DNA insertion mutagenesis in a Pseudomonas aeruginosa R plasmid.
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Carbenicillin↗
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Results for “Mutagenesis, Insertional”
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Functional synapses between neurons and small cell lung cancer.
Small cell lung cancer (SCLC) is a highly aggressive type of lung cancer, characterized by rapid proliferation, early metastatic spread, frequent early relapse and a high mortality rate1-3. Recent evidence has suggested that innervation has an important role in the development and progression of several types of cancer4,5. Cancer-to-neuron synapses have been reported in gliomas6,7, but whether peripheral tumours can form such structures is unknown. Here we show that SCLC cells can form functional synapses and receive synaptic transmission. Using in vivo insertional mutagenesis screening in conjunction with cross-species genomic and transcriptomic validation, we identified neuronal, synaptic and glutamatergic signalling gene sets in mouse and human SCLC. Further experiments revealed the ability of SCLC cells to form synaptic structures with neurons in vitro and in vivo. Electrophysiology and optogenetic experiments confirmed that cancer cells can receive NMDA receptor- and GABAA receptor-mediated synaptic inputs. Fitting with a potential oncogenic role of neuron-SCLC interactions, we showed that SCLC cells derive a proliferation advantage when co-cultured with vagal sensory or cortical neurons. Moreover, inhibition of glutamate signalling had therapeutic efficacy in an autochthonous mouse model of SCLC. Therefore, following malignant transformation, SCLC cells seem to hijack synaptic signalling to promote tumour growth, thereby exposing a new route for therapeutic intervention.
Animals↗
Barcoded mutant library enables high-throughput functional genomics in a filamentous fungus.
Advances in sequencing technology enabling rapid and inexpensive whole-genome sequencing highlight how few genes are functionally characterized. This problem is particularly acute in filamentous fungi, where even in the best studied organisms upward of half of genes are poorly characterized or unannotated. High-throughput tools to identify gene function exist for single-celled organisms, like yeast and bacteria. However, filamentous fungi present challenges to high-throughput gene characterization, including low transformation efficiency and multinucleate cells. Filamentous fungi are critical components of nutrient cycling in ecosystems, form symbioses with plants that improve nutrient uptake, and are devastating human, plant, and animal pathogens causing millions of deaths and substantial crop loss each year. Thus, it is critical to overcome challenges to rapid gene characterization in filamentous fungi. We generated a library of hundreds of millions of uniquely barcoded plasmids containing a broad host-range drug resistance marker for ectopic insertion into filamentous fungal genomes by Agrobacterium tumefaciens. We then optimized A. tumefaciens mediated transformation of the biocontrol agent Trichoderma atroviride and made an insertional mutagenesis library containing 83,311 barcoded insertions, disrupting 5,331 of 11,863 predicted genes. This library enables high-throughput screens to rapidly connect genotype to phenotype. Quantifying relative barcode abundance in the pooled library before and after exposure to experimental conditions identified candidate genes and recovered known pathway components in amino acid biosynthetic, fructose utilization, and xylose utilization pathways. This resource establishes a scalable platform for high-throughput functional genomics in filamentous fungi, enabling investigations of fungal biology to improve medical outcomes, biotechnology, and sustainable agriculture.
Genomics↗
Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality.
