RESUMEN
Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P. falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P. falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination.
Asunto(s)
Vacunas contra la Malaria , Malaria Falciparum , Humanos , Animales , Plasmodium falciparum , Epítopos , Proteínas Protozoarias , Antígenos de Protozoos , Anticuerpos Monoclonales , Anticuerpos Antiprotozoarios , Malaria Falciparum/prevención & controlRESUMEN
Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1-D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2 µg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.
Asunto(s)
Culicidae , Vacunas contra la Malaria , Malaria Falciparum , Malaria , Animales , Humanos , Plasmodium falciparum , Culicidae/metabolismo , Proteínas Protozoarias , Anticuerpos Monoclonales , Malaria Falciparum/prevención & control , Anticuerpos AntiprotozoariosRESUMEN
The gastrointestinal (GI) microbiota is a complex community of microorganisms residing within the mammalian gastrointestinal tract. The GI microbiota is vital to the development of the host immune system and plays a crucial role in human health and disease. The composition of the GI microbiota differs immensely among individuals yet specific shifts in composition and diversity have been linked to inflammatory bowel disease, obesity, atopy, and susceptibility to infection. In this review, we describe the GI microbiota and its role in enteric diseases caused by pathogenic Escherichia coli, Salmonella enterica, and Clostridium difficile. We discuss the central role of the GI microbiota in protective immunity, resistance to enteric pathogens, and resolution of enteric colitis.
Asunto(s)
Colitis/genética , Tracto Gastrointestinal/microbiología , Microbiota/genética , Animales , Clostridioides difficile/inmunología , Clostridioides difficile/patogenicidad , Colitis/inmunología , Colitis/microbiología , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Tracto Gastrointestinal/inmunología , Humanos , Microbiota/inmunología , Salmonella enterica/inmunología , Salmonella enterica/patogenicidadRESUMEN
Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citrobacter rodentium/crecimiento & desarrollo , Citrobacter rodentium/genética , Infecciones por Enterobacteriaceae/microbiología , Regulación Bacteriana de la Expresión Génica , Proteínas Quinasas/metabolismo , Regulón , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Ratones , Proteoma/análisisRESUMEN
Transcriptional signal cointegrators associate with transcription factors or nuclear receptors and coregulate tissue-specific gene transcription. We report on recessive loss-of-function mutations in two genes (TRIP4 and ASCC1) that encode subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. We used autozygosity mapping and whole-exome sequencing to search for pathogenic mutations in four families. Affected individuals presented with prenatal-onset spinal muscular atrophy (SMA), multiple congenital contractures (arthrogryposis multiplex congenita), respiratory distress, and congenital bone fractures. We identified homozygous and compound-heterozygous nonsense and frameshift TRIP4 and ASCC1 mutations that led to a truncation or the entire absence of the respective proteins and cosegregated with the disease phenotype. Trip4 and Ascc1 have identical expression patterns in 17.5-day-old mouse embryos with high expression levels in the spinal cord, brain, paraspinal ganglia, thyroid, and submandibular glands. Antisense morpholino-mediated knockdown of either trip4 or ascc1 in zebrafish disrupted the highly patterned and coordinated process of α-motoneuron outgrowth and formation of myotomes and neuromuscular junctions and led to a swimming defect in the larvae. Immunoprecipitation of the ASC-1 complex consistently copurified cysteine and glycine rich protein 1 (CSRP1), a transcriptional cofactor, which is known to be involved in spinal cord regeneration upon injury in adult zebrafish. ASCC1 mutant fibroblasts downregulated genes associated with neurogenesis, neuronal migration, and pathfinding (SERPINF1, DAB1, SEMA3D, SEMA3A), as well as with bone development (TNFRSF11B, RASSF2, STC1). Our findings indicate that the dysfunction of a transcriptional coactivator complex can result in a clinical syndrome affecting the neuromuscular system.
