RESUMO
Bacteroides spp. are prominent gut commensals that are believed to modulate the intestinal environment, in part, by producing outer membrane vesicles (OMVs). Bacteroides OMVs have been ascribed many functions in vitro, but the genetic underpinnings behind OMV biogenesis and regulation are unclear. Understanding the mechanism of OMV biogenesis is required to determine the importance of Bacteroides OMVs in vivo. Here, we describe our methodology for screening Bacteroides thetaiotaomicron VPI-5482 to identify genes required for OMV biogenesis and regulation in a high-throughput format. This protocol is easily adaptable and can potentially be employed to further our knowledge of OMV biogenesis in other bacteria.
Assuntos
Bacteroides thetaiotaomicron , Bacteroides thetaiotaomicron/genética , Bacteroides thetaiotaomicron/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismoRESUMO
Circular dichroism (CD) is a spectroscopic technique commonly used for the analysis of proteins. Particularly, it allows the determination of protein secondary structure content in various media, including the membrane environment. In this chapter, we present how CD applications can be used to analyze the interaction of proteins with bacterial outer membrane vesicles (OMVs). Most CD studies characterizing the structure of proteins inserted into membranes rely on artificial lipid bilayers, mimicking natural membranes. Nevertheless, these artificial models lack the important features of the true membrane, especially for the outer membrane of Gram-negative bacteria. These features include lipid diversity, glycosylation, and asymmetry. Here, we show how to analyze the interactions of proteins, either integral or peripheral, with OMVs in solution and with supported membranes of OMVs, using conventional CD and orientated circular dichroism (OCD). We explain how to decipher the spectroscopic signals to obtain information on the molecular structure of the protein upon its interaction with an OMV and through its potential insertion into an OMV membrane.
Assuntos
Proteínas da Membrana Bacteriana Externa , Dicroísmo Circular , Síncrotrons , Dicroísmo Circular/métodos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Estrutura Secundária de Proteína , Bicamadas Lipídicas/metabolismo , Bicamadas Lipídicas/químicaRESUMO
Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.
Assuntos
Antígenos de Bactérias , Biotinilação , Vesículas Extracelulares , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Vacinas Bacterianas/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Desenvolvimento de Vacinas , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/imunologiaRESUMO
Outer membrane vesicles (OMVs) are small, spherical, nanoscale proteoliposomes released from Gram-negative bacteria that play an important role in cellular defense, pathogenesis, and signaling, among other functions. The functionality of OMVs can be enhanced by engineering developed for biomedical and biochemical applications. Here, we describe methods for directed packaging of enzymes into bacterial OMVs of E. coli using engineered molecular systems, such as localizing proteins to the inner or outer surface of the vesicle. Additionally, we detail some modification strategies for OMVs such as lyophilization and surfactant conjugation that enable the protection of activity of the packaged enzyme when exposed to non-physiological conditions such as elevated temperature, organic solvents, and repeated freeze/thaw that otherwise lead to a substantial loss in the activity of the free enzyme.
Assuntos
Escherichia coli , Proteolipídeos , Escherichia coli/metabolismo , Escherichia coli/genética , Proteolipídeos/metabolismo , Membrana Externa Bacteriana/metabolismo , Liofilização/métodos , Proteínas da Membrana Bacteriana Externa/metabolismo , Enzimas/metabolismo , Enzimas/químicaRESUMO
Developing programmable bacterial cell-cell adhesion is of significant interest due to its versatile applications. Current methods that rely on presenting cell adhesion molecules (CAMs) on bacterial surfaces are limited by the lack of a generalizable strategy to identify such molecules targeting bacterial membrane proteins in their natural states. Here, we introduce a whole-cell screening platform designed to discover CAMs targeting bacterial membrane proteins within a synthetic bacteria-displayed nanobody library. Leveraging the potency of the bacterial type IV secretion system-a contact-dependent DNA delivery nanomachine-we have established a positive feedback mechanism to selectively enrich for bacteria displaying nanobodies that target antigen-expressing cells. Our platform successfully identified functional CAMs capable of recognizing three distinct outer membrane proteins (TraN, OmpA, OmpC), demonstrating its efficacy in CAM discovery. This approach holds promise for engineering bacterial cell-cell adhesion, such as directing the antibacterial activity of programmed inhibitor cells toward target bacteria in mixed populations.
