RESUMO
Glycoproteins, in which polysaccharides are usually attached to proteins, are an important class of biomolecules that are widely used as therapeutic agents in clinical treatments for decades. Uropathogenic Escherichia coli (UPEC) O21 has been identified as a serogroup that induces urinary tract infections, with a global increasing number among women and young children. Therefore, there is an urgent need to establish protective vaccines against UPEC infection. Herein, we engineered non-pathogenic E. coli MG1655 to achieve robust, cost-effective de novo biosynthesis of O21 O-antigen polysaccharide-based glycoprotein against UPEC O21. Specifically, this glycoengineered E. coli MG1655 was manipulated for high-efficient glucose-glycerol co-utilization and for the gene cluster installation and O-glycosylation machinery assembly. The key pathways of UDP-sugar precursors were also strengthened to enforce more carbon flux towards the glycosyl donors, which enhanced the glycoprotein titer by 5.6-fold. Further optimization of culture conditions yielded glycoproteins of up to 35.34 mg/L. Glycopeptide MS confirmed the preciset biosynthesis of glycoprotein. This glycoprotein elicited antigen-specific IgG immune responses and significantly reduced kidney and bladder colonization. This bacterial cell-based glyco-platform and optimized strategies can provide a guideline for the biosynthesis of other value-added glycoproteins.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Criança , Feminino , Humanos , Pré-Escolar , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismo , Antígenos O/genética , Antígenos O/metabolismo , Proteínas de Escherichia coli/metabolismo , Infecções Urinárias/microbiologia , Infecções por Escherichia coli/microbiologia , Glicoproteínas/genética , Glicoproteínas/metabolismoRESUMO
Extraintestinal pathogenic Escherichia coli O1 is a frequently identified serotype that causes serious infections and is often refractory to antimicrobial therapy. Glycoconjugate vaccine represents a promising measure to reduce ExPEC infections. Herein, we designed an O1-specific glyco-optimized chassis strain for manufacture of O-polysaccharide (OPS) antigen and OPS-based bioconjugate. Specifically, OPS and OPS-based glycoprotein were synthesized in glyco-optimized chassis strain, when compared to the unmeasurable level of the parent strain. The optimal expression of oligosaccharyltransferase and carrier protein further improved the titer. MS analysis elucidated the correct structure of resulting bioconjugate at routine and unreported glycosylation sequons of carrier protein, with a higher glycosylation efficiency. Finally, purified bioconjugate stimulated mouse to generate specific IgG antibodies and protected them against virulent ExPEC O1 challenge. The plug-and-play glyco-optimized platform is suitable for bioconjugate synthesis, thus providing a potential platform for future medical applications.
Assuntos
Antibacterianos/farmacologia , Escherichia coli Extraintestinal Patogênica/efeitos dos fármacos , Glicoconjugados/farmacologia , Polissacarídeos/farmacologia , Engenharia de Proteínas , Vacinas Conjugadas/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Desenvolvimento de Medicamentos , Feminino , Glicoconjugados/síntese química , Glicoconjugados/química , Camundongos , Testes de Sensibilidade Microbiana , Polissacarídeos/síntese química , Polissacarídeos/química , Vacinas Conjugadas/químicaRESUMO
Cronobacter spp. are foodborne pathogens that can cause severe infections in neonates through contaminated powdered infant formula. Accurate and rapid pathogen identification and serotyping are crucial to limit the detrimental effects of bacterial infections, and to prevent outbreaks and sporadic infections. Conventional serotyping is tedious, laborious, and time-consuming; however, with whole-genome sequencing (WGS) becoming faster and cheaper, WGS has vast potential in routine typing and surveillance. Hence, in this study, we developed a publicly available tool, CroTrait (CronobacterTraits), for in silico species identification and O serotyping of Cronobacter isolates based on WGS data. CroTrait showed excellent performance in species identification and O serotyping when 810 genomes with known species identities and 276 genomes with known O serotype were tested. Moreover, CroTrait allows rapid prediction of new potential O serotypes. We identified 11 novel potential O serotypes of Cronobacter using CroTrait. Therefore, CroTrait is a convenient and promising tool for species identification and O serotyping of Cronobacter isolates.
Assuntos
Cronobacter sakazakii , Cronobacter , Simulação por Computador , Cronobacter/genética , Cronobacter sakazakii/genética , Humanos , Lactente , Recém-Nascido , Sorogrupo , Sorotipagem , Sequenciamento Completo do GenomaRESUMO
Nitrogen in different chemical forms is critical for metabolic alterations in Monascus strains and associated pigment diversity. In this study, we observed that ammonium-form nitrogen was superior in promoting the biosynthesis of Monascus pigments (MPs) when compared with nitrate and organic forms. Moreover, with any nitrogen source, the production of yellow and orange pigments was highly synchronized but distantly related to red pigments. However, transcriptional analyses of MP gene clusters suggested a low contribution to MP accumulation, suggesting that MP-limiting factors were located outside the gene cluster. Our metabolomic analyses demonstrated that red pigment biosynthesis was closely related to intracellular amino acids, whereas orange and yellow pigments were associated with nucleotides. In addition, weighted gene coexpression network analyses (WGCNA) based on transcriptomic data showed that multiple primary metabolic pathways were closely related to red pigment production, while several secondary pathways were related to orange pigments, and others were involved with yellow pigment regulation. These findings demonstrate that pigment diversity in Monascus is under combined regulation at metabolomic and transcriptomic levels. IMPORTANCE Natural MPs containing a mixture of red, orange, and yellow pigments are widely used as food coloring agents. MP diversity provides foods with versatile colors and health benefits but, in turn, complicate efforts to achieve maximum yield or desirable combination of pigments during the manufacturing process. Apart from the MP biosynthetic gene cluster, interactions between the main biosynthetic pathways and other intracellular genes/metabolites are critical to our understanding of MP differentiation. The integrative multiomics analytical strategy provides a technical platform and new perspectives for the identification of metabolic shunting mechanisms in MP biosynthesis. Equally, our research highlights the influence of intracellular metabolic alterations on MP differentiation, which will facilitate the rational engineering and optimization of MP production in the future.
