Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the d-Ala-d-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. β-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a β-lactamase and is not trapped as an acyl intermediate with β-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.Penicillin binding proteins (PBPs) are critical components of the cell wall synthesis machinery in bacteria. These membrane-associated proteins are broadly classified as low-molecular-mass (LMM) PBPs that are monofunctional d,d-carboxypeptidase enzymes or multimodular high-molecular-mass (HMM) PBPs with multiple functional roles. PBPs, in general, are anchored to the cytoplasmic membrane by a noncleavable pseudo-signal peptide. In the case of the HMM PBPs, the cytoplasmic C-terminal domain binds penicillin and catalyzes peptidoglycan cross-linking, whereas the juxtamembrane N-terminal domain participates in transglycosylation (12). The catalytic penicillin-binding (PB) module also occurs as part of penicillin sensor transducers, such as Staphylococcus aureus MecR and Bacillus licheniformis BlaR (15). The transpeptidase activity in HMM PBPs is based on a conserved lysine residue located in the so-called catalytic S-X-X-K motif, whereas the other conserved S-X-N and K(H)-T(S)-G motifs govern carboxypeptidase activity and bind penicillin (20). The carboxypeptidase domain of PBPs is the target for β-lactam antibiotics in susceptible staphylococci (with penicillin MICs as low as 1 μg/ml).The transpeptidase activity of the PBPs occurs at the d-Ala-d-Ala terminus of a precursor disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Ala-l-Lys-d-Ala-d-Ala. This reaction is initiated by acylation involving a nucleophilic attack by the active-site serine on the penultimate d-Ala residue to form an acyl-enzyme complex. The C-terminal d-Ala is subsequently released from the peptide chain, followed by deacylation. In the case of HMM PBPs, deacylation occurs when an amino group on a second peptide substrate acts as an acceptor, resulting in a peptide cross-link between two adjacent peptidoglycan strands. The carboxypeptidase activity of LMM PBPs follows a similar reaction scheme, except that the acceptor in this case is a water molecule. β-Lactam antibiotics mimic the substrates of the PBPs. However, unlike the natural substrate, the β-lactam-PBP acyl adduct is stable and results in irreversible inhibition of PBP function. The β-lactam-PBP acyl adduct has been characterized extensively, with over 50 protein-antibiotic complexes reported to date (37). Thus, in contrast to the nonessential LMM PBPs, HMM PBPs constitute lethal targets for β-lactam antibiotics (6).Staphylococcus aureus is a gram-positive coccus and is one of the leading causes of high morbidity and mortality associated with both community- and hospital-associated infections (42, 46). This coccus shows extensive genomic variation, with over 22% of the genome dedicated to dispensable regions. A genome-scale analysis of a clinical strain of S. aureus is of particular interest in this context, wherein the conversion of a susceptible strain of S. aureus to a multidrug-resistant phenotype was shown to involve just 35 mutations in 13 loci, achieved within 3 months (36). Of the five PBPs in S. aureus, an acquired PBP, PBP2a, is the most extensively examined, as it was noted to be a specific marker for methicillin-resistant S. aureus (MRSA) strains. Among the intrinsic PBPs, PBP1 has been shown to play a key role in cell growth and division (2). PBP2 is a dual-function enzyme with both transglycosylase and transpeptidase activities, and inhibition of this protein leads to restrained peptidoglycan elongation and subsequent leakage of cytoplasmic contents due to cell lysis (34, 40). Inactivation of PBP3 neither changes the muropeptide composition of the cell wall nor significantly decreases the rate of autolysis. However, cells of abnormal size and shape and with disoriented septa are produced when bacteria with inactivated PBP3 are grown with sub-MIC levels of methicillin (29).S. aureus PBP4 is a carboxypeptidase and is needed for the secondary cross-linking of peptidoglycan (19). However, it is not essential for cell growth under laboratory conditions, because mutants of S. aureus defective in PBP4 are viable (48). Overexpression of PBP4 was noted to result in an increase in β-lactam resistance and in greater cross-linking of the peptidoglycan (18). S. aureus PBP4 is similar to other LMM PBPs and is grouped within the superfamily of penicillin-susceptible and penicillin-interacting enzymes. However, homologues of PBP4 have a different phenotype in other species (1, 15). For example, a mutation of PBP4 in Pseudomonas aeruginosa triggers an AmpR-dependent overproduction of the chromosomal β-lactamase AmpC. The P. aeruginosa PBP4 mutant also activates CreBC, a two-component regulator, thereby mediating β-lactam resistance (33). Indeed, S. aureus PBP4 has been suggested to have different functions in strains with different genetic backgrounds (26). However, based on in vitro and genetic data, S. aureus PBP4 is primarily a transpeptidase and has little d,d-carboxypeptidase activity. This is also supported by the observation that increased carboxypeptidase activity decreases cell wall cross-linking due to loss of the free d-Ala-d-Ala termini necessary for transpeptidation (10). In this context, it is pertinent that pbp4 gene knockout strains of S. aureus were more resistant to the glycopeptide antibiotic vancomycin (46).Here we present the biochemical and structural characteristics of PBP4 from S. aureus strain COL. S. aureus PBP4 is a β-lactamase. A comparison of the crystal structure of S. aureus PBP4 in complex with antibiotic with that of its Escherichia coli homologue, PBP5, provides a conformational and biochemical rationale for the β-lactamase activity of PBP4. Monitoring the expression of PBP4 in the MRSA strain COL and representative clinical strains of S. aureus suggested that the expression level of PBP4 does not fluctuate substantially across these strains. Together, these data on the structure, expression, activity, and regulation of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.     