Assuntos
Aderência Bacteriana , Moléculas de Adesão Celular , Anticorpos de Domínio Único , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Anticorpos de Domínio Único/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/metabolismo , Bactérias/metabolismoRESUMO
Acinetobacter baumannii is a critical opportunistic pathogen associated with nosocomial infections. The high rates of antibiotic-resistance acquisition make most antibiotics ineffective. Thus, new medical countermeasures are urgently needed. Outer membrane proteins (OMPs) are prime candidates for developing novel drug targets and antibacterial strategies. However, there are substantial gaps in our knowledge of A. baumannii OMPs. This study reports the impact of OmpA-like protein on bacterial physiology and virulence in A. baumannii strain AB5075. We found that PsaB (ABUW_0505) negatively correlates to stress tolerance, while ArfA (ABUW_2730) significantly affects bacterial stiffness, cell shape, and cell envelope thickness. Furthermore, we expand our knowledge on YiaD (ABUW_3045), demonstrating structural and virulence roles of this porin, in addition to meropenem resistance. This study provides solid foundations for understanding how uncharacterized OMPs contribute to A. baumannii's physiological and pathological processes, aiding the development of innovative therapeutic strategies against A. baumannii infections.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas da Membrana Bacteriana Externa , Porinas , Acinetobacter baumannii/patogenicidade , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Virulência , Antibacterianos/farmacologia , Porinas/metabolismo , Porinas/genética , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Animais , Farmacorresistência Bacteriana , Camundongos , Testes de Sensibilidade Microbiana , FemininoRESUMO
Helicobacter pylori has been recognized not only as a causative agent of a spectrum of gastroduodenal diseases including chronic gastritis, peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer, but also as the culprit in several extra-gastric diseases. However, the association of H. pylori infection with extra-gastric diseases remains elusive, prompting a reevaluation of the role of H. pylori-derived outer membrane vesicles (OMVs). Like other gram-negative bacteria, H. pylori constitutively sheds biologically active OMVs for long-distance delivery of bacterial virulence factors in a concentrated and protected form, averting the need of direct bacterial contact with distant host cells to induce extra-gastric diseases associated with this gastric pathogen. Additionally, H. pylori-derived OMVs contribute to bacterial survival and chronic gastric pathogenesis. Moreover, the immunogenic activity, non-replicable nature, and anti-bacterial adhesion effect of H. pylori OMVs make them a desirable vaccine candidate against infection. The immunogenic potency and safety concerns of the OMV contents are challenges in the development of H. pylori OMV-based vaccines. In this review, we discuss recent advances regarding H. pylori OMVs, focusing on new insights into their biogenesis mechanisms and biological functions.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Humanos , Animais , Fatores de Virulência/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismoRESUMO
The translocation and assembly module (TAM) has been proposed to play a crucial role in the assembly of a small subset of outer membrane proteins (OMPs) in Proteobacteria based on experiments conducted in vivo using tamA and tamB mutant strains and in vitro using biophysical methods. TAM consists of an OMP (TamA) and a periplasmic protein that is anchored to the inner membrane by a single α helix (TamB). Here we examine the function of the purified E. coli complex in vitro after reconstituting it into proteoliposomes. We find that TAM catalyzes the assembly of four model OMPs nearly as well as the ß-barrel assembly machine (BAM), a universal heterooligomer that contains a TamA homolog (BamA) and that catalyzes the assembly of almost all E. coli OMPs. Consistent with previous results, both TamA and TamB are required for significant TAM activity. Our study provides direct evidence that TAM can function as an independent OMP insertase and describes a new method to gain insights into TAM function.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Proteolipídeos/metabolismo , Transporte ProteicoRESUMO
Chlamydia psittaci-a zoonotic pathogen in birds-may be transmitted to humans, causing severe respiratory disease. Individuals working in or living near poultry farms are highly susceptible to C. psittaci infection. In this study, we assessed the prevalence and genotypes of C. psittaci in poultries and humans in three cities of China by collecting fecal samples from different poultry species and throat swab samples and serum samples from workers in poultry farms and zoos. These samples were screened by real-time fluorescence quantitative PCR (qPCR) targeting C. psittaci ompA. The positive samples were subjected to PCR amplification and sequencing of ompA. The strains detected in the samples were genotyped on the basis of the phylogenetic analysis of ompA sequences. In total, 3.13% (40/1278) poultry fecal samples were positive in the qPCR assay, whereas 3.82% (6/157) of throat swab samples and 42.59% (46/108) of serum samples from the workers were positive in the qPCR and indirect fluorescent antibody assays, respectively. The strains detected in the 32 poultry samples and 6 human samples were genotyped as type A, indicating that the workers were infected with C. psittaci that originated in poultry birds in farms. Additionally, eight peacocks showed strains with the genotype CPX0308, which was identified in China for the first time. Elucidating the distribution of C. psittaci in animals and poultry-related workers may provide valuable insights for reducing the risk of C. psittaci infection within a population.
