Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to next-generation ß-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance, is classically mediated by PBP2a, a penicillin-binding protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus spp. serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA-deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as methicillin-resistant lacking mec (MRLM), are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs, can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach toward diagnosis and treatment of ß-lactam-resistant S. aureus infections. IMPORTANCE: In Staphylococcus aureus, high-level, broad-spectrum resistance to ß-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA. This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to ß-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA, as seen by increased bacterial survival in the presence of ß-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and GGDEF domain protein containing phosphodiesterase (gdpP) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of ß-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by Methicillin-Resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to Next Generation ß-lactams (NGB) such as Methicillin, Nafcillin, Oxacillin etc. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance is classically mediated by PBP2a, a Penicillin-Binding Protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as Methicillin-Resistant Lacking mec (MRLM) are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance towards NGBs at levels comparable to that of MRSAs. Our study, provides a fresh new perspective about alternative mechanisms of NGBs resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach towards diagnosis and treatment of ß-lactam resistant S. aureus infections.

Identification and Preliminary Characterization of a Novel Single-Stranded DNA Binding Protein of Staphylococcus aureus Phage Phi11 Expressed in Escherichia coli.

Bacteriophage Phi11 harbors a gene, gp13, encoding the putative SSB protein (GenBank accession no. NC_004615.1). SSB proteins bind to and protect the single-stranded DNA molecules from nuclease digestion and are essential for the growth and metabolic activities of the organisms encoding them. In this investigation, we have carried out the cloning, recombinant expression, and purification of rGp13 for the first time in Escherichia coli. EMSA data indicated that the purified recombinant Gp13 protein was capable of binding to single-stranded DNA. The protein exhibited maximum binding activity at 32 °C. Furthermore, our bioinformatic analysis has revealed that Gp13 consists of an OB-fold, a characteristic of SSB proteins. However, the arrangement of the OB-fold is unique, being located in the C-terminal domain of Gp13. Despite the importance of SSB proteins in various metabolic processes as well as in various types of PCR, there are no reports on the purification and characterization of SSB proteins from staphylococcal bacteriophages. We expect that the purification and characterization of recombinant Gp13 will help us gain a better insight into its biological activity and make it available in large quantities for molecular biology work.

Biochemical and biophysical analysis of the interaction of a recombinant form of Staphylococcus aureus enolase with plasminogen.

Aim: Pathogenic invasion of Staphylococcus aureus is critically dependent on host plasminogen activation. Materials & methods: The pathophysiological implications of the interactions between S. aureus recombinant enolase and host plasminogen were investigated. The effects of mutation and small synthetic peptide inhibitors on interactions were assessed. Results: In vitro, the S. aureus recombinant enolase exists as a catalytically active fragile octamer and a robust dimer. The dimer interacts with the host plasminogen on the S. aureus surface. Conclusion: The interaction of host plasminogen and S. aureus enolase might mediate bacterial adherence to the host, activate the plasminogen with the help of plasminogen activators and prevent α2-antiplasmin-mediated inhibition of plasmin. Incorporating mutant and synthetic peptides inhibited the interactions and their associated pathophysiological consequences.

A coumarin coupled tetraphenylethylene based multi-targeted AIEgen for cyanide ion and nitro explosive detection, and cellular imaging.

A coumarin coupled tetraphenylethylene based AIEgen (TPE-Lac) with an intense greenish-yellow emission has been synthesized and utilized for multipurpose sensing and imaging applications. TPE-Lac acts as a sensitive sensor for the detection of cyanide ions (CN-) with an immediate turn-off response in the presence of many other interfering cations and anions. The limit of detection (LOD) was as low as 33 nM, which is well below the permissible limit set by the World Health Organization (WHO). Cyanide detection in the solid phase was successfully demonstrated by drop-casting the solution of the TPE-Lac probe on TLC plates and measuring and analysing the fluorescence response by ImageJ analysis. TPE-Lac was further employed in the detection of explosive nitroaromatics in solution and solid phases. Also, TPE-Lac was found suitable as an imaging agent and could easily percolate into live H520 cells giving bright fluorescence from the intra-cellular region. Easy and cost-effective synthesis, fast response and low LODs are some of the advantages of this AIEgen over available molecular probes for the same purpose.

Dramatic Changes in Oligomerization Property Caused by Single Residue Deletion in Staphylococcus aureus Enolase.

