Emergence of methicillin resistance predates the clinical use of antibiotics.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

Structural and kinetic analysis of the monofunctional Staphylococcus aureus PBP1.

Staphylococcus aureus, an ESKAPE pathogen, is a major clinical concern due to its pathogenicity and manifold antimicrobial resistance mechanisms. The commonly used ß-lactam antibiotics target bacterial penicillin-binding proteins (PBPs) and inhibit crosslinking of peptidoglycan strands that comprise the bacterial cell wall mesh, initiating a cascade of effects leading to bacterial cell death. S. aureus PBP1 is involved in synthesis of the bacterial cell wall during division and its presence is essential for survival of both antibiotic susceptible and resistant S. aureus strains. Here, we present X-ray crystallographic data for S. aureus PBP1 in its apo form as well as acyl-enzyme structures with distinct classes of ß-lactam antibiotics representing the penicillins, carbapenems, and cephalosporins, respectively: oxacillin, ertapenem and cephalexin. Our structural data suggest that the PBP1 active site is readily accessible for substrate, with little conformational change in key structural elements required for its covalent acylation of ß-lactam inhibitors. Stopped-flow kinetic analysis and gel-based competition assays support the structural observations, with even the weakest performing ß-lactams still having comparatively high acylation rates and affinities for PBP1. Our structural and kinetic analysis sheds insight into the ligand-PBP interactions that drive antibiotic efficacy against these historically useful antimicrobial targets and expands on current knowledge for future drug design and treatment of S. aureus infections.

Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Escherichia coli has robust regulatory mechanisms against elevated peptidoglycan cleavage by lytic transglycosylases.

Peptidoglycan (PG) is an essential and conserved exoskeletal component in all bacteria that protects cells from lysis. Gram-negative bacteria such as Escherichia coli encode multiple redundant lytic transglycosylases (LTs) that engage in PG cleavage, a potentially lethal activity requiring proper regulation to prevent autolysis. To elucidate the potential effects and cellular regulatory mechanisms of elevated LT activity, we individually cloned the periplasmic domains of two membrane-bound LTs, MltA and MltB, under the control of the arabinose-inducible system for overexpression in the periplasmic space in E. coli. Interestingly, upon induction, the culture undergoes an initial period of cell lysis followed by robust growth restoration. The LT-overexpressing E. coli exhibits altered morphology with larger spherical cells, which is in line with the weakening of the PG layer due to aberrant LT activity. On the other hand, the restored cells display a similar rod shape and PG profile that is indistinguishable from the uninduced control. Quantitative proteomics analysis of the restored cells identified significant protein enrichment in the regulator of capsule synthesis (Rcs) regulon, a two-component stress response known to be specifically activated by PG damage. We showed that LT-overexpressing E. coli with an inactivated Rcs system partially impairs the growth restoration process, supporting the involvement of the Rcs system in countering aberrant PG cleavage. Furthermore, we demonstrated that the elevated LT activity specifically potentiates ß-lactam antibiotics against E. coli with a defective Rcs regulon, suggesting the dual effects of augmented PG cleavage and blocked PG synthesis as a potential antimicrobial strategy.

Mutagenesis and structural analysis reveal the CTX-M ß-lactamase active site is optimized for cephalosporin catalysis and drug resistance.

CTX-M ß-lactamases are a widespread source of resistance to ß-lactam antibiotics in Gram-negative bacteria. These enzymes readily hydrolyze penicillins and cephalosporins, including oxyimino-cephalosporins such as cefotaxime. To investigate the preference of CTX-M enzymes for cephalosporins, we examined eleven active-site residues in the CTX-M-14 ß-lactamase model system by alanine mutagenesis to assess the contribution of the residues to catalysis and specificity for the hydrolysis of the penicillin, ampicillin, and the cephalosporins cephalothin and cefotaxime. Key active site residues for class A ß-lactamases, including Lys73, Ser130, Asn132, Lys234, Thr216, and Thr235, contribute significantly to substrate binding and catalysis of penicillin and cephalosporin substrates in that alanine substitutions decrease both kcat and kcat/KM. A second group of residues, including Asn104, Tyr105, Asn106, Thr215, and Thr216, contribute only to substrate binding, with the substitutions decreasing only kcat/KM. Importantly, calculating the average effect of a substitution across the 11 active-site residues shows that the most significant impact is on cefotaxime hydrolysis while ampicillin hydrolysis is least affected, suggesting the active site is highly optimized for cefotaxime catalysis. Furthermore, we determined X-ray crystal structures for the apo-enzymes of the mutants N106A, S130A, N132A, N170A, T215A, and T235A. Surprisingly, in the structures of some mutants, particularly N106A and T235A, the changes in structure propagate from the site of substitution to other regions of the active site, suggesting that the impact of substitutions is due to more widespread changes in structure and illustrating the interconnected nature of the active site.

