scholarly journals Lugdulysin of Staphylococcus lugdunensis, a metalloprotease that inhibits and disrupts protein biofilm of Staphylococcus aureus

2021 ◽  
Author(s):  
Juan Pinheiro Oliveira Martinez ◽  
Leonardo Vazquez ◽  
Matheus Mikio Takeyama ◽  
Tarcizio José Santos Filho ◽  
Fernanda Sampaio Cavalcante ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biochemical Characterization ◽  
Homology Modelling ◽  
Clinical Importance ◽  
Coagulase Negative Staphylococci ◽  
Staphylococcus Lugdunensis ◽  
Competition Mechanism ◽  
Metal Cofactor ◽  
Optimal Ph

Abstract Background Staphylococcus lugdunensis is a commensal skin microorganism that, unlike other coagulase-negative staphylococci, presents increasing clinical importance. This species yields a metalloprotease called lugdulysin that may contribute to its higher degree of virulence. This study aimed to determine the biochemical characterization of the lugdulysin produced by S. lugdunensis clinical isolates and investigate its effect on the formation and disruption of biofilm of Staphylococcus aureus isolates. The protease was isolated and characterized for its optimal pH and temperature, activity in the presence of inhibitors and enzymatic kinetics. The influence of metal cofactor supplementation on proteolysis was also evaluated, with and without inhibitors. Finally, the protease capacity to inhibit and disrupt biofilms of different S. aureus lineages and biofilm matrix was analyzed. Results The protease optimal pH and temperature were 7.0 and 37° C, respectively. EDTA inhibited the protease, and the activity was not recovered by divalent ion supplementation. In addition, divalent ions did not change enzymatic activity without inhibitors, which was stable for up to 3 hours. Its structure was determined via homology modelling. The protease significantly inhibited the formation and disrupted established biofilms of S. aureus isolates with protein biofilm. Conclusions This study confirmed features of the lugdulysin metalloprotease and showed that this S. lugdunensis virulence factor may be a new competition mechanism and/or modulation of the staphylococcal biofilm.

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

scholarly journals Genotypic Characterization of Coagulase-negative Staphylococci Isolated from Sheep Milk in Slovakia

2010 ◽  
Vol 79 (2) ◽  
pp. 269-275 ◽  
Author(s):  
Ivana Pilipčincová ◽  
Mangesh Bhide ◽  
Eva Dudriková ◽  
Milan Trávniček
Keyword(s):  
Molecular Characterization ◽  
Gel Electrophoresis ◽  
Biochemical Characterization ◽  
Pulse Field ◽  
Coagulase Negative Staphylococci ◽  
Pulse Field Gel Electrophoresis ◽  
Milk Samples ◽  
Sheep Milk ◽  
Epidemiological Situation

Hitherto very few reports are available presenting identification and molecular characterization of the coagulase negative staphylococci (CNS) from sheep milk in the subclinical stage of mastitis. Furthermore, very scanty data are available on the epidemiological status of CNS in different Slovak provinces. Milk samples from 54 sheep farms located in eastern Slovak region were screened. A total 240 CNS were identified with series of biochemical testes (STAPH-API) and subjected further for genotyping with the help of pulse field gel electrophoresis (PFGE). The most frequently occurring CNS species according the biochemical characterization were:S. epidermidis(36.3 %),S. caprae(21.3 %),S. hominis(6.6 %),S. chromogenes(6.3 %),S. xylosus(5.8 %),S. warneri(5.0 %) andS. capitis(4.6 %). Further PFGE-based characterization of these isolates revealed six pulsotypes of theS. epidermidis, two ofS. caprae, three ofS. chromogenes, nine ofS. hominis, five ofS. capitisand seven ofS. xylosus. These results contribute to knowledge of the epidemiological situation of the CNS from the subclinical form of mastitis in Slovakia.

scholarly journals Characterization of Novel Phages Isolated in Coagulase-Negative Staphylococci Reveals Evolutionary Relationships with Staphylococcus aureus Phages

2012 ◽  
Vol 194 (21) ◽  
pp. 5829-5839 ◽  
Author(s):  
M. Deghorain ◽  
L.-M. Bobay ◽  
P. R. Smeesters ◽  
S. Bousbata ◽  
M. Vermeersch ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Evolutionary Relationships ◽  
Coagulase Negative Staphylococci

scholarly journals Physical and Biochemical Characterization of the qacA Gene Encoding Antiseptic and Disinfectant Resistance in Staphylococcus aureus

Microbiology ◽  
1989 ◽  
Vol 135 (1) ◽  
pp. 1-10 ◽  
Author(s):  
J. M. TENNENT ◽  
B. R. LYON ◽  
M. MIDGLEY ◽  
G. JONES ◽  
A. S. PUREWAL ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biochemical Characterization ◽  
Gene Encoding ◽  
Disinfectant Resistance

Characterization of staphylococci contaminating automated teller machines in Hong Kong

2011 ◽  
Vol 140 (8) ◽  
pp. 1366-1371 ◽  
Author(s):  
M. ZHANG ◽  
M. O'DONONGHUE ◽  
M. V. BOOST
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Benzalkonium Chloride ◽  
Tetracycline Resistance ◽  
Chlorhexidine Digluconate ◽  
Coagulase Negative Staphylococci ◽  
Automated Teller Machines ◽  
Resistance Determinants

