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ABSTRACT: Nonmuscle cell contractility is an essential feature underlying diverse cellular processes such as motility, morphogenesis, division and genome replication, intracellular transport, and secretion. Blood clot contraction is a well-studied process driven by contracting platelets. Megakaryocytes (MKs), which are the precursors to platelets, can be found in bone marrow and lungs. Although they express many of the same proteins and structures found in platelets, little is known about their ability to engage with extracellular proteins such as fibrin and contract. Here, we have measured the ability of MKs to compress plasma clots. Megakaryocytes derived from human induced pluripotent stem cells (iPSCs) were suspended in human platelet-free blood plasma and stimulated with thrombin. Using real-time macroscale optical tracking, confocal microscopy, and biomechanical measurements, we found that activated iPSC-derived MKs (iMKs) caused macroscopic volumetric clot shrinkage, as well as densification and stiffening of the fibrin network via fibrin-attached plasma membrane protrusions undergoing extension-retraction cycles that cause shortening and bending of fibrin fibers. Contraction induced by iMKs involved 2 kinetic phases with distinct rates and durations. It was suppressed by inhibitors of nonmuscle myosin IIA, actin polymerization, and integrin αIIbß3-fibrin interactions, indicating that the molecular mechanisms of iMK contractility were similar or identical to those in activated platelets. Our findings provide new insights into MK biomechanics and suggest that iMKs can be used as a model system to study platelet contractility. Physiologically, the ability of MKs to contract plasma clots may play a role in the mechanical remodeling of intravascular blood clots and thrombi.
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Células Madre Pluripotentes Inducidas , Trombosis , Humanos , Megacariocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Plaquetas/metabolismo , Trombosis/metabolismo , Fibrina/metabolismo , PlasmaRESUMEN
Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.
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Células Madre Pluripotentes Inducidas , Enfermedad por Deficiencia de Múltiples Sulfatasas , Humanos , Leucocitos Mononucleares/metabolismo , Neuronas/patología , Sulfatasas , Oxidorreductasas actuantes sobre Donantes de Grupos SulfuroRESUMEN
Red blood cell (RBC) transfusion is one of the most common medical treatments, with more than 10 million units transfused per year in the United States alone. Alloimmunization to foreign Rh proteins (RhD and RhCE) on donor RBCs remains a challenge for transfusion effectiveness and safety. Alloantibody production disproportionately affects patients with sickle cell disease who frequently receive blood transfusions and exhibit high genetic diversity in the Rh blood group system. With hundreds of RH variants now known, precise identification of Rh antibody targets is hampered by the lack of appropriate reagent RBCs with uncommon Rh antigen phenotypes. Using a combination of human-induced pluripotent stem cell (iPSC) reprogramming and gene editing, we designed a renewable source of cells with unique Rh profiles to facilitate the identification of complex Rh antibodies. We engineered a very rare Rh null iPSC line lacking both RHD and RHCE. By targeting the AAVS1 safe harbor locus in this Rh null background, any combination of RHD or RHCE complementary DNAs could be reintroduced to generate RBCs that express specific Rh antigens such as RhD alone (designated D--), Goa+, or DAK+. The RBCs derived from these iPSCs (iRBCs) are compatible with standard laboratory assays used worldwide and can determine the precise specificity of Rh antibodies in patient plasma. Rh-engineered iRBCs can provide a readily accessible diagnostic tool and guide future efforts to produce an alternative source of rare RBCs for alloimmunized patients.