Potential involvement of beta-lactamase homologous proteins in resistance to beta-lactam antibiotics in gram-negative bacteria of the ESKAPEE group.

BACKGROUND: Enzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens. In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher's exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems. We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.

Staphylococcus aureus AbcA transporter enhances persister formation under ß-lactam exposure.

We evaluated the role of Staphylococcus aureus AbcA transporter in bacterial persistence and survival following exposure to the bactericidal agents nafcillin and oxacillin at both the population and single-cell levels. We show that AbcA overexpression resulted in resistance to nafcillin but not oxacillin. Using distinct fluorescent reporters of cell viability and AbcA expression, we found that over 6-14 hours of persistence formation, the proportion of AbcA reporter-expressing cells assessed by confocal microscopy increased sixfold as cell viability reporters decreased. Similarly, single-cell analysis in a high-throughput microfluidic system found a strong correspondence between antibiotic exposure and AbcA reporter expression. Persister cells grown in the absence of antibiotics showed neither an increase in nafcillin MIC nor in abcA transcript levels, indicating that survival was not associated with stable mutational resistance or abcA overexpression. Furthermore, persister cell levels on exposure to 1×MIC and 25×MIC of nafcillin decreased in an abcA knockout mutant. Survivors of nafcillin and oxacillin treatment overexpressed transporter AbcA, contributing to an enrichment of the number of persisters during treatment with pump-substrate nafcillin but not with pump-non-substrate oxacillin, indicating that efflux pump expression can contribute selectively to the survival of a persister population.

Penicillin-Binding Protein 5/6 Acting as a Decoy Target in Pseudomonas aeruginosa Identified by Whole-Cell Receptor Binding and Quantitative Systems Pharmacology.

The ß-lactam antibiotics have been successfully used for decades to combat susceptible Pseudomonas aeruginosa, which has a notoriously difficult to penetrate outer membrane (OM). However, there is a dearth of data on target site penetration and covalent binding of penicillin-binding proteins (PBP) for ß-lactams and ß-lactamase inhibitors in intact bacteria. We aimed to determine the time course of PBP binding in intact and lysed cells and estimate the target site penetration and PBP access for 15 compounds in P. aeruginosa PAO1. All ß-lactams (at 2 × MIC) considerably bound PBPs 1 to 4 in lysed bacteria. However, PBP binding in intact bacteria was substantially attenuated for slow but not for rapid penetrating ß-lactams. Imipenem yielded 1.5 ± 0.11 log10 killing at 1h compared to <0.5 log10 killing for all other drugs. Relative to imipenem, the rate of net influx and PBP access was ~ 2-fold slower for doripenem and meropenem, 7.6-fold for avibactam, 14-fold for ceftazidime, 45-fold for cefepime, 50-fold for sulbactam, 72-fold for ertapenem, ~ 249-fold for piperacillin and aztreonam, 358-fold for tazobactam, ~547-fold for carbenicillin and ticarcillin, and 1,019-fold for cefoxitin. At 2 × MIC, the extent of PBP5/6 binding was highly correlated (r2 = 0.96) with the rate of net influx and PBP access, suggesting that PBP5/6 acted as a decoy target that should be avoided by slowly penetrating, future ß-lactams. This first comprehensive assessment of the time course of PBP binding in intact and lysed P. aeruginosa explained why only imipenem killed rapidly. The developed novel covalent binding assay in intact bacteria accounts for all expressed resistance mechanisms.

Penicillin-Binding Proteins and Alternative Dual-Beta-Lactam Combinations for Serious Enterococcus faecalis Infections with Elevated Penicillin MICs.

