Pub Date : 2024-09-12DOI: 10.1101/2024.09.10.612307
Azad Alizada, Aline Martins, Nolwenn Mouniee, Julia Rodriguez Suarez, Benjamin Bertin, Nathalie Gueguen, Vincent Mirouse, Stephanie Maupetit Mehouas, Austin J Rivera, Nelson C. Lau, Gregory J Hannon, Benjamin Czech Nicholson, Emilie Brasset
Transposable elements (TEs) pose a threat to genome integrity, and the piRNA pathway in animal gonads plays a crucial role in silencing TE activity. While the transcriptional regulation of the piRNA pathway components in germ cells has been documented in mice and flies, the mechanisms orchestrating the transcriptional program of the somatic piRNA pathway in Drosophila ovaries remains unresolved. Here, we demonstrate that Traffic jam (Tj), an orthologue of a large Maf transcription factor in mammals, is a master regulator of the piRNA pathway in ovarian somatic cells, playing a crucial role in maintaining TE silencing and genomic integrity in somatic tissues. We show that Tj directly binds to the promoters of somatic-enriched piRNA factors such as fs(1)Yb, nxf2, panx, and armi, as well as the flamenco piRNA cluster, a major locus for TE silencing in somatic cells. Depletion of Tj in somatic follicle cells results in a significant downregulation of these piRNA factors, a complete loss of flam expression and de-repression of gypsy-family TEs, which have gained the ability to activate in ovarian somatic cells allowing them to infect germ cells and be transmitted to future generations. We have identified an enhancer carrying Tj binding motifs located downstream of the flam promoter that is essential for robust and tissue-specific flam expression in somatic follicle cells of the adult ovary. This work uncovers a previously unappreciated layer of transcriptional regulation of the piRNA pathway, and we propose that the arms race between the host and TEs has driven the evolution of promoters in piRNA genes and clusters to respond to a unique transcription factor thereby ensuring efficient silencing of gypsy-family TEs
可转座元件（TE）对基因组完整性构成威胁，而动物性腺中的 piRNA 通路在沉默 TE 活性方面起着至关重要的作用。虽然小鼠和苍蝇的生殖细胞中 piRNA 通路成分的转录调控已被记录，但果蝇卵巢中体细胞 piRNA 通路转录程序的协调机制仍未解决。在这里，我们证明了Traffic jam（Tj）是哺乳动物中大型Maf转录因子的直系同源物，是卵巢体细胞中piRNA通路的主调控因子，在维持体细胞组织中的TE沉默和基因组完整性方面起着至关重要的作用。我们的研究表明，Tj 直接与体细胞丰富的 piRNA 因子（如 fs(1)Yb、nxf2、panx 和 armi）的启动子以及 flamenco piRNA 簇（体细胞中 TE 沉默的主要位点）结合。体细胞卵泡中 Tj 的缺失会导致这些 piRNA 因子的显著下调、flam 表达的完全丧失以及吉普赛家族 TEs 的去抑制，而吉普赛家族 TEs 已获得在卵巢体细胞中激活的能力，使其能够感染生殖细胞并传给后代。我们发现了一个位于flam启动子下游、携带Tj结合基序的增强子，它对于flam在成体卵巢体细胞卵泡中的强健表达和组织特异性表达至关重要。这项工作揭示了 piRNA 通路转录调控的一个以前未被认识的层次，我们认为宿主和 TE 之间的军备竞赛推动了 piRNA 基因和基因簇启动子的进化，使其能够对一种独特的转录因子做出反应，从而确保对吉普赛家族 TE 的有效沉默。
{"title":"The transcription factor Traffic jam orchestrates the somatic piRNA pathway in Drosophila ovaries","authors":"Azad Alizada, Aline Martins, Nolwenn Mouniee, Julia Rodriguez Suarez, Benjamin Bertin, Nathalie Gueguen, Vincent Mirouse, Stephanie Maupetit Mehouas, Austin J Rivera, Nelson C. Lau, Gregory J Hannon, Benjamin Czech Nicholson, Emilie Brasset","doi":"10.1101/2024.09.10.612307","DOIUrl":"https://doi.org/10.1101/2024.09.10.612307","url":null,"abstract":"Transposable elements (TEs) pose a threat to genome integrity, and the piRNA pathway in animal gonads plays a crucial role in silencing TE activity. While the transcriptional regulation of the piRNA pathway components in germ cells has been documented in mice and flies, the mechanisms orchestrating the transcriptional program of the somatic piRNA pathway in Drosophila ovaries remains unresolved. Here, we demonstrate that Traffic jam (Tj), an orthologue of a large Maf transcription factor in mammals, is a master regulator