Functional and structural characterization of Streptococcus pneumoniae pyruvate kinase involved in fosfomycin resistance.

Glycolysis is the primary metabolic pathway in the strictly fermentative Streptococcus pneumoniae, which is a major human pathogen associated with antibiotic resistance. Pyruvate kinase (PYK) is the last enzyme in this pathway that catalyzes the production of pyruvate from phosphoenolpyruvate (PEP) and plays a crucial role in controlling carbon flux; however, while S. pneumoniae PYK (SpPYK) is indispensable for growth, surprisingly little is known about its functional properties. Here, we report that compromising mutations in SpPYK confers resistance to the antibiotic fosfomycin, which inhibits the peptidoglycan synthesis enzyme MurA, implying a direct link between PYK and cell wall biogenesis. The crystal structures of SpPYK in the apo and ligand-bound states reveal key interactions that contribute to its conformational change as well as residues responsible for the recognition of PEP and the allosteric activator fructose 1,6-bisphosphate (FBP). Strikingly, FBP binding was observed at a location distinct from previously reported PYK effector binding sites. Furthermore, we show that SpPYK could be engineered to become more responsive to glucose 6-phosphate instead of FBP by sequence and structure-guided mutagenesis of the effector binding site. Together, our work sheds light on the regulatory mechanism of SpPYK and lays the groundwork for antibiotic development that targets this essential enzyme.

A Time-Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW.

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.

Coordination of bacterial cell wall and outer membrane biosynthesis.

Gram-negative bacteria surround their cytoplasmic membrane with a peptidoglycan (PG) cell wall and an outer membrane (OM) with an outer leaflet composed of lipopolysaccharide (LPS)1. This complex envelope presents a formidable barrier to drug entry and is a major determinant of the intrinsic antibiotic resistance of these organisms2. The biogenesis pathways that build the surface are also targets of many of our most effective antibacterial therapies3. Understanding the molecular mechanisms underlying the assembly of the Gram-negative envelope therefore promises to aid the development of new treatments effective against the growing problem of drug-resistant infections. Although the individual pathways for PG and OM synthesis and assembly are well characterized, almost nothing is known about how the biogenesis of these essential surface layers is coordinated. Here we report the discovery of a regulatory interaction between the committed enzymes for the PG and LPS synthesis pathways in the Gram-negative pathogen Pseudomonas aeruginosa. We show that the PG synthesis enzyme MurA interacts directly and specifically with the LPS synthesis enzyme LpxC. Moreover, MurA was shown to stimulate LpxC activity in cells and in a purified system. Our results support a model in which the assembly of the PG and OM layers in many proteobacterial species is coordinated by linking the activities of the committed enzymes in their respective synthesis pathways.

Validation of indications for enucleation for benign parotid gland tumors.

BACKGROUND: Enucleation has been reported as a minimally invasive surgery for Warthin's tumor (WT). However, the definite indications for enucleation have not been clarified. METHODS: Enucleation was indicated by the following findings: findings of WT, cystic fluid, or benign leukocytes by fine-needle aspiration cytology; a well-margined and homogeneous pattern on imaging; and a tumor location in the tail or preauricular area of the parotid gland. We reviewed 552 cases treated with parotid gland surgery in our hospital. RESULTS: A total of 108 tumors were treated with enucleation and included no malignant solid tumors or pleomorphic adenoma. Enucleation demonstrated low invasiveness and complication rates. Revision surgery for WT reappearance after enucleation was rare and showed minimal scarring, with a lower risk of facial weakness. CONCLUSIONS: The indication criteria for enucleation were validated. Such enucleation is useful, as it is associated with minimal invasiveness, low complication rates, and safety in revision surgery.

Masters of Misdirection: Peptidoglycan Glycosidases in Bacterial Growth.

The dynamic composition of the peptidoglycan cell wall has been the subject of intense research for decades, yet how bacteria coordinate the synthesis of new peptidoglycan with the turnover and remodeling of existing peptidoglycan remains elusive. Diversity and redundancy within peptidoglycan synthases and peptidoglycan autolysins, enzymes that degrade peptidoglycan, have often made it challenging to assign physiological roles to individual enzymes and determine how those activities are regulated. For these reasons, peptidoglycan glycosidases, which cleave within the glycan strands of peptidoglycan, have proven veritable masters of misdirection over the years. Unlike many of the broadly conserved peptidoglycan synthetic complexes, diverse bacteria can employ unrelated glycosidases to achieve the same physiological outcome. Additionally, although the mechanisms of action for many individual enzymes have been characterized, apparent conserved homologs in other organisms can exhibit an entirely different biochemistry. This flexibility has been recently demonstrated in the context of three functions critical to vegetative growth: (i) release of newly synthesized peptidoglycan strands from their membrane anchors, (ii) processing of peptidoglycan turned over during cell wall expansion, and (iii) removal of peptidoglycan fragments that interfere with daughter cell separation during cell division. Finally, the regulation of glycosidase activity during these cell processes may be a cumulation of many factors, including protein-protein interactions, intrinsic substrate preferences, substrate availability, and subcellular localization. Understanding the true scope of peptidoglycan glycosidase activity will require the exploration of enzymes from diverse organisms with equally diverse growth and division strategies.

