Antibacterial activities of beta-lactamase inhibitors associated with morphological changes of cell wall in Helicobacter pylori

Background. Recent study has demonstrated that β‐lactamase inhibitors including clavulanate, sulbactam and tazobactam have an vitro antibacterial effect on Helicobacter pylori. Here we describe the relationship between viability and cell profiles of H. pylori exposed to β‐lactamase inhibitors and some antibiotics in a short‐time course. Materials and methods. The antibacterial effects of β‐lactamase inhibitors including clavulanate, sulbactam and tazobactam on the bacterial viability of and morphological changes in H. pylori ATCC43504 were examined. Results. The β‐lactamase inhibitors such as clavulanate and sulbactam alone decreased the viable counts of H. pylori, depending on the antibiotic concentrations. Exposure to these β‐lactamase inhibitors resulted in morphological changes of cell shape, cell‐wall disintegration and cell lysis. Among these β‐lactamase inhibitors, clavulanate was the most active, causing a decrease in viable counts and morphological changes such as short filamentous to sphaeroplast formation and lysis. One × minimum inhibitory concentration (MIC) of amoxicillin plus 1 × MIC of clavulanate decreased viable counts effectively compared with 1 × MIC of amoxicillin or 1 × MIC of clavulanate alone, and induced morphological changes of cell shape and cell wall. Conclusion. Our results suggest that the β‐lactamase inhibitors alone have concentration‐dependent antibacterial activities against H. pylori and affect the morphology of the cell shape and the cell wall in vitro.     

Development of a LytE‐based high‐density surface display system in Bacillus subtilis

The three N‐terminal, tandemly arranged LysM motifs from a Bacillus subtilis cell wall hydrolase, LytE, formed a cell wall‐binding module. This module, designated CWBM_LytE, was demonstrated to have tight cell wall‐binding capability and could recognize two classes of cell wall binding sites with fivefold difference in affinity. The lower‐affinity sites were approximately three times more abundant. Fusion proteins with β‐lactamase attached to either the N‐ or C‐terminal end of CWBM_LytE showed lower cell wall‐binding affinity. The number of the wall‐bound fusion proteins was less than that of CWBM_LytE. These effects were less dramatic with CWBM_LytE at the N‐terminal end of the fusion. Both CWBM_LytE and β‐lactamase were essentially functional whether they were at the N‐ or C‐terminal end of the fusion. In the optimal case, 1.2 × 10⁷ molecules could be displayed per cell. As cells overproducing CWBM_LytE and its fusions formed filamentous cells (with an average of nine individual cells per filamentous cell), 1.1 × 10⁸β‐lactamase molecules could be displayed per filamentous cell. Overproduced CWBM_LytE and its fusions were distributed on the entire cell surface. Surface exposure and accessibility of these proteins were confirmed by immunofluorescence microscopy.     

Extracellular β‐nicotinamide adenine dinucleotide (β‐NAD) promotes the endothelial cell barrier integrity via PKA‐ and EPAC1/Rac1‐dependent actin cytoskeleton rearrangement

Extracellular β‐NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5‐trisphate and cAMP. Recently, β‐NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since β‐NAD can be released extracellularly from endothelial cells (EC), we have proposed its involvement in the regulation of EC permeability. Here we show, for the first time, that endothelial integrity can be enhanced in EC endogenously expressing β‐NAD‐activated purinergic receptors upon β‐NAD stimulation. Our data demonstrate that extracellular β‐NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration‐dependent manner indicating endothelial barrier enhancement. Importantly, β‐NAD significantly attenuated thrombin‐induced EC permeability as well as the barrier‐compromising effects of Gram‐negative and Gram‐positive bacterial toxins representing the barrier‐protective function of β‐NAD. Immunofluorescence microscopy reveals more pronounced staining of cell–cell junctional protein VE‐cadherin at the cellular periphery signifying increased tightness of the cell‐cell contacts after β‐NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in β‐NAD‐induced TER increase. β‐NAD‐treatment attenuates the lipopolysaccharide (LPS)‐induced phosphorylation of myosin light chain (MLC) indicating its involvement in barrier protection. Our studies also show the involvement of cAMP‐dependent protein kinase A and EPAC1 pathways as well as small GTPase Rac1 in β‐NAD‐induced EC barrier enhancement. With these results, we conclude that β‐NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1‐ and MLCP‐ dependent signaling pathways. J. Cell. Physiol. 223: 215–223, 2010. © 2010 Wiley‐Liss, Inc.     