BACKGROUND: Gene therapy (GT) using retroviral vectors (RVs) is efficacious in treating monogenic diseases. However, there is an inherent risk for severe adverse effects due to insertional mutagenesis. Preclinical safety assessment and patient monitoring are inevitable in GT. To assess the genotoxic risk of novel RV vectors, mainly murine hematopoietic stem and progenitor cells (HPSCs) are routinely used, because human HSPCs cannot be immortalized in vitro using mutagenic vectors. In this study, we aim to identify early signs of clonal outgrowth by performing integration site analyses (ISA). METHODS: The small molecules A83-01, pomalidomide, and UM171 (APU) were used for the ex vivo expansion, lentiviral transduction, and long-term cultivation of umbilical cord blood-derived HSPCs. We determined the influence of APU on the stemness of HSPCs and their differentiation capacity via single-cell RNA sequencing (scRNA seq) and in xenotransplantation studies. To track vector insertion site dynamics, we transduced 7-day expanded HSPCs with a mutagenic or a safer RV. ISA was conducted in human HSPCs over a 5-week cultivation in vitro and compared to the bone marrow of xenotransplanted mice to assess clonal skewings. RESULTS: APU supported the expansion of CD34+CD38-CD45RA-CD90+EPCR+ HSPCs. scRNA seq confirmed the enrichment of HSC signature genes in APU-expanded HSPCs compared to the clinically used medium SFT3 (SCF, FLT3-L, TPO, IL-3). After RV transduction, APU still maintained around 30% of CD34+ cells for 5 more weeks. Without the compounds, already 2 weeks post-transduction, less than 10% of cells were CD34+. The long-term culture allowed the detection of high-risk integrations of the mutagenic SIN-LV.SF in MEIS1 or SUSD6 due to their increasing abundance over time. Bone marrow of xenotransplanted mice was less clonal but did not support the outgrowth of insertional mutants. Overall, APU increased clonal diversity. CONCLUSIONS: Our findings propose that long-term cultivation of transduced HSPC in APU allows for outgrowth of clonal integration sites. The decrease of clonality has been observed in gene therapy patient's years after treatment. Thus, the in vitro model could be used to develop novel human HSPC-based genotoxicity assays that predict insertional mutagenesis, in addition to existing preclinical biosafety assays.
Humans↗
Systematic mapping of insertion-tolerant regions enables capsid engineering of an infectious RNA phage.
RNA phages are attractive platforms for the design of programmable bioparticles, but their development has been constrained by limited knowledge of genomic sites that can tolerate sequence insertion. Here, we combined MuA transposase-mediated in vitro insertion mutagenesis with our established reverse genetics systems to systematically identify insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. Screening of 4,555 MS2 and 2,228 PP7 random insertion clones identified 29 and 26 non-redundant ITRs, respectively. We further analyzed and compared these ITRs in the context of RNA genome organization and virion architecture. Both phages contained ITRs within the maturation protein, whereas only PP7 tolerated insertions within the coat protein (CP). On the basis of structural location and plaque-forming capacity, an ITR situated between Gly74 and Glu75 (GGC^GAG) in the PP7 CP was selected for further study. Infectious phage particles generated from complementary DNA clones retained the 15-bp insertion at both the RNA and protein levels. Engineered PP7 phages carrying an Arg-Gly-Asp motif inserted into the CP at this ITR displayed enhanced in vivo clearance in a Drosophila model, despite having in vitro stability comparable to that of the wild type. These findings provide the first example of CP engineering in an infectious RNA phage and establish a framework for engineering RNA phages for biological and biotechnological applications.IMPORTANCEA major obstacle to developing RNA phages as synthetic biology platforms is the lack of design principles for genomic insertion. Here, we address this limitation by establishing a mutagenesis-and-recovery workflow that systematically identifies insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. The resulting maps reveal distinct structural constraints in the two phages and enable rational engineering of a peptide-display site in the PP7 capsid. Using this approach, we generated an engineered infectious phage with a modified capsid, thereby providing the first demonstration of capsid engineering in an infectious RNA phage, to our knowledge. This study lays the groundwork for the rational design of live RNA phage virions as tractable and engineerable scaffolds for future biological and biotechnological applications.
Animals↗
Two CENH3 paralogs in the green alga Chlamydomonas reinhardtii have a redundantly essential function and associate with ZeppL-LINE1 elements.