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Antígenos de Grupos Sanguíneos , Células Madre Pluripotentes , Medicina Transfusional , Alelos , Antígenos de Grupos Sanguíneos/genética , Humanos , Sistema del Grupo Sanguíneo Rh-Hr/genéticaRESUMEN
Trisomy 21 (T21), or Down syndrome (DS), is associated with baseline macrocytic erythrocytosis, thrombocytopenia, and neutrophilia, and transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). TAM and ML-DS blasts both arise from an aberrant megakaryocyte-erythroid progenitor and exclusively express GATA1s, the truncated isoform of GATA1 , while germline GATA1s mutations in a non-T21 context lead to congenital cytopenias without a leukemic predisposition. This suggests that T21 and GATA1s perturb hematopoiesis independently and synergistically, but this interaction has been challenging to study in part due to limited human cell and murine models. To dissect the developmental impacts of GATA1s on hematopoiesis in euploid and T21 cells, we performed a single-cell RNA-sequencing timecourse on hematopoietic progenitors (HPCs) derived from isogenic human induced pluripotent stem cells differing only by chromosome 21 and/or GATA1 status. These HPCs were surprisingly heterogeneous and displayed spontaneous lineage skew apparently dictated by T21 and/or GATA1s. In euploid cells, GATA1s nearly eliminated erythropoiesis, impaired MK maturation, and promoted an immature myelopoiesis, while in T21 cells, GATA1s appeared to compete with the enhanced erythropoiesis and suppressed megakaryopoiesis driven by T21 to give rise to immature erythrocytes, MKs, and myeloid cells. T21 and GATA1s both disrupted temporal regulation of lineage-specific transcriptional programs and specifically perturbed cell cycle genes. These findings in an isogenic system can thus be attributed specifically to T21 and GATA1s and suggest that these genetic changes together enhance HPC proliferation at the expense of maturation, consistent with a pro-leukemic phenotype.
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The HtrA protease of Streptococcus pneumoniae functions both in a general stress response role and as an error sensor that specifically represses genetic competence when the overall level of biosynthetic errors in cellular proteins is low. However, the mechanism through which HtrA inhibits development of competence has been unknown. We found that HtrA digested the pneumococcal competence-stimulating peptide (CSP) and constituted the primary extracytoplasmic CSP-degrading activity in cultures of S. pneumoniae. Mass spectrometry demonstrated that cleavage predominantly followed residue Phe-8 of the CSP-1 isoform of the peptide within its central hydrophobic patch, and in competition assays, both CSP-1 and CSP-2 interacted with HtrA with similar efficiencies. More generally, analysis of ß-casein digestion and of digestion within HtrA itself revealed a preference for substrates with non-polar residues at the P1 site. Consistent with a specificity for exposed hydrophobic residues, competition from native BSA only weakly inhibited digestion of CSP, but denaturation converted BSA into a strong competitive inhibitor of such proteolysis. Together these findings support a model in which digestion of CSP by HtrA is reduced in the presence of other unfolded proteins that serve as alternative targets for degradation. Such competition may provide a mechanism by which HtrA functions in a quality control capacity to monitor the frequency of biosynthetic errors that result in protein misfolding.
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Péptido Hidrolasas/química , Serina Proteasas/fisiología , Streptococcus pneumoniae/enzimología , Fenómenos Fisiológicos Bacterianos , Sitios de Unión , Dominio Catalítico , Transferencia Resonante de Energía de Fluorescencia , Eliminación de Gen , Cinética , Espectrometría de Masas/métodos , Mutación , Péptidos/química , Reacción en Cadena de la Polimerasa/métodos , Desnaturalización Proteica , Proteolisis , Percepción de Quorum , Serina Proteasas/química , Transducción de SeñalRESUMEN
Visceral myopathies are debilitating conditions characterized by dysfunction of smooth muscle in visceral organs (bowel, bladder, and uterus). Individuals affected by visceral myopathy experience feeding difficulties, growth failure, life-threatening abdominal distension, and may depend on intravenous nutrition for survival. Unfortunately, our limited understanding of the pathophysiology of visceral myopathies means that current therapies remain supportive, with no mechanism-based treatments. We developed a patient-derived iPSC line with a c.769C > T p.R257C/+ mutation, the most common genetic cause of visceral myopathy. This cell line will facilitate studies of how the ACTG2 R257C heterozygous variant affects smooth muscle development and function.