The interplay between effector binding and allostery in an engineered protein switch

The protein design rules for engineering allosteric regulation are not well understood. A fundamental understanding of the determinants of ligand binding in an allosteric context could facilitate the design and construction of versatile protein switches and biosensors. Here, we conducted extensive in vitro and in vivo characterization of the effects of 285 unique point mutations at 15 residues in the maltose‐binding pocket of the maltose‐activated β‐lactamase MBP317‐347. MBP317‐347 is an allosteric enzyme formed by the insertion of TEM‐1 β‐lactamase into the E. coli maltose binding protein (MBP). We find that the maltose‐dependent resistance to ampicillin conferred to the cells by the MBP317‐347 switch gene (the switch phenotype) is very robust to mutations, with most mutations slightly improving the switch phenotype. We identified 15 mutations that improved switch performance from twofold to 22‐fold, primarily by decreasing the catalytic activity in the absence of maltose, perhaps by disrupting interactions that cause a small fraction of MBP in solution to exist in a partially closed state in the absence of maltose. Other notable mutations include K15D and K15H that increased maltose affinity 30‐fold and Y155K and Y155R that compromised switching by diminishing the ability of maltose to increase catalytic activity. The data also provided insights into normal MBP physiology, as select mutations at D14, W62, and F156 retained high maltose affinity but abolished the switch's ability to substitute for MBP in the transport of maltose into the cell. The results reveal the complex relationship between ligand binding and allostery in this engineered switch.     

Specificity and cooperativity at β-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions

Establishing a quantitative understanding of the determinants of affinity in protein–protein interactions remains challenging. For example, TEM‐1/β‐lactamase inhibitor protein (BLIP) and SHV‐1/BLIP are homologous β‐lactamase/β‐lactamase inhibitor protein complexes with disparate K_d values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV‐1, results in a 1000‐fold enhancement in binding affinity. In TEM‐1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV‐1 D104 does not in the wild type SHV‐1/BLIP co‐structure. Here, we present a 1.6 Å crystal structure of the SHV‐1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV‐1 D104 and BLIP K74 and a 400‐fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Structural/mechanistic insights into the efficacy of nonclassical β‐lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates

Clavulanic acid and avibactam are clinically deployed serine β‐lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo β‐lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine β‐lactamase) but not L1 (metallo β‐lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X‐ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam‐like β‐lactams plus nonclassical β‐lactamase inhibitors, particularly avibactam‐like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.     

A rapid protein folding assay for the bacterial periplasm

An array of genetic screens and selections has been developed for reporting protein folding and solubility in the cytoplasm of living cells. However, there are currently no analogous folding assays for the bacterial periplasm, despite the significance of this compartment for the expression of recombinant proteins, especially those requiring important posttranslational modifications (e.g., disulfide bond formation). Here, we describe an engineered genetic selection for monitoring protein folding in the periplasmic compartment of Escherichia coli cells. In this approach, target proteins are sandwiched between an N‐terminal signal recognition particle (SRP)‐dependent signal peptide and a C‐terminal selectable marker, TEM‐1 β‐lactamase. The resulting chimeras are localized to the periplasmic space via the cotranslational SRP pathway. Using a panel of native and heterologous proteins, we demonstrate that the folding efficiency of various target proteins correlates directly with in vivo β‐lactamase activity and thus resistance to ampicillin. We also show that this reporter is useful for the discovery of extrinsic periplasmic factors (e.g., chaperones) that affect protein folding and for obtaining folding‐enhanced proteins via directed evolution. Collectively, these data demonstrate that our periplasmic folding reporter is a powerful tool for screening and engineering protein folding in a manner that does not require any structural or functional information about the target protein.     