Centromeres in eukaryotes are defined by the presence of histone H3 variant CENP-A/CENH3. Chlamydomonas encodes two predicted CENH3 paralogs, CENH3.1 and CENH3.2, that have not been previously characterized. We generated peptide antibodies to unique N-terminal epitopes for each of the two predicted Chlamydomonas CENH3 paralogs as well as an antibody against a shared CENH3 epitope. All three CENH3 antibodies recognized proteins of the expected size on immunoblots and had punctate nuclear immunofluorescence staining patterns. These results are consistent with both paralogs being expressed and localized to centromeres. CRISPR-Cas9-mediated insertional mutagenesis was used to generate predicted null mutations in either CENH3.1 or CENH3.2. Single mutants were viable but cenh3.1 cenh3.2 double mutants were not recovered, confirming that the function of CENH3 is essential. We sequenced and assembled two chromosome-scale Chlamydomonas genomes from strains CC-400 and UL-1690 (a derivative of CC-1690) with complete centromere sequences for 17/17 and 14/17 chromosomes respectively, enabling us to compare centromere evolution across four isolates with near complete assemblies. These data revealed significant changes across isolates between homologous centromeres including mobility and degeneration of ZeppL-LINE1 (ZeppL) transposons that comprise the major centromere repeat sequence in Chlamydomonas. We used cleavage under targets and tagmentation (CUT&Tag) to purify and map CENH3-bound genomic sequences and found enrichment of CENH3-binding almost exclusively at predicted centromere regions. An interesting exception was chromosome 2 in UL-1690, which had enrichment at its genetically mapped centromere repeat region as well as a second, distal location, centered around a single recently acquired ZeppL insertion. The CENH3-bound regions of the 17 Chlamydomonas centromeres ranged from 63.5 kb (average lower estimate) to 175 kb (average upper estimate). The relatively small size of its centromeres suggests that Chlamydomonas may be a useful organism for testing and deploying artificial chromosome technologies.
Chlamydomonas reinhardtii↗
Single-cell vector copy number analysis of phenotypically defined long-term hematopoietic stem cells for gene therapy safety assessment.
Hematopoietic stem cell (HSC)-based gene therapy has emerged as a transformative approach for the treatment of genetic diseases; however, accurate evaluation of vector copy number (VCN) remains critical for ensuring safety. Conventional bulk VCN assays, including quantitative PCR (qPCR) and droplet digital PCR (ddPCR), do not resolve clonal heterogeneity and cannot identify rare high-VCN cells that may contribute disproportionately to insertional mutagenesis risk. Here, we developed an accessible single-cell VCN profiling method by combining fluorescence-activated cell sorting (FACS) of phenotypically defined long-term HSCs (Lineage- CD34+ CD38- CD90+ CD45RA- cells) with whole-genome amplification followed by conventional qPCR. This approach enabled resolution of VCN distributions at single-cell level using standard laboratory techniques. Notably, single-cell analysis revealed a high VCN tail that bulk VCN analysis could not resolve. Furthermore, in a humanized mouse transplantation model, single-cell VCN profiling demonstrated that overall VCN distributions could be analyzed after engraftment, although inter-donor and inter-mouse variability was observed. Collectively, this method provides a rapid, cost-effective, and phenotypically resolved strategy for assessing VCN heterogeneity in gene-modified HSCs. Single-cell VCN profiling offers complementary insights beyond conventional bulk assays and may enhance preclinical safety evaluation of gene and cell therapy products.
lentiviral vector↗
AAV-mediated genome editing is influenced by the formation of R-loops.
Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.
Dependovirus↗
AAV-mediated genome editing is influenced by the formation of R-loops.
Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.
Preprint↗
mRNA therapy: A novel approach for retinal neurodegenerative diseases.