Ampicillin-ceftriaxone has become a first-line therapy for Enterococcus faecalis endocarditis. We characterized the penicillin-binding protein (PBP) profiles of various E. faecalis strains and tested for synergy to better inform beta-lactam options for the treatment of E. faecalis infections. We assessed the affinity of PBP2B from elevated-MIC strain E. faecalis LS4828 compared to type strain JH2-2 using the fluorescent beta-lactam Bocillin FL. We also characterized pbp4 and pbpA structures and PBP4 and PBP2B expression and used deletion and complementation studies to assess the impact of PBP2B on the levels of resistance. We tested penicillin-susceptible and -resistant E. faecalis isolates against ceftriaxone or ceftaroline combinations with other beta-lactams in 24-h time-kill studies. Two penicillin-susceptible strains (JH2-2 and L2052) had identical pbp sequences and similar PBP expression levels. One reduced-penicillin-susceptibility strain (L2068) had pbp sequences identical to those of the susceptible strains but expressed more PBP4. The second decreased-penicillin-susceptibility strain (LS4828) had amino acid substitutions in both PBP4 and PBP2B and expressed increased quantities of both proteins. PBP2B did not appear to contribute significantly to the elevated beta-lactam MICs. No synergy was demonstrable against the strains with both mutated PBPs and increased expression (L2068 and LS4828). Meropenem plus ceftriaxone or ertapenem plus ceftriaxone demonstrated the most consistent synergistic activity. PBP2B of strain LS4828 does not contribute significantly to reduced penicillin susceptibility. Neither the MIC nor the level of PBP expression correlated directly with the identified synergistic combinations when tested at static subinhibitory concentrations.

DNA Nanoflower Eye Drops with Antibiotic-Resistant Gene Regulation Ability for MRSA Keratitis Target Treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) biofilm-associated bacterial keratitis is highly intractable, with strong resistance to ß-lactam antibiotics. Inhibiting the MRSA resistance gene mecR1 to downregulate penicillin-binding protein PBP2a has been implicated in the sensitization of ß-lactam antibiotics to MRSA. However, oligonucleotide gene regulators struggle to penetrate dense biofilms, let alone achieve efficient gene regulation inside bacteria cells. Herein, an eye-drop system capable of penetrating biofilms and targeting bacteria for chemo-gene therapy in MRSA-caused bacterial keratitis is developed. This system employed rolling circle amplification to prepare DNA nanoflowers (DNFs) encoding MRSA-specific aptamers and mecR1 deoxyribozymes (DNAzymes). Subsequently, ß-lactam antibiotic ampicillin (Amp) and zinc oxide (ZnO) nanoparticles are sequentially loaded into the DNFs (ZnO/Amp@DNFs). Upon application, ZnO on the surface of the nanosystem disrupts the dense structure of biofilm and fully exposes free bacteria. Later, bearing encoded aptamer, the nanoflower system is intensively endocytosed by bacteria, and releases DNAzyme under acidic conditions to cleave the mecR1 gene for PBP2a down-regulation, and ampicillin for efficient MRSA elimination. In vivo tests showed that the system effectively cleared bacterial and biofilm in the cornea, suppressed proinflammatory cytokines interleukin 1ß ï¼ˆIL-1ß） and tumor neocrosis factor-alpha （TNF-α）, and is safe for corneal epithelial cells. Overall, this design offers a promising approach for treating MRSA-induced keratitis.

Temoneira-1 ß-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography.

ß-lactamases protect bacteria from ß-lactam antibiotics. Temoneira (TEM) is a class A serine ß-lactamase and its coding sequence is designed into DNA vectors, such as pET-21a (+), to provide antibiotic resistance. TEM-1 ß-lactamase was overexpressed efficiently from this vector upon inducing protein expression by IPTG in BL21(DE3) cells. Immobilized metal ion affinity chromatography (IMAC) was used based on the three native putative metal ion binding sites of TEM-1 ß-lactamase, each consisting of a pair of histidine sidechains. Elution was achieved at low concentrations of imidazole (∼15-200 mM). Two steps of IMAC and a subsequent anion exchange purification produced highly pure TEM-1 ß-lactamase with a yield of 1.9 mg/g of wet bacterial pellet weight. Mass spectrometry revealed that the mature form of ß-lactamase (without the signal sequence) was obtained. The secondary structure composition, calculated from the circular dichroism spectrum, showed that the target protein was folded similar to the published crystal structure. Ni(II) binding to the enzyme was also investigated. Increasing amounts of Ni(II) ions had only a small effect on the protein structure. Mass spectrometry detected up to three bound metal ions at 10:1 Ni(II):protein molar ratio, but the major peak was assigned to the monometallated ß-lactamase indicating the presence of a paramount metal ion binding site formed by the H151/H156 pair.

ß-Lactams against the Fortress of the Gram-Positive Staphylococcus aureus Bacterium.

