Cold-shock proteins (Csps), proteins expressed when the ambient temperature drops below the growth-supporting temperature, bind to single-stranded nucleic acids and act as RNA chaperones to regulate translation. Listeria monocytogenes is a psychrophilic food-borne pathogen that is problematic for the food industry. Structures of Csps from psychrophilic bacteria have not yet been studied. Despite dramatic differences in the thermostability of Csps of various thermophilic microorganisms, these proteins share a high degree of primary sequence homology and a high degree of three-dimensional structural similarity. Here, we investigated the structural and dynamic features as well as the thermostability of L. monocytogenes CspA (Lm-CspA). Lm-CspA has a five-stranded β-barrel structure with hydrophobic core packing and two salt bridges. When heptathymidine (dT(7)) binds, values for the heteronuclear nuclear Overhauser effect and order parameters of residues in surface loop regions near nucleic acid binding sites increase dramatically. Moreover, Carr-Purcell-Meiboom-Gill experiments showed that slow motions observed for the nucleic acid binding residues K7, W8, F15, F27, and R56 disappeared in Lm-CspA-dT(7). Lm-CspA is less thermostable than mesophilic and thermophilic Csps, with a lower melting temperature (40 °C). The structural flexibility that accompanies longer surface loops and less hydrophobic core packing and a number of salt bridges and unfavorable electrostatic repulsion are likely key factors in the low thermostability of Lm-CspA. This implies that the large conformational flexibility of psychrophilic Lm-CspA, which more easily accommodates nucleic acids at low temperature, is required for RNA chaperone function under cold-shock conditions and for the cold adaptation of L. monocytogenes.
For the timely treatment of patients with infections in bloodstream and cerebrospinal fluid, a rapid antimicrobial susceptibility test (AST) is urgently needed. Here, we describe a direct and rapid antimicrobial susceptibility testing (dRAST) system, which can determine the antimicrobial susceptibility of bacteria from a positive blood culture bottle (PBCB) in six hours. The positive blood culture sample is directly mixed with agarose and inoculated into a micropatterned plastic microchip with lyophilized antibiotic agents. Using microscopic detection of bacterial colony formation in agarose, the total time to result from a PBCB for dRAST was only six hours for a wide range of bacterial concentrations in PBCBs. The results from the dRAST system were consistent with the results from a standard AST, broth microdilution test. In tests of clinical isolates (n = 206) composed of 16 Gram-negative species and seven Gram-positive species, the dRAST system was accurate compared to the standard broth microdilution test, with rates of 91.11% (2613/2868) categorical agreement, 6.69% (192/2868) minor error, 2.72% (50/1837) major error and 1.45% (13/896) very major error. Thus, the dRAST system can be used to rapidly identify appropriate antimicrobial agents for the treatment of blood stream infection (BSI) and antibiotic-resistant strain infections.
Phosphatases of regenerating liver (PRLs) constitute a novel class of small, prenylated phosphatases with oncogenic activity. PRL-3 is particularly important in cancer metastasis and represents a potential therapeutic target. The flexibility of the WPD loop as well as the P-loop of protein tyrosine phosphatases is closely related to their catalytic activity. Using nuclear magnetic resonance spectroscopy, we studied the structure of vanadate-bound PRL-3, which was generated by addition of sodium orthovanadate to PRL-3. The WPD loop of free PRL-3 extended outside of the active site, forming an open conformation, whereas that of vanadate-bound PRL-3 was directed into the active site by a large movement, resulting in a closed conformation. We suggest that vanadate binding induced structural changes in the WPD loop, P-loop, helices α4-α6, and the polybasic region. Compared to free PRL-3, vanadate-bound PRL-3 has a longer α4 helix, where the catalytic R110 residue coordinates with vanadate in the active site. In addition, the hydrophobic cavity formed by helices α4-α6 with a depth of 14-15 Å can accommodate a farnesyl chain at the truncated prenylation motif of PRL-3, i.e., from R169 to M173. Conformational exchange data suggested that the WPD loop moves between open and closed conformations with a closing rate constant k(close) of 7 s(-1). This intrinsic loop flexibility of PRL-3 may be related to their catalytic rate and may play a role in substrate recognition.
Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Previously, we reported that triamterene (Trm) inhibits VEGF-amyloid β (Aβ) interactions without affecting other biological activities of VEGF or Aβ [Jeong, K.-W., et al. (2011) Biochemistry 50, 4843-4854]. We further showed that molecular motions in the N-terminal disordered loop region of the heparin-binding domain (HBD) are important for interaction with Trm. To investigate the importance of motion at the C-terminal domain of HBD, we constructed a binding model of HBD with heparin octasaccharide (HOS) based on measurements of chemical shift changes and docking studies. Furthermore, the dynamic properties of the HBD-HOS and HBD-Trm-HOS complexes were assessed by measuring spin relaxation rates. The results showed that the HOS-binding site is composed of two basic clusters consisting of side chains of residues R13, R14, and K15 and residues K30, R35, and R49. When HOS binds, values for the heteronuclear nuclear Overhauser effect near HOS-binding sites increased dramatically. CPMG (Carr-Purcell-Meiboom-Gill sequence) experiments as well as an R2 relaxation experiment were undertaken to understand millisecond time-scale motions in HBD. There is large relaxation dispersion of residues at Trm- and HOS-binding sites in free HBD. C-Terminal residues such as S34, C48, and D51 near the HOS-binding sites continued to exhibit slow conformational motions in the HBD-Trm complex, while those slow motions disappeared in the bound conformation of HBD with HOS. Collectively, our results demonstrate that the inherent structural flexibilities of the C-terminal region of the HBD are important in the heparin binding process and that Trm does not inhibit VEGF-heparin interactions necessary for the biological activities of VEGF.
Background: Timely intervention in the treatment of bloodstream infection is important for prescription of appropriate antimicrobials. With prompt determination of the antimicrobial susceptibility of a causative agent, rapid antimicrobial susceptibility test (AST) can help select the appropriate antimicrobial therapy. This clinical study is for evaluation of the clinical performance of the QMAC-dRAST for rapid AST directly from positive blood culture (PBC)s with Gram-positive cocci. Methods: A total of 115 PBC samples with Grampositive organisms (76 Staphylococcus spp. and 39 Enterococcus spp.) were evaluated by the QMACdRAST system, and their pure culture isolates were evaluated by the MicroScan WalkAway (Beckman Coulter, USA) as the comparative AST system. Thirteen antimicrobial agents were included, and the agreement and discrepancy rates of the QMACdRAST system (Quantamatrix Inc., Republic of Korea) compared to the MicroScan WalkAway were calculated. To resolve discrepancies, the broth microdilution method was performed. Results:The QMAC-dRAST system exhibited a categorical agreement rate of 94.9% (1,126/1,187) and an essential agreement rate of 98.3% (1,167/1,187). The QMAC-dRAST system yielded very major (falsesusceptible) errors at 1.0% (5/485), major (false-resistant) errors at 1.3% (9/693), and minor errors at 4.0% (47/1,187) compared to the MicroScan WalkAway. The QMAC-dRAST system significantly eliminated 30 hours of total turnaround time by combination of direct inoculation of PBC and an image-based approach. Conclusion:The results of the QMAC-dRAST system were highly accurate. Thereby, the QMAC-dRAST may provide essential information to accelerate therapeutic decisions for earlier and adequate antibiotic treatment and patient management in clinical settings.
There is a high demand for novel approaches to counter the various challenges of conventional drug susceptibility testing (DST) for tuberculosis, the most prevalent infectious disease with significant global mortality. The QMAC-DST system was recently developed for rapid DST using image technology to track the growth of single cells of Mycobacterium tuberculosis (MTB). The purpose of this study was to clinically validate the QMAC-DST system compared to conventional DST. In total, 178 MTB isolates recovered from clinical specimens in Asan Medical Center in 2016 were tested by both QMAC-DST and absolute concentration methods using Lowenstein-Jensen media (LJ-DST). Among the isolates, 156 were subjected to DST using BACTEC MGIT 960 SIRE kits (BD, Sparks, MD, United States) (MGIT-DST). The susceptibility/resistance results obtained by QMAC-DST were read against 13 drugs after 7 days of incubation and compared with those of LJ-DST. Based on the gold standard LJ-DST, the agreement rates of QMAC-DST for all drugs were 97.8%, 97.9%, and 97.8% among susceptible, resistant, and total isolates, respectively, while the overall agreement of MGIT-DST tested for 156 isolates against first-line drugs was 95.5%. QMAC-DST showed the highest major error of 6.4% for rifampin, however, it could be corrected by a revised threshold of growth since false-resistant isolates showed grew only half than the true-resistant isolates. The rapid and accurate performance of QMAC-DST warrants ideal phenotypic DST for a wide range of first-line and second-line drugs.
tients who receive inadequate antimicrobial treatment [1]. The case fatality rate for bacteremia is 30-40% [2]. In Korea, bacteremia caused by major antimicrobial-resistant pathogens, especially among patients hospitalized in intensive care units, has a high incidence [3, 4]. Furthermore, extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae, vancomycin-resistant Enterococcus faecium, and imipenem-resistant Acinetobacter baumannii have been on the rise [5-7]. Rapid antimicrobial susceptibility testing (AST) results are important for the selection of suitable anti-bacterial treatments for bacteremia. The main problem with current AST methods is the long turnaround time (TAT). In most cases, conducting AST requires overnight incubation and usually requires 48-72 hours to complete, depending on the drug-organism combination [8].
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