The Polycyclic Polyprenylated Acylphloroglucinol Antibiotic PPAP 23 Targets the Membrane and Iron Metabolism in Staphylococcus aureus

Recently, a series of endo-type B polycyclic polyprenylated acylphloroglucinols (PPAP) derivatives with high antimicrobial activities were chemically synthesized. One of the derivatives, PPAP 23, which showed high antimicrobial activity and low cytotoxicity, was chosen for further investigation of its bactericidal profiles and mode of action. PPAP 23 showed a better efficacy in killing methicillin resistant Staphylococcus aureus (MRSA) and decreasing the metabolic activity of 5-day-old biofilm cells than vancomycin. Moreover, S. aureus did not appear to develop resistance against PPAP 23. The antimicrobial mechanism of PPAP 23 was investigated by RNA-seq combined with phenotypic and biochemical approaches. RNA-seq suggested that PPAP 23 signaled iron overload to the bacterial cells because genes involved in iron transport were downregulated and iron storage gene was upregulated by PPAP 23. PPAP 23 affected the membrane integrity but did not induce pore formation; it inhibited bacterial respiration. PPAP 23 preferentially inhibited Fe–S cluster enzymes; it has a mild iron chelating activity and supplementation of exogenous iron attenuated its antimicrobial activity. PPAP 23 was more effective in inhibiting the growth of S. aureus under iron-restricted condition. The crystal structure of a benzylated analog of PPAP 23 showed a highly defined octahedral coordination of three PPAP ligands around a Fe (3+) core. This suggests that PPAPs are generally capable of iron chelation and are able to form defined stable complexes. PPAP 23 was found to induce reactive oxygen species (ROS) and oxidative stress. Fluorescence microscopic analysis showed that PPAP 23 caused an enlargement of the bacterial cells, perturbed the membrane, and dislocated the nucleoid. Taken together, we postulate that PPAP 23 interacts with the cytoplasmic membrane with its hydrophobic pocket and interferes with the iron metabolism to exert its antimicrobial activity in Staphylococcus aureus

Polycyclic Polyprenylated Acylphloroglucinols: An Emerging Class of Non-Peptide-Based MRSA- and VRE-Active Antibiotics

Методы оценки экономической безопасности позволяют эффективно выявлять и устранять негативные последствия экономической безопасности предприятия. Стратегическое планирование эффективно для решения будущих проблем и рисков, а тактическое планирование помогает осуществить четкие тактические решения, для снижения опасности и угроз, в т.ч. возможности их избежать. Методы оценки экономической безопасности нужно совершенствовать, адаптируя к современным вызовам экономической безопасности

Non-classical Protein Excretion Is Boosted by PSMα-Induced Cell Leakage

Release of cytoplasmic proteins into the supernatant occurs both in bacteria and eukaryotes. Because the underlying mechanism remains unclear, the excretion of cytoplasmic proteins (ECP) has been referred to as “non-classical protein secretion.” We show that none of the known specific protein transport systems of Gram-positive bacteria are involved in ECP. However, the expression of the cationic and amphipathic α-type phenol-soluble modulins (PSMs), particularly of PSMα2, significantly increase ECP, while PSMβ peptides or δ-toxin have no effect on ECP. Because psm expression is strictly controlled by the accessory gene regulator (agr), ECP is also reduced in agr-negative mutants. PSMα peptides damage the cytoplasmic membrane, as indicated by the release of not only CPs but also lipids, nucleic acids, and ATP. Thus, our results show that in Staphylococcus aureus, PSMα peptides non-specifically boost the translocation of CPs by their membrane-damaging activity

Excretion of Cytoplasmic Proteins (ECP) in Staphylococcus Aureus

Excretion of cytoplasmic proteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG-binding protein, which is secreted via the Sec-pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan-binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non-excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism

Excreted Cytoplasmic Proteins Contribute to Pathogenicity in Staphylococcus aureus

Excretion of cytoplasmic proteins in pro- and eukaryotes, also referred to as “nonclassical protein export,” is a well-known phenomenon. However, comparatively little is known about the role of the excreted proteins in relation to pathogenicity. Here, the impact of two excreted glycolytic enzymes, aldolase (FbaA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), on pathogenicity was investigated in Staphylococcus aureus. Both enzymes bound to certain host matrix proteins and enhanced adherence of the bacterial cells to host cells but caused a decrease in host cell invasion. FbaA and GAPDH also bound to the cell surfaces of staphylococcal cells by interaction with the major autolysin, Atl, that is involved in host cell internalization. Surprisingly, FbaA showed high cytotoxicity to both MonoMac 6 (MM6) and HaCaT cells, while GAPDH was cytotoxic only for MM6 cells. Finally, the contribution of external FbaA and GAPDH to S. aureus pathogenicity was confirmed in an insect infection model