Molecular Characterization of the Interaction of Staphylococcal Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMM) ClfA and Fbl with Fibrinogen

The ligand-binding domain of Fbl (the fibrinogen binding protein from Staphylococcus lugdunensis) shares 60% sequence identity with ClfA (clumping factor A) of Staphylococcus aureus. Recombinant Fbl corresponding to the minimum fibrinogen-binding region (subdomains N2N3) was compared with ClfA for binding to fibrinogen. Fbl and ClfA had very similar affinities for fibrinogen by surface plasmon resonance. The binding site for Fbl in fibrinogen was localized to the extreme C terminus of the fibrinogen γ-chain at the same site recognized by ClfA. Isothermal titration calorimetry showed that Fbl and ClfA had very similar affinities for a peptide mimicking the C-terminal segment of the fibrinogen γ-chain. The peptide also inhibited binding of Fbl and ClfA to fibrinogen. A series of substituted γ-chain variant peptides behaved very similarly when used to inhibit ClfA and Fbl binding to immobilized fibrinogen. Both ClfA and Fbl bound to bovine fibrinogen with a lower affinity compared with human fibrinogen and did not bind detectably to ovine fibrinogen. The structure of the N2N3 subdomains of Fbl in complex with the fibrinogen γ-chain peptide was modeled based on the crystal structure of the N2N3 subdomains of the ClfA-γ-chain peptide complex. Residues in the putative binding trench likely to be involved in fibrinogen binding were identified. Fbl variant proteins with alanine substitutions in key residues had reduced affinities for fibrinogen. Thus Fbl and ClfA bind the same site in fibrinogen by similar mechanisms.     

Mycobacterium tuberculosis Prokaryotic Ubiquitin-like Protein-deconjugating Enzyme Is an Unusual Aspartate Amidase

Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.     

Molecular Characterization of a Novel Staphylococcus Aureus Surface Protein (SasC) Involved in Cell Aggregation and Biofilm Accumulation

Background

Staphylococci belong to the most important pathogens causing implant-associated infections. Colonization of the implanted medical devices by the formation of a three-dimensional structure made of bacteria and host material called biofilm is considered the most critical factor in these infections. To form a biofilm, bacteria first attach to the surface of the medical device, and then proliferate and accumulate into multilayered cell clusters. Biofilm accumulation may be mediated by polysaccharide and protein factors.

Methology/Principal Findings

The information on Staphylococcus aureus protein factors involved in biofilm accumulation is limited, therefore, we searched the S. aureus Col genome for LPXTG-motif containing potential surface proteins and chose the so far uncharacterized S. aureus surface protein C (SasC) for further investigation. The deduced SasC sequence consists of 2186 amino acids with a molecular mass of 238 kDa and has features typical of Gram-positive surface proteins, such as an N-terminal signal peptide, a C-terminal LPXTG cell wall anchorage motif, and a repeat region consisting of 17 repeats similar to the domain of unknown function 1542 (DUF1542). We heterologously expressed sasC in Staphylococcus carnosus, which led to the formation of huge cell aggregates indicative of intercellular adhesion and biofilm accumulation. To localize the domain conferring cell aggregation, we expressed two subclones of sasC encoding either the N-terminal domain including a motif that is found in various architectures (FIVAR) or 8 of the DUF1542 repeats. SasC or its N-terminal domain, but not the DUF1542 repeat region conferred production of huge cell aggregates, higher attachment to polystyrene, and enhanced biofilm formation to S. carnosus and S. aureus. SasC does not mediate binding to fibrinogen, thrombospondin-1, von Willebrand factor, or platelets as determined by flow cytometry.

Conclusions/Significance

Thus, SasC represents a novel S. aureus protein factor involved in cell aggregation and biofilm formation, which may play an important role in colonization during infection with this important pathogen.     