SUMMARYEnvironmental staphylococcal contamination was investigated by culture of 400 automated teller machines (ATMs). Isolates were characterized for antibiotic and antiseptic susceptibility, carriage of antiseptic resistance genes (QAC genes), and spa types. MRSA, which was similar to local clinical isolates, was present on two (0·5%) of the 62 (15·5%) ATMs that yielded Staphylococcus aureus. QAC genes were more common in coagulase-negative staphylococci (qacA/B 26·0%, smr 14%) than S. aureus (11·3% qacA/B, 1·6% smr). QAC-positive isolates had significantly higher minimum inhibitory concentrations/minimum bactericidal concentrations to benzalkonium chloride and chlorhexidine digluconate. QAC gene presence was significantly associated with methicillin and tetracycline resistance. Survival of staphylococci, including MRSA, on common access sites may be facilitated by low disinfectant concentrations, which select for disinfectant-tolerant strains, while co-selecting for antibiotic-resistance determinants. Disinfection procedures should be performed correctly to help prevent spread of resistant pathogens from reservoirs in the community.

scholarly journals Inhibition and Biochemical Characterization of Methicillin-Resistant Staphylococcus aureus Shikimate Dehydrogenase: An in Silico and Kinetic Study

Molecules ◽  
2014 ◽  
Vol 19 (4) ◽  
pp. 4491-4509 ◽  
Author(s):  
Claudia Avitia-Domínguez ◽  
Erick Sierra-Campos ◽  
José Salas-Pacheco ◽  
Hugo Nájera ◽  
Arturo Rojo-Domínguez ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Kinetic Study ◽  
In Silico ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Biochemical Characterization ◽  
Shikimate Dehydrogenase ◽  
Methicillin Resistant

scholarly journals Optical Mapping Reveals a Large Genetic Inversion between Two Methicillin-Resistant Staphylococcus aureus Strains

2009 ◽  
Vol 191 (18) ◽  
pp. 5717-5723 ◽  
Author(s):  
Sanjay K. Shukla ◽  
Jennifer Kislow ◽  
Adam Briska ◽  
John Henkhaus ◽  
Colin Dykes
Keyword(s):  
Staphylococcus Aureus ◽  
Optical Mapping ◽  
Bacterial Genome ◽  
Clinical Importance ◽  
Restriction Map ◽  
Nucleotide Polymorphisms ◽  
Specific Pcr ◽  
Genetic Inversion

ABSTRACT Staphylococcus aureus is a highly versatile and evolving bacterium of great clinical importance. S. aureus can evolve by acquiring single nucleotide polymorphisms and mobile genetic elements and by recombination events. Identification and location of novel genomic elements in a bacterial genome are not straightforward, unless the whole genome is sequenced. Optical mapping is a new tool that creates a high-resolution, in situ ordered restriction map of a bacterial genome. These maps can be used to determine genomic organization and perform comparative genomics to identify genomic rearrangements, such as insertions, deletions, duplications, and inversions, compared to an in silico (virtual) restriction map of a known genome sequence. Using this technology, we report here the identification, approximate location, and characterization of a genetic inversion of ∼500 kb of a DNA element between the NRS387 (USA800) and FPR3757 (USA300) strains. The presence of the inversion and location of its junction sites were confirmed by site-specific PCR and sequencing. At both the left and right junction sites in NRS387, an IS1181 element and a 73-bp sequence were identified as inverted repeats, which could explain the possible mechanism of the inversion event.

scholarly journals Crystallization and biochemical characterization of the human spliceosomal Aar2–Prp8RNaseHcomplex

2015 ◽  
Vol 71 (11) ◽  
pp. 1421-1428
Author(s):  
Karine Santos ◽  
Marco Preussner ◽  
Anna Christina Heroven ◽  
Gert Weber
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Molecular Replacement ◽  
Noncoding Sequences ◽  
Internal Loop ◽  
Catalytic Activation ◽  
Maturation Factor ◽  
Spliceosome Activation

In eukaryotes, the removal of nuclear noncoding sequences (pre-mRNA splicing) is catalyzed by the spliceosome, which consists of five ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs, each with a respective snRNA) and a plethora of protein factors that aid spliceosomal maturation, assembly, activation and disassembly. Recently, the U5 snRNP maturation factor Aar2p from baker's yeast has been characterized structurally and biochemically. Aar2p binds to the RNaseH (RH) and Jab1/MPN domains of the highly conserved U5-specific Prp8p, which forms a framework for the spliceosomal catalytic centre. Thereby, Aar2p sterically excludes Brr2p, a helicase essential for the catalytic activation of the spliceosome, from Prp8p binding. At the same time, Aar2p blocks U4/U6 di-snRNA binding to Prp8p. Aar2p therefore prevents premature spliceosome activation and its functions are regulated by reversible phosphorylation. To date, little is known about the hypothetical human Aar2 (hsAar2) orthologue C20ORF4. This study identifies C20ORF4 (i) as part of the HeLa proteome by Western blotting and (ii) as a true Aar2 orthologue which binds to the RH domain (hsRH) of Prp8 and corroborates an evolutionary link between yeast and human Aar2 function. An elaborate strategy was devised to crystallize hsAar2 in complex with hsRH. The analysis of initial weakly diffracting crystals obtained byin situproteolysis and homology modelling guided the design of an hsAar2 construct in which an internal loop was replaced by three serines (hsAar2Δloop). A complex of hsAar2Δloopand hsRH crystallized in space groupC2; the crystals diffracted to 2.35 Å resolution and were suitable for structure determination by molecular-replacement approaches. The study presented here suggests a connection between Aar2 and the spliceosome in human cells and paves the way for structural studies of human Aar2.

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