RESUMO
In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen.
Assuntos
Anticorpos Monoclonais , Eletroforese Capilar/métodos , Focalização Isoelétrica/métodos , Proteínas Recombinantes , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/químicaRESUMO
Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering and evaluating data based on scientific understanding and clinical experience of the relationship between product quality attributes and their impact on safety and efficacy. This review summarizes the current understanding of various modifications of recombinant monoclonal antibodies. It further outlines the critical steps in designing and executing successful comparability studies to support process changes at different stages of a product's lifecycle.
Assuntos
Anticorpos Monoclonais/química , Química Farmacêutica/métodos , Química Farmacêutica/normas , Garantia da Qualidade dos Cuidados de Saúde/métodos , Proteínas Recombinantes/química , Descoberta de Drogas , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Forced degradation studies have become integral to the development of recombinant monoclonal antibody therapeutics by serving a variety of objectives from early stage manufacturability evaluation to supporting comparability assessments both pre- and post- marketing approval. This review summarizes the regulatory guidance scattered throughout different documents to highlight the expectations from various agencies such as the Food and Drug Administration and European Medicines Agency. The various purposes for forced degradation studies, commonly used conditions and the major degradation pathways under each condition are also discussed.
Assuntos
Anticorpos Monoclonais/metabolismo , Química Farmacêutica/métodos , Proteínas Recombinantes/metabolismo , Tecnologia Farmacêutica/métodos , Anticorpos Monoclonais/genética , Guias como Assunto , Humanos , Concentração de Íons de Hidrogênio , Estabilidade Proteica , Proteólise , TemperaturaRESUMO
Glycosylation of the conserved asparagine residue in the CH2 domain is the most common posttranslational modification of recombinant monoclonal antibodies. Ideally, a consistent oligosaccharide profile should be maintained from early clinical material to commercial material for the development of recombinant monoclonal therapeutics, though variation in the profile is a typical result of process changes. The risk of oligosaccharide variation posed to further development is required to be thoroughly evaluated based on its impact on antibody structure, stability, efficacy and safety. The variation should be controlled within a range so that there is no detrimental impact on safety and efficacy and thus allowing the use of early phase safety and efficacy data to support project advancement to later phase. This review article focuses on the current scientific understanding of the commonly observed oligosaccharides found in recombinant monoclonal antibodies and their impact on structure, stability and biological functions, which are the basis to evaluate safety and efficacy. It also provides a brief discussion on critical quality attribute (CQA) assessment with regard to oligosaccharides based on the mechanism of action (MOA). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1173-1181, 2017.
Assuntos
Anticorpos Monoclonais , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Oligossacarídeos/química , Proteínas Recombinantes , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Células CHO , Cricetinae , Cricetulus , Humanos , Estabilidade Proteica , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/normas , Proteínas Recombinantes/uso terapêuticoRESUMO
LC-MS peptide mapping is the most commonly used method to analyze protein modifications. The proteins are generally digested using trypsin at a slightly basic pH at 37 °C from several hours to overnight. Assay-induced artifacts can be generated during this procedure, potentially causing false-positive or false-negative results for a given modification. Unfortunately, for the analysis of succinimide, both false-negative and false-positive results can be generated within the same procedure. This study evaluates the stability of succinimide during the peptide mapping procedure and has demonstrated that up to 13% of pre-existing succinimide was lost during a 4 h trypsin digestion at pH 5.0 which was previously determined to be optimal for the detection of succinimide. The same procedure was able to simultaneously generate approximately 3% succinimide. Using the optimized procedure, it was also found that two aspartate residues that are followed by glycine residues in the conserved Fc region of a recombinant monoclonal antibody were not prone to isomerization. On the other hand, an aspartate residue followed by a glycine in the heavy chain variable domain was highly susceptible to isomerization. Interestingly, the antibody containing the succinimide eluted from an SEC column after the monomer peak.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Muramidase/química , Muramidase/metabolismo , Succinimidas/urina , Tripsina/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Ácido Aspártico/química , Humanos , Mapeamento de PeptídeosRESUMO
An efficient strategy to characterize recombinant monoclonal antibody charge variants was established using weak anion exchange chromatography, LC-MS and IdeS digestion to allow subunit level characterization. Significantly higher resolution was achieved at subunit levels by weak anion exchange chromatography and LC-MS. In addition, subunit analysis localized potential modifications to either F(ab')2 or Fc fragments to facilitate further characterization. Peptide mapping of fractions from various charge variants after IdeS digestion identified aspartate isomerization, asparagine deamidation and glycation as the modifications. Although, aspartate isomerization does not generate net charge difference directly, it does generate antibody basic species. Antibodies with either isoaspartate or aspartate from deamidation showed different retention times by chromatography. Even more interestingly, the antibody contained succinimide as the isomerization intermediate, which though more basic compared to aspartate, eluted off the weak anion exchange column as an acidic species. The results demonstrated not only the utility of subunit level characterization but also the unpredictable chromatographic behavior of antibody charge variants.
