The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenosintrifosfatasy genetika metabolismus MeSH
- bakteriální proteiny * metabolismus MeSH
- nukleotidy metabolismus MeSH
- proteiny SecA metabolismus MeSH
- proteiny z Escherichia coli * metabolismus MeSH
- translokační kanály SEC chemie MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Antimicrobial resistance is well-known to be a global health and development threat. Due to the decrease of effective antimicrobials, re-evaluation in clinical practice of old antibiotics, as fosfomycin (FOS), have been necessary. FOS is a phosphonic acid derivate that regained interest in clinical practice for the treatment of complicated infection by multi-drug resistant (MDR) bacteria. Globally, FOS resistant Gram-negative pathogens are raising, affecting the public health, and compromising the use of the antibiotic. In particular, the increased prevalence of FOS resistance (FOSR) profiles among Enterobacterales family is concerning. Decrease in FOS effectiveness can be caused by i) alteration of FOS influx inside bacterial cell or ii) acquiring antimicrobial resistance genes. In this review, we investigate the main components implicated in FOS flow and report specific mutations that affect FOS influx inside bacterial cell and, thus, its effectiveness. FosA enzymes were identified in 1980 from Serratia marcescens but only in recent years the scientific community has started studying their spread. We summarize the global epidemiology of FosA/C2/L1-2 enzymes among Enterobacterales family. To date, 11 different variants of FosA have been reported globally. Among acquired mechanisms, FosA3 is the most spread variant in Enterobacterales, followed by FosA7 and FosA5. Based on recently published studies, we clarify and represent the molecular and genetic composition of fosA/C2 genes enviroment, analyzing the mechanisms by which such genes are slowly transmitting in emerging and high-risk clones, such as E. coli ST69 and ST131, and K. pneumoniae ST11. FOS is indicated as first line option against uncomplicated urinary tract infections and shows remarkable qualities in combination with other antibiotics. A rapid and accurate identification of FOSR type in Enterobacterales is difficult to achieve due to the lack of commercial phenotypic susceptibility tests and of rapid systems for MIC detection.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- Escherichia coli genetika MeSH
- fosfomycin * farmakologie MeSH
- Klebsiella pneumoniae genetika MeSH
- proteiny z Escherichia coli * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Wild birds including raptors can act as vectors of clinically relevant bacteria with antibiotic resistance. The aim of this study was to investigate the occurrence of antibiotic-resistant Escherichia coli in black kites (Milvus migrans) inhabiting localities in proximity to human-influenced environments in southwestern Siberia and investigate their virulence and plasmid contents. A total of 51 E. coli isolates mostly with multidrug resistance (MDR) profiles were obtained from cloacal swabs of 35 (64%, n = 55) kites. Genomic analyses of 36 whole genome sequenced E. coli isolates showed: (i) high prevalence and diversity of their antibiotic resistance genes (ARGs) and common association with ESBL/AmpC production (27/36, 75%), (ii) carriage of mcr-1 for colistin resistance on IncI2 plasmids in kites residing in proximity of two large cities, (iii) frequent association with class one integrase (IntI1, 22/36, 61%), and (iv) presence of sequence types (STs) linked to avian-pathogenic (APEC) and extra-intestinal pathogenic E. coli (ExPEC). Notably, numerous isolates had significant virulence content. One E. coli with APEC-associated ST354 carried qnrE1 encoding fluoroquinolone resistance on IncHI2-ST3 plasmid, the first detection of such a gene in E. coli from wildlife. Our results implicate black kites in southwestern Siberia as reservoirs for antibiotic-resistant E. coli. It also highlights the existing link between proximity of wildlife to human activities and their carriage of MDR bacteria including pathogenic STs with significant and clinically relevant antibiotic resistance determinants. IMPORTANCE Migratory birds have the potential to acquire and disperse clinically relevant antibiotic-resistant bacteria (ARB) and their associated antibiotic resistance genes (ARGs) through vast geographical regions. The opportunistic feeding behavior associated with some raptors including black kites and the growing anthropogenic influence on their natural habitats increase the transmission risk of multidrug resistance (MDR) and pathogenic bacteria from human and agricultural sources into the environment and wildlife. Thus, monitoring studies investigating antibiotic resistance in raptors may provide essential data that facilitate understanding the fate and evolution of ARB and ARGs in the environment and possible health risks for humans and animals associated with the acquisition of these resistance determinants by wildlife.