Lentinan protects pancreatic β cells from STZ‐induced damage

Pancreatic β‐cell death or dysfunction mediated by oxidative stress underlies the development and progression of diabetes mellitus (DM). In this study, we evaluated the effect of lentinan (LNT), an active ingredient purified from the bodies of Lentinus edodes, on pancreatic β‐cell apoptosis and dysfunction caused by streptozotocin (STZ) and the possible mechanisms implicated. The rat insulinoma cell line INS‐1 were pre‐treated with the indicated concentration of LNT for 30 min. and then incubated for 24 hrs with or without 0.5 mM STZ. We found that STZ treatment causes apoptosis of INS‐1 cells by enhancement of intracellular reactive oxygen species (ROS) accumulation, inducible nitric oxide synthase (iNOS) expression and nitric oxide release and activation of the c‐jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK) signalling pathways. However, LNT significantly increased cell viability and effectively attenuated STZ‐induced ROS production, iNOS expression and nitric oxide release and the activation of JNK and p38 MAPK in a dose‐dependent manner in vitro. Moreover, LNT dose‐dependently prevented STZ‐induced inhibition of insulin synthesis by blocking the activation of nuclear factor kappa beta and increasing the level of Pdx‐1 in INS‐1 cells. Together these findings suggest that LNT could protect against pancreatic β‐cell apoptosis and dysfunction caused by STZ and therefore may be a potential pharmacological agent for preventing pancreatic β‐cell damage caused by oxidative stress associated with diabetes.     

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

A large number of β‐lactamases have emerged that are capable of conferring bacterial resistance to β‐lactam antibiotics. Comparison of the structural and functional features of this family has refined understanding of the catalytic properties of these enzymes. An arginine residue present at position 244 in TEM‐1 β‐lactamase interacts with the carboxyl group common to penicillin and cephalosporin antibiotics and thereby stabilizes both the substrate and transition state complexes. A comparison of class A β‐lactamase sequences reveals that arginine at position 244 is not conserved, although a positive charge at this structural location is conserved and is provided by an arginine at positions 220 or 276 for those enzymes lacking arginine at position 244. The plasticity of the location of positive charge in the β‐lactamase active site was experimentally investigated by relocating the arginine at position 244 in TEM‐1 β‐lactamase to positions 220, 272, and 276 by site‐directed mutagenesis. Kinetic analysis of the engineered β‐lactamases revealed that removal of arginine 244 by alanine mutation reduced catalytic efficiency against all substrates tested and restoration of an arginine at positions 272 or 276 partially suppresses the catalytic defect of the Arg244Ala substitution. These results suggest an evolutionary mechanism for the observed divergence of the position of positive charge in the active site of class A β‐lactamases.     

Modeling spatial distribution of oxygen in 3d culture of islet beta‐cells

Three‐dimensional (3D) scaffold culture of pancreatic β‐cell has been proven to be able to better mimic physiological conditions in the body. However, one critical issue with culturing pancreatic β‐cells is that β‐cells consume large amounts of oxygen, and hence insufficient oxygen supply in the culture leads to loss of β‐cell mass and functions. This becomes more significant when cells are cultured in a 3D scaffold. In this study, in order to understand the effect of oxygen tension inside a cell‐laden collagen culture on β‐cell proliferation, a culture model with encapsulation of an oxygen‐generator was established. The oxygen‐generator was made by embedding hydrogen peroxide into nontoxic polydimethylsiloxane to avoid the toxicity of a chemical reaction in the β‐cell culture. To examine the effectiveness of the oxygenation enabled 3D culture, the spatial‐temporal distribution of oxygen tension inside a scaffold was evaluated by a mathematical modeling approach. Our simulation results indicated that an oxygenation‐aided 3D culture would augment the oxygen supply required for the β‐cells. Furthermore, we identified that cell seeding density and the capacity of the oxygenator are two critical parameters in the optimization of the culture. Notably, cell‐laden scaffold cultures with an in situ oxygen supply significantly improved the β‐cells' biological function. These β‐cells possess high insulin secretion capacity. The results obtained in this work would provide valuable information for optimizing and encouraging functional β‐cell cultures. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:221–228, 2017     

Protein�Cprotein binding affinities by pulse proteolysis: Application to TEM-1/BLIP protein complexes

Efficient methods for quantifying dissociation constants have become increasingly important for high‐throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein–ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein–protein complex involving the β‐lactamase TEM‐1 and various β‐lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C_m, of TEM‐1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein–protein complexes. From a small set (n = 4) of TEM‐1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC_m was observed. From this “calibration curve,” accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis‐derived ΔC_m values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high‐throughput mutagenesis binding studies.     