Asunto(s)
Fracturas Óseas/genética , Regulación del Desarrollo de la Expresión Génica , Atrofia Muscular Espinal/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Artrogriposis/diagnóstico , Artrogriposis/genética , Proteínas Portadoras/genética , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Fracturas Óseas/diagnóstico , Perfilación de la Expresión Génica , Homocigoto , Humanos , Proteínas con Dominio LIM/genética , Ratones , Datos de Secuencia Molecular , Atrofia Muscular Espinal/diagnóstico , Mutación , Proteínas Nucleares/genética , Linaje , Fenotipo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
In a number of families with colorectal adenomatous polyposis or suspected Lynch syndrome/HNPCC, no germline alteration in the APC, MUTYH, or mismatch repair (MMR) genes are found. Missense mutations in the polymerase genes POLE and POLD1 have recently been identified as rare cause of multiple colorectal adenomas and carcinomas, a condition termed polymerase proofreading-associated polyposis (PPAP). The aim of the present study was to evaluate the clinical relevance and phenotypic spectrum of polymerase germline mutations. Therefore, targeted sequencing of the polymerase genes POLD1, POLD2, POLD3, POLD4, POLE, POLE2, POLE3 and POLE4 was performed in 266 unrelated patients with polyposis or fulfilled Amsterdam criteria. The POLE mutation c.1270C>G;p.Leu424Val was detected in four unrelated patients. The mutation was present in 1.5% (4/266) of all patients, 4% (3/77) of all familial cases and 7% (2/30) of familial polyposis cases. The colorectal phenotype in 14 affected individuals ranged from typical adenomatous polyposis to a HNPCC phenotype, with high intrafamilial variability. Multiple colorectal carcinomas and duodenal adenomas were common, and one case of duodenal carcinoma was reported. Additionally, various extraintestinal lesions were evident. Nine further putative pathogenic variants were identified. The most promising was c.1306C>T;p.Pro436Ser in POLE. In conclusion, a PPAP was identified in a substantial number of polyposis and familial colorectal cancer patients. Screening for polymerase proofreading mutations should therefore be considered, particularly in unexplained familial cases. The present study broadens the phenotypic spectrum of PPAP to duodenal adenomas and carcinomas, and identified novel, potentially pathogenic variants in four polymerase genes.
Asunto(s)
Adenoma/genética , Neoplasias Colorrectales/genética , ADN Polimerasa II/genética , Predisposición Genética a la Enfermedad/genética , Mutación de Línea Germinal , Mutación Missense , Adenoma/enzimología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Neoplasias Colorrectales/enzimología , ADN Polimerasa Dirigida por ADN/genética , Salud de la Familia , Femenino , Frecuencia de los Genes , Humanos , Isoenzimas/genética , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Fosfolipasa D/genética , Proteínas de Unión a Poli-ADP-Ribosa , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Adulto JovenRESUMEN
To uncover novel causative genes in patients with unexplained adenomatous polyposis, a model disease for colorectal cancer, we performed a genome-wide analysis of germline copy number variants (CNV) in a large, well characterized APC and MUTYH mutation negative patient cohort followed by a targeted next generation sequencing (NGS) approach. Genomic DNA from 221 unrelated German patients was genotyped on high-resolution SNP arrays. Putative CNVs were filtered according to stringent criteria, compared with those of 531 population-based German controls, and validated by qPCR. Candidate genes were prioritized using in silico, expression, and segregation analyses, data mining and enrichment analyses of genes and pathways. In 27% of the 221 unrelated patients, a total of 77 protein coding genes displayed rare, nonrecurrent, germline CNVs. The set included 26 candidates with molecular and cellular functions related to tumorigenesis. Targeted high-throughput sequencing found truncating point mutations in 12% (10/77) of the prioritized genes. No clear evidence was found for autosomal recessive subtypes. Six patients had potentially causative mutations in more than one of the 26 genes. Combined with data from recent studies of early-onset colorectal and breast cancer, recurrent potential loss-of-function alterations were detected in CNTN6, FOCAD (KIAA1797), HSPH1, KIF26B, MCM3AP, YBEY and in three genes from the ARHGAP family. In the canonical Wnt pathway oncogene CTNNB1 (ß-catenin), two potential gain-of-function mutations were found. In conclusion, the present study identified a group of rarely affected genes which are likely to predispose to colorectal adenoma formation and confirmed previously published candidates for tumor predisposition as etiologically relevant.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Variaciones en el Número de Copia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Adolescente , Adulto , Anciano , Niño , ADN Glicosilasas/genética , Estudio de Asociación del Genoma Completo , Proteínas del Choque Térmico HSP110/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Cinesinas/genética , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/genética , beta Catenina/genéticaRESUMEN