- MeSH
- antagonisté receptorů pro angiotenzin MeSH
- antibakteriální látky farmakologie MeSH
- divoká zvířata MeSH
- Escherichia coli * MeSH
- inhibitory ACE MeSH
- lidé MeSH
- mnohočetná bakteriální léková rezistence genetika MeSH
- proteiny z Escherichia coli * genetika MeSH
- ptáci mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Sibiř MeSH
ArdB proteins are known to inhibit the activity of the type I restriction-modification (RM-I) system, in particular EcoKI (IA family). The mechanism of ArdB's activity still remains unknown; the spectrum of targets inhibited has been poorly studied. In this work, it was shown that the presence of the ardB gene from the R64 plasmid could suppress the activity of EcoAI endonuclease (IB family) in Escherichia coli TG1 cells. Due to the absence of specificity of ArdB to a certain RM-I system (it inhibits both the IA- and IB-family), it can be assumed that the mechanism of the anti-restriction activity of this protein does not depend on the sequence DNA at the recognition site nor the structure of the restriction enzyme of the RM-I systems.
Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to aminoglycosides under anaerobiosis and engaged in cell membrane regulation.
- MeSH
- adenosintrifosfatasy * metabolismus MeSH
- aminoglykosidy * farmakologie MeSH
- antibakteriální látky farmakologie MeSH
- ATPázy spojené s různými buněčnými aktivitami metabolismus MeSH
- Escherichia coli * účinky léků enzymologie MeSH
- fosfolipidy MeSH
- fumaráty MeSH
- gentamiciny MeSH
- kyslík metabolismus MeSH
- membránové lipidy MeSH
- proteiny z Escherichia coli * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Aim: Colistin resistance poses a serious clinical problem. This study aims to investigate the presence of plasmid-mediated resistance and heteroresistance among Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) clinical isolates at intensive care units of Main Alexandria University Hospital. Material and Methods: Seventy colistin-resistant and 30 colistin-susceptible K. pneumoniae and E. coli isolates constituted the material of the study. The rapid polymyxin NP (Nordmann Poirel) test was performed. Heteroresistance in colistin-susceptible isolates was investigated by population analysis profile. mcr-1 and mcr-2 genes were amplified by polymerase chain reaction (PCR). Results: The rapid polymyxin NP test showed 100% concordance with the results of BMD (broth microdilution method). Heteroresistance to colistin was detected in 23.3% of colistin-susceptible K. pneumoniae isolates. All colistin-resistant isolates were negative for mcr-1 and mcr-2 genes. Conclusions: The rapid polymyxin NP test is a reliable screening tool for colistin resistance, but not for heteroresistance. Other colistin resistance mechanisms should be investigated.
- MeSH
- antibakteriální látky imunologie MeSH
- bakteriální léková rezistence * MeSH
- Escherichia coli * genetika MeSH
- jednotky intenzivní péče MeSH
- Klebsiella pneumoniae * genetika MeSH
- klinická studie jako téma MeSH
- kolistin farmakologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- polymyxiny MeSH
- proteiny z Escherichia coli genetika MeSH
- Check Tag
- lidé MeSH
BACKGROUND: Conjugative plasmids play a major role in the dissemination of antibiotic resistance genes. Knowledge of the plasmid characteristics and behaviour can allow development of control strategies. Here we focus on the IncX group of plasmids carrying genes conferring quinolone resistance (PMQR), reporting their transfer and persistence within host bacteria of various genotypes under distinct conditions and levels of induced stress in form of temperature change and various concentrations of ciprofloxacin supplementation. METHODS: Complete nucleotide sequences were determined for eight qnr-carrying IncX-type plasmids, of IncX1 (3), IncX2 (3) and a hybrid IncX1-2 (2) types, recovered from Escherichia coli of various origins. This data was compared with further complete sequences of IncX1 and IncX2 plasmids carrying qnr genes (n = 41) retrieved from GenBank and phylogenetic tree was constructed. Representatives of IncX1 (pHP2) and IncX2 (p194) and their qnrS knockout mutants, were studied for influence of induced stress and genetic background on conjugative transfer and maintenance. RESULTS: A high level of IncX core-genome similarity was found in plasmids of animal, environmental and clinical origin. Significant differences were found between the individual IncX plasmids, with IncX1 subgroup plasmids showing higher conjugative transfer rates than IncX2 plasmids. Knockout of qnr modified transfer frequency of both plasmids. Two stresses applied simultaneously were needed to affect transfer rate of wildtype plasmids, whereas a single stress was sufficient to affect the IncX ΔqnrS plasmids. The conjugative transfer was shown to be biased towards the host phylogenetic proximity. A long-term cultivation experiment pointed out the persistence of IncX plasmids in the antibiotic-free environment. CONCLUSIONS: The study indicated the stimulating effect of ciprofloxacin supplementation on the plasmid transfer that can be nullified by the carriage of a single PMQR gene. The findings present the significant properties and behaviour of IncX plasmids carrying antibiotic resistance genes that are likely to play a role in their dissemination and stability in bacterial populations.