Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus

Bacteria of the genus Staphylococcus are common pathogens responsible for a broad spectrum of human and animal infections and belong to the most important etiological factors causing food poisoning. Because of rapid increase in the prevalence of isolation of staphylococci resistant to many antibiotics, there is an urgent need for the development of new alternative chemotherapeutics. A number of studies have recently demonstrated the strong potential of peptidoglycan hydrolases (PHs) to control and treat infections caused by this group of bacteria. PHs cause rapid lysis and death of bacterial cells. The review concentrates on enzymes hydrolyzing peptidoglycan of staphylococci. Usually, they are characterized by high specificity to only Staphylococcus aureus cell wall components; however, some of them are also able to lyse cells of other staphylococci, e.g., Staphylococcus epidermidis-human pathogen of growing importance and also other groups of bacteria. Some PHs strengthen the bactericidal or bacteriostatic activity of common antibiotics, and as a result, they should be considered as component of combined therapy which could definitely reduced the development of bacterial resistance to both enzymes and antibiotics. The preliminary research revealed that most of these enzymes can be produced using heterologous, especially Escherichia coli expression systems; however, still much effort is required to develop more efficient and large-scale production technologies. This review discusses current state on knowledge with emphasis on the possibilities of application of PHs in the context of therapeutics for infections caused by staphylococci.     

Growth inhibitory effect of antibiotic tetaine on yeast and mycelial forms of Candida albicans

The mycelial (M) form of Candida albicans is more sensitive to the action of the antibiotic tetaine than the yeast (Y) form. Tetaine, at low concentrations about 1 microgram/ml also inhibits Y-M transition. It causes severe deformation of cells, agglutination and inhibits septum formation in the yeast forms. Tetaine action is reversed by dipeptides in both forms and by tripeptides in M form. N-acetyl glucosamine is a powerful antagonist of tetaine action on both morphological forms. Tetaine action on mycelial forms is slightly antagonised by N-acetyl mannosamine and very powerfully by glutamine.     

The composition of the polyglutamine‐containing proteins influences their co‐aggregation properties

The sequestration of crucial cellular proteins into insoluble aggregates formed by the polypeptides containing expanded polyglutamine tracts has been proposed to be the key mechanism responsible for the abnormal cell functioning in the so‐called polyglutamine diseases. To evaluate to what extent the ability of polyglutamine sequences to recruit other proteins into the intracellular aggregates depends on the composition of the aggregating peptide, we analysed the co‐aggregation properties of the N‐terminal fragment of huntingtin fused with unrelated non‐aggregating and/or self‐aggregating peptides. We show that the ability of the mutated N‐terminal huntingtin fragment to sequester non‐related proteins can be significantly increased by fusion with the non‐aggregating reporter protein [GFP (green fluorescence protein)]. By contrast, fusion with the self‐aggregating C‐terminal fragment of the CFTR (cystic fibrosis transmembrane conductance regulator) dramatically reduces the sequestration of related non‐fused huntingtin fragments. We also demonstrate that the co‐aggregation of different non‐fused N‐terminal huntingtin fragments depends on their length, with long fragments of the wild‐type huntingtin not only excluded from the nuclear inclusions, but also very inefficiently sequestered into the cytoplasmic aggregates formed by the short fragments of mutant protein. Additionally, our results suggest that atypical intracellular aggregation patterns, which include unusual distribution and/or morphology of protein aggregates, are associated with altered ability of accumulating proteins to co‐aggregate with other peptides.     

Phosphorylation of glucosamine-6-phosphate synthase is important but not essential for germination and mycelial growth of Candida albicans

A site-directed mutagenesis of the GFA1 gene encoding Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase afforded its GFA1S208A version. A product of the modified gene, lacking the putative phosphorylation site for protein kinase A (PKA), exhibited all the basic properties identical to those of the wild-type enzyme but was no longer a substrate for PKA. Comparison of the C. albicans Deltagfa1/GFA1 and Deltagfa1/GFA1S208A cells, grown under conditions stimulating yeast-to-mycelia transformation, revealed that the latter demonstrated lower GlcN-6-P synthase specific activity, decreased chitin content and formed much fewer mycelial forms. All these findings, as well as the observed effects of specific inhibitors of protein kinases, suggest that a loss of the possibility of GlcN-6-P synthase phosphorylation by PKA strongly reduces but not completely eliminates the germinative response of C. albicans cells.     