RESUMO
Monascus yellow pigments (MYPs), as food colorants, are of great interest to the food industry, because of their beneficial biological activities. In this study, a comparative metabolomics strategy revealed the metabolic regulatory mechanism of MYP overproduction, comparing ammonium chloride with peptone as nitrogen sources. Metabolomics-based multivariate regression modeling showed that metabolic biomarkers/modules, such as glucose, lactate, and the pentose phosphate (PP) pathway, were closely associated with the biosynthesis of MYPs. Exogenous addition of glucose increased production of MYPs, whereas lactate reduced it. Inhibition of the PP pathway with dehydroepiandrosterone decreased MYP production, while increasing the shunting production of orange and red pigments. All these treatments significantly changed the expression profiles of the pigment biosynthetic gene cluster and the mycelial morphology. Overall, this study demonstrates the feasibility of elucidating the mechanism of MYP biosynthesis by comprehensive metabolomics analysis, as well as discovering potential engineering targets of efficiency improvements to commercial MYP production. KEY POINTS: ⢠Comparative metabolomics revealed the biomarkers/modules of MYP production. ⢠A rational exogenously adding strategy was implemented to regulate MYP synthesis. ⢠Expression profiles of gene cluster and mycelial morphology were characterized.
Assuntos
Monascus , Cloreto de Amônio , Metabolômica , Nitrogênio , Pigmentos BiológicosRESUMO
Enterohemorrhagic Escherichia coli serotype O157:H7 (O157) is a critical, foodborne, human intestinal pathogen that causes severe acute hemorrhagic diarrhea, abdominal cramping, and even death. Small RNAs (sRNAs) are noncoding regulatory molecules that sense environmental changes and trigger various virulence-related signaling pathways; however, few such sRNAs have been identified in O157. Here, we report a novel sRNA, EsrF that senses high ammonium concentrations in the colon and enhances O157 pathogenicity by promoting bacterial motility and adhesion to host cells. Specifically, EsrF was found to directly interact with the 5' untranslated regions of the flagellar biosynthetic gene, flhB, mRNA and increase its abundance, thereby upregulating expression of essential flagellar genes, including flhD, flhC, fliA, and fliC, leading to elevated O157 motility and virulence. Meanwhile, an infant rabbit model of O157 infection showed that deletion of esrF and flhB significantly attenuates O157 pathogenicity. Furthermore, NtrC-the response regulator of the NtrC/B two-component system-was found to exert direct, negative regulation of esrF expression. Meanwhile, high ammonium concentrations in the colon release the inhibitory effect of NtrC on esrF, thereby enhancing its expression and subsequently promoting bacterial colonization in the host colon. Our work reveals a novel, sRNA-centered, virulence-related signaling pathway in O157 that senses high ammonium concentrations. These findings provide novel insights for future research on O157 pathogenesis and targeted treatment strategies.IMPORTANCE The process by which bacteria sense environmental cues to regulate their virulence is complex. Several studies have focused on regulating the expression of the locus of enterocyte effacement pathogenicity island in the typical gut pathogenic bacterium, O157. However, few investigations have addressed the regulation of other virulence factors in response to intestinal signals. In this study, we report our discovery of a novel O157 sRNA, EsrF, and demonstrate that it contributed to bacterial motility and virulence in vitro and in vivo through the regulation of bacterial flagellar synthesis. Furthermore, we show that high ammonium concentrations in the colon induced esrF expression to promote bacterial virulence by releasing the repression of esrF by NtrC. This study highlights the importance of sRNA in regulating the motility and pathogenicity of O157.
Assuntos
Compostos de Amônio/metabolismo , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , RNA/genética , Compostos de Amônio/análise , Animais , Animais Recém-Nascidos , Aderência Bacteriana , Colo/química , Colo/microbiologia , Colo/patologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Escherichia coli O157/metabolismo , Movimento , Coelhos , Ativação Transcricional , Fatores de Virulência/genéticaRESUMO
Extraintestinal pathogenic Escherichia coli (ExPEC) has presented a major clinical infection emerged in the past decades. O-polysaccharide (OPS)-based glycoconjugate vaccines produced using the bacterial glycosylation machinery can be utilized to confer protection against such infection. However, constructing a low-cost microbial cell factory for high-efficient production of OPS-based glycoconjugate vaccines remains challenging. Here, we engineered a glyco-optimized chassis strain by reprogramming metabolic network. The yield was enhanced to 38.6 mg L-1, the highest level reported so far. MS analysis showed that designed glycosylation sequon was modified by target polysaccharide with high glycosylation efficiency of 90.7 % and 76.7 % for CTB-O5 and CTB-O7, respectively. The glycoconjugate vaccines purified from this biosystem elicited a marked increase in protection against ExPEC infection in mouse model, compared to a non-optimized system. The glyco-optimized platform established here is broadly suitable for polysaccharide-based conjugate production against ExPEC and other surface-polysaccharide-producing pathogens.