The biological diversity of the unicellular bacteria-whether assessed by shape, food, metabolism, or ecological niche-surely rivals (if not exceeds) that of the multicellular eukaryotes. The relationship between bacteria whose ecological niche is the eukaryote, and the eukaryote, is often symbiosis or stasis. Some bacteria, however, seek advantage in this relationship. One of the most successful-to the disadvantage of the eukaryote-is the small (less than 1 µm diameter) and nearly spherical Staphylococcus aureus bacterium. For decades, successful clinical control of its infection has been accomplished using ß-lactam antibiotics such as the penicillins and the cephalosporins. Over these same decades S. aureus has perfected resistance mechanisms against these antibiotics, which are then countered by new generations of ß-lactam structure. This review addresses the current breadth of biochemical and microbiological efforts to preserve the future of the ß-lactam antibiotics through a better understanding of how S. aureus protects the enzyme targets of the ß-lactams, the penicillin-binding proteins. The penicillin-binding proteins are essential enzyme catalysts for the biosynthesis of the cell wall, and understanding how this cell wall is integrated into the protective cell envelope of the bacterium may identify new antibacterials and new adjuvants that preserve the efficacy of the ß-lactams.

The Antibiotic Resistome: A Guide for the Discovery of Natural Products as Antimicrobial Agents.

The use of life-saving antibiotics has long been plagued by the ability of pathogenic bacteria to acquire and develop an array of antibiotic resistance mechanisms. The sum of these resistance mechanisms, the antibiotic resistome, is a formidable threat to antibiotic discovery, development, and use. The study and understanding of the molecular mechanisms in the resistome provide the basis for traditional approaches to combat resistance, including semisynthetic modification of naturally occurring antibiotic scaffolds, the development of adjuvant therapies that overcome resistance mechanisms, and the total synthesis of new antibiotics and their analogues. Using two major classes of antibiotics, the aminoglycosides and tetracyclines as case studies, we review the success and limitations of these strategies when used to combat the many forms of resistance that have emerged toward natural product-based antibiotics specifically. Furthermore, we discuss the use of the resistome as a guide for the genomics-driven discovery of novel antimicrobials, which are essential to combat the growing number of emerging pathogens that are resistant to even the newest approved therapies.

ß-Lactams from the Ocean.

The title of this essay is as much a question as it is a statement. The discovery of the ß-lactam antibiotics-including penicillins, cephalosporins, and carbapenems-as largely (if not exclusively) secondary metabolites of terrestrial fungi and bacteria, transformed modern medicine. The antibiotic ß-lactams inactivate essential enzymes of bacterial cell-wall biosynthesis. Moreover, the ability of the ß-lactams to function as enzyme inhibitors is of such great medical value, that inhibitors of the enzymes which degrade hydrolytically the ß-lactams, the ß-lactamases, have equal value. Given this privileged status for the ß-lactam ring, it is therefore a disappointment that the exemplification of this ring in marine secondary metabolites is sparse. It may be that biologically active marine ß-lactams are there, and simply have yet to be encountered. In this report, we posit a second explanation: that the value of the ß-lactam to secure an ecological advantage in the marine environment might be compromised by its close structural similarity to the ß-lactones of quorum sensing. The steric and reactivity similarities between the ß-lactams and the ß-lactones represent an outside-of-the-box opportunity for correlating new structures and new enzyme targets for the discovery of compelling biological activities.

Effects of Inactivation of d,d-Transpeptidases of Acinetobacter baumannii on Bacterial Growth and Susceptibility to ß-Lactam Antibiotics.

Resistance to ß-lactams, the most used antibiotics worldwide, constitutes the major problem for the treatment of bacterial infections. In the nosocomial pathogen Acinetobacter baumannii, ß-lactamase-mediated resistance to the carbapenem family of ß-lactam antibiotics has resulted in the selection and dissemination of multidrug-resistant isolates, which often cause infections characterized by high mortality rates. There is thus an urgent demand for new ß-lactamase-resistant antibiotics that also inhibit their targets, penicillin-binding proteins (PBPs). As some PBPs are indispensable for the biosynthesis of the bacterial cell wall and survival, we evaluated their importance for the growth of A. baumannii by performing gene inactivation studies of d,d-transpeptidase domains of high-molecular-mass (HMM) PBPs individually and in combination with one another. We show that PBP3 is essential for A. baumannii survival, as deletion mutants of this d,d-transpeptidase were not viable. The inactivation of PBP1a resulted in partial cell lysis and retardation of bacterial growth, and these effects were further enhanced by the additional inactivation of PBP2 but not PBP1b. Susceptibility to ß-lactam antibiotics increased 4- to 8-fold for the A. baumannii PBP1a/PBP1b/PBP2 triple mutant and 2- to 4-fold for all remaining mutants. Analysis of the peptidoglycan structure revealed a significant change in the muropeptide composition of the triple mutant and demonstrated that the lack of d,d-transpeptidase activity of PBP1a, PBP1b, and PBP2 is compensated for by an increase in the l,d-transpeptidase-mediated cross-linking activity of LdtJ. Overall, our data showed that in addition to essential PBP3, the simultaneous inhibition of PBP1a and PBP2 or PBPs in combination with LdtJ could represent potential strategies for the design of novel drugs against A. baumannii.

