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.     

Characterization of an alkaline active-thiol forming extracellular serine keratinase by the newly isolated Bacillus pumilus

Aims: The aim of the study was to optimize microbial degradation of keratinous waste and to characterize the alkaline active keratinase showing its biotechnological importance. Method and Results: An extracellular keratinase enzyme was purified from the culture medium of a bacterial isolate and the conditions were optimized. The molecular weight of DEAE‐Sepharose‐purified keratinase was determined by SDS‐PAGE. Instrumental analyses were investigated to study the mechanism of bovine hair hydrolysis. Isolate was identified as Bacillus pumilus based on phenotypic characteristics and 16S rDNA sequence. The optimized condition for its growth was pH 8 and 35°C. The molecular weight of the keratinase was estimated as 65 kDa. Activity inhibition by phenyl methyl sulphonyl fluoride confirmed keratinase as serine protease type. Instrumental analysis revealed the sulphitolysis and proteolysis involved mechanism in bovine hair hydrolysis. Conclusion: This study indicates that the isolated keratinase is an alkaline active serine protease with a high degree of activity towards bovine hair. Significance and Impact of the Study: This study examines a serine protease with high keratinolytic activity and degradation mechanism for bovine hair. The keratinolytic activity of the isolated strain and its reaction mechanism on bovine hair could show biotechnological potential in the leather industry.     

Identification and partial characterization of juvenile hormone esterase from cotton pest Dysdercus cingulatus

Juvenile hormone esterase (JHE), a selective enzyme that hydrolyzes the methyl ester of insect juvenile hormone plays an important role in regulating metamorphosis in nymphs as well as reproduction in adults. Studies on JH degradation provide insight into the possibilities of physiological disruption in the insects. In the present study, the JH degrading enzyme, JHE from the cotton pest Dysdercus cingulatus (Heteroptera) is characterized. Electrophoretic analysis of haemolymph during various developmental stages showed the JHE bands prominent only on the final day of 5th instar nymph, and the esterase substrate specificity confirmed the presence of JHE isoforms. In an attempt to clone cDNA of JHE gene from the final instar nymphs, mRNA isolated from fat bodies was coupled with JHE gene-specific primers and the cDNA was synthesized using RT-PCR. The PCR amplified cDNA showed the presence of JHE isoforms in D. cingulatus.     

DNA methyltransferase homologue TRDMT1 in Plasmodium falciparum specifically methylates endogenous aspartic acid tRNA

In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite.     

Primitive neuroectodermal tumor of the kidney. A report of two cases diagnosed by fine needle aspiration cytology

BACKGROUND: Primitive neurocetodermal tumors (PNETs) constitute a family of neoplasms of presumed neuroectrodermal origin most often presenting as bone or soft tissue masses. There are very few reported cases of PNET of the kidney and none diagnosed by fine needle aspiration cytology (FNAC), to the best of our knowledge, in the world literature. We present two cases of renal PNET diagnosed on cytology. CASES: Two patients with renal masses were diagnosed as having PNET on FNAC. Cytologically the tumors showed a dispersed population of malignant small round cells with focal rosette formation and perivascular arrangement of tumor cells. Immunohistochemistry on the cell blocks in both cases showed strong membrane positivity for CD99 (MIC2). Cytogenetic studies in both cases showed the characteristic t(11;22)(q24;q12) translocation, with additional chromosomal abnormalities in case 2. CONCLUSION: PNET of the kidney is a distinct entity and can be diagnosed on fine needle aspiration smears and confirmed with immunohistochemistry and cytogenetic studies. A diagnosis of PNET must be included in the differential diagnosis of renal masses in adolescents and young adults.     

Different functional properties of troponin T mutants that cause dilated cardiomyopathy

The effects of Troponin T (TnT) mutants R141W and DeltaK210, the only two currently known mutations in TnT that cause dilated cardiomyopathy(DCM) independent of familial hypertrophic cardiomyopathy (FHC), and TnT-K273E, a mutation that leads to a progression from FHC to DCM, were investigated. Studies on the Ca2+ sensitivity of force development in porcine cardiac fibers demonstrated that TnT-DeltaK210 caused a significant decrease in Ca2+ sensitivity, whereas the TnT-R141W did not result in any change in Ca2+ sensitivity when compared with human cardiac wild-type TnT (HCWTnT). TnT-DeltaK210 also caused a decrease in maximal force when compared with HCWTnT and TnT-R141W. In addition, the TnT-DeltaK210 mutant decreased maximal ATPase activity in the presence of Ca2+. However, the TnT-K273E mutation caused a significant increase in Ca2+ sensitivity but behaved similarly to HCWTnT in actomyosin activation assays. Inhibition of ATPase activity in reconstituted actin-activated myosin ATPase assays was similar for all three TnT mutants and HCWTnT. Additionally, circular dichroism studies suggest that the secondary structure of all three TnT mutants was similar to that of the HCWTnT. These results suggest that a rightward shift in Ca2+ sensitivity is not the only determinant for the phenotype of DCM.     

Antifungal constituents of the stem bark of Bridelia retusa

Antifungal activity guided fractionation of solvent extracts of the stem bark of Bridelia retusa of the family Euphorbiaceae against Cladosporium cladosporioides, furnished new bisabolane sesquiterpenes, (E)-4-(1,5-dimethyl-3-oxo-1-hexenyl)benzoic acid, (E)-4-(1,5-dimethyl-3-oxo-1,4-hexadienyl) benzoic acid, (R)-4-(1,5-dimethyl-3-oxo-4-hexenyl)benzoic acid and (-)-isochaminic acid, together with the known (R)-4-(1,5-dimethyl-3-oxohexyl)benzoic acid (ar-todomatuic acid), 5-allyl-1,2,3-trimethoxybenzene (elemicin), (+)-sesamin and 4-isopropylbenzoic acid (cumic acid). All these compounds showed fungicidal activity on TLC bioautography method at very low concentrations except elemicin.     

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.