RESUMO
Enterobacteriaceae are widely present in many environments related to humans, including the human body and the food that they consume, from both plant or animal origin. Hence, they are considered relevant members of the gastrointestinal tract microbiota. On the other hand, these bacteria are also recognized as putative pathogens, able to impair human health and, in food, they are considered indicators for the microbiological quality and hygiene status of a production process. Nevertheless, beneficial properties have also been associated with Enterobacteriaceae, such as the ability to synthesize peptides and proteins, which can have a role in the structure of microbial communities. Among these antimicrobial molecules, those with higher molecular mass are called colicins, while those with lower molecular mass are named microcins. In recent years, some studies show an emphasis on molecules that can help control the development of pathogens. However, not enough data are available on this subject, especially related to microcins. Hence, this review gathers and summarizes current knowledge on colicins and microcins, potential usage in the treatment of pathogen-associated diseases and cancer, as well as putative applications in food biotechnology.
Assuntos
Anti-Infecciosos , Colicinas , Animais , Bacteriocinas , Colicinas/química , Colicinas/metabolismo , Colicinas/farmacologia , Enterobacteriaceae , Humanos , PeptídeosRESUMO
The Escherichia coli ZP strain (ZP) was constructed based on the known probiotic E. coli strain Nissle 1917. It was genetically modified to carry the colicin E7 synthesis gene encoding DNase on a conjugative plasmid and the colicin E7 immunity gene in the chromosome. The aim of this study was to evaluate the effects of the daily ZP per oral administration (5 × 108 or 5 × 1010 CFU per bird) on the growth performance, hematological, biochemical, histological parameters, gut microbiota, and nonspecific immunity of the 4-24 days old broilers. The ZP administration increased the abundance of genera Bacillus, Butyrivibrio, and Clostridium and did not influence the weight gain of 4-16 days old broilers. The biochemical parameters were within normal ranges for poultry in experimental and control groups. The ZP administration had no effect on the erythrocyte numbers, hemoglobin and immunoglobulin Y concentrations, but significantly increased the serum lysozyme concentration, leukocyte numbers, and reactive oxygen species production by phagocytes compared with the control group. It did not cause inflammatory changes in intestinal mucosa, Peyer's patches, and spleen. Thus, the ZP had no detrimental effects on broiler health and could be an efficient probiotic for the broiler colibacillosis prophylaxis.
Assuntos
Colicinas , Infecções por Escherichia coli , Microbioma Gastrointestinal , Probióticos , Animais , Colicinas/farmacologia , Escherichia coli/genética , Galinhas , Infecções por Escherichia coli/prevenção & controle , Probióticos/farmacologiaRESUMO
In the bid to survive and thrive in an environmental setting, bacterial species constantly interact and compete for resources and space in the microbial ecosystem. Thus, they have adapted to use various antibiotics and toxins to fight their rivals. Simultaneously, they have evolved an ability to withstand weapons that are directed against them. Several bacteria harbor colicinogenic plasmids which encode toxins that impair the translational apparatus. One of them, colicin E3 ribotoxin, mediates cleavage of the 16S rRNA in the decoding center of the ribosome. In order to thrive upon deployment of such ribotoxins, competing bacteria may have evolved counter-conflict mechanisms to prevent their demise. A recent study demonstrated the role of PrfH and the RtcB2 module in rescuing a damaged ribosome and the subsequent re-ligation of the cleaved 16S rRNA by colicin E3 in vitro. The rtcB2-prfH genes coexist as gene neighbors in an operon that is sporadically spread among different bacteria. In the current study, we report that the RtcB2-PrfH module confers resistance to colicin E3 toxicity in E. coli ATCC25922 cells in vivo. We demonstrated that the viability of E. coli ATCC25922 strain that is devoid of rtcB2 and prfH genes is impaired upon action of colicin E3, in contrast to the parental strain which has intact rtcB2 and prfH genes. Complementation of the rtcB2 and prfH gene knockout with a high copy number-plasmid (encoding either rtcB2 alone or both rtcB2-prfH operon) restored resistance to colicin E3. These results highlight a counter-conflict system that may have evolved to thwart colicin E3 activity.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Colicinas , Proteínas de Escherichia coli/metabolismo , Colicinas/genética , Colicinas/farmacologia , Ecossistema , Escherichia coli/genética , Óperon , Plasmídeos/genética , RNA Ribossômico 16SRESUMO
The double membrane architecture of Gram-negative bacteria forms a barrier that is impermeable to most extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed proteins embedded in the outer membrane to deliver cytotoxic cargo. Colicin E1 is a bacteriocin produced by, and lethal to, Escherichia coli that hijacks the outer membrane proteins (OMPs) TolC and BtuB to enter the cell. Here, we capture the colicin E1 translocation domain inside its membrane receptor, TolC, by high-resolution cryo-electron microscopy to obtain the first reported structure of a bacteriocin bound to TolC. Colicin E1 binds stably to TolC as an open hinge through the TolC pore-an architectural rearrangement from colicin E1's unbound conformation. This binding is stable in live E. coli cells as indicated by single-molecule fluorescence microscopy. Finally, colicin E1 fragments binding to TolC plug the channel, inhibiting its native efflux function as an antibiotic efflux pump, and heightening susceptibility to three antibiotic classes. In addition to demonstrating that these protein fragments are useful starting points for developing novel antibiotic potentiators, this method could be expanded to other colicins to inhibit other OMP functions.
