Ramoplanin at bactericidal concentrations induces bacterial membrane depolarization in Staphylococcus aureus

Ramoplanin is an actinomycetes-derived antibiotic with broad-spectrum activity against Gram-positive bacteria that has been evaluated in clinical trials for the treatment of gastrointestinal vancomycin-resistant enterococci (VRE) and Clostridium difficile infections. Recent studies have proposed that ramoplanin binds to bacterial membranes as a C2 symmetrical dimer that can sequester Lipid II, which causes inhibition of cell wall peptidoglycan biosynthesis and cell death. In this study, ramoplanin was shown to bind to anionic and zwitterionic membrane mimetics with a higher affinity for anionic membranes and to induce membrane depolarization of methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 25923 at concentrations at or above the minimal bactericidal concentration (MBC). The ultrastructural effects of ramoplanin on S. aureus were also examined by transmission electron microscopy (TEM), and this showed dramatic changes to bacterial cell morphology. The correlation observed between membrane depolarization and bacterial cell viability suggests that this mechanism may contribute to the bactericidal activity of ramoplanin

A building block of Antibiotic Ramoplanin A2

학위논문(석사) -- 서울대학교대학원 : 공과대학 화학생물공학부, 2021.8. 김영규.This paper covers the contents of the synthesis of building block of ramoplanin A2 based on the stereoselectivity of the L-threo-β-hydroxy aspartate. Ramoplanin A2, synthesized by Actinoplanes, is a type of glycolipodepsipeptide and exhibits clinical effects. In particular, ramoplanin A2 plays an effective role in the treatment of vancomycin-resistant E. faecium (VREF) in the gastrointestinal tract by inhibiting the biosynthesis of bacterial cell wall in Gram-positive bacteria. According to the currently published papers, ramoplanin A2 is composed of three key subunits and can be synthesized through coupling reactions and cyclization reactions. Among them, the structure of L-threo-β-hydroxy asparagine is considered to be important in terms of stereochemistry as the backbone of the second key subunit, pentadepsipeptide. In our group, we have studied the stereoselective synthesis of trans-oxazolidine. N-hydroxymethyl-α-amino aldehyde has a stable structure by shifting its equilibrium to hemiacetal and participates in the reaction. Trans-oxazolidine is synthesized through the reaction of N-hydroxymethyl-α-amino aldehyde with phenylsulfonyl-nitromethane, and H-eclipsed formation of transition state enhances the stereoselectivity of trans-oxazolidine structure in the ratio of 20:1. Through this procedure, L-threo-β-hydroxy aspartate was synthesized in a total of 11 steps, 11% from D-serine. This product was synthesized to contain different protection groups on each terminal, which allows selective conversions to other form such as L-threo-β-hydroxy asparagine. Moreover, the possibility of synthesizing ramoplanin A2 was confirmed through Yamaguchi coupling reaction with N-phenylalanine.본 논문은 L-트레오-β-하이드록시 아스파라진 구조의 입체선택적 특징을 기본으로 하는 라모플라닌 에이2의 전구체의 합성에 관한 내용을 다루고 있다. 악티노플라네스에 의해 합성되는 라모플라닌 에이2는 당지질 뎁시펩타이드의 한 종류로 임상효과를 나타낸다. 특히 라모플라닌 에이2는 그람 양성 박테리아에서 박테리아의 세포벽 생합성을 저해하여 소화기관에 존재하는 반코마이신 내성 장구균의 처리에 효과적인 역할을 한다. 현재 발표된 논문에 따르면 라모플라닌 에이2는 3개의 소단위체로 구성되어 커플링 반응과 고리화 반응을 통해 합성할 수 있다고 한다. 그 중 L-트레오-β-하이드록시 아스파라진 구조는 두 번째 주요 소단위인 펜타뎁시펩타이드의 뼈대로써 입체화학적 측면에서 중요하게 여겨진다. 우리 그룹은 트랜스-옥사졸리딘 구조를 형성할 때 일어날 수 있는 입체선택적 합성 방법에 대해 보고한 평형이 이동하여 안정한 구조를 가지고 반응에 참여한다. 트랜스-옥사졸리딘은 N-하이드록시메틸-α-아미노 알데하이드와 페닐설포닐나이트로메테인의 반응을 통해 합성되고, 전이상태에서의 H-가리움 형태로 인한 입체장애로 인해 트랜스-옥사졸리딘의 입체 선택성이 20:1의 비율로 높아진다. 이를 통하여 D-세린으로부터 L-트레오-β-하이드록시 아스팔테이트를 총 11단계, 11%로 합성하였다. 이 합성물은 각 말단이 서로 다른 종류의 보호기로 보호되어 L-트레오-β-하이드록시 아스파라진과 같은 형태로의 선택적 전환이 용이하게 합성되었다. 또한, N-페닐알라닌과의 야마구치 커플링 반응을 통한 뎁시펩타이드의 형성으로 라모플라닌 에이2의 합성 가능성을 확인하였다.1. Introduction 1 1.1 Introduction of Depsipeptide 1 1.2 Introduction of Ramoplanin A2 3 1.3 Introduction of building block of Ramoplanin A2 5 1.4 Introduction of N-protected-α-amino aldehyde 8 1.5 Introduction of trans-oxazolidine 10 2. Results and Discussion 13 2.1 Retrosynthesis of building block of Ramoplanin A2 13 2.2 Formation of D-serinal 14 2.3 Formation of stereoselective trans-oxazolidine 16 2.4 Formation of building block for Ramoplanin A2 18 3. Conclusion 21 4. Experimental Details 22 REFERENCES 30 APPENDICES 33 ABSTRACT IN KOREAN 50석