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Células Madre Pluripotentes Inducidas , Seudoobstrucción Intestinal , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Actinas/metabolismo , Seudoobstrucción Intestinal/genética , Seudoobstrucción Intestinal/metabolismo , Intestinos , MutaciónRESUMEN
Dysfunction of visceral smooth muscle ("visceral myopathy") impairs bowel, bladder, and uterine function. Symptoms of this life-threatening condition include massive intestinal distension with slow transit, vomiting, feeding intolerance, growth failure, poor bladder emptying, and difficult vaginal delivery. The most common genetic cause of visceral myopathy is a heterozygous point mutation (R257C) in gamma smooth muscle actin (ACTG2). We genetically modified the WAe0009-A human embryonic stem cell line to carry the c.769C>T p.R257C/+ mutation. This cell line will facilitate studies of how the ACTG2 R257C heterozygous variant affects smooth muscle development and function.
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Células Madre Embrionarias , Enfermedades Musculares , Humanos , Femenino , Línea Celular , Heterocigoto , Desarrollo de Músculos , Actinas/genéticaRESUMEN
The patterning of excitatory cortical neurons from human pluripotent stem cells (hPSCs) is a desired technique for the study of neurodevelopmental disorders, as neurons can be created and compared from control hPSC lines, hPSC lines generated from patients, and CRISPR-modified hPSC lines. Therefore, this technique allows for the examination of disease phenotypes and assists in the development of potential new therapeutics for neurodevelopmental disorders. Many protocols, however, are optimized for use with specific hPSC lines or within a single laboratory, and they often provide insufficient guidance on how to identify positive stages in the differentiation or how to troubleshoot. Here, we present an efficient and reproducible directed differentiation protocol to generate two-dimensional cultures of hPSC-derived excitatory cortical neurons without intermediary embryoid body formation. This novel protocol is supported by our data generated with five independent hPSC lines and in two independent laboratories. Importantly, as neuronal differentiations follow a long time course to reach maturity, we provide extensive guidance regarding morphological and flow cytometry checkpoints allowing for early indications of successful differentiation. We also include extensive troubleshooting tips and support protocols to assist the operator. The goal of this protocol is to assist others in the successful differentiation of excitatory cortical neurons from hPSCs. © 2023 Wiley Periodicals LLC. Basic Protocol: Directed differentiation of hPSCs into excitatory cortical neurons Support Protocol 1: Harvesting and fixing cells for flow cytometry analyses Support Protocol 2: Performing flow cytometry analyses Support Protocol 3: Thawing NPCs from a cryopreserved stock Alternate Protocol 1: Continuing Expansion of NPCs Alternate Protocol 2: Treatment of neurons with Ara-C to ablate radial glia Support Protocol 4: Experimental methods for validation of excitatory cortical neurons.
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Técnicas de Cultivo de Célula , Células Madre Pluripotentes , Humanos , Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes/fisiología , Neuronas/fisiología , Diferenciación Celular/fisiología , Cuerpos EmbrioidesRESUMEN
Alternative splicing of neuronal genes is controlled partly by the coordinated action of polypyrimidine tract binding proteins (PTBPs). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal. Here, we define the PTBP2 footprint in the human transcriptome using brain tissue and human induced pluripotent stem cell-derived neurons (iPSC-neurons). We map PTBP2 binding sites, characterize PTBP2-dependent alternative splicing events, and identify novel PTBP2 targets including SYNGAP1, a synaptic gene whose loss-of-function leads to a complex neurodevelopmental disorder. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and nonsense-mediated decay, and that antisense oligonucleotides (ASOs) that disrupt PTBP binding redirect splicing and increase SYNGAP1 mRNA and protein expression. In SYNGAP1 haploinsufficient iPSC-neurons generated from two patients, we show that PTBP2-targeting ASOs partially restore SYNGAP1 expression. Our data comprehensively map PTBP2-dependent alternative splicing in human neurons and cerebral cortex, guiding development of novel therapeutic tools to benefit neurodevelopmental disorders.