Bacteria are constantly warring with each other for space and resources. As a result, they have developed a range of molecular weapons to poison, damage or disable other cells. For instance, bacteriocins are proteins that can latch onto structures at the surface of enemy bacteria and push toxins through their outer membrane. Bacteria are increasingly resistant to antibiotics, representing a growing concern for modern healthcare. One way that they are able to survive is by using 'efflux pumps' studded through their external membranes to expel harmful drugs before these can cause damage. Budiardjo et al. wanted to test whether bacteriocins could interfere with this defence mechanism by blocking efflux pumps. Bacteriocins are usually formed of binding elements (which recognise specific target proteins) and of a 'killer tail' that can stab the cell. Experiments showed that the binding parts of a bacteriocin could effectively 'plug' efflux pumps in Escherichia coli bacteria: high-resolution molecular microscopy revealed how the bacteriocin fragment binds to the pump, while fluorescent markers showed that it attached to the surface of E. coli and stopped the efflux pumps from working. As a result, lower amounts of antibiotics were necessary to kill the bacteria when bacteriocins were present. The work by Budiardjo et al. could lead to new ways to combat bacteria that will reduce the need for current antibiotics. In the future, bacteriocins could also be harnessed to target other proteins than efflux pumps, allowing scientists to manipulate a range of bacterial processes.
Assuntos
Bacteriocinas , Colicinas , Proteínas de Escherichia coli , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriocinas/metabolismo , Colicinas/química , Colicinas/metabolismo , Colicinas/farmacologia , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte ProteicoRESUMO
In light of an increasing number of antibiotic-resistant bacterial strains, it is essential to understand an action imposed by various antimicrobial agents on bacteria at the molecular level. One of the leading mechanisms of killing bacteria is related to the alteration of their plasmatic membrane. We study bio-inspired peptides originating from natural antimicrobial proteins colicins, which can disrupt membranes of bacterial cells. Namely, we focus on the α-helix H1 of colicin U, produced by bacterium Shigella boydii, and compare it with analogous peptides derived from two different colicins. To address the behavior of the peptides in biological membranes, we employ a combination of molecular simulations and experiments. We use molecular dynamics simulations to show that all three peptides are stable in model zwitterionic and negatively charged phospholipid membranes. At the molecular level, their embedment leads to the formation of membrane defects, membrane permeation for water, and, for negatively charged lipids, membrane poration. These effects are caused by the presence of polar moieties in the considered peptides. Importantly, simulations demonstrate that even monomeric H1 peptides can form toroidal pores. At the macroscopic level, we employ experimental co-sedimentation and fluorescence leakage assays. We show that the H1 peptide of colicin U incorporates into phospholipid vesicles and disrupts their membranes, causing leakage, in agreement with the molecular simulations. These insights obtained for model systems seem important for understanding the mechanisms of antimicrobial action of natural bacteriocins and for future exploration of small bio-inspired peptides able to disrupt bacterial membranes.