Retention of native-like structure in an acyclic counterpart of a β-sheet antibiotic

AbstractAn acyclic derivative of the cyclic peptide antibiotic, ramoplanin, has been prepared. In aqueous solution, two-dimensional NMR spectroscopy indicates that the acyclic form adopts a threshold population of conformers in which at least part of the β-sheet characteristic of the intact ramoplanin persists. Thus, despite losing the entropic benefit which the macrocycle must lend to β-sheet formation, the polypeptide chain of the acyclic ramoplanin appears to display an innate tendency to adopt a native-like conformation

The absence of a mature cell wall sacculus in stable Listeria monocytogenes L-form cells is independent of peptidoglycan synthesis

L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan

New advances in the treatment of Clostridium difficile infection (CDI)

Clostridium difficile infections (CDI) have increased in frequency throughout the world. In addition to an increase in frequency, recent CDI epidemics have been linked to a hypervirulent C. difficile strain resulting in greater severity of disease. Although most mild to moderate cases of CDI continue to respond to metronidazole or vancomycin, refractory and recurrent cases of CDI may require alternative therapies. This review provides a brief overview of CDI and summarizes studies involving alternative antibiotics, toxin binders, probiotics, and immunological therapies that can be considered for treatment of acute and recurrent CDI in severe and refractory situations

Decolonization of gastrointestinal carriage of vancomycin-resistant Enterococcus faecium: case series and review of literature

Background: Prolonged asymptomatic carriage of vancomycin-resistant enterococci (VRE) in the gastrointestinal tract and the lack of effective decolonization regimen perpetuate the endemicity of VRE in the healthcare settings.Case presentation: We report a regimen for decolonization of gastrointestinal carriage of VRE by a combination of environmental disinfection, patient isolation, bowel preparation to wash-out the fecal bacterial population using polyethylene glycol, a five-day course of oral absorbable linezolid and non-absorbable daptomycin to suppress any remaining VRE, and subsequent oral Lactobacillus rhamnosus GG to maintain the colonization resistance in four patients, including two patients with end-stage liver cirrhosis, one patient with complication post liver transplant, and one patient with complicated infective endocarditis. All patients had clearance of VRE immediately after decolonization, and 3 of them remained VRE-free for 23 to 137 days of hospitalization, despite subsequent use of intravenous broad-spectrum antibiotics without anti-VRE activity.Conclusion: This strategy should be further studied in settings of low VRE endemicity with limited isolation facilities. © 2014 Cheng et al.; licensee BioMed Central Ltd.published_or_final_versio