Interaction of the Rho-ADP-ribosylating C3 exoenzyme with RalA

RhoA, -B, and -C are ADP-ribosylated and biologically inactivated by Clostridium botulinum C3 exoenzyme and related C3-like transferases. We report that RalA GTPase, which is not ADP-ribosylated by C3, inhibits ADP-ribosylation of RhoA by C3 from C. botulinum (C3bot), Clostridium limosum (C3lim), and Bacillus cereus (C3cer) but not from Staphylococcus aureus (C3stau) in human platelet membranes and rat brain lysate. Inhibition by RalA occurs with the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound forms of RalA and is overcome by increasing concentrations of C3. A direct interaction of RalA with C3 was verified by precipitation of the transferase with GST-RalA-Sepharose. The affinity constant (K(d)) of the binding of RalA to C3lim was 12 nm as determined by fluorescence titration. RalA increased the NAD glycohydrolase activity of C3bot by about 5-fold. Although RalA had no effect on glucosylation of Rho GTPases by Clostridium difficile toxin B, C3bot and C3lim inhibited glucosylation of RalA by Clostridium sordellii lethal toxin. Furthermore, C3bot decreased activation of phospholipase D by RalA. The data indicate that several C3 exoenzymes directly interact with RalA without ADP-ribosylating the GTPase. The interaction is of high affinity and interferes with essential functions of C3 and RalA.     

The crystal structure of C3stau2 from Staphylococcus aureus and its complex with NAD

The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase that ADP-ribosylates not only RhoA-C but also RhoE/Rnd3. In this study we have crystallized and determined the structure of C3stau2 in both its native form and in complex with NAD at 1.68- and 2.02-A resolutions, respectively. The topology of C3stau2 is similar to that of C3bot1 from Clostridium botulinum (with which it shares 35% amino acid sequence identity) with the addition of two extra helices after strand beta1. The native structure also features a novel orientation of the catalytic ARTT loop, which approximates the conformation seen for the "NAD bound" form of C3bot1. C3stau2 orients NAD similarly to C3bot1, and on binding NAD, C3stau2 undergoes a clasping motion and a rearrangement of the phosphate-nicotinamide binding loop, enclosing the NAD in the binding site. Comparison of these structures with those of C3bot1 and related toxins reveals a degree of divergence in the interactions with the adenine moiety among the ADP-ribosylating toxins that contrasts with the more conserved interactions with the nicotinamide. Comparison with C3bot1 gives some insight into the different protein substrate specificities of these enzymes.     

Structure-function analysis of the C3 binding region of Staphylococcus aureus immune subversion protein Sbi

Among the recently discovered Staphylococcus aureus immune evasion proteins, Sbi is unique in its ability to interact with components of both the adaptive and innate immune systems of the host. Sbi domains I and II (Sbi-I and Sbi-II) bind IgG. Sbi domain IV (residues 198-266) binds the central complement protein C3. When linked to Sbi-III, Sbi-IV induces a futile consumption of complement via alternative pathway activation, whereas isolated Sbi-IV specifically inhibits the alternative pathway without complement consumption. Here we have determined the three-dimensional structure of Sbi-IV by NMR spectroscopy, showing that Sbi-IV adopts a three-helix bundle fold similar to those of the S. aureus complement inhibitors Efb-C, Ehp, and SCIN. The (1)H-(15)N HSQC spectrum of Sbi-III indicates that this domain, essential for futile complement consumption, is natively unfolded, at least when isolated from the rest of Sbi. Sbi-IV and Sbi-III-IV both bind C3dg with 1:1 stoichiometry and submicromolar affinity. Despite low overall sequence identity, Sbi possesses the same residues as Efb at two positions essential for Efb-C binding to C3d. Mutation to alanine of either of these residues, Arg-231 and Asn-238, abolishes both Sbi-IV binding to C3dg and Sbi-IV alternative pathway inhibition. The almost complete conservation of Sbi-III and Sbi-IV amino acid sequences across more than 30 strains isolated from human and animal hosts indicates that the unique mechanism of Sbi in complement system subversion is a feature of infections of both humans and economically important animals.     

Structure-function analysis of the SaPIbov1 replication origin in Staphylococcus aureus

The SaPIs and their relatives are phage satellites and are unique among the known bacterial pathogenicity islands in their ability to replicate autonomously. They possess a phage-like replicon, which is organized as two sets of iterons arrayed symmetrically to flank an AT-rich region that is driven to melt by the binding of a SaPI-specific initiator (Rep) to the flanking iterons. Extensive deletion analysis has revealed that Rep can bind to a single iteron, generating a simple shift in a gel mobility assay; when bound on both sides, a second retarded band is seen, suggesting independent binding. Binding to both sites of the ori is necessary but not sufficient to melt the AT-rich region and initiate replication. For these processes, virtually the entire origin must be present. Since SaPI replication can be initiated on linear DNA, it is suggested that bilateral binding may be necessary to constrain the intervening DNA to enable Rep-driven melting.     