Retinal neurodegeneration remains a major cause of irreversible vision loss, yet current therapeutic options are limited in effectiveness. Although gene therapies have shown clinical potential, the overexpression platforms they rely on, such as adeno-associated virus DNA, are constrained by safety concerns, limited efficacy, and cargo size restrictions. In contrast, mRNA therapy has gained recognition as a compelling alternative, enabling rapid and efficient protein expression without the risk of genomic integration. This review synthesizes recent advances in mRNA engineering, delivery systems, and administration routes for retinal applications, and highlight strategies to enhance targeting, penetration, and controlled release through interdisciplinary collaboration between ophthalmology and bioengineering. In recent years, engineered mRNA formats, including chemically modified linear, circular, and self-amplifying RNA, can achieve higher translation efficiency within a tunable expression window. The transient nature and relatively low immunogenicity of in vitro transcribed mRNA support repeat dosing without insertional mutagenesis. Advances in nanocarriers, particularly lipid nanoparticles, have enabled preferential delivery to retinal neurons, Müller glia, and pigment epithelium via intraocular administration, while improving mRNA stability and transfection efficiency. In preclinical studies, mRNA has been widely used to deliver gene-editing tools, transcription factors, and supplementary functional proteins. In disease models such as optic nerve crush and laser-induced choroidal neovascularization, mRNA-based therapies enhance neuroprotection and suppress pathological angiogenesis in the injured retina, with favorable ocular safety profiles. However, it remains largely unexplored how the intrinsic advantages of mRNA therapy can be leveraged to develop tailored strategies for complex retinal disorders. Consistent with this gap, mRNA platforms have not yet been widely incorporated into retinal research or clinical practice. In parallel, clinical translation also lags: despite encouraging outcomes of lipid nanoparticle-mRNA formulations in preclinical models, no candidates have progressed into retinal clinical trials. This review draws on the complex pathology and therapeutic logic of retinal neurodegeneration. It proposes that mRNA therapy enables multitarget, repeatable, stage-specific interventions that align with the dynamic evolution of diseases and the requirements of combination therapy in retinal diseases. It may be used to support neuroprotection, axon regeneration, and neurovascular regulation. By integrating data across experimental models and modalities, this review outlines representative cases and experimental paradigms to guide rational trial design and carrier selection. Taken together, technical progress and evolving application strategies position mRNA therapy as a compelling therapeutic avenue for retinal neurodegeneration.
administration↗
Replication and gene functions of the bacteriocinogenic plasmid CloDF13.
The replication and genetic constitution of plasmid CloDF13 was studied using mutants of CloDF13 obtained by NTG mutagenesis, insertion of the ampicillin transposon Tn901, or deletion of particular CloDF13 DNA regions. Analysis of the polypeptides encoded by these mutant plasmids enabled us to locate six genes on the CloDF13 physical map. These genes cover about 60% of the coding capacity of CloDF13. A large part of the CloDF13 genome (about 30%) is involved in the conjugal transfer of this plasmid. This transfer region codes for at least two polypeptides, polypeptide B (61,000 daltons) and C (24,000 daltons). Those CloDF13 DNA regions responsible for the synthesis of the cloacin protein and immunity protein were also mapped on the plasmid genome. In addition we were able, using a copy mutant of CloDF13, CloDF13-cop3, to locate those DNA sequences involved in interaction with male-specific RNA phages and bacteriophage P1. For replication of CloDF13, two regions are essential. One region, from 43% to 64%, affects the stability of CloDF13-cop3 plasmids. In the case of the CloDF13-cop3 mutant, deletion of this region results in the generation of multimeric plasmid molecules accompanied by an impaired segregation of plasmid DNA molecules to daughter cells. The second region, from about 1.8% to 11.5%, contains an origin of replication as well as well as DNA sequences involved in the control of CloDF13 replication. The replication of CloDF13 starts at about 3% on the CloDF13 physical map and proceeds unidirectionally counter-clockwise.
Bacterial Proteins↗
A technique for mutagenesis by transposon insertion, applicable to most Gram-negative bacteria [proceedings].
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Bacteria↗
A Nine-Nucleotide Insertion Causes the Novel HLA-C*06:402 Allele in a Family Detected by Next-Generation Sequencing.