Assuntos
Colicinas/metabolismo , Fosfolipídeos/química , Lipossomas Unilamelares/metabolismo , Sequência de Aminoácidos , Colicinas/química , Colicinas/farmacologia , Simulação de Dinâmica Molecular , Permeabilidade/efeitos dos fármacos , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Conformação Proteica em alfa-Hélice , Shigella boydii/metabolismo , Lipossomas Unilamelares/químicaRESUMO
Currently, it is extremely important to identify and describe new alternative compounds with potential antimicrobial properties. Since various natural biological systems are capable of producing active compounds with such properties, many of them have been the subject of intensive study. The aim of this work was to heterologously overexpress, purify and preliminarily investigate the antimicrobial activity of a novel bacteriocin found in Salmonella species. Overexpressed protein shows an amino acid structure homologous to the well-known colicin M and was never expressed previously in the E. coli platform. Purified salmocin M showed an inhibition spectrum against Salmonella and E. coli strains. To determine its potential as an antimicrobial agent for use in medicine or the food industry, preliminary antimicrobial tests against pathogenic bacteria were carried out. Our research demonstrates that bacteriocin can be produced efficiently in bacterial expression systems, which are one of the cheapest and the most popular platforms for recombinant protein production. Moreover, preliminary results of microbiological tests showed its activity against most of the bacterial strains in a dose-dependent manner.
Assuntos
Anti-Infecciosos , Bacteriocinas , Colicinas , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Colicinas/genética , Colicinas/farmacologia , Escherichia coli/genética , Salmonella/genéticaRESUMO
Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter. IMPORTANCE Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coli. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Colicinas/farmacologia , DNA/metabolismo , Ferro/metabolismo , Receptores de Superfície Celular/metabolismo , Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Bacteriocinas/genética , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Colicinas/química , Colicinas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Periplasma/metabolismo , Proteínas Periplásmicas/metabolismo , Conformação Proteica , Transporte Proteico , Receptores de Superfície Celular/genéticaRESUMO
Bacteriophages and bacterial toxins are promising antibacterial agents to treat infections caused by multidrug-resistant (MDR) bacteria. In fact, bacteriophages have recently been successfully used to treat life-threatening infections caused by MDR bacteria (Schooley RT, Biswas B, Gill JJ, Hernandez-Morales A, Lancaster J, Lessor L, Barr JJ, Reed SL, Rohwer F, Benler S, et al. 2017. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents Chemother. 61(10); Chan BK, Turner PE, Kim S, Mojibian HR, Elefteriades JA, Narayan D. 2018. Phage treatment of an aortic graft infected with Pseudomonas aeruginosa. Evol Med Public Health. 2018(1):60-66; Petrovic Fabijan A, Lin RCY, Ho J, Maddocks S, Ben Zakour NL, Iredell JR, Westmead Bacteriophage Therapy Team. 2020. Safety of bacteriophage therapy in severe Staphylococcus aureus infection. Nat Microbiol. 5(3):465-472). One potential problem with using these antibacterial agents is the evolution of resistance against them in the long term. Here, we studied the fitness landscape of the Escherichia coli TolC protein, an outer membrane efflux protein that is exploited by a pore forming toxin called colicin E1 and by TLS phage (Pagie L, Hogeweg P. 1999. Colicin diversity: a result of eco-evolutionary dynamics. J Theor Biol. 196(2):251-261; Andersen C, Hughes C, Koronakis V. 2000. Chunnel vision. Export and efflux through bacterial channel-tunnels. EMBO Rep. 1(4):313-318; Koronakis V, Andersen C, Hughes C. 2001. Channel-tunnels. Curr Opin Struct Biol. 11(4):403-407; Czaran TL, Hoekstra RF, Pagie L. 2002. Chemical warfare between microbes promotes biodiversity. Proc Natl Acad Sci U S A. 99(2):786-790; Cascales E, Buchanan SK, Duché D, Kleanthous C, Lloubès R, Postle K, Riley M, Slatin S, Cavard D. 2007. Colicin biology. Microbiol Mol Biol Rev. 71(1):158-229). By systematically assessing the distribution of fitness effects of â¼9,000 single amino acid replacements in TolC using either positive (antibiotics and bile salts) or negative (colicin E1 and TLS phage) selection pressures, we quantified evolvability of the TolC. We demonstrated that the TolC is highly optimized for the efflux of antibiotics and bile salts. In contrast, under colicin E1 and TLS phage selection, TolC sequence is very sensitive to mutations. Finally, we have identified a large set of mutations in TolC that increase resistance of E. coli against colicin E1 or TLS phage without changing antibiotic susceptibility of bacterial cells. Our findings suggest that TolC is a highly evolvable target under negative selection which may limit the potential clinical use of bacteriophages and bacterial toxins if evolutionary aspects are not taken into account.