Expanded profiling of ß-lactam selectivity for penicillin-binding proteins in Streptococcus pneumoniae D39.

Penicillin-binding proteins (PBPs) are integral to bacterial cell division as they mediate the final steps of cell wall maturation. Selective fluorescent probes are useful for understanding the role of individual PBPs, including their localization and activity during growth and division of bacteria. For the development of new selective probes for PBP imaging, several ß-lactam antibiotics were screened, as they are known to covalently bind PBP in vivo. The PBP inhibition profiles of 16 commercially available ß-lactam antibiotics were evaluated in an unencapsulated derivative of the D39 strain of Streptococcus pneumoniae, IU1945. These ß-lactams have not previously been characterized for their PBP inhibition profiles in S. pneumoniae and these data augment those obtained from a library of 20 compounds that we previously reported. We investigated seven penicillins, three carbapenems, and six cephalosporins. Most of these ß-lactams were found to be co-selective for PBP2x and PBP3, as was noted in our previous studies. Six out of 16 antibiotics were selective for PBP3 and one molecule was co-selective for PBP1a and PBP3. Overall, this work expands the chemical space available for development of future ß-lactam-based probes for specific pneumococcal PBP labeling and these methods can be used for the development of probes for PBP labelling in other bacterial species.

Biosynthesis of ß-lactam nuclei in yeast.

We have engineered brewer's yeast as a general platform for de novo synthesis of diverse ß-lactam nuclei starting from simple sugars, thereby enabling ready access to a number of structurally different antibiotics of significant pharmaceutical importance. The biosynthesis of ß-lactam nuclei has received much attention in recent years, while rational engineering of non-native antibiotics-producing microbes to produce ß-lactam nuclei remains challenging. Benefited by the integration of heterologous biosynthetic pathways and rationally designed enzymes that catalyze hydrolysis and ring expansion reactions, we succeeded in constructing synthetic yeast cell factories which produce antibiotic cephalosporin C (CPC, 170.1 ± 4.9 µg/g DCW) and the downstream ß-lactam nuclei, including 6-amino penicillanic acid (6-APA, 5.3 ± 0.2 mg/g DCW), 7-amino cephalosporanic acid (7-ACA, 6.2 ± 1.1 µg/g DCW) as well as 7-amino desacetoxy cephalosporanic acid (7-ADCA, 1.7 ± 0.1 mg/g DCW). This work established a Saccharomyces cerevisiae platform capable of synthesizing multiple ß-lactam nuclei by combining natural and artificial enzymes, which serves as a metabolic tool to produce valuable ß-lactam intermediates and new antibiotics.

The Roles of the Two-Component System, MtrAB, in Response to Diverse Cell Envelope Stresses in Dietzia sp. DQ12-45-1b.

