Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the β3–β4 loop to favor a low-affinity drug-binding state

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.Keywords: PBP2, Neisseria gonorrhoeae, beta-lactam, conformational dynamics, antibiotic resistance

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea, with nearly 80 million cases worldwide each year (1). Without antibiotic treatment, infections persist as a chronic disease and can cause serious sequelae, including pelvic inflammatory disease, infertility, arthritis, and disseminated infections (2). For many years, N. gonorrhoeae was treated with a single dose of penicillin, and more recently, ceftriaxone. In 2012, the emergence of several high-level ceftriaxone-resistant strains led the Centers for Disease Control and Prevention to change its recommended treatment for gonorrhea from monotherapy to dual therapy with ceftriaxone and azithromycin (3, 4, 5). However, treatment failures have been reported for both agents, and in 2018, a strain with high-level resistance to both ceftriaxone and azithromycin was identified (6, 7). Concern about azithromycin resistance led the Centers for Disease Control and Prevention recently to drop the recommendation of dual therapy in favor of an increased dose (500 mg) of ceftriaxone alone (8). Both penicillin and ceftriaxone inhibit cell wall biosynthesis in N. gonorrhoeae by targeting penicillin-binding protein 2 (PBP2).PBP2 is an essential peptidoglycan transpeptidase (TPase) that crosslinks the peptide chains from adjacent peptidoglycan strands during cell-wall synthesis (9). β-lactam antibiotics, including the extended-spectrum cephalosporin (ESC) ceftriaxone, are analogs of the d-Ala-d-Ala C terminus of the peptidoglycan substrate and as such target PBP2 by binding to and reacting with the active-site serine nucleophile (Ser310 in N. gonorrhoeae PBP2) to form a covalently acylated complex (10, 11). The acylation reaction (Equation 1) proceeds first through formation of a noncovalent complex with the β-lactam (defined by the equilibrium constant, Ks), which is then attacked by the serine nucleophile to form a covalent acyl-enzyme complex (k₂). For PBPs, hydrolysis of the acyl-enzyme (k₃) is very slow compared with its formation, and the enzyme is essentially irreversibly inactivated. The acylation of PBPs by β-lactam antibiotics is therefore defined by a second-order rate constant, k₂/Ks (M⁻¹ s⁻¹), which reflects both the noncovalent binding affinity (Ks) and the first-order acylation rate (k₂):E+S↔KsE⋅S→k2E−S→k3E+P(1)The emergence of resistance to penicillin and ceftriaxone in N. gonorrhoeae occurs primarily via the acquisition of mutant alleles of the penA gene encoding PBP2 (12). These alleles are referred to as mosaic because they arise through multiple homologous recombination events with DNA released by commensal Neisseria species. PBP2 from the high-level ceftriaxone-resistant strain, H041, contains 61 mutations compared with PBP2 from the antibiotic-susceptible strain, FA19 (13, 14). Determining how these mutations lower the k₂/Ks of ceftriaxone for PBP2 by over 10,000-fold while still preserving essential TPase activity is fundamental for understanding the evolution of antibiotic resistance.Toward this goal, we have identified a subset of these mutations that, when incorporated into the penA gene from FA19, confer ∼80% of the increase in minimum inhibitory concentration for ceftriaxone relative to that of the penA gene from H041 (penA41) (15, 16). We recently reported the structures of apo and ceftriaxone-acylated PBP2 at high resolution and have detailed conformational changes in β3 and the β3–β4 loop involved in antibiotic binding and acylation (17). Intriguingly, although present in the active site region, most of the mutations conferring resistance are not in direct contact with ceftriaxone in the crystal structure of acylated PBP2 (17, 18). We have proposed that these mutations alter the binding and acylation kinetics of PBP2 with ceftriaxone by restricting protein dynamics (18).To understand further the structural and biochemical mechanisms by which these mutations lower the acylation rates of β-lactam antibiotics, we utilized a combination of solution ¹⁹F NMR, X-ray crystallography, and biochemical approaches to investigate PBP2. We report that the β3–β4 loop in the TPase domain of WT PBP2, which is known to adopt markedly different conformations in the apo versus acylated crystal structures (17), samples two major conformational states in solution. Substitutions of WT PBP2 residues with mutations in H041 that confer ceftriaxone resistance alter the conformational landscape of PBP2 by destabilizing the high-affinity state containing the inward conformation of the β3–β4 loop and stabilizing a low-affinity conformation containing an extended β3–β4 loop conformation, thereby restricting access to the inward conformation required for high-affinity drug binding. Our combined solution NMR and crystallographic analyses of PBP2 and its preacylation drug complexes further support the notion that mutations in PBP2 from ceftriaxone-resistant strains of N. gonorrhoeae confer antibiotic resistance by hindering conformational changes required to form a productive drug-binding state (18).     