Assuntos
Chlamydophila psittaci , Genótipo , Filogenia , Psitacose , Animais , Chlamydophila psittaci/genética , Chlamydophila psittaci/isolamento & purificação , China/epidemiologia , Humanos , Psitacose/epidemiologia , Psitacose/veterinária , Psitacose/microbiologia , Prevalência , Aves Domésticas/microbiologia , Cidades/epidemiologia , Fezes/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/epidemiologiaRESUMO
Efflux-mediated ß-lactam resistance is a major public health concern, reducing the effectiveness of ß-lactam antibiotics against many bacteria. Structural analyses show the efflux protein TolC in Gram-negative bacteria acts as a channel for antibiotics, impacting bacterial susceptibility and virulence. This study examines ß-lactam drug efflux mediated by TolC using experimental and computational methods. Molecular dynamics simulations of drug-free TolC reveal essential movements and key residues involved in TolC opening. A whole-gene-saturation mutagenesis assay, mutating each TolC residue and measuring fitness effects under ß-lactam selection, is performed. Here we show the TolC-mediated efflux of three antibiotics: oxacillin, piperacillin, and carbenicillin. Steered molecular dynamics simulations identify general and drug-specific efflux mechanisms, revealing key positions at TolC's periplasmic entry affecting efflux motions. Our findings provide insights into TolC's structural dynamics, aiding the design of new antibiotics to overcome bacterial efflux mechanisms.
Assuntos
Antibacterianos , Proteínas da Membrana Bacteriana Externa , Simulação de Dinâmica Molecular , Resistência beta-Lactâmica , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Antibacterianos/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Conformação ProteicaRESUMO
Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella Typhimurium increases OMV production inside the acidic vacuoles of host cells by changing expression of its outer membrane proteins and modifying the composition of lipid A. However, the molecular mechanisms that translate pH changes into OMV production are not completely understood. Here, we show that the outer membrane protein PagC promotes OMV production through pH-dependent interactions between its extracellular loops and surrounding lipopolysaccharide (LPS). Structural comparisons and mutational studies indicate that a pH-responsive amino acid motif in PagC extracellular loops, containing PagC-specific histidine residues, is crucial for OMV formation. Molecular dynamics simulations suggest that protonation of histidine residues leads to changes in the structure and flexibility of PagC extracellular loops and their interactions with the surrounding LPS, altering membrane curvature. Consistent with that hypothesis, mimicking acidic pH by mutating those histidine residues to lysine increases OMV production. Thus, our findings reveal a mechanism for sensing and responding to environmental pH and for control of membrane dynamics by outer membrane proteins.