Two-component systems (TCSs) act as common regulatory systems allowing bacteria to detect and respond to multiple environmental stimuli, including cell envelope stress. The MtrAB TCS of Actinobacteria is critical for cell wall homeostasis, cell proliferation, osmoprotection, and antibiotic resistance, and thus is found to be highly conserved across this phylum. However, how precisely the MtrAB TCS regulates cellular homeostasis in response to environmental stress remains unclear. Here, we show that the MtrAB TCS plays an important role in the tolerance to different types of cell envelope stresses, including environmental stresses (i.e., oxidative stress, lysozyme, SDS, osmotic pressure, and alkaline pH stresses) and envelope-targeting antibiotics (i.e., isoniazid, ethambutol, glycopeptide, and ß-lactam antibiotics) in Dietzia sp. DQ12-45-1b. An mtrAB mutant strain exhibited slower growth compared to the wild-type strain and was characterized by abnormal cell shapes when exposed to various environmental stresses. Moreover, deletion of mtrAB resulted in decreased resistance to isoniazid, ethambutol, and ß-lactam antibiotics. Further, Cleavage under targets and tagmentation sequencing (CUT&Tag-seq) and electrophoretic mobility shift assays (EMSAs) revealed that MtrA binds the promoters of genes involved in peptidoglycan biosynthesis (ldtB, ldtA, murJ), hydrolysis (GJR88_03483, GJR88_4713), and cell division (ftsE). Together, our findings demonstrated that the MtrAB TCS is essential for the survival of Dietzia sp. DQ12-45-1b under various cell envelope stresses, primarily by controlling multiple downstream cellular pathways. Our work suggests that TCSs act as global sensors and regulators in maintaining cellular homeostasis, such as during episodes of various environmental stresses. The present study should shed light on the understanding of mechanisms for bacterial adaptivity to extreme environments. IMPORTANCE The multilayered cell envelope is the first line of bacterial defense against various extreme environments. Bacteria utilize a large number of sensing and regulatory systems to maintain cell envelope homeostasis under multiple stress conditions. The two-component system (TCS) is the main sensing and responding apparatus for environmental adaptation. The MtrAB TCS highly conserved in Actinobacteria is critical for cell wall homeostasis, cell proliferation, osmoprotection, and antibiotic resistance. However, how MtrAB works with regard to signals impacting changes to the cell envelope is not fully understood. Here, we found that in the Actinobacterium Dietzia sp. DQ12-45-1b, a TCS named MtrAB is pivotal for ensuring normal cell growth as well as maintaining proper cell morphology in response to various cell envelope stresses, namely, by regulating the expression of cell envelope-related genes. Our findings should greatly advance our understanding of the adaptive mechanisms responsible for maintaining cell integrity in times of sustained environmental shocks.

Antibacterial Mechanisms of Zinc Oxide Nanoparticle against Bacterial Food Pathogens Resistant to Beta-Lactam Antibiotics.

The increase in ß-lactam-resistant Gram-negative bacteria is a severe recurrent problem in the food industry for both producers and consumers. The development of nanotechnology and nanomaterial applications has transformed many features in food science. The antibacterial activity of zinc oxide nanoparticles (ZnO NPs) and their mechanism of action on ß-lactam-resistant Gram-negative food pathogens, such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Klebsiella pneumoniae, and Proteus mirabilis, are investigated in the present paper. The study results demonstrate that ZnO NPs possesses broad-spectrum action against these ß-lactamase-producing strains. The minimal inhibitory and minimal bactericidal concentrations vary from 0.04 to 0.08 and 0.12 to 0.24 mg/mL, respectively. The ZnO NPs elevate the level of reactive oxygen species (ROS) and malondialdehyde in the bacterial cells as membrane lipid peroxidation. It has been confirmed from the transmission electron microscopy image of the treated bacterial cells that ZnO NPs diminish the permeable membrane, denature the intracellular proteins, cause DNA damage, and cause membrane leakage. Based on these findings, the action of ZnO NPs has been attributed to the fact that broad-spectrum antibacterial action against ß-lactam-resistant Gram-negative food pathogens is mediated by Zn2+ ion-induced oxidative stress, actions via lipid peroxidation and membrane damage, subsequently resulting in depletion, leading to ß-lactamase enzyme inhibition, intracellular protein inactivation, DNA damage, and eventually cell death. Based on the findings of the present study, ZnO NPs can be recommended as potent broad-spectrum antibacterial agents against ß-lactam-resistant Gram-negative pathogenic strains.

Activity-Based Protein Profiling of Bile Salt Hydrolysis in the Human Gut Microbiome with Beta-Lactam or Acrylamide-Based Probes.

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine- and glycine-linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health. Bile salt metabolism has also been associated with disease phenotypes such as liver and colorectal cancer. In order to reshape the gut microbiome to optimize bile salt metabolism, tools to characterize and quantify these processes must exist to enable a much-improved understanding of how metabolism goes awry in the face of disease, and how it can be improved through an altered lifestyle and environment. Furthermore, it is necessary to attribute metabolic activity to specific members and BSHs within the microbiome. To this end, we have developed activity-based probes with two different reactive groups to target bile salt hydrolases. These probes bind similarly to the authentic bile salt substrates, and we demonstrate enzyme labeling of active bile salt hydrolases by using purified protein, cell lysates, and in human stool.