Elucidating the role of Trp105 in the KPC-2 ��-lactamase

The molecular basis of resistance to β‐lactams and β‐lactam‐β‐lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective β‐lactam therapy. Recent crystal structures of KPC‐2 and other class A β‐lactamases suggest that Ambler position Trp105 may be of importance in binding β‐lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC‐2 by conducting site‐saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, ‐Tyr, ‐Asn, and ‐His KPC‐2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC‐2. Interestingly, most of the variants showed increased MICs to ampicillin‐clavulanic acid but not to ampicillin‐sulbactam or piperacillin‐tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, ‐Asn, ‐Leu, and ‐Val) were studied in detail. Consistent with the MIC data, the Trp105Phe β‐lactamase displayed improved catalytic efficiencies, k_cat/K_m, toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k_cat/K_m toward cefotaxime and imipenem when compared to WT β‐lactamase. The Trp105Asn variant exhibited increased K_ms for all substrates. In contrast, the Trp105Leu and ‐Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k_cat/K_m) for all substrates. With respect to clavulanic acid, the K_is and partition ratios were increased for the Trp105Phe, ‐Asn, and ‐Val variants. We conclude that interactions between Trp105 of KPC‐2 and the β‐lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in β‐lactam and β‐lactamase inhibitor discrimination.     

Selection of broad‐spectrum cephalosporin‐resistant Escherichia coli in the feces of healthy dogs after administration of first‐generation cephalosporins

Although antimicrobial products are essential for treating diseases caused by bacteria, antimicrobial treatment selects for antimicrobial‐resistant (AMR) bacteria. The aim of this study was to determine the effects of administration of first‐generation cephalosporins on development of resistant Escherichia coli in dog feces. The proportions of cephalexin (LEX)‐resistant E. coli in fecal samples of three healthy dogs treated i.v. with cefazolin before castration and then orally with LEX for 3 days post‐operation (PO) were examined using DHL agar with or without LEX (50 µg/mL). LEX‐resistant E. coli were found within 3 days PO, accounted for 100% of all identified E. coli 3–5 days PO in all dogs, and were predominantly found until 12 days PO. LEX‐resistant E. coli isolates on DHL agar containing LEX were subjected to antimicrobial susceptibility testing, pulsed‐field gel electrophoresis (PFGE) genotyping, β‐lactamase typing and plasmid profiling. All isolates tested exhibited cefotaxime (CTX) resistance (CTX minimal inhibitory concentration ≥4 µg/mL). Seven PFGE profiles were classified into five groups and three β‐lactamase combinations (bla_CMY‐4‐bla_TEM‐1, bla_TEM‐1‐bla_CTX‐M‐15 and bla_TEM‐1‐bla_CTX‐M‐15‐bla_CMY‐4). All isolates exhibited identical PFGE profiles in all dogs on four days PO and subsequently showed divergent PFGE profiles. Our results indicate there are two selection periods for AMR bacteria resulting from the use of antimicrobials. Thus, continuing hygiene practices are necessary to prevent AMR bacteria transfer via dog feces after antimicrobial administration.     

The effect of a beta‐lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study

Co‐administration of beta‐lactam antibiotics and beta‐lactamase inhibitors has been a favored treatment strategy against beta‐lactamase‐mediated bacterial antibiotic resistance, but the emergence of beta‐lactamases resistant to current inhibitors necessitates the discovery of novel non‐beta‐lactam inhibitors. Peptides derived from the Ala46–Tyr51 region of the beta‐lactamase inhibitor protein are considered as potent inhibitors of beta‐lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell‐penetrating peptides could guide the design of beta‐lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta‐lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell‐penetrating peptide pVEC, our approach involved the addition of the N‐terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta‐lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta‐lactam antibiotic ampicillin, and the beta‐lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N‐terminus of the beta‐lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N‐terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration

Aims: The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla_TEM‐52‐carrying plasmid (lactose‐negative mutant of B1‐54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. Methods and Results: Fresh faeces from a healthy volunteer, negative for cephalosporin‐resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed‐field gel electrophoresis profiles (PFGE). The avian extended‐spectrum β‐lactamase‐positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1‐54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla_TEM‐52‐carrying plasmid. Conclusions: These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. Significance and Impact of the Study: The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.     