Buccal fat pad flap for transoral repair of radiation-induced necrosis in the lateral oropharyngeal wall.

Soft tissue necrosis (STN) is a late toxicity after radiotherapy. Extensive tissue defects due to STN near the carotid artery, such as in the lateral oropharyngeal wall, may lead to infectious pseudoaneurysms associated with fatal bleeding. Such defects are usually treated with transcervical reconstructive surgeries, which are highly invasive and technically difficult. We report a case in which a buccal fat pad (BFP) flap was used for minimally invasive transoral repair of tissue defects due to radiation-induced STN in the lateral oropharyngeal wall. The BFP flap covered the tissue defect, and the wound epithelialized completely. The patient had no dysfunctional mouth opening, speech, or swallowing. The BFP flap can be easily harvested via a minimally invasive transoral approach and is expected to be further utilized for radiation-induced STN in the lateral oropharyngeal wall.

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall.

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG's ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

A New Device for Fine-Needle Aspiration Cytology Consisting of a Vibrating Linear Resonant Actuator.

OBJECTIVE: To evaluate the efficacy of a new device for fine-needle aspiration cytology (FNAC) consisting of a vibrating linear resonant actuator (LRA). STUDY DESIGN: Prospective clinical study. METHODS: The LRA frequency was optimized by visualization of the needle motion using a high-speed camera. The FNAC device consists of a vibrating motor fixed to the stopper of a 5-ml syringe and piston. Upon insertion of the syringe needle into a thyroid nodule (with the stopper attached to the syringe piston), sufficient negative pressure with 1-ml suction was maintained. Subsequently, samples were obtained using vibration generated by an LRA or an eccentric rotating mass (ERM). Surgically resected thyroid specimens from 10 patients were evaluated. The number of follicular groups required for adequate diagnosis and the number of larger follicular groups were counted. Next, 254 thyroid nodules from 187 patients were also evaluated by FNAC. The inadequacy rate was determined, and final cytology was classified according to thyroid Bethesda categories. RESULTS: The optimized LRA frequency was 155 Hz. Both the LRA and ERM devices resulted in sufficient amounts of diagnostic material and achieved low inadequacy rates. The number of large follicular groups obtained was significantly greater with the LRA device compared with the ERM device. CONCLUSIONS: The vibrating device using an LRA for thyroid FNAC resulted in sufficient amounts of thyroid follicular groups and achieved low inadequacy rates. In addition, the LRA device allowed for collection of larger follicular groups sufficient to diagnose appropriate thyroid Bethesda categories. LEVEL OF EVIDENCE: 2 Laryngoscope, 131:E1393-E1399, 2021.

Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae.

Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid-shaped Streptococcus pneumoniae (Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Labeling with fluorescent D-amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced "nodes" of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.

Successful treatment of eosinophilic chronic rhinosinusitis and eosinophilic otitis media using the anti-IL-5 receptor monoclonal antibody benralizumab: A case report.

Eosinophilic chronic rhinosinusitis (ECRS) is characterized by the presence of nasal polyps, dominant ethmoid shadows in computed tomography (CT) scans, and elevated levels of eosinophil infiltration into the nasal polyps and peripheral blood. ECRS is often accompanied by severe asthma. The recent development of monoclonal antibody-based biologics, including benralizumab, has offered new therapeutic approaches for the treatment of asthma and allergic diseases. Asthma and ECRS are closely related; hence, benralizumab could provide clinical benefit in ECRS patients with severe asthma. Herein, we report a case of a 47-year-old female patient with severe asthma that presented with nasal obstruction and hearing impairment. Nasal endoscopic and otoscopic examinations indicated the presence of bilateral nasal polyps in the middle nasal meatus, as well as a bilateral effusion in the tympanic cavity. Sinus and temporal CT images showed dominant ethmoid sinus and tympanic cavity shadows. Biopsy of nasal polyps revealed high numbers of eosinophils, which led to the diagnosis of ECRS; eosinophilic otitis media (EOM) with hypereosinophilia was also suspected. Treatment with benralizumab reduced the number of peripheral blood eosinophils and improved asthma symptoms. Prolonged benralizumab administration also resulted in a remarkable size reduction in bilateral middle nasal polyps and aeration of the tympanic cavity. In conclusion, benralizumab treatment improved the symptoms of severe asthma, ECRS, and EOM. Eosinophil depletion could be an important mechanism by which benralizumab improves ECRS and EOM. The use of benralizumab for the treatment of ECRS and EOM patients with severe asthma merits further investigation in large-cohort studies.

