RESUMEN
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.
RESUMEN
Lantibiotics are antimicrobial peptides that contain non-proteinogenic amino acids lanthionine and 3-methyllanthionine and are produced by Gram-positive bacteria. Here we addressed the pros and cons of lantibiotic production for its producing strains. Two staphylococcal strains, S. gallinarum Tü3928 and S. epidermidis Tü3298 producing gallidermin and epidermin respectively were selected. In each of these parental strains, the structural genes gdmA and epiA were deleted; all the other biosynthetic genes including the immunity genes were left intact. Comparative analysis of the lantibiotic-producing strains with their non-producing mutants revealed that lantibiotic production is a burden for the cells. The production affected growth, caused release of ATP, lipids and increased the excretion of cytoplasmic proteins (ECP). The epidermin and gallidermin immunity genes were insufficient to protect the cells from their own product. Co-cultivation studies showed that the ΔgdmA mutant has an advantage over the parental strain; the latter was outcompeted. On the one hand, the production of staphylococcal lantibiotics is beneficial by suppressing competitors, but on the other hand they impose a burden on the producing-strains when they accumulate in higher amounts. Our observations explain why antibiotic-producing strains occur as a minority on our skin and other ecological niches, but retain corresponding antibiotic resistance.
Asunto(s)
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Péptidos/metabolismo , Staphylococcus epidermidis/crecimiento & desarrollo , Staphylococcus/crecimiento & desarrollo , Adenosina Trifosfato/metabolismo , Humanos , Infecciones Estafilocócicas/microbiología , Staphylococcus/metabolismo , Staphylococcus epidermidis/metabolismoRESUMEN
In the past 20 years, peptide-based antibiotics, such as vancomycin, teicoplanin, and daptomycin, have often been considered as second-line antibiotics. However, in recent years, an increasing number of reports on vancomycin resistance in pathogens appeared, which forces researchers to find novel lead structures for potent new antibiotics. Herein, we report the total synthesis of a defined endo-typeâ B PPAP library and their antibiotic activity against multiresistant S.â aureus and various vancomycin-resistant Enterococci. Four new compounds that combine high activities and low cytotoxicity were identified, indicating that the PPAP core might become a new non-peptide-based lead structure in antibiotic research.
Asunto(s)
Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Floroglucinol/farmacología , Compuestos Policíclicos/farmacología , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Floroglucinol/síntesis química , Floroglucinol/química , Compuestos Policíclicos/síntesis química , Compuestos Policíclicos/químicaRESUMEN
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 PSMß 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.
Asunto(s)
Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Toxinas Bacterianas/genética , Mutación , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
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.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Interacciones Huésped-Patógeno , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Staphylococcus aureus/patogenicidad , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/toxicidad , Animales , Adhesión Bacteriana , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/toxicidad , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/microbiología , Larva/microbiología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Leucocitos/microbiología , Mariposas Nocturnas/microbiología , N-Acetil Muramoil-L-Alanina Amidasa/genética , Unión Proteica , Transducción de Señal , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Análisis de Supervivencia , VirulenciaRESUMEN
In bacteria, extracellular signals are transduced into the cell predominantly by two-component systems (TCSs) comprising a regulatory unit triggered by a specific signal. Some of the TCSs control executing units such as ABC transporters involved in antibiotic resistance. For instance, inStaphylococcus aureus, activation of BraSR leads to the upregulation ofvraDEexpression that encodes an ABC transporter playing a role in bacitracin and nisin resistance. In this study, we show that the small staphylococcal transmembrane protein VraH forms, together with VraDE, a three-component system. Although the expression ofvraHin the absence ofvraDEwas sufficient to mediate low-level resistance, only this VraDEH entity conferred high-level resistance against daptomycin and gallidermin. In most staphylococcal genomes,vraHis located immediately downstream ofvraDE, forming an operon, whereas in some species it is localized differently. In an invertebrate infection model, VraDEH significantly enhancedS. aureuspathogenicity. In analogy to the TCS connectors, VraH can be regarded as an ABC connector that modulates the activity of ABC transporters involved in antibiotic resistance.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Proteínas de la Membrana/genética , Staphylococcus aureus/genética , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Bacteriocinas/farmacología , Clonación Molecular , Daptomicina/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Larva/microbiología , Proteínas de la Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas/microbiología , Operón , Péptidos/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/patogenicidad , Análisis de Supervivencia , VirulenciaRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico , Pared Celular/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Unión ProteicaRESUMEN
The Staphylococcus epidermidis derived epidermin was the first lantibiotic that has been shown to be ribosomally synthesized and posttranslationally modified. Together with gallidermin, produced by Staphylococcus gallinarum, they belong to the large class of cationic antimicrobial peptides (CAMPs) that act against a broad spectrum of Gram-positive bacteria. Here we describe the genetic organization, biosynthesis and modification, excretion, extracellular activation of the modified pre-peptide by proteolytic processing, self-protection of the producer, gene regulation, structure, and the mode of action of gallidermin and epidermin. We also address mechanisms of bacterial tolerance to these lantibiotics and other CAMPs. Particularly gallidermin has a high potential for therapeutic application, as it is active against methicillin-resistant Staphylococcus aureus strains (MRSA) and as it is able to prevent biofilm formation at sublethal concentrations.