Functional Characterization of <i>Staphylococcus epidermidis</i> IcaB, a De‑<i>N</i>‑acetylase Important for Biofilm Formation

A polymer of partially de-<i>N</i>-acetylated β-1,6-linked <i>N</i>-acetylglucosamine (dPNAG), also known as the polysaccharide intercellular adhesin (PIA), is an important component of many bacterial biofilm matrices. In <i>Staphyloccocus epidermidis</i>, the poly-<i>N</i>-acetylglucosamine polymer is partially de-<i>N</i>-acetylated by the extracellular protein IcaB. To understand the mechanism of action of IcaB, the enzyme was overexpressed and purified. IcaB demonstrates metal-dependent de-<i>N</i>-acetylase activity on β-1,6-linked <i>N</i>-acetylglucosamine oligomers with a broad preference for divalent metals. Steady-state kinetic analysis reveals the low catalytic efficiency (pentasaccharide <i>k</i>_cat/<i>K</i>_M 0.03 M^–1 s^–1) of the enzyme toward the oligomeric substrates. While IcaB displays similar rates of de-<i>N</i>-acetylation with tri- through hexasaccharide PNAG oligomers, position specific de-<i>N</i>-acetylation was only observed with penta- and hexasaccharides. The enzyme preferentially de-<i>N</i>-acetylates the second residue from the reducing terminus in the pentasaccharide and second and third residues from the reducing terminus in the hexasaccharide. The data described here represent an important step toward a detailed understanding of dPNAG biosynthesis in <i>S. epidermidis</i>, an important nosocomial pathogen, as well as in other Gram-positive bacteria. The low catalytic activity of IcaB is consistent with reports of other enzymes which act on biofilm-related polysaccharides, and this emerging trend may indicate a common feature among this group of polysaccharide processing enzymes

Functional Mapping of PilF and PilQ in the <i>Pseudomonas aeruginosa</i> Type IV Pilus System

<i>Pseudomonas aeruginosa</i> uses type IV pili (T4P) to interact with the environment and as key virulence factors when acting as an opportunistic pathogen. Assembly of the outer membrane PilQ secretin channel through which the pili are extruded is essential for pilus biogenesis. The <i>P. aeruginosa</i> T4P pilotin, PilF, is required for PilQ outer membrane localization and assembly into secretins and contains six tetratricopeptide (TPR) protein–protein interaction motifs, suggesting that the two proteins interact. In this study, we found that the first four TPR motifs of PilF are sufficient for PilQ outer membrane targeting, oligomerization, and function. Guided by our structure of PilF, site-directed mutagenesis of the protein surface revealed that a hydrophobic groove on the first TPR is required for PilF-mediated PilQ assembly. Deletion of individual domains within PilQ suggests that the N0, KH-like, or secretin domain, but not the C-terminus, interacts with PilF. Purified PilQ was found to pull down PilF from <i>Pseudomonas</i> cell lysates. Together, these data allow us to propose a model for PilF function in the T4P system. PilF interacts directly or indirectly with the PilQ monomer after translocation of both proteins through the inner membrane and acts as a co-chaperone with the Lol system to facilitate transit across the periplasm to the outer membrane. The mechanism of PilQ insertion and assembly, which appears to be independent of the Bam system, remains to be determined

Molecular Basis for the Attachment of S‑Layer Proteins to the Cell Wall of <i>Bacillus anthracis</i>

Bacterial surface (S) layers are paracrystalline arrays of protein assembled on the bacterial cell wall that serve as protective barriers and scaffolds for housekeeping enzymes and virulence factors. The attachment of S-layer proteins to the cell walls of the <i>Bacillus cereus sensu lato</i>, which includes the pathogen <i>Bacillus anthracis</i>, occurs through noncovalent interactions between their S-layer homology domains and secondary cell wall polysaccharides. To promote these interactions, it is presumed that the terminal <i>N</i>-acetylmannosamine (ManNAc) residues of the secondary cell wall polysaccharides must be ketal-pyruvylated. For a few specific S-layer proteins, the O-acetylation of the penultimate <i>N</i>-acetylglucosamine (GlcNAc) is also required. Herein, we present the X-ray crystal structure of the SLH domain of the major surface array protein Sap from <i>B. anthracis</i> in complex with 4,6-<i>O</i>-ketal-pyruvyl-β-ManNAc-(1,4)-β-GlcNAc-(1,6)-α-GlcN. This structure reveals for the first time that the conserved terminal SCWP unit is the direct ligand for the SLH domain. Furthermore, we identify key binding interactions that account for the requirement of 4,6-<i>O</i>-ketal-pyruvyl-ManNAc while revealing the insignificance of the O-acetylation on the GlcNAc residue for recognition by Sap

Factors associated with death and limitation of life-sustaining therapies in patients with traumatic brain injury