A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3

Clostridium botulinum C3 is the prototype of the family of the C3-like transferases that ADP-ribosylate exclusively RhoA, -B and -C. The ADP-ribose at Asn-41 results in functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton. We report on a new C3-like transferase produced by a pathogenic Staphylococcus aureus strain. The transferase designated C3(Stau) was cloned from the genomic DNA. At the amino acid level, C3(Stau) revealed an identity of 35% to C3 from C. botulinum and Clostridium limosum exoenzyme, respectively, and of 78% to EDIN from S. aureus. In addition to RhoA, which is the target of the other C3-like transferases, C3(Stau) modified RhoE and Rnd3. RhoE was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA. RhoE and Rnd3 are members of the Rho subfamily, which are deficient in intrinsic GTPase activity and possess a RhoA antagonistic cell function. The protein substrate specificity found with recombinant Rho proteins was corroborated by expression of RhoE in Xenopus laevis oocytes showing that RhoE was also modified in vivo by C3(Stau) but not by C3 from C. botulinum. The poor cell accessibility of C3(Stau) was overcome by generation of a chimeric toxin recruiting the cell entry machinery of C. botulinum C2 toxin. The chimeric C3(Stau) caused the same morphological and cytoskeletal changes as the chimeric C. botulinum C3. C3(Stau) is a new member of the family of the C3-like transferases but is also the prototype of a subfamily of RhoE/Rnd modifying transferases.     

Crystal structure of the Clostridium limosum C3 exoenzyme

C3-like toxins ADP-ribosylate and inactivate Rho GTPases. Seven C3-like ADP-ribosyltransferases produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus were identified and two representatives - C3bot from C. botulinum and C3stau2 from S. aureus - were crystallized. Here we present the 1.8 Å structure of C. limosum C3 transferase C3lim and compare it to the structures of other family members. In contrast to the structure of apo-C3bot, the canonical ADP-ribosylating turn turn motif is observed in a primed conformation, ready for NAD binding. This suggests an impact on the binding mode of NAD and on the transferase reaction. The crystal structure explains why auto-ADP-ribosylation of C3lim at Arg41 interferes with the ADP-ribosyltransferase activity of the toxin.     

C3stau, a new member of the family of C3-like ADP-ribosyltransferases.

C3-like ADP-ribosyltransferases, which are produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus, are exoenzymes lacking a translocation unit. These enzymes specifically inactivate Rho GTPases in host target cells. Recently, a novel C3-like transferase from S. aureus with new properties was identified, raising questions regarding its function. As Rho GTPases are master regulators of several eukaryotic signal processes and S. aureus can invade eukaryotic cells, C3 might play a role as a virulence factor.     

Recognition of RhoA by Clostridium botulinum C3 exoenzyme

The C3-like ADP-ribosyltransferases exhibit a very confined substrate specificity compared with other Rho-modifying bacterial toxins; they selectively modify the RhoA, -B, and -C isoforms but not other members of the Rho or Ras subfamilies. In this study, the amino acid residues involved in the RhoA substrate recognition by C3 from Clostridium botulinum are identified by applying mutational analyses of the nonsubstrate Rac. First, the minimum domain responsible for the recognition by C3 was identified as the N-terminal 90 residues. Second, the combination of the N-terminal basic amino acids ((Rho)Arg(5)-Lys(6)), the acid residues (Rho)Glu(47) and (Rho)Glu(54) only slightly increases ADP-ribosylation but fully restores the binding of the respective mutant Rac to C3. Third, the residues (Rho)Glu(40) and (Rho)Val(43) also participate in binding to C3 but they are mainly involved in the correct formation of the ternary complex between Rho, C3, and NAD(+). Thus, these six residues (Arg(5), Lys(6), Glu(40), Val(43), Glu(47), and Glu(54)) distributed over the N-terminal part of Rho are involved in the correct binding of Rho to C3. Mutant Rac harboring these residues shows a kinetic property with regard to ADP-ribosylation, which is identical with that of RhoA. Differences in the conformation of Rho given by the nucleotide occupancy have only minor effects on ADP-ribosylation.     

美洲大蠊肠道内生放线菌抗金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌活性分析

昆虫肠道共生放线菌作为一种特境放线菌,因与土壤中的放线菌生存环境的不同,研究其种类和抗菌活性具有重要意义.将金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)这两种致病菌作为指示细菌,对分离自美洲大蠊Periplaneta ame...     