Studies were conducted to understand the role of C-terminal lysine residues in the catalytic activity, structural stability and oligomeric properties of Staphylococcus aureus enolase. Interestingly, the S. aureus enolase, in solution, shows its presence as a stable dimer as well as the catalytically active fragile octamer. Compared to the hexa-histidine tagged S. aureus enolase (rSaeno), the deletion mutant showed the negligible difference in Km, but approximately 20-25% reduction in maximum reaction velocity (Vmax) and 2% reduction in turnover number were observed. These kinetic parameters indicate that K-434Δ deletion mutation does not drastically compromise the enzyme efficiency. The secondary structure and the octameric conformation of both the rSaeno and the K-434Δ mutant are very much stable between pH ranging from 6 to 9, temperatures ranging from 20 to 40 °C and in the presence of divalent metal ions Mg2+, Zn2+ and Mn2+. Under these conditions, the recombinant enzyme and the mutant are also catalytically very active. Intrinsic tryptophan fluorescence (320-380 nm) and CD spectral (195-260 nm) analysis revealed that the secondary structure and the surface architecture of the proteins are not majorly altered by the mutation. But, a significant correlation was observed between the time-dependent decrease in the catalytic activity and the oligomeric stability of rSaeno and K-434Δ mutant. The C-terminal lysine residues in the inter-dimer groove aid in folding and oligomerization of S. aureus enolase.

An overview of moonlighting proteins in Staphylococcus aureus infection.

Staphylococcus aureus is responsible for numerous instances of superficial, toxin-mediated, and invasive infections. The emergence of methicillin-resistant (MRSA), as well as vancomycin-resistant (VRSA) strains of S. aureus, poses a massive threat to human health. The tenacity of S. aureus to acquire resistance against numerous antibiotics in a very short duration makes the effort towards developing new antibiotics almost futile. S. aureus owes its destructive pathogenicity to the plethora of virulent factors it produces among which a majority of them are moonlighting proteins. Moonlighting proteins are the multifunctional proteins in which a single protein, with different oligomeric conformations, perform multiple independent functions in different cell compartments. Peculiarly, proteins involved in key ancestral functions and metabolic pathways typically exhibit moonlighting functions. Pathogens mainly employ those proteins as virulent factors which exhibit high structural conservation towards their host counterparts. Consequentially, the host immune system counteracts these invading bacterial virulent factors with minimal protective action. Additionally, many moonlighting proteins also play multiple roles in various stages of pathogenicity while augmenting the virulence of the bacterium. This has necessitated elaborative studies to be conducted on moonlighting proteins of S. aureus that can serve as drug targets. This review is a small effort towards understanding the role of various moonlighting proteins in the pathogenicity of S. aureus.

Studies on the gene regulation involved in the lytic-lysogenic switch in Staphylococcus aureus temperate bacteriophage Phi11.

Antirepressor proteins of bacteriophages are chiefly involved in interfering with the function of the repressor protein and forcing the bacteriophage to adopt the lytic cycle. The genome of Staphylococcus aureus phage, Phi11 has already been sequenced; from the genome sequence, we amplified gp07 gene and analysed its involvement in the developmental pathway of Phi11. Our results indicate that Gp07 functions as a novel antirepressor and regulates the developmental pathway of Phi11 by enhancing the binding of the Cro repressor protein to its cognate operator. We also report our finding that the CI repressor protein of Phi11 binds to the putative operator of Gp07 and regulates its expression. We further report that S.aureus transcriptional repressor LexA and coprotease RecA play a crucial role in the lytic-lysogenic switching in Phi11. We also identified that the N-terminal domain (Bro-N) of Gp07 is actually responsible for enhancing the binding of Cro repressor to its cognate operator. Our results suggest that Phi11 prophage induction is different from other bacteriophages. This study furnishes a first-hand report regarding the regulation involved in the developmental pathway of Phi11.

Effect of ions and inhibitors on the catalytic activity and structural stability of S. aureus enolase.

The glycolytic enzyme enolase of Staphylococcus aureus is a highly conserved enzyme which binds to human plasminogen thereby aiding the infection process. The cloning, over expression and purification of S. aureus enolase as well as the effect of various metals upon the catalytic activity and structural stability of the enzyme have been reported. The recombinant enzyme (rSaeno) has been purified to homogeneity in abundant amounts (60 mg/L of culture) and the kinetic parameters (Km = 0.23 +/- 0.013 x 10-3 M; Vmax = 90.98 +/- 0.00052 U/mg) and the optimum pH were calculated. This communication further reports that increasing concentrations of Na+ ions inhibit the enzyme while increasing concentrations of K+ ions were stimulatory. In case of divalent cations, it was found that Mg2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn2+, Mn2+, Fe2+, Cu2+, Ni2+ and Ca2+) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg2+ and Cr2+. The circular dichroism data indicate that other than Hg2+, Ni2+ and to a certain extent Cu2+, none of the other ions destabilized rSaeno. The inhibitory roles of fluorides, as well as neurotoxic compounds upon the catalytic activity of rSaeno, have also been studied. Conformational changes in rSaeno (induced by ions) were studied using partial trypsin digestion.