of the piRNA pathway in ovarian somatic cells, playing a crucial role in maintaining TE silencing and genomic integrity in somatic tissues. We show that Tj directly binds to the promoters of somatic-enriched piRNA factors such as fs(1)Yb, nxf2, panx, and armi, as well as the flamenco piRNA cluster, a major locus for TE silencing in somatic cells. Depletion of Tj in somatic follicle cells results in a significant downregulation of these piRNA factors, a complete loss of flam expression and de-repression of gypsy-family TEs, which have gained the ability to activate in ovarian somatic cells allowing them to infect germ cells and be transmitted to future generations. We have identified an enhancer carrying Tj binding motifs located downstream of the flam promoter that is essential for robust and tissue-specific flam expression in somatic follicle cells of the adult ovary. This work uncovers a previously unappreciated layer of transcriptional regulation of the piRNA pathway, and we propose that the arms race between the host and TEs has driven the evolution of promoters in piRNA genes and clusters to respond to a unique transcription factor thereby ensuring efficient silencing of gypsy-family TEs","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"100 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212723","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1101/2024.08.27.609932
Saraladevi Muthusamy, Ramesh Raju Vetukuri, Anneli Lundgren, Sungyong Kim, Pruthvi B Kalyandurg, Ake Strid, Li-Hua Zhu, Selvaraju Kanagarajan, Peter Brodelius
The aqueous extract of Cynara cardunculus flowers is traditionally used in cheese production across Mediterranean countries. To meet the growing industrial demand for plant-based milk-clotting enzymes and to explore potential biotechnological applications, we initiated a study to heterologously produce cyprosin B (CYPB), a key milk-clotting enzyme from C. cardunculus, in Nicotiana benthamiana. We also investigated the role of its plant-specific insert (PSI) domain in the CYPBs activity and its localization. In this study, full-length CYPB and a PSI domain deleted CYPB (CYPBΔPSI) were transiently expressed in N. benthamiana leaves using Agrobacterium-mediated infiltration. The leaves were harvested nine days post-infiltration, and proteins were purified, yielding approximately 81 mg/kg (CYPB) and 60 mg/kg (CYPBΔPSI) fresh weight. CYPBΔPSI showed significantly higher proteolytic activity (156.72 IU/mg) than CYPB (57.2 IU/mg), indicating that the PSI domain is not essential for enzymatic activity and that its removal results in enhanced enzymatic efficiency. In the milk-clotting activity assay, CYPBΔPSI demonstrated a significantly faster clotting time than full-length CYPB, indicating enhanced milk-clotting efficiency for CYPBΔPSI. Subcellular localization studies revealed that CYPB and PSI were localized in the vacuole and endocytic vesicles. In contrast, CYPBΔPSI was primarily localized in the endoplasmic reticulum (ER) and the tonoplast, suggesting that the PSI domain is critical for vacuolar targeting and membrane permeabilization that affects overall protein yield. This study demonstrates the feasibility of using N. benthamiana as a platform for the scalable production of more efficient recombinant CYPB. It highlights the multifunctional role of the PSI domain in vacuolar sorting without impairing its functionality. These results underscore the potential of plant-based expression systems as a viable alternative for the industrial production of plant milk-clotting enzymes, with significant implications for sustainable cheese production.
