Molecular basis of ß-lactam antibiotic resistance of ESKAPE bacterium E. faecium Penicillin Binding Protein PBP5.

Penicillin-binding proteins (PBPs) are essential for the formation of the bacterial cell wall. They are also the targets of ß-lactam antibiotics. In Enterococcus faecium, high levels of resistance to ß-lactams are associated with the expression of PBP5, with higher levels of resistance associated with distinct PBP5 variants. To define the molecular mechanism of PBP5-mediated resistance we leveraged biomolecular NMR spectroscopy of PBP5 - due to its size (>70 kDa) a challenging NMR target. Our data show that resistant PBP5 variants show significantly increased dynamics either alone or upon formation of the acyl-enzyme inhibitor complex. Furthermore, these variants also exhibit increased acyl-enzyme hydrolysis. Thus, reducing sidechain bulkiness and expanding surface loops results in increased dynamics that facilitates acyl-enzyme hydrolysis and, via increased ß-lactam antibiotic turnover, facilitates ß-lactam resistance. Together, these data provide the molecular basis of resistance of clinical E. faecium PBP5 variants, results that are likely applicable to the PBP family.

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into ß-lactam resistance.

The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of ß-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the ß-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three ß-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

Structural and Regulatory Changes in PBP4 Trigger Decreased ß-Lactam Susceptibility in Enterococcus faecalis.

Enterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 -35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to ß-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.IMPORTANCEEnterococcus faecalis is an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report an E. faecalis strain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to "suppress" infections in prosthetic joints and offer novel insights into the interaction of ß-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.

(1)H, (15)N and (13)C resonance assignments and secondary structure prediction of Q4D059, a conserved and kinetoplastid-specific hypothetical protein from Trypanosoma cruzi.

Trypanosoma cruzi is a human parasite that causes Chagas disease, an illness affecting millions of people and without an efficient treatment available. Sequencing the pathogen genome has revealed that near half of protein-coding genes correspond to hypothetical proteins of unknown function, increasing the possibilities for novel target discovery. Q4D059 is a putative essential hypothetical protein from T. cruzi and it is specific and conserved among the trypanosomatid genomes. Here, we report the sequential backbone and side chain resonance assignments and secondary structure analysis of Q4D059, as first step for protein structure determination, function elucidation and drug screening.

Docking, synthesis and anti-diabetic activity of novel sulfonylhydrazone derivatives designed as PPAR-gamma agonists.

Diabetes is a metabolic disorder characterized by hyperglycemia. When not properly controlled, complications include neuropathy, coronary artery disease, and renal failure. Several drugs are approved for diabetes treatment; however their use is associated with side effects and lack of efficacy in attenuating the development of long-term complications. This work describes the virtual screening and synthesis of a novel series of sulfonylhydrazone derivatives designed as peroxisome proliferator-activated receptor gamma (PPARγ) agonists and investigation of the analogs for hypoglycemic activity in a murine model of diabetes. Docking studies identified LASSBio-331 (5) as having theoretical affinity for PPARγ similar to the prototype (S)-rosiglitazone. Several structural modifications were proposed for the structure of LASSBio-331, resulting in the synthesis of five novel compounds, which showed experimental affinity for PPARγ. Among these new compounds, LASSBio-1471 (15) had the best theoretical binding energy for PPARγ and was selected for testing in STZ-induced diabetes. Four weeks after single intravenous injection of STZ (60 mg/kg), Wistar rats were treated with vehicle (DMSO) or LASSBio-1471 (20 mg/kg, i.p.) for 7 days. The blood glucose levels of rats treated with LASSBio-1471 were reduced from 548.4 ± 26.0 mg/dL before treatment to 259.6 ± 73.1 mg/dL (P < 0.05). Paw withdrawal threshold was significantly reduced in diabetic rats and was restored from 21.9 ± 1.7 g to 36.7 ± 1.2 g after 7 days of treatment with LASSBio-1471 (P < 0.05). Thus, the novel sulfonylhydrazone derivative is a PPARγ ligand that is effective for treatment of diabetic neuropathy in STZ-injected rats.

Efeitos agudos da aplicação endovenosa do cogumelo-do-sol (Agaricus blazei Murill) sobre a pressão arterial média e a freqüência cardíaca de ratos anestesiados / Acute effects of the endovenous application of the royal mushroom (Agaricus blazei Murill) on mean arterial pressure and heart rate of anesthetized rats

Este trabalho objetivou verificar os efeitos agudos da aplicação endovenosa do extrato aquoso do Agaricus blazei Murill sobre a pressão arterial média (PAM) e a freqüência cardíaca (FC) de ratos anestesiados. Foram usados Rattus novergicus albinus, n = 6, anestesiados com tiopental sódico, traqueostomizados e canulados através da veia jugular e da artéria carótida. Foram injetadas as concentrações de 1,25 mg/kg, 2,50 mg/kg e 5,00 mg/kg do extrato aquoso em volume de 0,2 mL. A PAM foi registrada com um sistema Biopac, modelo MP100, e a FC com um eletrocardiógrafo ECG-4 Funbec. Os resultados foram obtidos no controle e nos tempos 15, 30, 45, 60 e 120s após a aplicação dos extratos. Os valores foram expressos em média ± EPM e analisados estatisticamente pelos testes "t" de Student-Newman-Keuls e Tukey (p<0,05). O extrato aquoso de A. blazei reduziu a PAM de maneira concentração dependente, sendo que a concentração de 1,25 mg/kg não provocou modificações significativas na PAM nem na FC; a de 2,50 mg/kg provocou diminuição da PAM aos 15s (p<0,01) e da FC aos 30s (p<0,001) e a de 5,00 mg/kg diminuiu a PAM aos 15s (p<0,001) e a FC aos 15 e 30s (p<0,001).

The aim of this paper was to verify the acute effects of the endovenous application of the aqueous extract of Agaricus blazei Murill on mean arterial pressure and heart rate of the anesthetized rats. The injected concentrations were: 1.25 mg/kg, 2.50 mg/kg and 5.00 mg/kg, in volume of 0.2 mL. The rats were anesthetized with sodium thiopental and, after tracheotomy, both jugular vein and carotid artery were cannulated. The MAP was recorded with a Biopac System, model MP100. The HR was obtained with an electrocardiograph model ECG-4 (Funbec). The records were made in the control and 15, 30, 45, 60 and 120s after the application of the different concentrations of the extracts. The values were expressed by mean ± SEM and by paired "t"-Student-Newman-Keuls and Tukey tests (p<0.05). The aqueous extract of the A. blazei decreased the MAP of dependent manner. The concentration of 1.25 mg/kg did not provoke effects; 2.50 mg/kg provoked decrease of the PAM at 15s (p<0.01) and of the HR at 30s (p<0.001) and 5.00 mg/kg provoked decrease of the PAM at 15s (p<0.001) and of the HR at 15 and 30s (p<0.001).