Assuntos
Anticorpos Monoclonais/química , Cromatografia por Troca Iônica , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida de Alta Pressão , Imunoglobulina G/química , Imunoglobulina G/genética , Isomerismo , Espectrometria de Massas , Peptídeos/análise , Peptídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103-1112, 2016.
Assuntos
Anticorpos Monoclonais/metabolismo , Técnicas de Cultura de Células , Oligossacarídeos/metabolismo , Animais , Anticorpos Monoclonais/química , Humanos , Oligossacarídeos/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
A recombinant monoclonal antibody with trisulfide bonds and cysteinylation was thoroughly characterized in the current study. Trisulfide bonds and cysteinylation were first detected when the recombinant monoclonal antibody was analyzed by LC-MS to determine the molecular weights of the intact antibody and its F(ab')2 fragment generated from IdeS digestion. LC-MS analysis of nonreduced tryptic peptides indicated trisulfide bonds are associated with the interchain disulfide bonds of both A isoform and A/B isoform and cysteinylation is associated only with the A isoform. A low percentage of trisulfide bonds was detected in between the light chain and heavy chain disulfide bond of the A and A/B forms. While the majority of trisulfide bonds and cysteinylation is associated with the hinge region peptide that involves the four closely spaced cysteine residues of the heavy chain. The locations of trisulfide bond and cysteinylation were determined using a combination of Edman sequencing and LC-MS. In the A isoform, the major site of the trisulfide bond and cysteinylation is between the first disulfide bond in the hinge region. In the A/B isoform, the trisulfide was also located in between the disulfide bond that is formed by the second pair of cysteine residues.
Assuntos
Anticorpos Monoclonais/química , Cisteína/química , Sulfetos/química , Espectrometria de Massas em Tandem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Mapeamento de Peptídeos , Peptídeos/análise , Peptídeos/isolamento & purificação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Light exposure is one of several conditions used to study the degradation pathways of recombinant monoclonal antibodies. Tryptophan is of particular interest among the 20 amino acids because it is the most photosensitive. Tryptophan degradation forms several products, including an even stronger photosensitizer and several reactive oxygen species. The current study reports a specific peptide mapping procedure to monitor tryptophan degradation. Instead of monitoring peptides using UV 214 nm, fluorescence detection with an excitation wavelength of 295 nm and an emission wavelength of 350 nm was used to enable specific detection of tryptophan-containing peptides. Peaks that decreased in area over time are likely to contain susceptible tryptophan residues. This observation can allow further liquid chromatography-mass spectrometry (LC-MS) analysis to focus only on those peaks to confirm tryptophan degradation products. After confirmation of tryptophan degradation, susceptibility of tryptophan residues can be compared based on the peak area decrease.
