthase (DHDPS) catalyzes the first step of this pathway. Unlike its homologues, Staphylococcus aureus DHDPS is a dimer both in solution and in the crystal and is not feedback inhibited by lysine. The crystal structure of S. aureus DHDPS in the free and substrate bound forms provides a structural rationale for its catalytic mechanism. The structure also reveals unique conformational features of the S. aureus enzyme that could be crucial for the design of specific non-competitive inhibitors.     

Structural characterization of the alpha-hemolysin monomer from Staphylococcus aureus

Alpha-hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states-from the soluble monomer to all potential "pre-pore" structures-are yet unknown. Here, we propose a model of the monomeric alpha-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the alpha-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates.     

Structural and evolutionary relationships of beta-lactamase transposons from Staphylococcus aureus

A comparison of the beta-lactamase elements detected on three classes of large plasmids together with the chromosomes of penicillin-resistant Staphylococcus aureus revealed substantial physical and genetic relatedness. In most cases, beta-lactamase production could be associated with the presence of a DNA segment of approximately 6.7 kb. Analysis showed that the plasmid-borne determinants constitute nearly identical transposons or transposon-like elements. An element indistinguishable from one of these, Tn4002, which is carried by the pSK1 family of plasmids in clinical isolates from Australian hospitals, was also identified on the staphylococcal chromosome and is implicated in an evolutionary cycle of transposition between chromosomal and extrachromosomal sites in Australian strains of multiresistant S. aureus.     

Structural analysis of Staphylococcus aureus serine/threonine kinase PknB

Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 ? resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.     

Structural insight into binding of Staphylococcus aureus to human fibronectin

Staphylococcus aureus possesses cell-wall attached proteins that bind the human protein fibronectin (Fn). An intermodule interface between the 4F1 and 5F1 modules in the N-terminal domain of Fn is maintained on bacterial peptide binding but there is a small change in the intermodule orientation and alignment of beta-strands that are predicted to bind the peptide. The module pair is elongated, as in the unbound state. Combined with evidence that residues in both 4F1 and 5F1 are directly involved in peptide binding, this observation supports the hypothesis that, when bound to intact Fn, the bacterial protein adopts an unusual, highly extended conformation.     

Structural and Functional analysis of Staphylococcus aureus NADP-dependent IDH and its comparison with Bacterial and Human NADPdependent IDH

Staphylococcus aureus a natural inhabitant of nasopharyngeal tract mainly survives as biofilms and possess complete Krebs cycle which plays major role in its pathogenesis. This TCA cycle is regulated by Isocitrate dehydrogenase (IDH) we have earlier cloned, sequenced ({"type":"entrez-nucleotide","attrs":{"text":"HM067707","term_id":"297186241","term_text":"HM067707"}}HM067707), expressed and characterized this enzyme from S. aureus ATCC12600. We have observed only one type of IDH in all the strains of S. aureus which dictates the flow of carbon thereby controlling the virulence and biofilm formation, this phenomenon is variable among bacteria. Therefore in the present study comparative structural and functional analysis of IDH was undertaken. As the crystal structure of S. aureus IDH was not available therefore using the deduced amino sequence of complete gene the 3D structure of IDH was built in Modeller 9v8. The PROCHECK and ProSAweb analysis showed the built structure was close to the crystal structure of Bacillus subtilis. This structure when superimposed with other bacterial IDH structures exhibited extensive structural variations as evidenced from the RMSD values correlating with extensive sequential variations. Only 24% sequence identity was observed with both human NADP dependent IDHs (PDB: 1T09 and 1T0L) and the structural comparative studies indicated extensive structural variations with an RMSD values of 14.284Å and 10.073Å respectively. Docking of isocitrate to both human IDHs and S. aureus IDH structures showed docking scores of -11.6169 and -10.973 respectively clearly indicating higher binding affinity of isocitrate to human IDH.     

Structural Insights into SraP-Mediated Staphylococcus aureus Adhesion to Host Cells

Staphylococcus aureus, a Gram-positive bacterium causes a number of devastating human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. S. aureus SraP, a surface-exposed serine-rich repeat glycoprotein (SRRP), is required for the pathogenesis of human infective endocarditis via its ligand-binding region (BR) adhering to human platelets. It remains unclear how SraP interacts with human host. Here we report the 2.05 Å crystal structure of the BR of SraP, revealing an extended rod-like architecture of four discrete modules. The N-terminal legume lectin-like module specifically binds to N-acetylneuraminic acid. The second module adopts a β-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differ in calcium coordination pattern. Under the conditions tested, small-angle X-ray scattering and molecular dynamic simulation indicated that the three C-terminal modules function as a relatively rigid stem to extend the N-terminal lectin module outwards. Structure-guided mutagenesis analyses, in addition to a recently identified trisaccharide ligand of SraP, enabled us to elucidate that SraP binding to sialylated receptors promotes S. aureus adhesion to and invasion into host epithelial cells. Our findings have thus provided novel structural and functional insights into the SraP-mediated host-pathogen interaction of S. aureus.     