Assuntos
Bacteriófagos , Colicinas , Proteínas de Escherichia coli , Escherichia coli , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas da Membrana Bacteriana Externa , Bacteriófagos/genética , Colicinas/química , Colicinas/metabolismo , Colicinas/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Cancer is one of the most causes of death all over the world, although improvements in its treatment and recognition. Due to the limitations of common anticancer methods, including surgery, chemotherapy, and radiotherapy, attention has been drawn to other anti-cancer compounds, especially natural peptides such as bacteriocins. In this study, we used a combination of two bacteriocins, colicin E1 and enterocin A, against AGS gastric cancer cell lines. In order to evaluate anticancer properties of fusion peptide, we applied MTT assay, real-time PCR, and flow cytometry tests. This is the first report to show the cell growth inhibitory activity of the enterocin A in combination with colicin E1 against AGS human cancer cells. The results of this study showed that this fusion peptide at a concentration of 60.4 µg/mL and 24 h was able to kill half of the tested cancer cells, and treatment of the cells with this concentration increased the expression of bax and caspase 3 genes and reduced the expression of bacl-2 in 24 h. Flow cytometry analysis of annexin V-FITC/propidium iodide results also showed that our peptide was able to induce apoptosis in treated cells compared with control. Taken together, enterocin A-colicin E1 (ent A-col E1) can be considered as a good candidate for anticancer therapies.
Assuntos
Antineoplásicos/farmacologia , Bacteriocinas/farmacologia , Colicinas/farmacologia , Neoplasias Gástricas , Linhagem Celular Tumoral , Humanos , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Neoplasias Gástricas/tratamento farmacológicoRESUMO
INTRODUCTION: The emergence and spread of antibiotic resistance among pathogenic bacteria drives the search for alternative antimicrobial therapies. Bacteriocins represent a potential alternative to antibiotic treatment. In contrast to antibiotics, bacteriocins are peptides or proteins that have relatively narrow spectra of antibacterial activities and are produced by a wide range of bacterial species. Bacteriocins of Escherichia coli are historically classified as microcins and colicins, and, until now, more than 30 different bacteriocin types have been identified and characterized. AREAS COVERED: We performed bibliographical searches of online databases to review the literature regarding bacteriocins produced by E. coli with respect to their occurrence, bacteriocin role in bacterial colonization and pathogenicity, and application of their antimicrobial effect. EXPERT OPINION: The potential use of bacteriocins for applications in human and animal medicine and the food industry includes (i) the use of bacteriocin-producing probiotic strains, (ii) recombinant production in plants and application in food, and (iii) application of purified bacteriocins.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Escherichia coli/metabolismo , Animais , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Bacteriocinas/biossíntese , Bacteriocinas/isolamento & purificação , Colicinas/biossíntese , Colicinas/isolamento & purificação , Colicinas/farmacologia , Humanos , Probióticos/farmacologiaRESUMO
Aim: The resident bacterial microbiome may shape and protect the health of vertebrate host. An array of molecules secreted by microbiome may contribute to the ecological stability of the microbiome itself. Material & methods: ELISA, radioactivity, immunofluorescence and cytokines measurements were used to observe the bioactivity and stability of colicin Ia level in oviparous and viviparous animal circulation. Results: Colicin Ia, a protein antimicrobial produced by Escherichia coli, is not present in animals at birth, but increases in concentration with the establishment of a stable gut microbiome and drops when the microbiome is experimentally disrupted. Colicin introduced in vivo is transported to tissues at concentrations able to prevent or eliminate bacterial infection. Conclusion: Our findings suggest an unexpected benefit provided by the presence of a resident microbiome in the form of active, circulating, bacterially-synthesized antimicrobial molecules.
Assuntos
Bactérias/efeitos dos fármacos , Colicinas/farmacologia , Escherichia coli/metabolismo , Microbioma Gastrointestinal , Vertebrados/sangue , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Bovinos , Colicinas/sangue , Colicinas/metabolismo , Escherichia coli/química , Fezes/microbiologia , Humanos , Coelhos , Vertebrados/microbiologiaRESUMO
Colicin M is an enzymatic bacteriocin produced by some Escherichia coli strains which provokes cell lysis of competitor strains by hydrolysis of the cell wall peptidoglycan undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) precursor. The overexpression of a gene, cbrA (formerly yidS), was shown to protect E. coli cells from the deleterious effects of this colicin, but the underlying resistance mechanism was not established. We report here that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance toward this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now displayed a saturated isoprene unit at the α-position, i.e., the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. In vitro and in vivo assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway.IMPORTANCE Overexpression of the chromosomal cbrA gene allows E. coli to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C55-carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C55-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.