Regulation of RNA N6-methyladenosine modification and its emerging roles in skeletal muscle development

N⁶-methyladenosine (m⁶A) is one of the most widespread and highly conserved chemical modifications in cellular RNAs of eukaryotic genomes. Owing to the development of high-throughput m⁶A sequencing, the functions and mechanisms of m⁶A modification in development and diseases have been revealed. Recent studies have shown that RNA m⁶A methylation plays a critical role in skeletal muscle development, which regulates myoblast proliferation and differentiation, and muscle regeneration. Exploration of the functions of m⁶A modification and its regulators provides a deeper understanding of the regulatory mechanisms underlying skeletal muscle development. In the present review, we aim to summarize recent breakthroughs concerning the global landscape of m⁶A modification in mammals and examine the biological functions and mechanisms of enzymes regulating m⁶A RNA methylation. We describe the interplay between m⁶A and other epigenetic modifications and highlight the regulatory roles of m⁶A in development, especially that of skeletal muscle. m⁶A and its regulators are expected to be targets for the treatment of human muscle-related diseases and novel epigenetic markers for animal breeding in meat production.     

天门冬提取液对小鼠心肌LPF、GSH-Px影响的实验研究

目的:观察天门冬不同极性提取组份对小鼠心肌IPF、GSH-Px的影响.方法:采用D-半乳糖衰老模型小鼠,分别给予天门冬氯仿、乙醇、水三种不同极性提取组份,观察其对小鼠心肌LPF、GSH-Px的影响.结果:天门冬脂溶性的提取液对小鼠心肌抗氧化作用优越于极性溶剂提取液.结论:天门冬提取液对抗氧化机能的调节是其延缓衰老的重要机制之一.     

褪黑素抑制小鼠卵母细胞的体外成熟

目的:探讨褪黑素(MT)对小鼠卵母细胞的体外成熟的影响.方法:通过卵母细胞自发、次黄嘌呤(HX)阻滞和激素诱导成熟三种体外培养模型研究了褪黑素(MT)对小鼠卵母细胞体外成熟的影响.结果:①0.1 g/L、0.02g/L、0.004 g/L及0.0008 g/L浓度的MT均能显著抑制小鼠卵丘卵母细胞复合体(CEOs)自发成熟过程中第一极体(PB1)的释放(P＜0.01);②动力曲线分析表明,MT对自发成熟的CEOs的GVBD和PB1有显著的推后作用,与对照组相比,处理组的GVBD和PB1分别被推后8～10 h和3～4 h;③0.1 g/L和0.02 g/L两有效浓度的MT还能显著抑制促性腺激素(FSH)诱导的HX阻滞的CEOsGVBD的发生(P＜0.05),对PB1的排出虽有一定的抑制作用,但没有统计学意义;④MT和次黄嘌呤(HX)对CEOs的自发成熟有协同抑制作用(P＜0.01),但在裸卵(DO)自发成熟的阻滞中没有协同效应.结论:MT是调节哺乳动物卵母细胞成熟的重要激素之一,其作用机制可能是通过卵丘细胞实现的.     

肝组织TGF-β1、TIMP-1及MMP-2表达与纤维化关系的实验研究

目的探讨免疫性肝纤维化肝组织TGF-β1、TIMP-1及MMP-2表达与肝纤维化间的关系.方法 24只雄性Wistar大鼠尾静脉注射白蛋白建立实验性免疫性肝纤维化模型,检测血清HA、ALT、AST、ALB,流式细胞仪定量分析肝组织TGF-β1、TIMP-1、MMP-2和α-SMA基因表达量,观察大鼠肝组织病理、肝组织Pollak三重染色和Gomori银染色、苦味酸-天狼红染色、Ⅳ型胶原免疫组化、α-SMA免疫组化、肝匀浆Hyp测定.结果随着肝纤维化程度的加重血清HA、ALT、AST、肝组织匀浆Hyp的浓度和TGF-β1、TIMP-1及α-SMA基因表达量均增加, MMP-2于肝纤维化早期明显升高,晚期明显降低.结论实验性肝纤维化过程中TGF-β1、TIMP-1促进肝纤维化的进展,MMP-2抑制其进展.     

Sequence and genomic structure of the human adult skeletal muscle sodium channel alpha subunit gene on 17q.

The amino acid sequence of the sodium channel alpha subunit from adult human skeletal muscle has been deduced by cross-species PCR-mediated cloning and sequencing of the cDNA. The protein consists of 1836 amino acid residues. The amino acid sequence shows 93% identity to the alpha subunit from rat adult skeletal muscle and 70% identity to the alpha subunit from other mammalian tissues. A 500 kb YAC clone containing the complete coding sequence and two overlapping lambda clones covering 68% of the cDNA were used to estimate the gene size at 35 kb. The YAC clone proved crucial for gene structure studies as the high conservation between ion channel genes made hybridization studies with total genomic DNA difficult. Our results provide valuable information for the study of periodic paralysis and paramyotonia congenita, two inherited neurological disorders which are caused by point mutations within this gene.     

Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients

Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.     

Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.

A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.