Gram-negative bacteria possess several envelope stress responses that detect and respond to damage to this critical cellular compartment. The σ(E) envelope stress response senses the misfolding of outer membrane proteins (OMPs), while the Cpx two-component system is believed to detect the misfolding of periplasmic and inner membrane proteins. Recent studies in several Gram-negative organisms found that deletion of hfq, encoding a small RNA chaperone protein, activates the σ(E) envelope stress response. In this study, we assessed the effects of deleting hfq upon activity of the σ(E) and Cpx responses in non-pathogenic and enteropathogenic (EPEC) strains of Escherichia coli. We found that the σ(E) response was activated in Δhfq mutants of all E. coli strains tested, resulting from the misregulation of OMPs. The Cpx response was activated by loss of hfq in EPEC, but not in E. coliâ K-12. Cpx pathway activation resulted in part from overexpression of the bundle-forming pilus (BFP) in EPEC Δhfq. We found that Hfq repressed expression of the BFP via PerA, a master regulator of virulence in EPEC. This study shows that Hfq has a more extensive role in regulating the expression of envelope proteins and horizontally acquired virulence genes in E. coli than previously recognized.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli Enteropatógena/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/metabolismo , Proteínas Quinasas/metabolismo , Factor sigma/metabolismo , Estrés Fisiológico , Análisis Mutacional de ADN , Escherichia coli Enteropatógena/fisiología , Proteínas de Escherichia coli/genética , Eliminación de Gen , Proteína de Factor 1 del Huésped/genéticaRESUMEN
Gastrointestinal pathogens must overcome many obstacles in order to successfully colonize a host, not the least of which is the presence of the gut microbiota, the trillions of commensal microorganisms inhabiting mammals' digestive tracts, and their products. It is well established that a healthy gut microbiota provides its host with protection from numerous pathogens, including Salmonella species, Clostridium difficile, diarrheagenic Escherichia coli, and Vibrio cholerae. Conversely, pathogenic bacteria have evolved mechanisms to establish an infection and thrive in the face of fierce competition from the microbiota for space and nutrients. Here, we review the evidence that gut microbiota-generated metabolites play a key role in determining the outcome of infection by bacterial pathogens. By consuming and transforming dietary and host-produced metabolites, as well as secreting primary and secondary metabolites of their own, the microbiota define the chemical environment of the gut and often determine specific host responses. Although most gut microbiota-produced metabolites are currently uncharacterized, several well-studied molecules made or modified by the microbiota are known to affect the growth and virulence of pathogens, including short-chain fatty acids, succinate, mucin O-glycans, molecular hydrogen, secondary bile acids, and the AI-2 quorum sensing autoinducer. We also discuss challenges and possible approaches to further study of the chemical interplay between microbiota and gastrointestinal pathogens.
Asunto(s)
Bacterias/efectos de los fármacos , Bacterias/metabolismo , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Metaboloma , Interacciones Microbianas , Animales , Humanos , MamíferosRESUMEN
The Escherichia coli genome encodes approximately 30 two-component systems that are required for sensing and responding to a variety of environmental and physiological cues. Recent studies have revealed numerous regulatory connections between two-component systems and small noncoding RNAs (sRNAs), which posttranscriptionally regulate gene expression by base pairing with target mRNAs. In this study, we investigated the role of sRNAs in the CpxAR two-component system, which detects and mediates an adaptive response to potentially lethal protein misfolding in the Gram-negative bacterial envelope. Here, we showed for the first time that sRNAs are members of the Cpx regulon. We found that CpxR binds to the promoter regions and regulates expression of two sRNA genes, cyaR and rprA. We also investigated the roles that these sRNAs play in the Cpx response. Cpx repression of cyaR expression creates a feed-forward loop, in which CpxAR increases expression of the inner membrane protein YqaE both directly at the transcriptional level and indirectly at the translational level. Moreover, we found that RprA exerts negative feedback on the Cpx response, reducing Cpx activity in a manner that is dependent on the response regulator CpxR but independent of all of RprA's previously described targets. sRNAs therefore permit the fine-tuning of Cpx pathway activity and its regulation of target genes, which could assist bacterial survival in the face of envelope stress.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas Quinasas/metabolismo , ARN Pequeño no Traducido/metabolismo , Estrés Fisiológico , Proteínas Bacterianas/genética , Proteínas de Escherichia coli/genética , Proteínas Quinasas/genética , ARN Pequeño no Traducido/genética , Transducción de SeñalRESUMEN
The gastrointestinal (GI) environment plays a critical role in shaping enteric infections. Host environmental factors create bottlenecks, restrictive events that reduce the genetic diversity of invading bacterial populations. However, the identity and impact of bottleneck events on bacterial infection are largely unknown. We used Citrobacter rodentium infection of mice, a model of human pathogenic Escherichia coli infections, to examine bacterial population dynamics and quantify bottlenecks to host colonization. Using Sequence Tag-based Analysis of Microbial Populations (STAMP) we characterized the founding population size (Nb') and relatedness of C. rodentium populations at relevant tissue sites during early- and peak-infection. We demonstrate that the GI environment severely restricts the colonizing population, with an average Nb' of only 12-43 lineages (of 2,000+ inoculated) identified regardless of time or biogeographic location. Passage through gastric acid and escape to the systemic circulation were identified as major bottlenecks during C. rodentium colonization. Manipulating such events by increasing gastric pH dramatically increased intestinal Nb'. Importantly, removal of the stomach acid barrier had downstream consequences on host systemic colonization, morbidity, and mortality. These findings highlight the capability of the host GI environment to limit early pathogen colonization, controlling the population of initial founders with consequences for downstream infection outcomes.