PDZ-binding motifs are unable to ensure correct polarized protein distribution in the absence of additional localization signals

The C-terminal PDZ-binding motifs are required for polarized apical/basolateral localization of many membrane proteins. To determine the specificity of the PDZ-binding motifs in establishing cellular distribution, we utilized a 111-amino acid region from the C-terminus of cystic fibrosis transmembrane conductance regulator (CFTR) that is able to direct apical localization of fused reporter proteins. Substitution of the C-terminal PDZ-binding motif of CFTR with corresponding motifs necessary for basolateral localization of other membrane proteins did not lead to the redistribution of the fusion protein to the basolateral membrane. Instead, some fusion proteins remained localized to the apical membrane, whereas others showed no specific distribution. The specificity of the PDZ-based interactions was substantially increased when specific amino acids located upstream of the classical PDZ-binding motifs were included. However, even the presence of a longer C-terminal motif from a basolateral protein could not ensure basolateral distribution of the fusion protein. Our results indicate that the C-terminal PDZ-binding motifs are not the primary signals for polarized protein distribution, although they are required for targeting and/or stabilization of protein at the given location.     

Structural analogues of reactive intermediates as inhibitors of glucosamine-6-phosphate synthase and phosphoglucose isomerase

The active centers of phosphoglucose isomerase (PGI) and the hexose phosphate isomerase domain (HPI) of glucosamine-6-P (GlcN-6-P) synthase demonstrate apparent similarity in spatial arrangement of critical amino acid residues, except Arg272 of the former and Lys603 and Lys485 of the latter. Ten derivatives of d-hexitol-6-P, 5-phosphoarabinoate, or 6-phosphogluconate, structural analogues of putative cis-enolamine or cis-enolate intermediates, were tested as inhibitors of fungal GlcN-6-P synthase and PGI. None of the investigated compounds demonstrated equally high inhibitory potential against both enzymes. 2-Amino-2-deoxy-D-mannitol 6-P was found to be the strongest GlcN-6-P synthase inhibitor in the series, with an inhibition constant equal to 9.0 (+/-1.0) x 10(-6)M. On the contrary, 5-phosphoarabinoate (5PA) exhibited specificity for PGI, with K(i)=2.2 (+/-0.1) x 10(-6) M. N-acetylation substantially lowered the GlcN-6-P synthase inhibitory potential of 2-amino-2-deoxy-D-glucitol-6-P but strongly enhanced inhibitory potential of this compound towards PGI. Molecular modeling studies revealed that interactions of the C1-C2 part of transition state analogue inhibitors with the respective areas demonstrating different distribution of molecular electrostatic potential (MEP) inside HPI and PGI active centers determined enzyme:ligand affinity. In Escherichia coli HPI, a patch of the negative potential created by Glu488 aided by Val399, supposed to stabilize a putative positively charged intermediate, especially attracts ligands containing 2-amino function. The Arg272, Lys210, and Gly271 peptide bond nitrogen system, present in the corresponding space of rabbit PGI, creates an area of positive MEP, stabilizing cis-enolate intermediate and attracting its structural mimics, such as 5PA.     

EFFICIENT PRODUCTION OF Staphylococcus simulans LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT Escherichia coli

Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary medicine, or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high cost of its production. In this study we have determined the optimal conditions for lysostaphin production in a 5-L batch bioreactor. The enzyme production was based on a heterologous, Escherichia coli expression system designated as pBAD2Lys and constructed earlier in our laboratory. An evident influence of physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. Efficiency of production of about 13,000 U/L has been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH 6, and temperature 37°C in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally, more than 80,000 units of lysostaphin were obtained from one (batch) bioreactor with 3 L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. To the best of our knowledge, this is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.     

Aggregation of amphotericin B in the presence of gamma-cyclodextrin

The macrolide antibiotic amphotericin B (AmB) forms an inclusion complex with gamma-cyclodextrin (gamma-CDx), resulting in a molecularly dispersed state of the drug. The state of aggregation of AmB in different solvents has been studied by absorption (uv-vis) and CD spectroscopy. While in aqueous solutions AmB forms colloid-like multimolecular aggregates, in the presence of gamma-CDx true solutions can be prepared, which show similar spectral properties as AmB dissolved in organic solvents. The AmB-gamma-CDx complex can be isolated as an amorphous, stable, water-soluble powder, indicating that gamma-CDx is a good carrier for the solubilization of this antibiotic. Using gamma-CDx as a carrier, the danger of precipitation of the drug during parenteral or intravenous administration can be largely reduced.