RESUMEN
The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.
Asunto(s)
Pared Celular/genética , Proteínas de Escherichia coli/genética , Lipopolisacáridos/genética , Oxidorreductasas/genética , Peptidoglicano/genética , División Celular/genética , Membrana Celular/genética , Membrana Celular/microbiología , Pared Celular/microbiología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Lipopolisacáridos/biosíntesis , Mutagénesis , Fosfolípidos/biosíntesis , Fosfolípidos/genéticaRESUMEN
Bacterial amidases are essential to split the shared envelope of adjunct daughter cells to allow cell separation. Their activity needs to be precisely controlled to prevent cell lysis. In Escherichia coli, amidase activity is controlled by three regulatory proteins NlpD, EnvC and ActS. However, recent studies linked the outer membrane lipoprotein DolP (formerly YraP) as a potential upstream regulator of NlpD. In this study we explored this link in further detail. To our surprise DolP did not modulate amidase activity in vitro and was unable to interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. Next, we excluded the hypothesis that ΔdolP phenocopied ΔnlpD in a range of envelope stresses. However, morphological analysis of double deletion mutants of amidases (AmiA, AmiB AmiC) and amidase regulators with dolP revealed that ΔamiAΔdolP and ΔenvCΔdolP mutants display longer chain length compared to their parental strains indicating a role for DolP in cell division. Overall, we present evidence that DolP does not affect NlpD function in vitro, implying that DolP is not an upstream regulator of NlpD. However, DolP may impact daughter cell separation by interacting directly with AmiA or AmiC, or by a yet undiscovered mechanism.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Separación Celular , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Peptidoglicano/metabolismoRESUMEN
Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Dinámicas Mitocondriales , Mitofagia , Músculo Esquelético/patología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Activación Enzimática , Células HeLa , Humanos , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Ionóforos de Protónes/farmacología , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The asymmetric Gram-negative outer membrane (OM) is the first line of defense for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here, we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active-site plug. Informed by our structure, we demonstrate that free movement of the active-site plug is essential for BepA function, suggesting that the protein is autoregulated by the active-site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the tetratricopeptide repeat (TPR) cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Last, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid.IMPORTANCE M48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Metaloproteasas/química , Metaloproteasas/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cristalografía por Rayos X , Proteínas de Escherichia coli/aislamiento & purificación , Metaloproteasas/aislamiento & purificación , Permeabilidad , Sensibilidad y Especificidad , Relación Estructura-ActividadRESUMEN
The activity of any bacterial promoter is generally supposed to be set by its base sequence and the different transcription factors that bind in the local vicinity. Here, we review recent data indicating that the activity of the Escherichia coli lac operon promoter also depends upon its chromosomal location. Factors that affect promoter activity include the binding of nucleoid-associated proteins to neighbouring sequences, supercoiling and the activity of neighbouring promoters. We suggest that many bacterial promoters might be susceptible to similar position-dependent effects and we review recent data showing that the expression of mobile genes encoding antibiotic-resistance determinants is also location-dependent, both when carried on a bacterial chromosome or a conjugative plasmid.
Asunto(s)
Efectos de la Posición Cromosómica , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Regiones Promotoras Genéticas , Cromosomas Bacterianos , Elementos Transponibles de ADN , Operón Lac , Plásmidos , Transcripción GenéticaRESUMEN
Mutations in σE-regulated lipoproteins have previously been shown to impact bacterial viability under conditions of stress and during in vivo infection. YraP is conserved across a number of Gram-negative pathogens, including Neisseria meningitidis, where the homolog is a component of the Bexsero meningococcal group B vaccine. Investigations using laboratory-adapted Escherichia coli K-12 have shown that yraP mutants have elevated sensitivity to a range of compounds, including detergents and normally ineffective antibiotics. In this study, we investigate the role of the outer membrane lipoprotein YraP in the pathogenesis of Salmonella enterica serovar Typhimurium. We show that mutations in S Typhimurium yraP result in a defective outer membrane barrier with elevated sensitivity to a range of compounds. This defect is associated with attenuated virulence in an oral infection model and during the early stages of systemic infection. We show that this attenuation is not a result of defects in lipopolysaccharide and O-antigen synthesis, changes in outer membrane protein levels, or the ability to adhere to and invade eukaryotic cell lines in vitro.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/patología , Salmonella typhimurium/patogenicidad , Factores de Virulencia/metabolismo , Animales , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Modelos Animales de Enfermedad , Células Epiteliales/microbiología , Humanos , Lipoproteínas/genética , Macrófagos/microbiología , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Mutación , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Virulencia , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome. RESULTS: We developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication. CONCLUSION: Regions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.