Assuntos
Proteínas da Membrana Bacteriana Externa , Lipopolissacarídeos , Simulação de Dinâmica Molecular , Salmonella typhimurium , Concentração de Íons de Hidrogênio , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/genética , Lipopolissacarídeos/metabolismo , Membrana Externa Bacteriana/metabolismo , Motivos de Aminoácidos , Histidina/metabolismoRESUMO
Siderophores are specialized molecules produced by bacteria and fungi to scavenge iron, a crucial nutrient for growth and metabolism. Catecholate-type siderophores are mainly produced by bacteria, while hydroxamates are mostly from fungi. This study investigates the capacity of nine hydroxamate-type siderophores from fungi and Streptomyces to facilitate iron acquisition by the human pathogen Pseudomonas aeruginosa. Growth assays under iron limitation and 55Fe incorporation tests showed that all nine siderophores promoted bacterial growth and iron transport. The study also aimed to identify the TonB-dependent transporters (TBDTs) involved in iron import by these siderophores. Using mutant strains lacking specific TBDT genes, it was found that iron is imported into P. aeruginosa cells by FpvB for coprogen, triacetylfusarinine, fusigen, ferrirhodin, and ferrirubin. Iron complexed by desferioxamine G is transported by FpvB and FoxA, ferricrocin-Fe and ferrichrycin-Fe by FpvB and FiuA, and rhodotoluric acid-Fe by FpvB, FiuA, and another unidentified TBDT. These findings highlight the effectiveness of hydroxamate-type siderophores in iron transport into P. aeruginosa and provide insights into the complex molecular mechanisms involved, which are important for understanding microbial interactions and ecological balance.
Assuntos
Proteínas de Bactérias , Ácidos Hidroxâmicos , Ferro , Pseudomonas aeruginosa , Sideróforos , Sideróforos/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/genética , Ferro/metabolismo , Ácidos Hidroxâmicos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Ferricromo/metabolismo , Ferricromo/análogos & derivados , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas da Membrana Bacteriana Externa , Proteínas de Membrana , Receptores de Superfície CelularRESUMO
At the end of 2022 into early 2023, the UK Health Security Agency reported unusually high levels of scarlet fever and invasive disease caused by Streptococcus pyogenes (StrepA or group A Streptococcus). During this time, we collected and genome-sequenced 341 non-invasive throat and skin S. pyogenes isolates identified during routine clinical diagnostic testing in Sheffield, a large UK city. We compared the data with that obtained from a similar collection of 165 isolates from 2016 to 2017. Numbers of throat-associated isolates collected peaked in early December 2022, reflecting the national scarlet fever upsurge, while skin infections peaked later in December. The most common emm-types in 2022-2023 were emm1 (28.7â%), emm12 (24.9â%) and emm22 (7.7â%) in throat and emm1 (22â%), emm12 (10â%), emm76 (18â%) and emm49 (7â%) in skin. While all emm1 isolates were the M1UK lineage, the comparison with 2016-2017 revealed diverse lineages in other emm-types, including emm12, and emergent lineages within other types including a new acapsular emm75 lineage, demonstrating that the upsurge was not completely driven by a single genotype. The analysis of the capsule locus predicted that only 51â% of throat isolates would produce capsule compared with 78% of skin isolates. Ninety per cent of throat isolates were also predicted to have high NADase and streptolysin O (SLO) expression, based on the promoter sequence, compared with only 56% of skin isolates. Our study has highlighted the value in analysis of non-invasive isolates to characterize tissue tropisms, as well as changing strain diversity and emerging genomic features which may have implications for spillover into invasive disease and future S. pyogenes upsurges.
Assuntos
Infecções Estreptocócicas , Streptococcus pyogenes , Streptococcus pyogenes/genética , Streptococcus pyogenes/classificação , Streptococcus pyogenes/isolamento & purificação , Humanos , Reino Unido , Infecções Estreptocócicas/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Antígenos de Bactérias/genética , Faringe/microbiologia , Escarlatina/microbiologia , Escarlatina/epidemiologia , Proteínas de Transporte/genética , Estreptolisinas/genética , Sequenciamento Completo do Genoma/métodos , Proteínas de Bactérias/genética , Filogenia , Criança , Adulto , NAD+ Nucleosidase/genética , NAD+ Nucleosidase/metabolismo , Pele/microbiologia , Pré-Escolar , MasculinoRESUMO
As other European countries, France is experiencing a resurgence of pertussis in 2024. Between 1 January and 31 May 2024, 5,616 (24.9%) positive Bordetella pertussis qPCR tests were identified, following a 3-year period of almost null incidence. Of 67 cultured and whole genome sequenced B. pertussis isolates, 66 produced pertactin and 56 produced FIM2, in contrast to pre-COVID-19 years. One isolate of genotype Bp-AgST4 was resistant to macrolides. Pertussis resurgence may favour isolates that produce FIM2 and pertactin.