Assuntos
Anticorpos Monoclonais/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Espectrometria de Fluorescência/métodos , Triptofano/metabolismo , Proteólise , Proteínas Recombinantes/metabolismoRESUMO
Limited proteolytic digestion is a method with a long history that has been used to study protein domain structures and conformational changes. A method of combining limited proteolytic digestion, stable isotope labeling, and mass spectrometry was established in the current study to investigate protein conformational changes. Recombinant monoclonal antibodies with or without the conserved oligosaccharides, and with or without oxidation of the conserved methionine residues, were used to test the newly proposed method. All of the samples were digested in ammonium bicarbonate buffer prepared in normal water. The oxidized deglycosylated sample was also digested in ammonium bicarbonate buffer prepared in (18)O-labeled water. The sample from the digestion in (18)O-water was spiked into each sample digested in normal water. Each mixed sample was subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). The molecular weight differences between the peptides digested in normal water versus (18)O-water were used to differentiate peaks from the samples. The relative peak intensities of peptides with or without the C-terminal incorporation of (18)O atoms were used to determine susceptibility of different samples to trypsin and chymotrypsin. The results demonstrated that the method was capable of detecting local conformational changes of the recombinant monoclonal antibodies caused by deglycosylation and oxidation.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cromatografia Líquida , Quimotripsina/metabolismo , Cricetulus , Glicosilação , Humanos , Imunoglobulina G/metabolismo , Marcação por Isótopo , Espectrometria de Massas , Metionina/análise , Metionina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Peptídeos/análise , Peptídeos/metabolismo , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tripsina/metabolismoRESUMO
Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies have been challenging to fully characterize despite the fact that several posttranslational modifications have already been identified. The acidic species of a recombinant monoclonal antibody were collected using weak cation exchange (WCX)-10 chromatography and characterized by LC-MS at multiple levels. In this study, methionine oxidation and asparagine deamidation are the only two modifications identified in the acidic species. Incubation of the collected main chromatographic peak with hydrogen peroxide generated acidic species, which confirmed that acidic species were enriched in oxidized antibody. Differences observed between the original acidic species and the oxidization-induced acidic species indicate that different mechanisms are involved in the formation of acidic species. Additionally, acidic species were generated by thermal stress of the collected main peak from the original sample. Thermal stress of the collected main peak in pH 9 buffer or ammonium bicarbonate generated chromatograms that are highly similar to those from the analysis of the original molecule. LC-MS analysis identified oxidation of the same methionine residue and deamidation of the same asparagine in the corresponding acidic fractions generated by thermal stress; however, relatively lower levels of methionine oxidation and higher levels of asparagine deamdiation were observed. The results support the use of stressed conditions to generate low abundance species for detailed characterization of recombinant monoclonal antibody charge variants, but with caution.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Animais , Células CHO , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cricetulus , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Proteínas Recombinantes/químicaRESUMO
Recombinant monoclonal antibody charge heterogeneity has been commonly observed as multiple bands or peaks when analyzed by charge-based analytical methods such as isoelectric focusing electrophoresis and cation or anion exchange chromatography. Those charge variants have been separated by some of the above-mentioned methods and used for detailed characterization. The utility of a combination of OFFGEL fractionation and weak anion exchange chromatography to separate the charge variants of a recombinant monoclonal antibody was demonstrated in the current study. Charge variants were separated into various fractions of high purity and then analyzed thoroughly by liquid chromatography mass spectrometry. Analysis of intact molecular weights identified the presence of heavy chain leader sequence, C-terminal lysine, and C-terminal amidation. The identified modifications were further localized into different regions of the antibody from analysis of antibody fragments obtained from FabRICATOR digestion. Analysis of tryptic peptides from various fractions further confirmed the previously identified modifications in the basic variants. Asparagine deamidation and aspartate isomerization were identified in acidic fractions from analysis of tryptic peptides. Basic variants have been fully accounted for by the identified modifications. However, only a portion of the acidic variants can be explained by deamidation and isomerization, suggesting that additional modifications are yet to be identified or acidic variants are an ensemble of molecules with different structures.
Assuntos
Anticorpos Monoclonais/análise , Animais , Células CHO , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cricetulus , Espectrometria de Massas , Peso Molecular , Proteínas Recombinantes/análiseRESUMO
Oligosaccharides are critical for structural integrity, stability, and biological functions of recombinant monoclonal antibodies. It is relatively easy to characterize, quantify, and determine the impact of major glycoforms. While challenging to detect and quantify, certain low abundance oligosaccharides are highly relevant to the stability and functions of recombinant monoclonal antibodies. Methods were established in this study based on enzymatic digestion to consolidate peaks of the same type of oligosaccharides by removing heterogeneity and thus increase detectability of low abundance peaks. Endo H was used to collapse high mannose oligosaccharides to a single peak of GlcNAc for ease of detection and quantitation. ß-Galactosidase and ß-N-acetylhexosaminidase were used to convert complex oligosaccharides into two peaks containing either GlcNAc2Man3Fuc or GlcNAc2Man3, which simplified the chromatograms and data analysis. More importantly, low abundance hybrid oligosaccharides can only be detected and qualified after ß-galactosidase and ß-N-acetylhexosaminidase digestion. Detection and quantitation of low abundance oligosaccharides can also be achieved using a combination of all three enzymes. These methods can be applied to the development of recombinant monoclonal antibody therapeutics.