Structural coordination of polymerization and crosslinking by a SEDS-bPBP peptidoglycan synthase complex.

The shape, elongation, division and sporulation (SEDS) proteins are a highly conserved family of transmembrane glycosyltransferases that work in concert with class B penicillin-binding proteins (bPBPs) to build the bacterial peptidoglycan cell wall1-6. How these proteins coordinate polymerization of new glycan strands with their crosslinking to the existing peptidoglycan meshwork is unclear. Here, we report the crystal structure of the prototypical SEDS protein RodA from Thermus thermophilus in complex with its cognate bPBP at 3.3 Å resolution. The structure reveals a 1:1 stoichiometric complex with two extensive interaction interfaces between the proteins: one in the membrane plane and the other at the extracytoplasmic surface. When in complex with a bPBP, RodA shows an approximately 10 Å shift of transmembrane helix 7 that exposes a large membrane-accessible cavity. Negative-stain electron microscopy reveals that the complex can adopt a variety of different conformations. These data define the bPBP pedestal domain as the key allosteric activator of RodA both in vitro and in vivo, explaining how a SEDS-bPBP complex can coordinate its dual enzymatic activities of peptidoglycan polymerization and crosslinking to build the cell wall.

Real-Time Acoustic Voice Analysis Using a Handheld Device Running Android Operating System.

OBJECTIVES: We developed a highly accessible acoustic voice analysis system (VArt) using a handheld device running Android operating system. To provide stable and reliable analysis using readily obtainable equipment under unfavorable conditions, we modified the fundamental frequency (F0) extraction algorithm and designed an intuitive user interface representing a new hoarseness index (real-time Ra: Rart), which is a derivative of harmonics-to-noise ratio developed by Kojima and Shoji (Ra2). Since Rart continues to display analysis results in real time, unlike conventional acoustic analysis, it can be used for evaluation such as during phonosurgery and speech therapy. We evaluated the agreement between the earlier version of acoustic voice analysis software (VA) running on a Windows personal computer and VArt running on a handheld device. METHODS: F0, Ra2, and Rart were measured in voice samples of sustained vowel phonation /a/ from 10 healthy volunteers and 22 patients with voice disorders using VA running on a Windows personal computer and VArt running on two types of handheld devices in a sound-treated room or in a medical examination room. Intraclass correlation coefficients were calculated for both systems under both conditions. RESULTS: All of the comparisons were highly correlated. CONCLUSIONS: Measurements obtained using our newly developed VArt were highly consistent with those using VA, indicating high reliability. Moreover, the new system increases the clinical feasibility of acoustic voice analysis.

Adrenal insufficiency following nivolumab therapy in patients with recurrent or metastatic head and neck cancer.

Nivolumab, an anti-programmed cell death-1 monoclonal antibody, is currently used to treat many types of advanced cancers including recurrent and metastatic head and neck cancer. However, there are increasing reports concerning immune-related adverse events related to nivolumab therapy. Here, we report three patients who presented with adrenal insufficiency following nivolumab therapy. Two were diagnosed as having isolated adrenocorticotropic hormone (ACTH) deficiency and one was diagnosed as having primary adrenal insufficiency. All three patients complained of progressive fatigue and appetite loss, so we measured their blood cortisol and ACTH levels and diagnosed them as having adrenal deficiency. Treatment with nivolumab was discontinued for all three patients, and replacement therapy using hydrocortisone was successful after a few days in all cases. Two patients subsequently resumed nivolumab therapy because their general condition had improved. Complaints of fatigue and appetite loss during cancer treatment are common and tend to be regarded as unimportant. Although adrenal insufficiency due to nivolumab is relatively rare, complaints of these symptoms could lead to the detection of adrenal insufficiency at an early stage. The present report highlights the importance of the early recognition of adrenal insufficiency.

Direction of Chain Growth and Substrate Preferences of Shape, Elongation, Division, and Sporulation-Family Peptidoglycan Glycosyltransferases.