Biophysical analysis of the putative acetyltransferase SACOL2570 from methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of a myriad of insidious and intractable infections in humans, especially in patients with compromised immune systems and children. Here, we report the apo- and CoA-bound crystal structures of a member of the galactoside acetyltransferase superfamily from methicillin-resistant S. aureus SACOL2570 which was recently shown to be down regulated in S. aureus grown in the presence of fusidic acid, an antibiotic used to treat MRSA infections. SACOL2570 forms a homotrimer in solution, as confirmed by small-angle X-ray scattering and dynamic light scattering. The protein subunit consists of an N-terminal alpha-helical domain connected to a C-terminal LβH domain. CoA binds in the active site formed by the residues from adjacent LβH domains. After determination of CoA-bound structure, molecular dynamics simulations were performed to model the binding of AcCoA. Binding of both AcCoA and CoA to SACOL2570 was verified by isothermal titration calorimetry. SACOL2570 most likely acts as an acetyltransferase, using AcCoA as an acetyl group donor and an as-yet-undetermined chemical moiety as an acceptor. SACOL2570 was recently used as a scaffold for mutations that lead the generation of cage-like assemblies, and has the potential to be used for the generation of more complex nanostructures.     

Staphylococcus aureus metalloprotease aureolysin cleaves complement C3 to mediate immune evasion

Complement is one of the first host defense barriers against bacteria. Activated complement attracts neutrophils to the site of infection and opsonizes bacteria to facilitate phagocytosis. The human pathogen Staphylococcus aureus has successfully developed ways to evade the complement system, for example by secretion of specific complement inhibitors. However, the influence of S. aureus proteases on the host complement system is still poorly understood. In this study, we identify the metalloprotease aureolysin as a potent complement inhibitor. Aureolysin effectively inhibits phagocytosis and killing of bacteria by neutrophils. Furthermore, we show that aureolysin inhibits the deposition of C3b on bacterial surfaces and the release of the chemoattractant C5a. Cleavage analyses show that aureolysin cleaves the central complement protein C3. Strikingly, there was a clear difference between the cleavages of C3 in serum versus purified conditions. Aureolysin cleaves purified C3 specifically in the α-chain, close to the C3 convertase cleavage site, yielding active C3a and C3b. However, in serum we observe that the aureolysin-generated C3b is further degraded by host factors. We pinpointed these factors to be factor H and factor I. Using an aureolysin mutant in S. aureus USA300, we show that aureolysin is essential and sufficient for C3 cleavage by bacterial supernatant. In short, aureolysin acts in synergy with host regulators to inactivate C3 thereby effectively dampening the host immune response.     

Two-Component Anti-Staphylococcus aureus Lantibiotic Activity Produced by Staphylococcus aureus C55

Staphylococcus aureus C55 was shown to produce bacteriocin activity comprising three distinct peptide components, termed staphylococcins C55α, C55β, and C55γ. The three peptides were purified to homogeneity by a simple four-step purification procedure that consisted of ammonium sulfate precipitation followed by XAD-2 and reversed-phase (C₈ and C₁₈) chromatography. The yield following C₈ chromatography was about 86%, with a more-than-300-fold increase in specific activity. When combined in approximately equimolar amounts, staphylococcins C55α and C55β acted synergistically to kill S. aureus or Micrococcus luteus but not S. epidermidis strains. The N-terminal amino acid sequences of all three peptides were obtained and staphylococcins C55α and C55β were shown to be lanthionine-containing (lantibiotic) molecules with molecular weights of 3,339 and 2,993, respectively. The C55γ peptide did not appear to be a lantibiotic, nor did it augment the inhibitory activities of staphylococcin C55α and/or C55β. Plasmids of 2.5 and 32.0 kb are present in strain C55, and following growth of this strain at elevated temperature (42°C), a large proportion of the progeny failed to produce strong bacteriocin activity and also lost the 32.0-kb plasmid. Protoplast transformation of these bacteria with purified 32-kb plasmid DNA regenerates the ability to produce the strong bacteriocin activity.     

Characterization and comparative analysis of a second thermonuclease from Staphylococcus aureus

Staphylococcal nuclease (here termed as Nuc1) is considered an important virulence factor and a unique marker widely used in the detection of Staphylococcus aureus. A second functional thermostable nuclease (here termed as Nuc2) in S. aureus was characterized after recombinant expression in Escherichia coli. Sequence alignment and phylogenetic analysis revealed that Nuc2 was a more conserved protein in the staphylococci group compared with Nuc1. Recombinant Nuc2 showed nuclease activity in the zymogram test and was able to degrade various types of nucleic acids. The optimal reaction temperature and pH for Nuc2 were 50 °C and pH 10, respectively. The enzymatic activity of Nuc2 was stimulated in the presence of Ca²⁺ (0.05 mM), Mg²⁺ (0.5 mM), dithiothreitol, β-mecaptoethanol, TritonX-100, Tween-20, and urea; however, activity decreased sharply when exposed to heavy metals such as Zn²⁺ and Mn²⁺, and in the presence of EDTA or SDS. Nuc2 showed weaker activity, lower thermostability and different sensitivity to these chemical agents compared with Nuc1, which was consistent with differences in the sequence pattern and structure predicted. Furthermore, a nuc1 and nuc2 double deletion mutant of S. aureus and respective complementation experiments suggest a major role for nuc1 in terms of thermonuclease activity in S. aureus.