{"title":"Heterologous Production of Cyprosin B in Nicotiana benthamiana: Unveiling the Role of the Plant-Specific Insert Domain in Protein Function and Subcellular Localization","authors":"Saraladevi Muthusamy, Ramesh Raju Vetukuri, Anneli Lundgren, Sungyong Kim, Pruthvi B Kalyandurg, Ake Strid, Li-Hua Zhu, Selvaraju Kanagarajan, Peter Brodelius","doi":"10.1101/2024.08.27.609932","DOIUrl":"https://doi.org/10.1101/2024.08.27.609932","url":null,"abstract":"The aqueous extract of Cynara cardunculus flowers is traditionally used in cheese production across Mediterranean countries. To meet the growing industrial demand for plant-based milk-clotting enzymes and to explore potential biotechnological applications, we initiated a study to heterologously produce cyprosin B (CYPB), a key milk-clotting enzyme from C. cardunculus, in Nicotiana benthamiana. We also investigated the role of its plant-specific insert (PSI) domain in the CYPBs activity and its localization. In this study, full-length CYPB and a PSI domain deleted CYPB (CYPBΔPSI) were transiently expressed in N. benthamiana leaves using Agrobacterium-mediated infiltration. The leaves were harvested nine days post-infiltration, and proteins were purified, yielding approximately 81 mg/kg (CYPB) and 60 mg/kg (CYPBΔPSI) fresh weight. CYPBΔPSI showed significantly higher proteolytic activity (156.72 IU/mg) than CYPB (57.2 IU/mg), indicating that the PSI domain is not essential for enzymatic activity and that its removal results in enhanced enzymatic efficiency. In the milk-clotting activity assay, CYPBΔPSI demonstrated a significantly faster clotting time than full-length CYPB, indicating enhanced milk-clotting efficiency for CYPBΔPSI. Subcellular localization studies revealed that CYPB and PSI were localized in the vacuole and endocytic vesicles. In contrast, CYPBΔPSI was primarily localized in the endoplasmic reticulum (ER) and the tonoplast, suggesting that the PSI domain is critical for vacuolar targeting and membrane permeabilization that affects overall protein yield. This study demonstrates the feasibility of using N. benthamiana as a platform for the scalable production of more efficient recombinant CYPB. It highlights the multifunctional role of the PSI domain in vacuolar sorting without impairing its functionality. These results underscore the potential of plant-based expression systems as a viable alternative for the industrial production of plant milk-clotting enzymes, with significant implications for sustainable cheese production.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612527
Savita Gupta, Gursimran Kaur Bajwa, Hadil El-Sammak, Kenny Mattonet, Stefan Gunther, Mario Looso, Didier Y.R. Stainier, Ruben Marin-Juez
Myocardial infarction occurs when coronary supply of oxygen and nutrients to part of the heart is interrupted. In contrast to adult mammals, adult zebrafish have a unique ability to regenerate their heart after cardiac injury. Several processes are involved in this regenerative response including inflammation, coronary endothelial cell proliferation and revascularization, endocardial expansion, cardiomyocyte repopulation, and transient scar formation. To identify potential regulators of zebrafish cardiac regeneration, we profiled the transcriptome of regenerating coronary endothelial cells at 7 days post cryoinjury (dpci) and observed the significant upregulation of dozens of genes including gpnmb. Gpnmb (glycoprotein non-metastatic melanoma protein B) is a transmembrane glycoprotein implicated in inflammation resolution and tissue regeneration. Transcriptomic profiling data of cryoinjured zebrafish hearts reveal that gpnmb is mostly expressed by macrophages. To investigate gpnmb function during zebrafish cardiac regeneration, we generated a full locus deletion (FLD) allele. We find that after cardiac cryoinjury, animals lacking gpnmb exhibit neutrophil retention and decreased macrophage recruitment as well as reduced myofibroblast numbers. Moreover, loss of gpnmb impairs coronary endothelial cell regeneration and cardiomyocyte dedifferentiation. Transcriptomic analyses of cryoinjured gpnmb mutant hearts identified enhanced collagen gene expression and the activation of extracellular matrix (ECM) related pathways. Furthermore, gpnmb mutant hearts exhibit larger fibrotic scars revealing additional defects in cardiac regeneration. Altogether, these data indicate that gpnmb expressing macrophages modulate inflammation and ECM deposition after cardiac cryoinjury in zebrafish and further highlight the importance of this subset of immune cells to support a regenerative response.