Assuntos
Antibacterianos/farmacologia , Colicinas/farmacologia , Farmacorresistência Bacteriana , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Flavoproteínas/metabolismo , Peptidoglicano/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Flavoproteínas/genética , Peptidoglicano/química , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5-15 µM selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10-15 µM) for 12 h. Moreover, 5-15 µM of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin ß1 and αV in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer.
Assuntos
Apoptose/efeitos dos fármacos , Colicinas/farmacologia , Neoplasias Pulmonares/metabolismo , Western Blotting , Caspase 8/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Integrinas/metabolismo , Propídio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismoRESUMO
The outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted by Escherichia coli, can target other E. coli cells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of various E. coli strains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why do E. coli strains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenic E. coli sequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen into E. coli K-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizing E. coli toward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coli infections can be a major health burden, especially with the organism becoming increasingly resistant to "last-resort" antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenic E. coli strain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity among E. coli organisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenic E. coli such as uropathogenic E. coli (UPEC).
Assuntos
Antibacterianos/farmacologia , Colicinas/farmacologia , Farmacorresistência Bacteriana , Antígenos O/metabolismo , Escherichia coli Uropatogênica/efeitos dos fármacos , Elementos de DNA Transponíveis , Inativação Gênica , Metabolismo/efeitos dos fármacos , Análise em Microsséries , Testes de Sensibilidade Microbiana , Mutagênese Insercional , Antígenos O/genética , Fenótipo , Análise de Sequência de DNA , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Escherichia coli Uropatogênica/metabolismoRESUMO
Colicins are natural non-antibiotic bacterial proteins with a narrow spectrum but an extremely high antibacterial activity. These proteins are promising food additives for the control of major pathogenic Shiga toxin-producing E. coli serovars in meats and produce. In the USA, colicins produced in edible plants such as spinach and leafy beets have already been accepted by the U. S. Food and Drug Administration (FDA) and U. S. Department of Agriculture (USDA) as food-processing antibacterials through the GRAS (generally recognized as safe) regulatory review process. Nicotiana benthamiana, a wild relative of tobacco, N. tabacum, has become the preferred production host plant for manufacturing recombinant proteins-including biopharmaceuticals, vaccines, and biomaterials-but the purification procedures that have been employed thus far are highly complex and costly. We describe a simple and inexpensive purification method based on specific acidic extraction followed by one chromatography step. The method provides for a high recovery yield of purified colicins, as well as a drastic reduction of nicotine to levels that could enable the final products to be used on food. The described purification method allows production of the colicin products at a commercially viable cost of goods and might be broadly applicable to other cost-sensitive proteins.
Assuntos
Antibacterianos/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Colicinas/isolamento & purificação , Aditivos Alimentares/isolamento & purificação , Carne/microbiologia , Nicotiana/genética , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Bovinos , Colicinas/biossíntese , Colicinas/química , Colicinas/farmacologia , Aditivos Alimentares/química , Aditivos Alimentares/metabolismo , Aditivos Alimentares/farmacologia , Testes de Sensibilidade Microbiana , Nicotina/antagonistas & inibidores , Nicotina/biossíntese , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/crescimento & desenvolvimento , Nicotiana/química , Nicotiana/metabolismoRESUMO
AIMS: The aim of this study was the coproduction in a single strain of the Gram-negative bacteriocin colicin V with other bacteriocins from lactic acid bacteria (LAB). METHODS AND RESULTS: Colicin V was expressed in Lactococcus and Enterococcus strains by replacing the colicin V leader peptide by the leader peptide and promoter of d-alanyl-d-alanine carboxypeptidase from Lactobacillus reuteri CECT925 in pNZ8048 (pNZ:LR-colV). The antimicrobial activity of colicin V against the indicator organism Escherichia coli DH5α in transformed strains was checked by agar diffusion assay and SDS-PAGE analysis. CONCLUSIONS: Lactococcus and Enterococcus transformed with pNZ:LR-colV were able to coproduce colicin V at high levels together with other LAB bacteriocins such as nisin A, nisin Z, lacticin 481 or enterocins A and B, obtaining broad-spectrum activity strains with large potential applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction showed in this work could be used for the heterologous expression of other bacteriocins active against Gram-negative bacteria or wide-spectrum bacteriocins from LAB.
