Attachment and detachment of bacteria on surfaces with tunable and switchable wettability

Controlling accumulations of unwanted biofilms requires an understanding of the mechanisms that organisms use to interact with submerged substrata. While the substratum properties influencing biofilm formation are well studied, those that may lead to cellular or biofilm detachment are not. Surface-grafted stimuli-responsive polymers, such as poly (N-isopropylacrylamide) (PNIPAAm) release attached cells upon induction of environmentally-triggered phase changes. Altering the physicochemical characteristics of such polymeric systems for systematically studying release, however, can alter the phase transition. The physico-chemical changes of thin films of PNIPAAm grafted from initiator-modified self-assembled monolayers (SAMs) of ω-substituted alkanethiolates on gold can be altered by changing the composition of the underlying SAM, without affecting the overlying polymer. This work demonstrates that the ability to tune such changes in substratum physico-chemistry allows systematic study of attachment and release of bacteria over a large range of water contact angles. Such surfaces show great promise for studying a variety of interactions at the biointerface. Understanding of the source of this tunability will require further studies into the heterogeneity of such films and further investigation of interactions beyond those of water wettability.     

Synthesis,characterization and in vitro activity of a surface-attached antimicrobial cationic peptide

Infection associated with implanted biomaterials is common and costly and such infections are extremely resistant to antibiotics and host defenses. Consequently, there is a need to develop surfaces which resist bacterial adhesion and colonization. The broad spectrum synthetic cationic peptide melimine has been covalently linked to a surface via two azide linkers, 4-azidobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA), and the resulting surfaces characterized by X-ray photoelectron spectroscopy and contact angle measurements. The quantity of bound peptide was estimated by a modified Bradford assay. The antimicrobial efficacy of the two melimine-modified surfaces against Pseudomonas aeruginosa and Staphylococcus aureus was compared by scanning electron microscopy (SEM) and fluorescence microscopy. Attachment of melimine via ABA gave an approximately 4-fold greater quantity of melimine bound to the surface than attachment via FNA. Surfaces melimine-modified by either attachment strategy showed significantly reduced bacterial adhesion for both strains of bacteria. P. aeruginosa exposed to ABA–melimine and FNA–melimine surfaces showed marked changes in cell morphology when observed by SEM and a reduction of approximately 15-fold (p < 0.001) in the numbers of adherent bacteria compared to controls. For the ABA–melimine surface there was a 33% increase in cells showing damaged membranes (p = 0.0016) while for FNA–melimine there was no significant difference. For S. aureus there were reductions in bacterial adhesion of approximately 40-fold (p < 0.0001) and 5-fold (p = 0.008) for surfaces modified with melimine via ABA or FNA, respectively. There was an increase in cells showing damaged membranes on ABA–melimine surfaces of approximately 87% (p = 0.001) compared to controls, while for FNA–melimine there was no significant difference observed. The data presented in this study show that melimine has excellent potential for development as a broad spectrum antimicrobial coating for biomaterial surfaces. Further, it was observed that the efficacy of antimicrobial activity is related to the method of attachment.     

Comparative studies of the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine against bovine mastitis using non-invasive mouse mastitis as a model system

This study was undertaken to compare the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine for the prevention of mastitis using the mouse as a model system. Mice immunized with formalin-killed whole cell vaccine of S. aureus residing in a biofilm when delivered via an intramammary route produced a cell mediated immune response. Mice immunized with this biofilm vaccine showed significant reductions in colonization by S. aureus in mammary glands, severity of clinical symptoms and tissue damage in mammary glands in comparison with the mice immunized with formalin-killed whole cells of planktonic S. aureus. The planktonic vaccine administered by a subcutaneous route produced a significantly higher humoral immune response (IgG₁ and IgG) than the biofilm vaccine. However, considering the host response, tissue damage, the clinical severity and colonization of S. aureus in mammary glands, the biofilm vaccine performed better in immunogenicity and protective potential when administered by the intramammary route.     

Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX,the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-l-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K_d = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.     

A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs

Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.     

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.     

Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.     

Methicillin-resistant Staphylococcus aureus Bacterial Nitric-oxide Synthase Affects Antibiotic Sensitivity and Skin Abscess Development

Staphylococcus aureus infections present an enormous global health concern complicated by an alarming increase in antibiotic resistance. S. aureus is among the few bacterial species that express nitric-oxide synthase (bNOS) and thus can catalyze NO production from l-arginine. Here we generate an isogenic bNOS-deficient mutant in the epidemic community-acquired methicillin-resistant S. aureus (MRSA) USA300 clone to study its contribution to virulence and antibiotic susceptibility. Loss of bNOS increased MRSA susceptibility to reactive oxygen species and host cathelicidin antimicrobial peptides, which correlated with increased MRSA killing by human neutrophils and within neutrophil extracellular traps. bNOS also promoted resistance to the pharmaceutical antibiotics that act on the cell envelope such as vancomycin and daptomycin. Surprisingly, bNOS-deficient strains gained resistance to aminoglycosides, suggesting that the role of bNOS in antibiotic susceptibility is more complex than previously observed in Bacillus species. Finally, the MRSA bNOS mutant showed reduced virulence with decreased survival and smaller abscess generation in a mouse subcutaneous infection model. Together, these data indicate that bNOS contributes to MRSA innate immune and antibiotic resistance phenotypes. Future development of specific bNOS inhibitors could be an attractive option to simultaneously reduce MRSA pathology and enhance its susceptibility to commonly used antibiotics.     

Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.     

Identification of potential targets in Staphylococcus aureus N315 using computer aided protein data analysis

Staphylococcus aureus is a gram positive bacterium, responsible for both community-acquired and hospital-acquired infection, resulting in a mortality rate of 39%. 43.2% resistance to methicilin and emerging resistance to Fluroquinolone and Oxazolidinone, have evoked the necessity of the establishment of alternative and effective therapeutic approach to treat this bacteria. In this computational study, various database and online software are used to determine some specific targets of Staphylococcus aureus N315 other than those used by Penicillin, Quinolone and Oxazolidinone. For this purpose, among 302 essential proteins, 101 nonhomologous proteins were accrued and 64 proteins which are unique in several metabolic pathways of S. aureus were isolated by using metabolic pathway analysis tools. Furthermore, 7 essentially unique enzymes involved in exclusive metabolic pathways were revealed by this research, which can be potential drug target. Along with these important enzymes, 15 non-homologous proteins located on membrane were identified, which can play a vital role as potential therapeutic targets for the future researchers.