{"title":"The transmembrane glycoprotein Gpnmb is required for the immune and fibrotic responses during zebrafish heart regeneration","authors":"Savita Gupta, Gursimran Kaur Bajwa, Hadil El-Sammak, Kenny Mattonet, Stefan Gunther, Mario Looso, Didier Y.R. Stainier, Ruben Marin-Juez","doi":"10.1101/2024.09.11.612527","DOIUrl":"https://doi.org/10.1101/2024.09.11.612527","url":null,"abstract":"Myocardial infarction occurs when coronary supply of oxygen and nutrients to part of the heart is interrupted. In contrast to adult mammals, adult zebrafish have a unique ability to regenerate their heart after cardiac injury. Several processes are involved in this regenerative response including inflammation, coronary endothelial cell proliferation and revascularization, endocardial expansion, cardiomyocyte repopulation, and transient scar formation. To identify potential regulators of zebrafish cardiac regeneration, we profiled the transcriptome of regenerating coronary endothelial cells at 7 days post cryoinjury (dpci) and observed the significant upregulation of dozens of genes including gpnmb. Gpnmb (glycoprotein non-metastatic melanoma protein B) is a transmembrane glycoprotein implicated in inflammation resolution and tissue regeneration. Transcriptomic profiling data of cryoinjured zebrafish hearts reveal that <em>gpnmb</em> is mostly expressed by macrophages. To investigate <em>gpnmb</em> function during zebrafish cardiac regeneration, we generated a full locus deletion (FLD) allele. We find that after cardiac cryoinjury, animals lacking gpnmb exhibit neutrophil retention and decreased macrophage recruitment as well as reduced myofibroblast numbers. Moreover, loss of <em>gpnmb</em> impairs coronary endothelial cell regeneration and cardiomyocyte dedifferentiation. Transcriptomic analyses of cryoinjured <em>gpnmb</em> mutant hearts identified enhanced collagen gene expression and the activation of extracellular matrix (ECM) related pathways. Furthermore, gpnmb mutant hearts exhibit larger fibrotic scars revealing additional defects in cardiac regeneration. Altogether, these data indicate that <em>gpnmb</em> expressing macrophages modulate inflammation and ECM deposition after cardiac cryoinjury in zebrafish and further highlight the importance of this subset of immune cells to support a regenerative response.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612416
Emilia A Zin, Melissa Desrosiers, Tommaso Ocari, Guillaume Labernede, Camille Robert, Charlotte Izabella, Bruno Saubamea, Ulisse Ferrari, Deniz Dalkara
Determining the concentration of recombinant adeno-associated virus (AAV) productions, also known as titering, is crucial not only for quality control purposes but also for comparative studies of preclinical and clinical gene therapy trials. Recently, several AAVs were engineered by inserting seven amino acids at the outermost tip of the capsid's protruding VR-VIII loop. These variants have demonstrated increased transduction capabilities over naturally occurring AAV serotypes in several studies. However, they have also been shown to produce lower yields when titered using standard techniques, raising questions about their adequacy for clinical development and use. Here, we investigated why peptide insertion onto AAV capsids reduces their titer by examining viral stocks using electron microscopy and PCR-based titering. We reveal that the DNAse digestion step, performed to eliminate free-floating DNA prior to qPCR or ddPCR, adversely impacts engineered capsid stability due to exposure to heat, artificially lowering viral titers of engineered serotypes. Titering without heating yields significantly higher titers for these variants which have melting temperatures (Tm) close to the DNAse inactivation temperature, while titers for parental serotypes with higher Tm remain unchanged. Our findings provide an important new perspective for titering engineered variants with lower thermostability, especially when comparing their effectiveness to their parental serotypes.