HLA-C*06:402 differs from HLA-C*06:02:01:01 by a nine-nucleotide (TCCTGGCTG) insertion in exon 5.
Humans↗
LINE-1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements.
LINE-1 (L1) retrotransposition is common in human cancers and rearrangements at insertion sites can contribute to cancer-driving oncogene amplifications and promote genome instability. However, the mechanisms underlying rearrangements of L1 retrotransposition intermediates are poorly understood. To address this gap, we developed GFP-based recombination reporter assays to study the formation of L1 retrotransposition-mediated rearrangements. Using these reporters combined with long-read sequencing, we find that L1 retrotransposition cDNA intermediates can recombine with distal DNA breaks to generate chromosomal rearrangements. We also find that two independent L1 insertion cDNA intermediates on distinct genomic loci can recombine with each other to generate chromosomal rearrangements. Both types of rearrangements depend on L1-encoded ORF2p endonuclease and reverse transcriptase activities. Using these reporters, we discover that L1 retrotransposition-mediated rearrangements are robustly induced when the recombining sequences share extensive homology and that their formation requires the homologous recombination factor BRCA1. In contrast, we find L1 retrotransposition-mediated rearrangements are suppressed by the mismatch repair factor MSH2 when the recombining sequences contain mismatches. Given the repetitive nature of our genome, these findings highlight the risk of L1 insertion intermediates becoming substrates for aberrant recombination and promoting genome instability.
Long Interspersed Nucleotide Elements↗
Prime assembly with linear DNA donors enables large genomic insertions.
Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy1,2. Twin prime-editing guide RNAs have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs3-6. Here we describe a prime assembly (PA) approach for the insertion of large DNA donor fragments, of which the ends are designed to overlap with the flaps generated by twin prime editing (twinPE). We used PA to insert one or multiple overlapping DNA fragments, with total insertion sizes ranging from 0.1 kb to 11 kb. An inhibitor of non-homologous end joining enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced, does not require co-delivery of exogenous DNA-dependent DNA polymerases and proceeds in non-cycling cells, suggesting independence from canonical homology-directed repair pathways. Our study demonstrates that PA can initiate Gibson-like assembly in cells to generate gene insertions without double-stranded DNA breaks, recombinases or homology-directed repair.
Animals↗
Identifying transgene insertions in Caenorhabditis elegans genomes with Oxford Nanopore sequencing.
Genetically modified organisms are commonly used in disease research and agriculture but the precise genomic alterations underlying transgenic mutations are often unknown. The position and characteristics of transgenes, including the number of independent insertions, influences the expression of both transgenic and wild-type sequences. We used long-read, Oxford Nanopore Technologies (ONT) to sequence and assemble two transgenic strains of Caenorhabditis elegans commonly used in the research of neurodegenerative diseases: BY250 (pPdat-1::GFP) and UA44 (GFP and human α-synuclein), a model for Parkinson's research. After scaffolding to the reference, the final assembled sequences were ∼102 Mb with N50s of 17.9 Mb and 18.0 Mb, respectively, and L90s of six contiguous sequences, representing chromosome-level assemblies. Each of the assembled sequences contained more than 99.2% of the Nematoda BUSCO genes found in the C. elegans reference and 99.5% of the annotated C. elegans reference protein-coding genes. We identified the locations of the transgene insertions and confirmed that all transgene sequences were inserted in intergenic regions, leaving the organismal gene content intact. The transgenic C. elegans genomes presented here will be a valuable resource for Parkinson's research as well as other neurodegenerative diseases. Our work demonstrates that long-read sequencing is a fast, cost-effective way to assemble genome sequences and characterize mutant lines and strains.
Caenorhabditis elegans↗
lpt6, a gene required for addition of phosphoethanolamine to inner-core lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae.