Non‐allosteric enzyme switches possess larger effector‐induced changes in thermodynamic stability than their non‐switch analogs

The ability to regulate cellular protein activity offers a broad range of biotechnological and biomedical applications. Such protein regulation can be achieved by modulating the specific protein activity or through processes that regulate the amount of protein in the cell. We have previously demonstrated that the nonhomologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β‐lactamase (BLA) can result in genes that confer maltose‐dependent resistance to β‐lactam antibiotics even though the encoded proteins are not allosteric enzymes. We showed that these phenotypic switches—named based on their conferral of a switching phenotype to cells—resulted from a specific interaction with maltose in the cell that increased the switches cellular accumulation. Since phenotypic switches represent an important class of engineered proteins for basic science and biotechnological applications in vivo, we sought to elucidate the phenomena behind the increased accumulation and switching properties. Here, we demonstrate the key role for the linker region between the two proteins. Experimental evidence supports the hypothesis that in the absence of their effector, some phenotypic switches possess an increased rate of unfolding, decreased conformational stability, and increased protease susceptibility. These factors alone or in combination serve to decrease cellular accumulation. The effector functions to increase cellular accumulation by alleviating one or more of these defects. This perspective on the mechanism for phenotypic switching will aid the development of design rules for switch construction for applications and inform the study of the regulatory mechanisms of natural cellular proteins.     

The extracellular β‐1,3‐endoglucanase EngA is involved in autolysis of Aspergillus nidulans

Aims: To elucidate the roles of the β‐1,3‐endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. Methods and Results: A β‐1,3‐endoglucanase was purified from carbon‐starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene‐expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. Conclusions: The β‐1,3‐endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall–degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. Significance and Impact of the Study: No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases.     

Exploring the potential of boronic acids as inhibitors of OXA‐24/40 β‐lactamase

β‐lactam antibiotics are crucial to the management of bacterial infections in the medical community. Due to overuse and misuse, clinically significant bacteria are now resistant to many commercially available antibiotics. The most widespread resistance mechanism to β‐lactams is the expression of β‐lactamase enzymes. To overcome β‐lactamase mediated resistance, inhibitors were designed to inactivate these enzymes. However, current inhibitors (clavulanic acid, tazobactam, and sulbactam) for β‐lactamases also contain the characteristic β‐lactam ring, making them susceptible to resistance mechanisms employed by bacteria. This presents a critical need for novel, non‐β‐lactam inhibitors that can circumvent these resistance mechanisms. The carbapenem‐hydrolyzing class D β‐lactamases (CHDLs) are of particular concern, given that they efficiently hydrolyze potent carbapenem antibiotics. Unfortunately, these enzymes are not inhibited by clinically available β‐lactamase inhibitors, nor are they effectively inhibited by the newest, non‐β‐lactam inhibitor, avibactam. Boronic acids are known transition state analog inhibitors of class A and C β‐lactamases, and are not extensively characterized as inhibitors of class D β‐lactamases. Importantly, boronic acids provide a novel way to potentially inhibit class D β‐lactamases. Sixteen boronic acids were selected and tested for inhibition of the CHDL OXA‐24/40. Several compounds were identified as effective inhibitors of OXA‐24/40, with K_i values as low as 5 μM. The X‐ray crystal structures of OXA‐24/40 in complex with BA3, BA4, BA8, and BA16 were determined and revealed the importance of interactions with hydrophobic residues Tyr112 and Trp115. These boronic acids serve as progenitors in optimization efforts of a novel series of inhibitors for class D β‐lactamases.