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Células Madre Pluripotentes Inducidas , Proteínas del Tejido Nervioso , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Empalme del ARN , Empalme Alternativo/genética , Encéfalo/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismoRESUMEN
Genomic imprinting is a rare form of gene expression in mammals in which a small number of genes are expressed in a parent-of-origin-specific manner. The aetiology of human imprinting disorders is diverse and includes chromosomal abnormalities, mutations, and epigenetic dysregulation of imprinted genes. The most common human imprinting disorder is Beckwith-Wiedemann syndrome (BWS), frequently caused by uniparental isodisomy and DNA methylation alterations. Because these lesions cannot be easily engineered, induced pluripotent stem cells (iPSC) are a compelling alternative. Here, we describe the first iPSC model derived from patients with BWS. Due to the mosaic nature of BWS patients, both BWS and non-BWS iPSC lines were derived from the same patient's fibroblasts. Importantly, we determine that DNA methylation and gene expression patterns of the imprinted region in the iPSC lines reflect the parental cells and are stable over time. Additionally, we demonstrate that differential expression in insulin signalling, cell proliferation, and cell cycle pathways was seen in hepatocyte lineages derived from BWS lines compared to controls. Thus, this cell based-model can be used to investigate the role of imprinting in the pathogenesis of BWS in disease-relevant cell types.
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Síndrome de Beckwith-Wiedemann , Síndrome de Beckwith-Wiedemann/genética , Metilación de ADN , Impresión Genómica , Humanos , MutaciónRESUMEN
The CHOPWT4 iPSC line was generated as a control for applications such as differentiation analyses to the three germ layers and derivative tissues. Human foreskin fibroblasts were reprogrammed using the non-integrating Sendai virus expressing Oct3/4, Sox2, c-myc, and Klf4.
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Células Madre Pluripotentes Inducidas , Diferenciación Celular , Células Epiteliales , Fibroblastos , Prepucio , Humanos , Factor 4 Similar a Kruppel , MasculinoRESUMEN
Remarkable strides have been made over the past decade on the development of pancreatic ß-cells from human stem cells through directed differentiation, allowing for modeling of ß-cell development, function and disease. However, in vitro models and future therapeutic applications will require the use of stem cell-derived islets with multiple monohormonal endocrine cells types, including α, ß, and δ cells. Using the previously reported Mel1 InsGFP/w human embryonic stem cell (hESC) line, we have knocked-in Red Fluorescence Protein (RFP) under the control of the endogenous somatostatin promoter using CRISPR/Cas9, generating a dual insulin and somatostatin reporter hESC line.
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Spinocerebellar Ataxia Type 2 (SCA2) is an autosomal dominant disease characterized by progressive degeneration of the cerebellum, brain stem, and spinal cord. SCA2 is caused by spontaneous misfolding and aggregate formation from abnormal CAG trinucleotide repeat expansion in the coding region of the ATXN2 gene. Here we describe the generation of two distinct iPSC lines from patients with SCA2.
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Ataxina-2/genética , Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/patología , Sistemas de Lectura Abierta/genética , Ataxias Espinocerebelosas/genética , Expansión de Repetición de Trinucleótido/genética , Adulto , Animales , Línea Celular , Humanos , Ratones , Persona de Mediana EdadRESUMEN
Juvenile myelomonocytic leukemia (JMML) is an uncommon myeloproliferative neoplasm driven by Ras pathway mutations and hyperactive Ras/MAPK signaling. Outcomes for many children with JMML remain dismal with current standard-of-care cytoreductive chemotherapy and hematopoietic stem cell transplantation. We used patient-derived induced pluripotent stem cells (iPSCs) to characterize the signaling profiles and potential therapeutic vulnerabilities of PTPN11-mutant and CBL-mutant JMML. We assessed whether MEK, JAK, and PI3K/mTOR kinase inhibitors (i) could inhibit myeloproliferation and aberrant signaling in iPSC-derived hematopoietic progenitors with PTPN11 E76K or CBL Y371H mutations. We detected constitutive Ras/MAPK and PI3K/mTOR signaling in PTPN11 and CBL iPSC-derived myeloid cells. Activated signaling and growth of PTPN11 iPSCs were preferentially inhibited in vitro by the MEKi PD0325901 and trametinib. Conversely, JAK/STAT signaling was selectively activated in CBL iPSCs and abrogated by the JAKi momelotinib and ruxolitinib. The PI3Kδi idelalisib and mTORi rapamycin inhibited signaling and myeloproliferation in both PTPN11 and CBL iPSCs. These findings demonstrate differential sensitivity of PTPN11 iPSCs to MEKi and of CBL iPSCs to JAKi, but similar sensitivity to PI3Ki and mTORi. Clinical investigation of mutation-specific kinase inhibitor therapies in children with JMML may be warranted.