We previously described a gene, lpt3, required for the addition of phosphoethanolamine (PEtn) at the 3 position on the beta-chain heptose (HepII) of the inner-core Neisseria meningitidis lipopolysaccharide (LPS), but it has long been recognized that the inner-core LPS of some strains possesses PEtn at the 6 position (PEtn-6) on HepII. We have now identified a gene, lpt6 (NMA0408), that is required for the addition of PEtn-6 on HepII. The lpt6 gene is located in a region previously identified as Lgt-3 and is associated with other LPS biosynthetic genes. We screened 113 strains, representing all serogroups and including disease and carriage strains, for the lpt3 and lpt6 genes and showed that 36% contained both genes, while 50% possessed lpt3 only and 12% possessed lpt6 only. The translated amino acid sequence of lpt6 has a homologue (72.5% similarity) in a product of the Haemophilus influenzae Rd genome sequence. Previous structural studies have shown that all H. influenzae strains investigated have PEtn-6 on HepII. Consistent with this, we found that, among 70 strains representing all capsular serotypes and nonencapsulated H. influenzae strains, the lpt6 homologue was invariably present. Structural analysis of LPS from H. influenzae and N. meningitidis strains where lpt6 had been insertionally inactivated revealed that PEtn-6 on HepII could not be detected. The translated amino acid sequences from the N. meningitidis and H. influenzae lpt6 genes have conserved residues across their lengths and are part of a family of proven or putative PEtn transferases present in a wide range of gram-negative bacteria.
Amino Acid Sequence↗
Adaptation for Staphylococcus aureus to hosts via insertion mutation in the accessory gene regulator agrC gene: decreased virulence and enhanced persistence capacity.
UNLABELLED: Staphylococcus aureus is an important human pathogen due to its vast array of virulence factors regulated by multiple regulatory mechanisms, including the accessory gene regulator. In this study, two S. aureus strains were simultaneously isolated from the blood of a febrile patient, belonging to the same clone, designated as 23H with a complete hemolytic phenotype, and 23B, exhibiting an incomplete hemolytic phenotype. The genomic comparison between strains 23B and 23H revealed that 23B had a single adenine base insertion at position 923 in the agrC gene, leading to a functional loss of the encoded AgrC. Experimental findings showed that strain 23B had decreased hemolytic activity, lower cytotoxicity against human alveolar epithelial A549 cells and in the Galleria mellonella model, and a reduced ability to survive intracellularly after infecting macrophages, in comparison to 23H. Conversely, 23B exhibited enhanced biofilm formation, greater adherence to A549 cells, and increased persistence in the face of vancomycin and daptomycin treatment. Transcriptomic analysis revealed that 23B upregulated surface protein-encoding genes while simultaneously reducing the expression levels of virulence factors, highlighting the intricate regulatory adjustments facilitating its persistence and reducing pathogenic potential. ATP assay results indicated that 23B maintained elevated ATP levels during the exponential phase yet exhibited reduced levels in the stationary phase when compared with 23H. Our findings suggested that the mutation in the agrC gene of S. aureus results in diminished virulence but markedly enhances persistence. This mutated strain warrants clinical attention because it may lead to treatment failures and persist in patients. IMPORTANCE: In clinical antimicrobial therapy, bacterial strains often develop resistance to antimicrobial agents. Additionally, mutations in their gene regulatory networks can increase their persistence, especially in immunocompromised patients. This study identified an insertion mutation in the accessory gene regulator, agrC gene, carried by a Staphylococcus aureus strain isolated from the blood of a febrile patient, leading to the functional loss of AgrC. Further research revealed that despite the reduced virulence of the mutated strain, it significantly bolstered the capacity to adapt and endure within the host during prolonged infections. This was evidenced by increased adhesion and biofilm formation capabilities, development of antimicrobial tolerance, and decreased ATP levels linked to persistence. Therefore, monitoring these mutations in S. aureus is crucial clinically, as they can complicate treatment strategies.
Staphylococcus aureus↗