The bacterial cell wall is composed of peptidoglycan, and its biosynthesis is an established target for antibiotics. Peptidoglycan is assembled from a glycopeptide precursor, Lipid II, that is polymerized by peptidoglycan glycosyltransferases into glycan strands that are subsequently cross-linked to form the mature cell wall. For decades bacteria were thought to contain only one family of enzymes that polymerize Lipid II, but recently, the ubiquitous Shape, Elongation, Division, and Sporulation (SEDS)-family proteins RodA and FtsW were shown to be peptidoglycan polymerases. Because RodA and FtsW are essential in nearly all bacteria, these enzymes are promising targets for new antibiotics. However, almost nothing is known about the mechanisms of these polymerases. Here, we report that SEDS proteins synthesize peptidoglycan by adding new Lipid II monomers to the reducing end of the growing glycan chain. Using substrates that can only react at the reducing end, we also show that the glycosyl donor and acceptor in the polymerization reaction have distinct lipid requirements. These findings provide the first fundamental insights into the mechanism of SEDS-family polymerases and lay the groundwork for future biochemical and structural studies.

Chemical tools to characterize peptidoglycan synthases.

The peptidoglycan cell wall is a unique macromolecular structure in bacteria that defines their shape and confers protection from the surrounding environment. Decades of research has focused on understanding the peptidoglycan synthesis pathway and exploiting its essentiality for antibiotic development. Recently, a new class of peptidoglycan polymerases known as the SEDS (shape, elongation, division and sporulation) proteins were identified; these polytopic membrane proteins function together with the better-known penicillin-binding proteins (PBPs) to build the cell wall. In this review, we will highlight recent developments in chemical tools and methods to label the bacterial cell wall and discuss how these developments are leading to a better understanding of peptidoglycan synthases and their cellular roles.

Quantitative Evaluation of Surface-Enhanced Raman Scattering Nanoparticles for Intracellular pH Sensing at a Single Particle Level.

Intracellular pH is one of the key factors for understanding various biological processes in biological cells. Plasmonic gold and silver nanoparticles (NPs) have been extensively studied for surface-enhanced Raman scattering (SERS) applications for pH sensing as a local pH probe in a living cell. However, the SERS performance of NPs depends on material, size, and shape, which can be controlled by chemical synthesis. Here, we synthesized 18 types of gold and silver NPs with different morphologies such as sphere, rod, flower, star, core/shell, hollow, octahedra, core/satellites, and chainlike aggregates, and quantitatively compared their SERS performance for pH sensing. The SERS intensity from the most commonly utilized SERS probe molecule ( para-mercaptobenzoic acid, p-MBA) for pH sensing was measured at the single nanoparticle level under the same measurement parameters such as low laser power (0.5 mW/µm2), short integration time (100 ms) at wavelengths of 405, 488, 532, 584, 676, and 785 nm. In our measurement, the Ag chain, Ag core/satellites, Ag@Au core/satellites, and Au core/satellites nanoassemblies showed efficient pH sensing at the single particle level. By using p-MBA-conjugated Au@Ag core/satellites, we performed time-lapse pH measurements during apoptosis of HeLa cells. These experimental results confirmed that the pH measurement using p-MBA-conjugated Au@Ag core/satellites can be applied for long-term measurements of intracellular pH during cellular events.

FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein.

The peptidoglycan cell wall is essential for the survival and morphogenesis of bacteria1. For decades, it was thought that only class A penicillin-binding proteins (PBPs) and related enzymes effected peptidoglycan synthesis. Recently, it was shown that RodA-a member of the unrelated SEDS protein family-also acts as a peptidoglycan polymerase2-4. Not all bacteria require RodA for growth; however, its homologue, FtsW, is a core member of the divisome complex that appears to be universally essential for septal cell wall assembly5,6. FtsW was previously proposed to translocate the peptidoglycan precursor lipid II across the cytoplasmic membrane7,8. Here, we report that purified FtsW polymerizes lipid II into peptidoglycan, but show that its polymerase activity requires complex formation with its partner class B PBP. We further demonstrate that the polymerase activity of FtsW is required for its function in vivo. Thus, our findings establish FtsW as a peptidoglycan polymerase that works with its cognate class B PBP to produce septal peptidoglycan during cell division.

Identification of a Functionally Unique Family of Penicillin-Binding Proteins.

Penicillin-binding proteins (PBPs) are enzymes involved in the assembly of the bacterial cell wall, a major target for antibiotics. These proteins are classified by mass into high-molecular-weight PBPs, which are transpeptidases that form peptidoglycan cross-links, and low-molecular-weight PBPs, which are typically hydrolases. We report a functionally unique family of low-molecular-weight PBPs that act as transpeptidases rather than hydrolases, but they do not cross-link peptidoglycan. We show that these PBPs can exchange d-amino acids bearing chemical tags or affinity handles into peptidoglycan precursors, including Lipid II, enabling biochemical studies of proteins involved in cell wall assembly. We report that, in two organisms, the PBPs incorporate lysine into cellular peptidoglycan and that, further, the PBPs have the unprecedented ability to transfer the primary ε-amine of lysine to peptidoglycan.