Assuntos
Antibacterianos , Bordetella pertussis , Macrolídeos , Coqueluche , Bordetella pertussis/genética , Bordetella pertussis/isolamento & purificação , Bordetella pertussis/efeitos dos fármacos , Humanos , França/epidemiologia , Macrolídeos/farmacologia , Coqueluche/epidemiologia , Coqueluche/microbiologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Proteínas da Membrana Bacteriana Externa/genética , Sequenciamento Completo do Genoma , Fatores de Virulência de Bordetella/genética , Genótipo , Adulto , Criança , Incidência , Pré-EscolarRESUMO
V. parahaemolyticus is a Gram-negative bacterium that causes gastroenteritis. Within the realm of bacterial interactions with the gut, the outer membrane protein MAM7 plays a key role. However, the precise function of MAM7 in intestinal inflammation, particularly its interactions with macrophages, remains unclear. In this study, we successfully expressed and purified recombinant MAM7. After optimization of the MAM7 expression condition, it was found that the optimal concentration and temperature were 0.75 mM and 15 °C, respectively, resulting in a 27-fold increase in its yield. Furthermore, RAW264.7 cytotoxicity assay was conducted. The CCK-8 results revealed that MAM7 substantially stimulated the proliferation of RAW264.7 cells, with its optimal concentration determined to be 7.5 µg/mL. Following this, the NO concentration of MAM7 was tested, revealing a significant increase (p < 0.05) in NO levels. Additionally, the relative mRNA levels of IL-1ß, IL-6, and TNF-α in RAW264.7 cells were measured by qRT-PCR, showing a remarkable elevation (p < 0.05). Moreover, ELISA results demonstrated that MAM7 effectively stimulated the secretion of IL-6 and TNF-α by RAW264.7 cells. In summary, these findings strongly suggest that MAM7 serves as a proinflammatory adhesion factor with the capacity to modulate immune responses.
Assuntos
Macrófagos , Proteínas Recombinantes , Vibrio parahaemolyticus , Animais , Células RAW 264.7 , Camundongos , Vibrio parahaemolyticus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Expressão GênicaRESUMO
Lyme disease is caused by the spirochete, Borrelia burgdorferi, which is transmitted by Ixodes spp ticks. The rise in Lyme disease cases since its discovery in the 1970s has reinforced the need for a vaccine. A vaccine based on B burgdorferi outer surface protein A (OspA) was approved by the Food and Drug Administration (FDA) several decades ago, but was pulled from the market a few years later, reportedly due to poor sales, despite multiple organizations concluding that it was safe and effective. Newer OspA-based vaccines are being developed and are likely to be available in the coming years. More recently, there has been a push to develop vaccines that target the tick vector instead of the pathogen to inhibit tick feeding and thus prevent transmission of tick-borne pathogens to humans and wildlife reservoirs. This review outlines the history of Lyme disease vaccines and this movement to anti-tick vaccine approaches.
Assuntos
Borrelia burgdorferi , Ixodes , Vacinas contra Doença de Lyme , Doença de Lyme , Doença de Lyme/prevenção & controle , Doença de Lyme/imunologia , Humanos , Animais , Borrelia burgdorferi/imunologia , Vacinas contra Doença de Lyme/imunologia , Ixodes/microbiologia , Vacinação , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Antígenos de Superfície/imunologia , Lipoproteínas/imunologiaRESUMO
Initiation of DNA replication in Escherichia coli is coupled to cell size via the DnaA protein, whose activity is dependent on its nucleotide-bound state. However, the oscillations in DnaA activity have never been observed at the single-cell level. By measuring the volume-specific production rate of a reporter protein under control of a DnaA-regulated promoter, we could distinguish two distinct cell-cycle oscillators. The first, driven by both DnaA activity and SeqA repression, shows a causal relationship with cell size and divisions, similarly to initiation events. The second one, a reporter of DnaA activity alone, loses the synchrony and causality properties. Our results show that transient inhibition of gene expression by SeqA keeps the oscillation of volume-sensing DnaA activity in phase with the subsequent division event and suggest that DnaA activity peaks do not correspond directly to initiation events.
