In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem

International audienceInfections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen

Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in; Corynebacterium glutamicum;

Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys; +1; residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys; +1; , which can eventually be further modified. Here, we identified the; lgt; and; lspA; genes from; Corynebacterium glutamicum; and found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, a; C. glutamicum; maltose-binding lipoprotein, and LppX, a; Mycobacterium tuberculosis; lipoprotein. However, Lgt is not required for these proteins' signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that in; C. glutamicum; the association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification

Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli

TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique α/β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of β-barrel OMPs, is dependent on BamA and BamD, two essential components of the β-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCΔ2–22). A significant amount of TolCΔ2–22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCΔ2–22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCΔ2–22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCΔ2–22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCΔ2–22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble α-helical domain

Getting drugs into gram-negative bacteria: rational rules for permeation through general porins

Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae and Klebsiella pneumoniae and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in-vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation

Research and Design of a Routing Protocol in Large-Scale Wireless Sensor Networks

无线传感器网络，作为全球未来十大技术之一，集成了传感器技术、嵌入式计算技术、分布式信息处理和自组织网技术，可实时感知、采集、处理、传输网络分布区域内的各种信息数据，在军事国防、生物医疗、环境监测、抢险救灾、防恐反恐、危险区域远程控制等领域具有十分广阔的应用前景。 本文研究分析了无线传感器网络的已有路由协议，并针对大规模的无线传感器网络设计了一种树状路由协议，它根据节点地址信息来形成路由，从而简化了复杂繁冗的路由表查找和维护，节省了不必要的开销，提高了路由效率，实现了快速有效的数据传输。 为支持此路由协议本文提出了一种自适应动态地址分配算——ADAR（AdaptiveDynamicAddre...As one of the ten high technologies in the future, wireless sensor network, which is the integration of micro-sensors, embedded computing, modern network and Ad Hoc technologies, can apperceive, collect, process and transmit various information data within the region. It can be used in military defense, biomedical, environmental monitoring, disaster relief, counter-terrorism, remote control of haz...学位：工学硕士院系专业：信息科学与技术学院通信工程系_通信与信息系统学号：2332007115216

Pompes d’efflux d’antibiotiques chez Enterobacter aerogenes: Identification, rôle dans la multirésistance et étude des relations structure-fonction

Over the past decade, Enterobacter aerogenes has become an important nosocomial pathogen in France, with many clinical isolates exhibiting a phenotype of multidrug resistance to antibiotics. This phenotype is generally associated with the overproduction of efflux pumps.First, I identified the eefABC locus encoding a tripartite efflux pump and showed that its expression was silenced by the repressive action of the H-NS protein.Second, I developed the conditions for overexpression and purification of the outer membrane proteins TolC and EefC to conduct functional studies. Reconstructed in artificial membranes, TolC and EefC form stable pores, reflecting a common resting conformation and low conductance. By this approach, I was also able to highlight the conserved blockage of the channels by divalent cations.Finally, I joined other team members to report the effectiveness of efflux-inhibiting molecules as a therapeutic alternative.Au cours de ces dix dernières années, Enterobacter aerogenes est devenu un pathogène nosocomial important en France, de nombreux isolats cliniques présentant un phénotype de multirésistance aux antibiotiques. Ce phénotype est généralement associé à la surproduction de pompes d'efflux.Premièrement, j'ai identifié le locus eefABC codant une pompe d'efflux tripartite et j'ai montré que son expression était rendue silencieuse par l'action répressive de la protéine H-NS.Deuxièmement, j'ai mis au point les conditions pour la surexpression et la purification des protéines de membrane externe TolC et EefC pour mener des études fonctionnelles. Reconstituées dans des membranes artificielles, TolC et EefC forment des pores stables, traduisant une conformation de repos commune et de faible conductance. Par cette approche, j'ai également pu mettre en évidence le blocage conservé des canaux par des cations divalents.Enfin, je me suis associée à d'autres membres de l'équipe pour rapporter l'efficacité de molécules inhibitrices de l'efflux comme alternative thérapeutique

Multiple Signals Direct the Assembly and Function of a Type 1 Secretion System▿ †

Type 1 secretion systems (T1SS) are present in a wide range of Gram-negative bacteria and are involved in the secretion of diverse substrates such as proteases, lipases, and hemophores. T1SS consist of three proteins: an inner membrane ABC (ATP binding cassette) protein, a periplasmic adaptor, and an outer membrane channel of the TolC family. Assembly of the tripartite complex is transient and induced upon binding of the substrate to the ABC protein. It is generally accepted that T1SS-secreted proteins have a C-terminal secretion signal required for secretion and that this signal interacts with the ABC protein. However, we have previously shown that for the Serratia marcescens hemophore HasA, interactions with the ABC protein and subsequent T1SS assembly require additional regions. In this work, we characterize these regions and demonstrate that they are numerous, distributed throughout the HasA polypeptide, and most likely linear. Together with the C-terminal signal, these elements maximize the secretion of HasA. The data also show that the C-terminal signal of HasA triggers HasD-driven ATP hydrolysis, leading to disassembly of the complex. These data support a model of type 1 secretion involving a multistep interaction between the substrate and the ABC protein that stabilizes the assembled secretion system until the C terminus is presented. This model also supports tight coupling between synthesis and secretion

Dual Regulation of the Small RNA MicC and the Quiescent Porin OmpN in Response to Antibiotic Stress in Escherichia coli

Antibiotic resistant Gram-negative bacteria are a serious threat for public health. The permeation of antibiotics through their outer membrane is largely dependent on porin, changes in which cause reduced drug uptake and efficacy. Escherichia coli produces two major porins, OmpF and OmpC. MicF and MicC are small non-coding RNAs (sRNAs) that modulate the expression of OmpF and OmpC, respectively. In this work, we investigated factors that lead to increased production of MicC. micC promoter region was fused to lacZ, and the reporter plasmid was transformed into E. coli MC4100 and derivative mutants. The response of micC–lacZ to antimicrobials was measured during growth over a 6 h time period. The data showed that the expression of micC was increased in the presence of β-lactam antibiotics and in an rpoE depleted mutant. Interestingly, the same conditions enhanced the activity of an ompN–lacZ fusion, suggesting a dual transcriptional regulation of micC and the quiescent adjacent ompN. Increased levels of OmpN in the presence of sub-inhibitory concentrations of chemicals could not be confirmed by Western blot analysis, except when analyzed in the absence of the sigma factor σE. We suggest that the MicC sRNA acts together with the σE envelope stress response pathway to control the OmpC/N levels in response to β-lactam antibiotics

Outer Membrane Porins

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Stress responses, outer membrane permeability control and antimicrobial resistance in Enterobacteriaceae 2 3

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