Structural analysis of the surface polysaccharide of Staphylococcus aureus M.

The chemical structure of the surface polysaccharide from Staphylococcus aureus M was investigated by a combination of methanolytic, hydrolytic, and chromatographic techniques. The repeating unit that was most consistent with the data was a hexasaccharide composed of N-acetyl-D-aminogalacturonic acid, N-acetyl-D-fucosamine, and taurine in molar ratios of 4:2:1. A disaccharide was isolated and characterized, by combined gas-liquid chromatography-mass spectrometry, as N-acetyl-D-aminogalacturonyl-(1 leads to 3)-N-acetyl-D-fucosamine. Taurine is linked to a carboxyl group of N-acetyl-D-aminogalacturonic acid via an amide bond.     

Relatedness between the coagulase gene 3'-end region and coagulase serotypes among Staphylococcus aureus strains

The 3'-end region of the coagulase gene from 22 strains of Staphylococcus aureus including 10 standard serotype strains was sequenced, and five subgroups with 4-8 tandem repeating units were distinguished among the tested strains. Phylogenetic analysis of the 3'-end region of the coagulase gene indicated that strains belonging to the same serotype were clustered in the same branch. A phylogenetic tree of the deduced amino acid sequences revealed that the C-terminal region might not be responsible for the epitope of the coagulase protein.     

Structural characterization of MepB from Staphylococcus aureus reveals homology to endonucleases

The MepRAB operon in Staphylococcus aureus has been identified to play a role in drug resistance. Although the functions of MepA and MepR are known, little information is available on the function of MepB. Here we report the X‐ray structure of MepB to 2.1 Å revealing its structural similarity to the PD‐(D/E)XK family of endonucleases. We further show that MepB binds DNA and RNA, with a higher affinity towards RNA and single stranded DNA than towards double stranded DNA. Notably, the PD‐(D/E)XK catalytic active site residues are not conserved in MepB. MepB's association with a drug resistance operon suggests that it plays a role in responding to antimicrobials. This role is likely carried out through MepB's interactions with nucleic acids.     

Structural basis of Staphylococcus aureus Cas9 inhibition by AcrIIA14

Bacteriophages have evolved a range of anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes, therefore Acrs can be used as switches to regulate gene editing. Herein, we report the crystal structure of a quaternary complex of AcrIIA14 bound SauCas9–sgRNA–dsDNA at 2.22 Å resolution, revealing the molecular basis for AcrIIA14 recognition and inhibition. Our structural and biochemical data analysis suggest that AcrIIA14 binds to a non-conserved region of SauCas9 HNH domain that is distinctly different from AcrIIC1 and AcrIIC3, with no significant effect on sgRNA or dsDNA binding. Further, our structural data shows that the allostery of the HNH domain close to the substrate DNA is sterically prevented by AcrIIA14 binding. In addition, the binding of AcrIIA14 triggers the conformational allostery of the HNH domain and the L1 linker within the SauCas9, driving them to make new interactions with the target-guide heteroduplex, enhancing the inhibitory ability of AcrIIA14. Our research both expands the current understanding of anti-CRISPRs and provides additional culues for the rational use of the CRISPR-Cas system in genome editing and gene regulation.     

Vancomycin-resistant Staphylococcus aureus

The evolution and molecular mechanisms of vancomycin resistance in Staphylococcus aureus were reviewed. Case reports and research studies on biochemestry, electron microscopy and molecular biology of Staphylococcus aureus were selected from Medline database and summarized in the following review. After almost 40 years of successful treatment of S. aureus with vancomycin, several cases of clinical failures have been reported (since 1997). S. aureus strains have appeared with intermediate susceptibility (MIC 8-16 microg/ml), as well as strains with heterogeneous resistance (global MIC < or =4 microg/ml), but with subpopulations of intermediate susceptibility. In these cases, resistance is mediated by cell wall thickening with reduced cross linking. This traps the antibiotic before it reaches its major target, the murein monomers in the cell membrane. In 2002, a total vancomycin resistant strain (MIC > or =32 microg/ml) was reported with vanA genes from Enterococcus spp. These genes induce the change of D-Ala-D-Ala terminus for D-Ala-D-lactate in the cell wall precursors, leading to loss of affinity for glycopeptides. Vancomycin resistance in S. aureus has appeared; it is mediated by cell wall modifications that trap the antibiotic before it reaches its action site. In strains with total resistance, Enterococcus spp. genes have been acquired that lead to modification of the glycopeptide target.