Asunto(s)
Infecciones por Enterobacteriaceae , Infecciones por Escherichia coli , Ratones , Humanos , Animales , Citrobacter rodentium/genética , Ácido Gástrico , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/patología , Tracto Gastrointestinal/microbiología , Ratones Endogámicos C57BLRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC's ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC's response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.
Asunto(s)
Escherichia coli Enterohemorrágica , Infecciones por Escherichia coli , Microbioma Gastrointestinal , Microbiota , Humanos , Escherichia coli Enterohemorrágica/genética , Biotina/metabolismo , ColonRESUMEN
Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli Enteropatógena/metabolismo , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/ultraestructura , Proteínas de Escherichia coli/genética , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/ultraestructura , Lipoproteínas/genética , Transporte de ProteínasRESUMEN
To uncover pathogenic deep intronic variants in patients with colorectal adenomatous polyposis, in whom no germline mutation in the APC or MUTYH genes can be identified by routine diagnostics, we performed a systematic APC messenger RNA analysis in 125 unrelated mutation-negative cases. Overall, we identified aberrant transcripts in 8% of the patients (familial cases 30%; early-onset manifestation 21%). In eight of them, two different out-of-frame pseudoexons were found consisting of a 167-bp insertion from intron 4 in five families with a shared founder haplotype and a 83-bp insertion from intron 10 in three patients. The pseudoexon formation was caused by three different heterozygous germline mutations, which are supposed to activate cryptic splice sites. In conclusion, a few deep intronic mutations contribute substantially to the APC mutation spectrum. Complementary DNA analysis and/or target sequencing of intronic regions should be considered as an additional mutation discovery approach in polyposis patients.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Intrones/genética , MutaciónRESUMEN
The type VI secretion system (T6SS) is a contractile nanomachine widely distributed among pathogenic and commensal Gram-negative bacteria. The T6SS is used for inter-bacterial competition to directly kill competing species; however, its importance during bacterial infection in vivo remains poorly understood. We report that the murine pathogen Citrobacter rodentium, used as a model for human pathogenic Escherichia coli, harbors two functional T6SSs. C. rodentium employs its T6SS-1 to colonize the murine gastrointestinal tract by targeting commensal Enterobacteriaceae. We identify VgrG1 as a C. rodentium T6SS antibacterial effector, which exhibits toxicity in E. coli. Conversely, commensal prey species E. coli Mt1B1 employs two T6SSs of its own to counter C. rodentium colonization. Collectively, these data demonstrate that the T6SS is a potent weapon during bacterial competition and is used by both invading pathogens and resident microbiota to fight for a niche in the hostile gut environment.