Assuntos
Anticorpos Monoclonais/química , Oligossacarídeos/análise , Proteínas Recombinantes/química , Cromatografia Líquida de Alta Pressão , Fucose/química , Humanos , Manose/química , Oligossacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Galactosidase/metabolismoRESUMO
Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos/imunologia , Imunoglobulina G/imunologia , Proteínas Recombinantes/imunologia , Animais , Anticorpos/genética , Anticorpos/metabolismo , Anticorpos Monoclonais/genética , Linhagem Celular , Glicosilação , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Sinais Direcionadores de Proteínas/genética , Ácido Pirrolidonocarboxílico/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Glycation is a common modification of proteins both in vitro and in vivo. To aid identification and comparative quantitation, a method of stable isotope labeling followed by LC-MS analysis was proposed. The samples were reduced using sodium borohydride or sodium borodeuteride. Reduction of the Schiff base between the amine group and the reducing sugars resulted in a molecular weight increase of 2Da using sodium borohydride or a molecular weight increase of 3Da using sodium borodeuteride. The molecular weight difference of 1Da between peptides containing glycated lysine residue reduced using sodium borohydride or sodium borodeuteride was used to identify glycated peptides and to calculate the glycation difference between samples. The method was used to investigate glycation of a recombinant human IgG1 antibody under native and denaturing conditions. The result demonstrated a good correlation between glycation propensity of lysine residues and their solvent exposure levels.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Imunoglobulina G/química , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Boroidretos/química , Glicosilação , Humanos , Marcação por Isótopo/métodos , Proteínas Recombinantes/químicaRESUMO
Methionine (Met) oxidation is a major modification of proteins, which converts Met to Met sulfoxide as the common product. It is challenging to determine the level of Met sulfoxide, because it can be generated during sample preparation and analysis as an artifact. To determine the level of Met sulfoxide in proteins accurately, an isotope labeling and LC-MS peptide mapping method was developed. Met residues in proteins were fully oxidized using hydrogen peroxide enriched with (18)O atoms before sample preparation. Therefore, it was impossible to generate Met sulfoxide as an artifact during sample preparation. The molecular weight difference of 2 Da between Met sulfoxide with the (16)O atom and Met sulfoxide with the (18)O atom was used to differentiate and calculate the level of Met sulfoxide in the sample originally. Using a recombinant monoclonal antibody as a model protein, much lower levels of Met sulfoxide were detected for the two susceptible Met residues with this new method compared to a typical peptide mapping procedure. The results demonstrated efficient elimination of the analytical artifact during LC-MS peptide mapping for the measurement of Met sulfoxide. This method can thus be used when accurate determination of the level of Met sulfoxide is critical.
Assuntos
Cromatografia Líquida/métodos , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Metionina/química , Metionina/metabolismo , Proteínas/química , Proteínas/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetulus , Oxirredução , Safrol/análogos & derivados , Safrol/química , Safrol/metabolismoRESUMO
Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.