Cerebellar control of visceral responses-possible mechanisms involved

It seems reasonable to assume that cerebellar autonomic control operates according to similar principles as those utilized in the somatomotor coordination. The unique and very uniform neuronal architecture throughout the cerebellum speaks in favour of such a view.     

Thiadiazolopiperazinyl ureas as inhibitors of fatty acid amide hydrolase

A series of thiadiazolopiperazinyl aryl urea fatty acid amide hydrolase (FAAH) inhibitors is described. The molecules were found to inhibit the enzyme by acting as mechanism-based substrates, forming a covalent bond with Ser241. SAR and PK properties are presented.     

Stereotactic cingulumotomy for drug addiction.

This paper analyzes the results of surgical treatment of 73 patients with drug addiction. They were treated by lesions in the anterior cingulum. Morphine, Pethidine (meperidine) and alcohol were the common drugs of addiction. Selection of cases, rationale for surgery and operative technique are discussed. Follow-up varied from 1 to 6 years. Results of surgery indicate this procedure as the most promising one for cure of drug addiction.     

Nanoscale Roughness and Morphology Affect the IsoElectric Point of Titania Surfaces

We report on the systematic investigation of the role of surface nanoscale roughness and morphology on the charging behaviour of nanostructured titania (TiO₂) surfaces in aqueous solutions. IsoElectric Points (IEPs) of surfaces have been characterized by direct measurement of the electrostatic double layer interactions between titania surfaces and the micrometer-sized spherical silica probe of an atomic force microscope in NaCl aqueous electrolyte. The use of a colloidal probe provides well-defined interaction geometry and allows effectively probing the overall effect of nanoscale morphology. By using supersonic cluster beam deposition to fabricate nanostructured titania films, we achieved a quantitative control over the surface morphological parameters. We performed a systematical exploration of the electrical double layer properties in different interaction regimes characterized by different ratios of characteristic nanometric lengths of the system: the surface rms roughness R_q, the correlation length ξ and the Debye length λ_D. We observed a remarkable reduction by several pH units of IEP on rough nanostructured surfaces, with respect to flat crystalline rutile TiO₂. In order to explain the observed behavior of IEP, we consider the roughness-induced self-overlap of the electrical double layers as a potential source of deviation from the trend expected for flat surfaces.     

MS Cortical Lesions on DIR: Not Quite What They Seem?