Asunto(s)
Sistemas de Secreción Tipo VI , Animales , Bacterias , Escherichia coli , Tracto Gastrointestinal/microbiología , Humanos , Ratones , SimbiosisRESUMEN
The stringent response is a regulatory system that allows bacteria to sense and adapt to nutrient-poor environments. The central mediator of the stringent response is the molecule guanosine 3',5'-bispyrophosphate (ppGpp), which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT. Our laboratory previously demonstrated that a relA mutant of Pseudomonas aeruginosa, the principal cause of lung infections in cystic fibrosis patients, was attenuated in virulence in a Drosophila melanogaster feeding model of infection. In this study, we examined the role of spoT in P. aeruginosa virulence. We generated an insertion mutation in spoT within the previously constructed relA mutant, thereby producing a ppGpp-devoid strain. The relA spoT double mutant was unable to establish a chronic infection in D. melanogaster and was also avirulent in the rat lung agar bead model of infection, a model in which the relA mutant is fully virulent. Synthesis of the virulence determinants pyocyanin, elastase, protease, and siderophores was impaired in the relA spoT double mutant. This mutant was also defective in swarming and twitching, but not in swimming motility. The relA spoT mutant and, to a lesser extent, the relA mutant were less able to withstand stresses such as heat shock and oxidative stress than the wild-type strain PAO1, which may partially account for the inability of the relA spoT mutant to successfully colonize the rat lung. Our results indicate that the stringent response, and SpoT in particular, is a crucial regulator of virulence processes in P. aeruginosa.
Asunto(s)
Proteínas Bacterianas/metabolismo , Drosophila melanogaster/microbiología , Regulación Bacteriana de la Expresión Génica , Respuesta al Choque Térmico/fisiología , Pulmón/microbiología , Pseudomonas aeruginosa/patogenicidad , Agar , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/fisiología , Conducta Alimentaria , Guanosina Pentafosfato/metabolismo , Humanos , Ligasas/genética , Ligasas/metabolismo , Mutación , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiología , Ratas , VirulenciaRESUMEN
The Cpx envelope stress response is induced by the misfolding of periplasmic proteins and restores envelope homeostasis by upregulating several periplasmic protein folding and degrading factors. The Cpx response also regulates the expression of a variety of envelope-spanning protein complexes, including flagella, secretion systems and pili, which play an important role in pathogenesis. In a previous study, we inactivated the Cpx response in enteropathogenic Escherichia coli (EPEC), a causative agent of infant diarrhoea, and observed decreased expression of its major adhesin, the bundle-forming pilus (BFP). Here, we examined the mechanism underlying this BFP expression defect, and found that this phenotype can be attributed to insufficient expression of periplasmic folding factors, such as DsbA, DegP and CpxP. Hence, a low level of Cpx pathway activity promotes BFP synthesis by upregulating factors important for folding of BFP component proteins. Conversely, we found that full induction of the Cpx response inhibits BFP expression, mainly by repressing transcription of the bfp gene cluster. In combination with a previous report examining EPEC type III secretion, our results demonstrate that the Cpx response co-ordinates the repression of cell-surface structures during periods of envelope stress.
Asunto(s)
Escherichia coli Enteropatógena/citología , Escherichia coli Enteropatógena/fisiología , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/metabolismo , Proteínas Quinasas/metabolismo , Estrés Fisiológico , Adhesión Bacteriana , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Humanos , Lactante , Pliegue de Proteína , Proteínas Quinasas/genética , Transcripción GenéticaRESUMEN
Enteropathogenic Escherichia coli (EPEC) are a major cause of infant morbidity and mortality due to diarrhoea in developing countries. The pathogenesis of EPEC is dependent on a coordinated multi-step process culminating in the intimate adherence of the organisms to the host's intestinal mucosa. During the initial stages of the EPEC colonization process, the fimbrial adhesin, bundle-forming pili (BFP), plays an integral role. We previously reported that the major BFP structural subunit, bundlin, displays lectin-like properties, which enables BFP to initially tether EPEC to N-acetyllactosamine (LacNAc) glycan receptors on host cell surfaces. We also reported that incubating EPEC with synthetic LacNAc-bearing neoglycoconjugates not only inhibits their adherence to host cells, but also induces BFP retraction and subsequent degradation of the bundlin subunits. Herein, we demonstrate that the periplasmic serine protease, DegP, is required for degrading bundlin during this process. We also show that DegP appears to act as a bundlin chaperone during BFP assembly and that LacNAc-BSA-induced BFP retraction is followed by transcriptional upregulation of the BFP operon and downregulation of the locus of enterocyte effacement operons in EPEC.