Assuntos
Aldeído Redutase/química , Cisteína/química , NADP/química , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Diabetes Mellitus/enzimologia , Diabetes Mellitus/genética , Ativação Enzimática/genética , Humanos , Ligação de Hidrogênio , Mutação de Sentido Incorreto , NADP/genética , NADP/metabolismo , Oxirredução , Estrutura Terciária de ProteínaRESUMO
Candida albicans utilizes mammalian cell-associated transglutaminase (TGase) activity to adhere covalently to human buccal epithelial cells (BECs) through Hyphal Wall Protein 1. Little is known about the factors leading to the identity and appearance of Hwp1 binding partners on cells lining the oral cavity. The observation that BECs vary in their ability to attach to C. albicans germ tubes and to bind recombinant Hwp1 (rHwp1) suggested that differentiation may play a role in affinity for germ tube attachment. Individual BECs were characterized for differentiation status and rHwp1 binding. rHwp1 bound to the more terminally differentiated cells displaying SPR3 and keratin 13 but not to less differentiated cells with abundant involucrin. Sequential expression of involucrin followed by SPR3 in oral keratinocytes was demonstrated using stratified organotypic cultures and a feeder layer system with the OKF6/TERT-2 cell line. Increased cross-linking of the lysine analogue 5-(biotinamido)pentylamine to cultured OKF6/TERT-2 cell proteins accompanied this increased expression of SPR3. Western blot analysis demonstrated the presence of rHwp1 cross-links to proteins from BECs or from OKF6/TERT-2 cells that had been mechanically dislodged from culture dishes. Therefore, the differentiation of SPR3 positive from involucrin positive cells is correlated with the acquisition of affinity for cross-linking to rHwp1 and covalent adhesion of germ tubes to BECs.
Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Glicoproteínas de Membrana/fisiologia , Mucosa Bucal/citologia , Aminas/metabolismo , Biotina/análogos & derivados , Biotina/metabolismo , Adesão Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas Fúngicas/análise , Humanos , Hifas/fisiologia , Queratina-13/metabolismo , Queratinócitos/metabolismo , Glicoproteínas de Membrana/análise , Mucosa Bucal/metabolismo , Ligação Proteica , Precursores de Proteínas/metabolismo , Transglutaminases/fisiologiaRESUMO
A culture-independent method called mRNA profiling has been developed for the analysis of fecal enterobacteria and their physiological status in environmental samples. This taxon-specific approach determines the single-cell content of selected gene transcripts whose abundance is either directly or inversely proportional to growth state. Fluorescence in situ hybridization using fluorochrome-labeled oligonucleotide probes was used to measure the cellular concentration of fis and dps mRNA. Relative levels of these transcripts provided a measure of cell growth state and the ability to enumerate fecal enterobacterial cell number. Orthologs were cloned by inverse PCR from several major enterobacterial genera, and probes specific for fecal enterobacteria were designed using multiple DNA sequence alignments. Probe specificity was determined experimentally using pure and mixed cultures of the major enterobacterial genera as well as secondary treated wastewater samples seeded with pure culture inocula. Analysis of the fecal enterobacterial community resident in unseeded secondary treated wastewater detected fluctuations in transcript abundance that were commensurate with incubation time and nutrient availability and demonstrated the utility of the method using environmental samples. mRNA profiling provides a new strategy to improve wastewater disinfection efficiency by accelerating water quality analysis.
Assuntos
Enterobacteriaceae/classificação , Enterobacteriaceae/citologia , Fezes/microbiologia , RNA Mensageiro/metabolismo , Microbiologia da Água , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Meios de Cultura , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/genética , Fator Proteico para Inversão de Estimulação/metabolismo , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , RNA Mensageiro/genética , Análise de Sequência de DNA , Esgotos/microbiologia , Eliminação de Resíduos Líquidos , Purificação da ÁguaRESUMO
The efficiency of enterobacterial disinfection is dependent largely on enterobacterial community physiology. However, the relationship between enterobacterial community physiology and wastewater processing is unclear. The purpose of this study was to investigate this relationship. The influence of wastewater treatment processes on enterobacterial community physiology was examined at the single-cell level by using culture-independent methods. Intracellular concentrations of two conserved proteins, the growth-related protein Fis and the stationary-phase protein Dps, were analyzed by epifluoresence microscopy of uncultivated cells by using enterobacterial group-specific polyclonal fluorochrome-coupled antibodies. Enterobacterial single-cell community protein profiles were distinct for different types of biological treatment. The differences were not apparent when bulk methods of protein analysis were used. Trickling filter wastewater yielded Fis-enriched communities compared to the communities in submerged aeration basin wastewater. Community differences in Fis and Dps contents were used to predict disinfection efficiency. Disinfection of community samples by heat exposure combined with cultivation in selective media confirmed that enterobacterial communities exhibited significant differences in sensitivity to disinfection. These findings provide strategies that can be used to increase treatment plant performance, reduce the enterobacterial content in municipal wastewater, and minimize the release of disinfection by-products into receiving water.