The Urokinase Receptor Homolog Haldisin Is a Novel Differentiation Marker of Stratum Granulosum in Squamous Epithelia

Several members of the Ly-6/uPAR (LU)-protein domain family are differentially expressed in human squamous epithelia. In some cases, they even play important roles in maintaining skin homeostasis, as exemplified by the secreted single domain member, SLURP-1, the deficiency of which is associated with the development of palmoplantar hyperkeratosis in the congenital skin disorder Mal de Meleda. In the present study, we have characterized a new member of the LU-protein domain family, which we find to be predominantly expressed in the stratum granulosum of human skin, thus resembling the expression of SLURP-1. In accordance with its expression pattern, we denote this protein product, which is encoded by the LYPD5 gene, as Haldisin (human antigen with LU-domains expressed in skin). Two of the five human glycolipid-anchored membrane proteins with multiple LU-domains characterized so far are predominantly confined to squamous epithelia (i.e., C4.4A), to stratum spinosum, and Haldisin to stratum granulosum under normal homeostatic conditions. Whether Haldisin is a prognostic biomarker for certain epithelial malignancies, like C4.4A and SLURP-1, remains to be explored.     

Direct Inhibition of Pumilo Activity by Bam and Bgcn in Drosophila Germ Line Stem Cell Differentiation

The fate of stem cells is intricately regulated by numerous extrinsic and intrinsic factors that promote maintenance or differentiation. The RNA-binding translational repressor Pumilio (Pum) in conjunction with Nanos (Nos) is required for self-renewal, whereas Bam (bag-of-marbles) and Bgcn (benign gonial cell neoplasm) promote differentiation of germ line stem cells in the Drosophila ovary. Genetic analysis suggests that Bam and Bgcn antagonize Pum/Nos function to promote differentiation; however, the molecular basis of this epistatic relationship is currently unknown. Here, we show that Bam and Bgcn inhibit Pum function through direct binding. We identified a ternary complex involving Bam, Bgcn, and Pum in which Bam, but not Bgcn, directly interacts with Pum, and this interaction is greatly increased by the presence of Bgcn. In a heterologous reporter assay to monitor Pum activity, Bam, but not Bgcn, inhibits Pum activity. Notably, the N-terminal region of Pum, which lacks the C-terminal RNA-binding Puf domain, mediates both the ternary protein interaction and the Bam inhibition of Pum function. These studies suggest that, in cystoblasts, Bam and Bgcn may directly inhibit Pum/Nos activity to promote differentiation of germ line stem cells.     

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

We have shown that Rpl3, a protein of the large ribosomal subunit from baker''s yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-³H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.     

Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.     

Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

N⁶-methyladenosine (m⁶A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m⁶A demethylases and cell-type and cell-state-dependent m⁶A patterns indicate that m⁶A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m⁶A modification include mRNA splicing, export, stability, and immune tolerance; but m⁶A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m⁶A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m⁶A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m⁶A modification. We applied the method to determine the m⁶A status at several sites in two human lncRNAs and three human mRNAs and found that m⁶A fraction varies between 6% and 80% among these sites. We also found that many m⁶A candidate sites in these RNAs are however not modified. The precise determination of m⁶A status in a long noncoding RNA also enables the identification of an m⁶A-containing RNA structural motif.     

Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r₂C1s₂ was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.     

Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte

The plasma membrane H⁺-ATPase provides the driving force for solute transport via an electrochemical gradient of H⁺ across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H⁺-ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H⁺-ATPase (pT H⁺-ATPase) and non-pT H⁺-ATPase as in the green algae, and that pT H⁺-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H⁺-ATPase genes, designated PpHA (Physcomitrella patens H⁺-ATPase). Six isoforms are the pT H⁺-ATPase; a remaining isoform is non-pT H⁺-ATPase. An apparent 95-kD protein was recognized by anti-H⁺-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H⁺-ATPase. Furthermore, we could not detect the pT H⁺-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H⁺-ATPase most likely appeared for the first time in bryophyte.     