Asunto(s)
Factor de Transcripción de AraC/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/metabolismo , Regiones Operadoras Genéticas , Factor de Transcripción de AraC/genética , Arabinosa/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Operón , Regiones Promotoras Genéticas , Proteína Fluorescente RojaRESUMEN
In eukaryotes, the location of a gene on the chromosome is known to affect its expression, but such position effects are poorly understood in bacteria. Here, using Escherichia coli K-12, we demonstrate that expression of a reporter gene cassette, comprised of the model E. coli lac promoter driving expression of gfp, varies by â¼300-fold depending on its precise position on the chromosome. At some positions, expression was more than 3-fold higher than at the natural lac promoter locus, whereas at several other locations, the reporter cassette was completely silenced: effectively overriding local lac promoter control. These effects were not due to differences in gene copy number, caused by partially replicated genomes. Rather, the differences in gene expression occur predominantly at the level of transcription and are mediated by several different features that are involved in chromosome organization. Taken together, our findings identify a tier of gene regulation above local promoter control and highlight the importance of chromosome position effects on gene expression profiles in bacteria.
Asunto(s)
Posicionamiento de Cromosoma , Escherichia coli K12/genética , Regulación Bacteriana de la Expresión Génica , Girasa de ADN/metabolismo , Escherichia coli K12/enzimología , Escherichia coli K12/metabolismo , Dosificación de Gen , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Escherichia coli has been the leading model organism for many decades. It is a fundamental player in modern biology, facilitating the molecular biology revolution of the last century. The acceptance of E. coli as model organism is predicated primarily on the study of one E. coli lineage; E. coli K-12. However, the antecedents of today's laboratory strains have undergone extensive mutagenesis to create genetically tractable offspring but which resulted in loss of several genetic traits such as O antigen expression. Here we have repaired the wbbL locus, restoring the ability of E. coli K-12 strain MG1655 to express the O antigen. We demonstrate that O antigen production results in drastic alterations of many phenotypes and the density of the O antigen is critical for the observed phenotypes. Importantly, O antigen production enables laboratory strains of E. coli to enter the gut of the Caenorhabditis elegans worm and to kill C. elegans at rates similar to pathogenic bacterial species. We demonstrate C. elegans killing is a feature of other commensal E. coli. We show killing is associated with bacterial resistance to mechanical shear and persistence in the C. elegans gut. These results suggest C. elegans is not an effective model of human-pathogenic E. coli infectious disease.
Asunto(s)
Caenorhabditis elegans/microbiología , Escherichia coli K12/metabolismo , Escherichia coli K12/patogenicidad , Antígenos O/biosíntesis , Animales , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Antígenos O/genéticaRESUMEN
Gram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in Escherichia coli, we probed the genetic interactions of mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆mrcB cells is significantly reduced under high salt stress and in vitro activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCEEscherichia coli and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in E. coli in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions.
Asunto(s)
Proteínas de Escherichia coli , Peptidoglicano Glicosiltransferasa , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Peptidoglicano Glicosiltransferasa/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Pared Celular/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismoRESUMEN
Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.
Asunto(s)
Elementos Transponibles de ADN , Klebsiella pneumoniae , Orina , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/crecimiento & desarrollo , Humanos , Elementos Transponibles de ADN/genética , Orina/microbiología , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/orina , Mutagénesis Insercional , Suero/microbiología , MutagénesisRESUMEN
The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD ß6-ß7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de Escherichia coli , Escherichia coli , Periplasma , Fosfolípidos , Fosfolípidos/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Periplasma/metabolismo , Transporte Biológico , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Modelos Moleculares , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de la MembranaRESUMEN
In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterized genes. To address this gap in functional annotation, many high-throughput screens have been devised to uncover novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered library where a gene has been replaced or disrupted by a uniform linear insertion (LI). We applied our method (LI-detector) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates, with a success rate of 99.7% for identifying the correct kanamycin cassette position. This data set provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of any linear insertion, such as an antibiotic resistance cassette in any ordered library. IMPORTANCE The construction of ordered gene replacement libraries requires significant investment of time and resources to create a valuable community resource. During construction, technical errors may result in a limited number of incorrect mutants being made. Such mutants may confound the output of subsequent experiments. Here, using the remarkable E. coli Keio knockout library, we describe a method to rapidly validate the construction of every mutant.
Asunto(s)
Elementos Transponibles de ADN , Infecciones por Escherichia coli , Escherichia coli/genética , Biblioteca de Genes , Humanos , Mutagénesis InsercionalRESUMEN
There is an increasing focus in healthcare environments on combatting antimicrobial resistant infections. While bacterial infections are well reported, infections caused by fungi receive less attention, yet have a broad impact on society and can be deadly. Fungi are eukaryotes with considerable shared biology with humans, therefore limited technologies exist to combat fungal infections and hospital infrastructure is rarely designed for reducing microbial load. In this study, a novel antimicrobial surface (AMS) that is modified with the broad-spectrum biocide chlorhexidine is reported. The surfaces are shown to kill the opportunistic fungal pathogens Candida albicans and Cryptococcus neoformans very rapidly (<15 min) and are significantly more effective than current technologies available on the commercial market, such as silver and copper.