Asunto(s)
Amino Azúcares/farmacología , Escherichia coli Enteropatógena , Fimbrias Bacterianas/efectos de los fármacos , Fimbrias Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Escherichia coli Enteropatógena/citología , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Lactante , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estructura Molecular , Operón , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND & AIMS: MUTYH-associated polyposis (MAP) is characterized by a lifetime risk of colorectal cancer of up to 100%. However, no systematic evaluation of extracolonic manifestations has been reported. METHODS: A large cohort of MAP patients was recruited from a European multicenter study. Data were collected on 276 cases from 181 unrelated families. Information on extracolonic tumor spectrum and incidence were evaluated to determine cumulative lifetime risk, which was compared with that of the general population to obtain standardized incidence ratios (SIRs). RESULTS: Duodenal polyposis occurred in 17% of cases; the relative risk (SIR) of duodenal cancer was 129 (95% confidence interval [CI]: 16-466), whereas the lifetime risk was 4%. The incidence of extraintestinal malignancies among cases was almost twice that of the general population (SIR: 1.9; 95% CI: 1.4-2.5), with a lifetime risk of 38%. We observed a significant increase in the incidence of ovarian, bladder, and skin cancers (SIR: 5.7, 7.2, and 2.8, respectively) and a trend of increased risk of breast cancer among cases. The median ages of onset of these 4 malignancies ranged from 51 to 61 years. In contrast to familial adenomatous polyposis, no desmoid tumors were observed, but sebaceous gland tumors, characteristic of the Muir-Torre variant of Lynch syndrome, occurred in 5 patients. CONCLUSIONS: The relative risks for several extraintestinal malignancies increased in patients with MAP, but based on the spectrum of cancers (which overlaps with that of Lynch syndrome) and the relatively advanced age at onset, intensive surveillance measures other than frequent endoscopy are unlikely to be helpful to patients with MAP.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Neoplasias de la Mama/genética , ADN Glicosilasas/genética , Neoplasias Gastrointestinales/genética , Neoplasias Ováricas/genética , Neoplasias de las Glándulas Sebáceas/genética , Neoplasias Cutáneas/genética , Neoplasias de la Vejiga Urinaria/genética , Poliposis Adenomatosa del Colon/epidemiología , Poliposis Adenomatosa del Colon/patología , Adolescente , Adulto , Edad de Inicio , Anciano , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/patología , Niño , Endoscopía , Europa (Continente)/epidemiología , Femenino , Neoplasias Gastrointestinales/epidemiología , Neoplasias Gastrointestinales/patología , Predisposición Genética a la Enfermedad , Humanos , Incidencia , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Neoplasias Ováricas/epidemiología , Neoplasias Ováricas/patología , Fenotipo , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Neoplasias de las Glándulas Sebáceas/epidemiología , Neoplasias de las Glándulas Sebáceas/patología , Neoplasias Cutáneas/epidemiología , Neoplasias Cutáneas/patología , Neoplasias de la Vejiga Urinaria/epidemiología , Neoplasias de la Vejiga Urinaria/patología , Adulto JovenRESUMEN
BACKGROUND & AIMS: Biallelic mutations in the base excision DNA repair gene MUTYH lead to MUTYH-associated polyposis (MAP) and predisposition to colorectal cancer (CRC). Functional studies have demonstrated significant differences in base recognition and glycosylase activity between various MUTYH mutations, notably for the 2 mutations most frequently reported in MAP patients: Y179C and G396D (previously annotated as Y165C and G382D). Our goal was to establish correlations between genotypes and colorectal phenotype of patients with MAP. METHODS: In this multicenter study, we analyzed genotype and phenotype data from 257 MAP patients. Data included age at presentation of MAP, polyp count, and the occurrence, location, and age at presentation of CRC. RESULTS: Patients with a homozygous G396D mutation or compound heterozygous G396D/Y179C mutations presented later with MAP and had a significantly lower hazard of developing CRC than patients with a homozygous Y179C mutation (P < .001). The mean ages of CRC diagnosis in patients were 58 years (homozygous G396D) and 52 years (compound heterozygous G396D/Y179C) versus 46 years (homozygous Y179C; P = .001, linear regression). CONCLUSIONS: Our study identified the phenotypic effects of Y179C as relatively severe and of G396D as relatively mild. These clinical data are in accord with findings from in vitro functional assays. Genotypic stratification may become useful in the development of guidelines for counseling, surveillance, and management of families with MAP.