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Células Madre Pluripotentes Inducidas/patología , Leucemia Mielomonocítica Juvenil/patología , Mutación , Células Madre Neoplásicas/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Proteínas Quinasas/genética , Niño , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Leucemia Mielomonocítica Juvenil/tratamiento farmacológico , Leucemia Mielomonocítica Juvenil/genética , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disorder of early childhood characterized by expansion of clonal myelomonocytic cells and hyperactive Ras/MAPK signaling. The disorder is caused by somatic and/or germline mutations in genes involved in the Ras/MAPK and JAK/STAT signaling pathways, including CBL. Here we describe the generation of an iPSC line with a homozygous CBL c.1111T->C (Y371H) mutation, designated CHOPJMML1854.
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Células Madre Pluripotentes Inducidas/metabolismo , Leucemia Mielomonocítica Juvenil/genética , Femenino , Humanos , Lactante , MutaciónRESUMEN
Induced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into ß-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient's disease. GATA6 mutant ß-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and ß-like cell functionality in humans.
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Endodermo/metabolismo , Factor de Transcripción GATA6/genética , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Páncreas/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Endodermo/efectos de los fármacos , Endodermo/embriología , Factor de Transcripción GATA6/metabolismo , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Modelos Biológicos , Familia de Multigenes , Mutación , Páncreas/embriología , Fenotipo , Tretinoina/farmacologíaRESUMEN
The CHOPWT10 iPS cell line was generated to be used as a control for applications such as in differentiation analyses to the three germ layers and derivative tissues. Peripheral blood mononuclear cells (PBMCs) obtained from a healthy adult male were reprogrammed using the non-integrating Sendai virus expressing Oct3/4, Sox2, c-Myc, and Klf4.
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Células Madre Pluripotentes Inducidas/citología , Leucocitos Mononucleares/citología , Adulto , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Cuerpos Embrioides/citología , Citometría de Flujo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Factor 4 Similar a Kruppel , Masculino , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Competence for genetic transformation in Streptococcus pneumoniae has previously been described as a quorum-sensing trait regulated by a secreted peptide pheromone. Recently we demonstrated that competence is also activated by reduction in the accuracy of protein biosynthesis. We have now investigated whether errors upstream of translation in the form of random genomic mutations can provide a similar stimulus. Here we show that generation of a mutator phenotype in S. pneumoniae through deletions of mutX, hexA or hexB enhanced the expression of competence. Similarly, chemical mutagenesis with the nucleotide analog dPTP promoted development of competence. To investigate the relationship between mutational load and the activation of competence, replicate lineages of the mutX strain were serially passaged under conditions of relaxed selection allowing random accumulation of secondary mutations. Competence increased with propagation in these lineages but not in control lineages having wild-type mutX. Resequencing of these derived strains revealed between 1 and 9 single nucleotide polymorphisms (SNPs) per lineage, which were broadly distributed across the genome and did not involve known regulators of competence. Notably, the frequency of competence development among the sequenced strains correlated significantly with the number of nonsynonymous mutations that had been acquired. Together, these observations provide support for the hypothesis that competence in S. pneumoniae is regulated in response to the accumulated burden of coding mutations in the bacterial genome. In contrast to previously described DNA damage response systems that are activated by physical lesions in the chromosome, this pneumococcal pathway may represent a unique stress response system that monitors the coding integrity of the genome.