Objective

Accurate identification and localization of cortical gray matter (CGM) lesions in MS is important when determining their clinical relevance. Double inversion recovery (DIR) scans have been widely used to detect MS CGM lesions. Phase sensitive inversion recovery (PSIR) scans have a higher signal to noise, and can therefore be obtained at a higher resolution within clinically acceptable times. This enables detection of more CGM lesions depicting a clearer cortical and juxtacortical anatomy. In this study, we systematically investigated if the use of high resolution PSIR scans changes the classification of CGM lesions, when compared with standard resolution DIR scans.

Methods

60 patients [30 RR(Relapsing remitting) and 15 each with PP(Primary progressive) and SP(Secondary progressive) MS] were scanned on a 3T Philips Achieva MRI scanner. Images acquired included DIR (1×1×3 mm resolution) and PSIR (0.5×0.5×2 mm). CGM lesions were detected and classified on DIR as intracortical (IC) or leucocortical (LC). We then examined these lesions on corresponding slices of the high resolution PSIR scans and categorized them as IC, LC, Juxtacortical white matter (JC-WM, abutting but not entering cortex) and other white matter (WM, not juxtacortical). Classifications using both scans were noted.

Results

282 IC and 483 LC were identified on DIR. Of the IC lesions, 61% were confirmed as IC on PSIR, 35.5% were reclassified as LC and 3.5% as JC-WM or other WM only. Of the LC DIR lesions, 43.9% were confirmed at LC on PSIR, 16.1% were reclassified as IC and 40% as JC-WM or other WM only. Overall, 50% (381/765) of CGM lesions seen on DIR were reclassified, and 26.5% (203/765) affected WM only.

Conclusions

When compared with higher resolution PSIR, a significant proportion of lesions classified as involving CGM on DIR appear to either contain more white matter than expected or to not involve CGM at all.     

Metabolic Proximity in the Order of Colonization of a Microbial Community

Microbial biofilms are often composed of multiple bacterial species that accumulate by adhering to a surface and to each other. Biofilms can be resistant to antibiotics and physical stresses, posing unresolved challenges in the fight against infectious diseases. It has been suggested that early colonizers of certain biofilms could cause local environmental changes, favoring the aggregation of subsequent organisms. Here we ask whether the enzyme content of different microbes in a well-characterized dental biofilm can be used to predict their order of colonization. We define a metabolic distance between different species, based on the overlap in their enzyme content. We next use this metric to quantify the average metabolic distance between neighboring organisms in the biofilm. We find that this distance is significantly smaller than the one observed for a random choice of prokaryotes, probably reflecting the environmental constraints on metabolic function of the community. More surprisingly, this metabolic metric is able to discriminate between observed and randomized orders of colonization of the biofilm, with the observed orders displaying smaller metabolic distance than randomized ones. By complementing these results with the analysis of individual vs. joint metabolic networks, we find that the tendency towards minimal metabolic distance may be counter-balanced by a propensity to pair organisms with maximal joint potential for synergistic interactions. The trade-off between these two tendencies may create a “sweet spot” of optimal inter-organism distance, with possible broad implications for our understanding of microbial community organization.     

Metabolism-guided discovery of a potent and orally bioavailable urea-based calcimimetic for the treatment of secondary hyperparathyroidism

A series of urea based calcimimetics was optimized for potency and oral bioavailability. Crucial to this process was overcoming the poor pharmacokinetic properties of lead thiazole 1. Metabolism-guided modifications, characterized by the use of metabolite identification (ID) and measurement of time dependent inhibition (TDI) of CYP3A4, were essential to finding a compound suitable for oral dosing. Calcimimetic 18 exhibited excellent in vivo potency in a 5/6 nephrectomized rat model and cross-species pharmacokinetics.     

A modular design of low‐background bioassays based on a high‐affinity molecular pair barstar:barnase

High‐affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (K_D) of the molecular pair, with avidin:biotin being the state‐of‐the‐art molecular pair (K_D ～ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high‐affinity protein pair, barstar:barnase (K_D ～ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non‐negligible background due to the non‐specific binding. A quantitative assessment of the non?specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin‐based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non‐specific binding, showing good prospects for high‐sensitivity rare biomolecular event nanoproteomic assays.