Assuntos
Ciclo Celular , Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Análise de Célula Única , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Replicação do DNA , Regiões Promotoras Genéticas , Proteínas da Membrana Bacteriana ExternaRESUMO
Previous observational studies found associations between Helicobacter pylori infection and autoimmune thyroid diseases (AITDs), but the causal nature of this association is still uncertain. We investigated the causal effect of six crucial antibodies against H. pylori on AITDs using a bidirectional Mendelian randomization (MR). We found that anti-H. pylori outer membrane protein (OMP) significantly increased the risk of hyperthyroidism and Graves' disease (GD). In addition, our reverse MR analysis indicated that hyperthyroidism could increase the levels of cytotoxin-associated gene A and OMP antibodies. We also observed causal roles of GD on anti-H. pylori OMP. Our analyses indicate the mutual effects of H. pylori infection and AITDs, suggesting the existence of a gut-thyroid axis. These results also provide evidence of the bidirectional causal association between anti-H. pylori OMP with hyperthyroidism and GD, resulting in a vicious circle.
Assuntos
Doença de Graves , Infecções por Helicobacter , Helicobacter pylori , Análise da Randomização Mendeliana , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/complicações , Humanos , Doença de Graves/genética , Doença de Graves/microbiologia , Hipertireoidismo/genética , Hipertireoidismo/microbiologia , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Doenças Autoimunes/genética , Doenças Autoimunes/microbiologia , Antígenos de Bactérias , Proteínas de BactériasRESUMO
Multidrug efflux pumps, especially those belonging to the class of resistance-nodulation-division (RND), are the key contributors to the rapidly growing multidrug resistance in Gram-negative bacteria. Understanding the role of efflux pumps in real-time drug transport dynamics across the complex dual-cell membrane envelope of Gram-negative bacteria is thus crucial for developing efficient antibiotics against them. Here, we employ second harmonic generation-based nonlinear spectroscopy to study the role of the tripartite efflux pump and its individual components. We systematically investigate the effect of periplasmic adaptor protein AcrA, inner membrane transporter protein AcrB, and outer membrane channel TolC on the overall drug transport in live Acr-type Escherichia coli and its mutant strain cells. Our results reveal that when one of its components is missing, the tripartite AcrAB-TolC efflux pump machinery in Escherichia coli can effectively function as a bipartite system, a fact that has never been demonstrated in live Gram-negative bacteria.
Assuntos
Antibacterianos , Proteínas de Escherichia coli , Escherichia coli , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Análise Espectral/métodos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Transporte Biológico , LipoproteínasRESUMO
Helicobacter pylori is a common resident in the stomach of at least half of the world's population and recent evidence suggest its emergence in other organs such as the pancreas. In this organ, the presence of H. pylori DNA has been reported in cats, although the functional implications remain unknown. In this work, we determined distinct features related to the H. pylori manifestation in pancreas in a rodent model, in order to analyse its functional and structural effect. Gerbils inoculated with H. pylori exhibited the presence of this bacterium, as revealed by the expression of some virulence factors, as CagA and OMPs in stomach and pancreas, and confirmed by urease activity, bacterial culture, PCR and immunofluorescence assays. Non-apparent morphological changes were observed in pancreatic tissue of infected animals; however, delocalization of intercellular junction proteins (claudin-1, claudin-4, occludin, ZO-1, E-cadherin, ß-catenin, desmoglein-2 and desmoplakin I/II) and rearrangement of the actin-cytoskeleton were exhibited. This structural damage was consistent with alterations in the distribution of insulin and glucagon, and a systemic inflammation, event demonstrated by elevated IL-8 levels. Overall, these findings indicate that H. pylori can reach the pancreas, possibly affecting its function and contributing to the development of pancreatic diseases.