Aim of the Study: A substantial proportion of deaths of patients in the Intensive Care Unit (ICU) follow a decision to limit life-sustaining therapies. Patients with moderate to severe Traumatic Brain Injury (TBI) differ from the general ICU population: They are usually younger, previously healthy, and often with no advance directives. The objective of this study was to identify factors associated with mortality and limitation of life-sustaining therapies in patients with moderate to severe traumatic brain injury in a Swiss academic tertiary care hospital. Methods: This study was a retrospective single center analysis of 170 non-elective admissions to the surgical ICU of a Swiss academic tertiary care hospital over a three-year period. Patients were eligible for the study if diagnosed with moderate to severe blunt TBI, and if the ICU length of stay was at least 48 hrs. Factors associated with mortality were investigated. Results: Mean age was 48 ± 21 years, 72.3% were male, and pre-existing medical conditions were overall rare. Forty-five patients (26.5%) died within 6 months after TBI (Non-survivors group). Most deaths (n=43, 95.5%) occurred after limitation of life-sustaining therapies. In the multiple binary logistic regression model age, Protestant religion, hypoxemia during the rescue phase, a higher category in the Marshall classification and a higher Injury Severity Score were independently associated with death. Conclusion: At our institution, most deaths of patients with moderate to severe TBI occurred after a deliberate decision to limit life-sustaining therapies. This decision was associated with age, spiritual belief of the patient, hypoxemia in the pre-hospital setting, radiological findings, and severity scores. Written advance directives should be encouraged to help surrogate decision makers and physicians in the acute and sudden setting of TBI to respect the patient’s willed

Structural comparison of <i>Sp</i>OatA_C with representative members of the SGNH/GDSL and AlgX-N/DHHW families of enzymes.

<p><b>A</b>. The cartoon representation of <i>Sp</i>OatA_C (gray) is superposed with <i>Bos taurus</i> platelet-activating factor acetylhydrolase (PAF-AH) (blue) and the N-terminal catalytic domain of <i>P</i>. <i>aeruginosa</i> AlgX (green). Right inset: Cartoons depicting the respective peptide backbones of the Block II-loop in the three enzymes. <b>B</b>. Sequence alignments of residues comprising the signature sequence Blocks of the SGNH/GDSL and AlgX-N/DHHW families of enzymes. Red lettering denotes invariant residues in the respective families.</p

<i>Sp</i>OatA_C and <i>Sa</i>OatA_C-catalyzed <i>O</i>-acetyltransferase reactions.

<p>ESI-MS analysis of reaction products of 2 mM chitotetraose (G₄) in 50 mM sodium phosphate buffer pH 6.5 incubated at 37 ^oC for 1 h in the absence (control) and presence of enzymes (5 μM, final concentration) with 1 mM concentrations of <b>A,</b> acetyl-CoA; <b>B,</b> 4MU-Ac; or <b>C,</b> <i>p</i>NP-Ac as potential donor acetyl substrates.</p

Specific activities of <i>Sp</i>OatA_C and <i>Sa</i>OatA_C variants.

<p>Specific activities of <i>Sp</i>OatA_C and <i>Sa</i>OatA_C variants.</p

Activity and domain structure of OatA.

<p><b>A</b>. PG is comprised of alternating GlcNAc (G) and MurNAc (M) residues with stem peptides (small circles). The lysozymes of innate immunity systems (LYZ) hydrolyze the linkage between M and G residues which results in cell rupture and death. OatA O-acetylates the C-6 hydroxyl group of MurNAc residues (red triangles) in PG of pathogenic Gram-positive bacteria which sterically inhibits the action of the lysozymes, thereby conferring resistance to this first line of the innate immune response. <b>B</b>. Domain organization of OatA. This bimodular protein is comprised of two domains, a predicted N-terminal Acyl_transferase_3 (Pfam PF01757) transmembrane domain and a C-terminal SGNH/GDSL extracytoplasmic domain. The genes encoding OatA from <i>S</i>. <i>aureus</i> and <i>S</i>. <i>pneumoniae</i> were engineered to produce the 25 kDa C-terminal SGNH/GDSL domains (OatA_C) as shown.</p

Active site structure of <i>Sp</i>OatA_C.

<p>The H-bonding network of catalytic and oxyanion hole residues in <b>A</b>, resting <i>Sp</i>OatA_C and <b>B</b>, <i>Sp</i>OatA_C in complex with MeS (<i>Sp</i>OatA_C-MeS). The water molecule w1 and the potential inter-residue interactions are depicted as a red sphere and black dashed lines, respectively. <b>C</b>. The <i>2F</i>_<i>o</i><i>-F</i>_<i>c</i> electron density map of the MeS-Ser438 adduct contoured at 1.0 σ. <b>D</b>. Superposition of the <i>Sp</i>OatA_C and <i>Sp</i>OatA_C-MeS active sites.</p

Stem peptide specificity of <i>Sp</i>OatA_C and <i>Sa</i>OatA_C.

<p><b>A</b>. Stacked and offset ESI-mass spectra of mutanolysin-treated products from reactions of 10 μg·mL^-1 of (left to right) muroglyan-5P, muroglycan-4P, and muroglycan-3P in 50 mM sodium phosphate buffer pH 6.5 incubated with 0.5 mM <i>p</i>NP-Ac in the absence (control) and presence of the respective enzyme (10 μM). The major O-acetylated products are labeled in blue which are 42.01 m/z units larger than the respective unmodified PG monomer. <b>B</b>. MS/MS analysis of the major product ions identified in the respective panels above and <b>C</b>, interpretation of the corresponding fragment ions.</p