RESUMEN
The COVID-19 pandemic has demonstrated the real need for mechanisms to control the spread of airborne respiratory pathogens. Thus, preventing the spread of disease from pathogens has come to the forefront of the public consciousness. This has brought an increasing demand for novel technologies to prioritise clean air. In this study we report on the efficacy of novel biocide treated filters and their antimicrobial activity against bacteria, fungi and viruses. The antimicrobial filters reported here are shown to kill pathogens, such as Candida albicans, Escherichia coli and MRSA in under 15 min and to destroy SARS-CoV-2 viral particles in under 30 s following contact with the filter. Through air flow rate testing, light microscopy and SEM, the filters are shown to maintain their structure and filtration function. Further to this, the filters are shown to be extremely durable and to maintain antimicrobial activity throughout the operational lifetime of the product. Lastly, the filters have been tested in field trials onboard the UK rail network, showing excellent efficacy in reducing the burden of microbial species colonising the air conditioning system.
Asunto(s)
Filtros de Aire/microbiología , Antiinfecciosos/química , Antivirales/química , Filtros de Aire/virología , Antiinfecciosos/farmacología , Antivirales/farmacología , COVID-19/epidemiología , COVID-19/virología , Candida albicans/efectos de los fármacos , Clorhexidina/análogos & derivados , Clorhexidina/química , Clorhexidina/farmacología , Escherichia coli/efectos de los fármacos , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Factores de TiempoRESUMEN
Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis.
Asunto(s)
Bacteriemia , Toxinas Bacterianas , Proteínas de Escherichia coli , Escherichia coli Uropatógena , Animales , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ratones , Serina Endopeptidasas/metabolismo , Serina Proteasas/genética , Sistemas de Secreción Tipo V/genética , Sistemas de Secreción Tipo V/metabolismoRESUMEN
AIM: The development of a polyarginine cell-penetrating peptide (CPP) could enable the treatment of age-related macular degeneration, with drugs like bevacizumab, to be administered using eye drops instead of intravitreal injections. Topical formulations have a vast potential impact on healthcare by increasing patient compliance while reducing the financial burden. However, as the ocular preparations may contain several doses, it is essential to understand the stability of the bevacizumab+CPP conjugate produced. MATERIALS AND METHODS: In this work, we examine the stability of a bevacizumab solution with and without cell-penetrating peptide using dynamic light scattering and circular dichroism to assess the physical stability. We use HPLC to assess the chemical stability and ELISA to assess its biological activity. We also examine the potential of the CPP to be used as an antimicrobial agent in place of preservatives in the eye drop. RESULTS: The structural stability of bevacizumab with and without the CPP was found not to be affected by temperature: samples stored at either 20°C or 4°C were identical in behavior. However, physical instability was observed after five weeks, leading to aggregation and precipitation. Further investigation revealed that the addition of the polypeptide led to increased aggregation, as revealed through dynamic light scattering and concentration analysis of the peptide through HPLC. Complexing the bevacizumab with CPP had no effect on biological stability or degradation. CONCLUSIONS: Our findings suggest that the shelf life of CPP+bevacizumab complexes is at least 38 days from its initial formulation. Currently, the mechanism for aggregation is not fully understood but does not appear to occur through chemical degradation.
Asunto(s)
Inhibidores de la Angiogénesis/química , Bevacizumab/química , Péptidos de Penetración Celular/química , Degeneración Macular/tratamiento farmacológico , Péptidos/química , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Sistemas de Liberación de Medicamentos , Estabilidad de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Luz , Soluciones Oftálmicas , Preparaciones Farmacéuticas , Dispersión de RadiaciónRESUMEN
Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Inmunización , Antígenos O/inmunología , Salmonella typhimurium/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Formación de Anticuerpos , Especificidad de Anticuerpos , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Protección Cruzada , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/inmunología , Inmunoglobulina G/sangre , Ratones , Modelos Moleculares , Antígenos O/química , Antígenos O/genética , Porinas/química , Porinas/genética , Porinas/inmunología , Conformación Proteica , Salmonelosis Animal/inmunología , Salmonelosis Animal/prevención & control , Análisis de Secuencia de ProteínaRESUMEN
The intimin protein is the major adhesin involved in the intimate adherence of atypical enteropathogenic Escherichia coli (aEPEC) strains to epithelial cells, but little is known about the structures involved in their early colonization process. A previous study demonstrated that the type III secretion system (T3SS) plays an additional role in the adherence of an Escherichia albertii strain. Therefore, we assumed that the T3SS could be related to the adherence efficiency of aEPEC during the first stages of contact with epithelial cells. To test this hypothesis, we examined the adherence of seven aEPEC strains and their eae (intimin) isogenic mutants in the standard HeLa adherence assay and observed that all wild-type strains were adherent while five isogenic eae mutants were not. The two eae mutant strains that remained adherent were then used to generate the eae/escN double mutants (encoding intimin and the T3SS ATPase, respectively) and after the adherence assay, we observed that one strain lost its adherence capacity. This suggested a role for the T3SS in the initial adherence steps of this strain. In addition, we demonstrated that this strain expressed the T3SS at significantly higher levels when compared to the other wild-type strains and that it produced longer translocon-filaments. Our findings reveal that the T3SS-translocon can play an additional role as an adhesin at the beginning of the colonization process of aEPEC.
RESUMEN
The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting ß-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.