CRISPRs: Molecular Signatures Used for Pathogen Subtyping

Rapid and accurate strain identification is paramount in the battle against microbial outbreaks, and several subtyping approaches have been developed. One such method uses clustered regular interspaced short palindromic repeats (CRISPRs), DNA repeat elements that are present in approximately half of all bacteria. Though their signature function is as an adaptive immune system against invading DNA such as bacteriophages and plasmids, CRISPRs also provide an excellent framework for pathogen tracking and evolutionary studies. Analysis of the spacer DNA sequences that reside between the repeats has been tremendously useful for bacterial subtyping during molecular epidemiological investigations. Subtyping, or strain identification, using CRISPRs has been employed in diverse Gram-positive and Gram-negative bacteria, including Mycobacterium tuberculosis, Salmonella enterica, and the plant pathogen Erwinia amylovora. This review discusses the several ways in which CRISPR sequences are exploited for subtyping. This includes the well-established spoligotyping methodologies that have been used for 2 decades to type Mycobacterium species, as well as in-depth consideration of newer, higher-throughput CRISPR-based protocols.     

Regeneration and assessment of genetic fidelity of the endangered tree Moringa peregrina (Forsk.) Fiori using Inter Simple Sequence Repeat (ISSR)

Moringa peregrinais an endangered species of Moringaceae.M. peregrinais a multipurpose tree with a wide variety of potential uses including its medicinal activity. In our study, a rapid and efficient micropropagation protocol for M. peregrina has been established. In vitro germinated seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different levels of either 6-benzyladenine (BA) or kinetin (Kin). The maximum shoot proliferation of 6.5 shoots per explant with 100 % shoot proliferation rate was observed on MS medium supplemented with 1.0 mg/l BA. On the other hand, MS medium supplemented with 1 mg/l indole-3-butyric acid (IBA) resulted in the maximum number of roots. Micropropagated plants were successfully acclimatized. Genetic stability of micropropagated plants was assessed using Inter-Simple Sequence Repeat (ISSR). The amplification products were monomorphic in all in vitro grown plants. No polymorphism was detected indicating the genetic integrity of in vitro propagated plants. This micropropagation protocol could be useful for raising genetically uniform plants for plant propagation and commercial cultivation.     

Identification of Cysteine Residues in Human Cationic Amino Acid Transporter hCAT-2A That Are Targets for Inhibition by N-Ethylmaleimide

In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y⁺L (y⁺LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y⁺ (most likely CAT-1), but not system y⁺L (Devés, R., Angelo, S., and Chávez, P. (1993) J. Physiol. 468, 753–766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y⁺LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y⁺LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced transporter expression in the plasma membrane, indicating that NEM reduces the intrinsic transporter activity. Individual mutation of each of the seven cysteine residues conserved in all CAT isoforms did not lead to NEM insensitivity of hCAT-2A. However, a cysteine-less mutant was no longer inhibited by NEM, suggesting that inhibition occurs through modification of more than one cysteine in hCAT-2A. Indeed, also the double mutant C33A/C273A was insensitive to NEM inhibition, whereas reintroduction of a cysteine at either position 33 or 273 in the cysteine-less mutant led to NEM sensitivity. We thus identified Cys-33 and Cys-273 in hCAT-2A as the targets of NEM inhibition. In addition, all proteins with Cys-33 mutations showed a pronounced reduction in transport activity, suggesting that, surprisingly, this residue, located in the cytoplasmic N terminus, is important for transporter function.     

Aminoquinoline Surfen Inhibits the Action of SEVI (Semen-derived Enhancer of Viral Infection)

In semen, proteolytic peptide fragments from prostatic acid phosphatase can form amyloid fibrils termed SEVI (semen-derived enhancer of viral infection). These fibrils greatly enhance human immunodeficiency virus (HIV) infectivity by increasing the attachment of virions to target cells. Therefore, SEVI may have a significant impact on whether HIV is successfully transmitted during sexual contact. Here, we demonstrate that surfen, a small molecule heparan sulfate proteoglycan antagonist, inhibits both SEVI- and semen-mediated enhancement of HIV type 1 infection. Surfen interferes with the binding of SEVI to both target cells and HIV type 1 virions but does not deaggregate SEVI fibrils. Because SEVI can increase HIV infectivity by several orders of magnitude, supplementing current HIV microbicide candidates with SEVI inhibitors, such as surfen, might greatly increase their potency.     

A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.     

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope,Endoplasmic Reticulum,and Mitochondria

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.     

Complementary Roles for Exonuclease 1 and Flap Endonuclease 1 in Maintenance of Triplet Repeats

Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5′-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5′-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5′-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5′-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5′-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.