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- ciprofloxacin farmakologie MeSH
- Escherichia coli * genetika MeSH
- fylogeneze MeSH
- genomika MeSH
- konjugace genetická MeSH
- plazmidy genetika MeSH
- proteiny z Escherichia coli * genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The heat-labile enterotoxins of Escherichia coli and cholera toxin of Vibrio cholerae are related in structure and function. Each of these oligomeric toxins is comprised of one A polypeptide and five B polypeptides. The B-subunits bind to gangliosides, which are followed by uptake into the intoxicated cell and activation of the host's adenylate cyclase by the A-subunits. There are two antigenically distinct groups of these toxins. Group I includes cholera toxin and type I heat-labile enterotoxin of E. coli; group II contains the type II heat-labile enterotoxins of E. coli. Three variants of type II toxins, designated LT-IIa, LT-IIb and LT-IIc have been described. Earlier studies revealed the crystalline structure of LT-IIb. Herein the carbohydrate binding specificity of LT-IIc B-subunits was investigated by glycosphingolipid binding studies on thin-layer chromatograms and in microtiter wells. Binding studies using a large variety of glycosphingolipids showed that LT-IIc binds with high affinity to gangliosides with a terminal Neu5Acα3Gal or Neu5Gcα3Gal, e.g. the gangliosides GM3, GD1a and Neu5Acα3-/Neu5Gcα3--neolactotetraosylceramide and Neu5Acα3-/Neu5Gcα3-neolactohexaosylceramide. The crystal structure of LT-IIc B-subunits alone and with bound LSTd/sialyl-lacto-N-neotetraose d pentasaccharide uncovered the molecular basis of the ganglioside recognition. These studies revealed common and unique functional structures of the type II family of heat-labile enterotoxins.
- MeSH
- bakteriální toxiny * chemie metabolismus MeSH
- cholerový toxin metabolismus MeSH
- enterotoxiny chemie metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- G(M1) gangliosid metabolismus MeSH
- gangliosidy metabolismus MeSH
- proteiny z Escherichia coli * metabolismus MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The stringent response enables bacteria to respond to nutrient limitation and other stress conditions through production of the nucleotide-based second messengers ppGpp and pppGpp, collectively known as (p)ppGpp. Here, we report that (p)ppGpp inhibits the signal recognition particle (SRP)-dependent protein targeting pathway, which is essential for membrane protein biogenesis and protein secretion. More specifically, (p)ppGpp binds to the SRP GTPases Ffh and FtsY, and inhibits the formation of the SRP receptor-targeting complex, which is central for the coordinated binding of the translating ribosome to the SecYEG translocon. Cryo-EM analysis of SRP bound to translating ribosomes suggests that (p)ppGpp may induce a distinct conformational stabilization of the NG domain of Ffh and FtsY in Bacillus subtilis but not in E. coli.
- MeSH
- bakteriální proteiny metabolismus MeSH
- Escherichia coli metabolismus MeSH
- guanosinpentafosfát metabolismus MeSH
- proteiny z Escherichia coli * metabolismus MeSH
- receptory cytoplazmatické a nukleární metabolismus MeSH
- signál-rozpoznávající částice * metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a β-barrel protein begins with formation of a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a β-hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl-transferase center suggest that protein folding could modulate ribosome activity.
- MeSH
- cirkulární dichroismus MeSH
- elektronová kryomikroskopie MeSH
- Escherichia coli genetika metabolismus MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- molekulární modely MeSH
- posttranslační úpravy proteinů MeSH
- proteiny a peptidy chladového šoku chemie genetika metabolismus MeSH
- proteiny z Escherichia coli chemie genetika metabolismus MeSH
- proteosyntéza MeSH
- ribozomy genetika metabolismus MeSH
- sbalování proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