Assuntos
Antibacterianos/metabolismo , Colicinas/biossíntese , Enterococcus/metabolismo , Ácido Láctico/metabolismo , Lactococcus/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Colicinas/química , Colicinas/farmacologia , Enterococcus/química , Escherichia coli/efeitos dos fármacos , Microbiologia Industrial , Lactococcus/química , Sinais Direcionadores de ProteínasRESUMO
Colicinogenic plasmids encode toxic proteins which have antagonistic activity against closely related bacteria. This study describes the molecular characterization of three colicinogenic plasmids designated as pSSE3, pSSE and pSSE2, each with a molecular size of â¼6 kb, identified in clinical isolates of Shigella sonnei. Sequence analysis revealed that pSSE and pSSE2 shared extensive sequence homology with each other and with Escherichia coli E-type colicinogenic plasmids. The plasmid pSSE3 lacked an additional gene imparting immunity to colicin E8, a unique feature not observed in any of the previously reported sequences of colicin E3 plasmids. Incomplete digestion of colicinogenic plasmids by restriction endonucleases, metachromatic staining with acridine orange and presence of single stranded initiation (ssi) region confirmed the coexistence of ssDNA along with dsDNA. Plasmid copy number as determined by real-time PCR was found to be about 20. Transmission electron microscopy revealed DNA impairment in test bacteria after colicin exposure. We hypothesize that S. sonnei has acquired E-group colicin plasmids from its close relative E. coli, with their sequences undergoing subtle changes depending on the cohabitation in the same milieu.
Assuntos
Colicinas/genética , Plasmídeos , Shigella sonnei/genética , Colicinas/farmacologia , Enzimas de Restrição do DNA/metabolismo , DNA de Cadeia Simples , Disenteria Bacilar/microbiologia , Escherichia coli/genética , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Shigella sonnei/efeitos dos fármacos , Shigella sonnei/ultraestruturaRESUMO
Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the "TolC box," was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. IMPORTANCE: The Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Colicinas/farmacologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Transporte ProteicoRESUMO
The Escherichia coli bacteriocin colicin M (ColM) acts via degradation of the cell wall precursor lipid II in target cells. ColM producers avoid self-inhibition by a periplasmic immunity protein anchored in the inner membrane. In this study, we identified colM-like bacteriocin genes in genomes of several ß-proteobacterial strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. Two selected Burkholderia ambifaria proteins, designated burkhocins M1 and M2, were produced recombinantly and showed antagonistic activity against Bcc strains. In their considerably sequence-diverged catalytic domain, a conserved aspartate residue equally proved pivotal for cytotoxicity. Immunity to M-type burkhocins is conferred upon susceptible strains by heterologous expression of a cognate gene located either upstream or downstream of the toxin gene. These genes lack homology with currently known ColM immunity genes and encode inner membrane-associated proteins of two distinct types, differing in predicted transmembrane topology and moiety exposed to the periplasm. The addition of burkhocins to the bacteriocin complement of Burkholderia reveals a wider phylogenetic distribution of ColM-like bacteriotoxins, beyond the γ-proteobacterial genera Escherichia, Pectobacterium and Pseudomonas, and illuminates the diversified nature of immunity-providing proteins.
Assuntos
Bacteriocinas/biossíntese , Complexo Burkholderia cepacia/metabolismo , Burkholderia pseudomallei/metabolismo , Burkholderia/metabolismo , Colicinas/biossíntese , Microbiologia do Solo , Sequência de Aminoácidos , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Burkholderia/classificação , Burkholderia/genética , Burkholderia/imunologia , Complexo Burkholderia cepacia/genética , Complexo Burkholderia cepacia/imunologia , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/imunologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colicinas/genética , Colicinas/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Mimetismo Molecular , Dados de Sequência Molecular , Filogenia , Pseudomonas/efeitos dos fármacos , Pseudomonas/genética , Pseudomonas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Alinhamento de SequênciaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing â¼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant- and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.