{"title":"The Role of Thermal Stability in AAV Titration of Engineered Variants","authors":"Emilia A Zin, Melissa Desrosiers, Tommaso Ocari, Guillaume Labernede, Camille Robert, Charlotte Izabella, Bruno Saubamea, Ulisse Ferrari, Deniz Dalkara","doi":"10.1101/2024.09.11.612416","DOIUrl":"https://doi.org/10.1101/2024.09.11.612416","url":null,"abstract":"Determining the concentration of recombinant adeno-associated virus (AAV) productions, also known as titering, is crucial not only for quality control purposes but also for comparative studies of preclinical and clinical gene therapy trials. Recently, several AAVs were engineered by inserting seven amino acids at the outermost tip of the capsid's protruding VR-VIII loop. These variants have demonstrated increased transduction capabilities over naturally occurring AAV serotypes in several studies. However, they have also been shown to produce lower yields when titered using standard techniques, raising questions about their adequacy for clinical development and use. Here, we investigated why peptide insertion onto AAV capsids reduces their titer by examining viral stocks using electron microscopy and PCR-based titering. We reveal that the DNAse digestion step, performed to eliminate free-floating DNA prior to qPCR or ddPCR, adversely impacts engineered capsid stability due to exposure to heat, artificially lowering viral titers of engineered serotypes. Titering without heating yields significantly higher titers for these variants which have melting temperatures (Tm) close to the DNAse inactivation temperature, while titers for parental serotypes with higher Tm remain unchanged. Our findings provide an important new perspective for titering engineered variants with lower thermostability, especially when comparing their effectiveness to their parental serotypes.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212745","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.10.612363
Bitong Li, Yew Yan Wong, Neftali Flores-Rodriguez, Tara Davidson, Matthew S Graus, Valeriia Smialkovska, Hiroaki Ohishi, Angelika Feldmann, Hiroshi Ochiai, Mathias Francois
The ability of stem cells to divide and self-renew depends on a complex choreography of molecular events that maintain the transcriptional oscillation of pluripotency genes. Only a handful of transcription factors (TFs) are necessary to preserve pluripotency and reprogram differentiated cells into stem cells. Paradoxically, while the protein players are known, the challenge remains to decipher the series of steps that TFs undertake to modulate on and off fluctuations of gene transcription. Here, we use single-molecule tracking combined with the STREAMING-tag transcriptional reporter systems to reveal temporal clustering patterns of endogenous SOX2 occupancy at the Nanog locus in relation to its nascent mRNA synthesis in live embryonic stem cells. These patterns distinctively outline multifaceted regulatory behaviours of SOX2 associated with various stages of the Nanog transcription cycle. This study exposes that SOX2 clustering activity is out-of-phase with regulatory factors that engage with transcription burst at the Nanog gene locus.
{"title":"Anti-phase clustering of regulatory factors shapes gene bursting","authors":"Bitong Li, Yew Yan Wong, Neftali Flores-Rodriguez, Tara Davidson, Matthew S Graus, Valeriia Smialkovska, Hiroaki Ohishi, Angelika Feldmann, Hiroshi Ochiai, Mathias Francois","doi":"10.1101/2024.09.10.612363","DOIUrl":"https://doi.org/10.1101/2024.09.10.612363","url":null,"abstract":"The ability of stem cells to divide and self-renew depends on a complex choreography of molecular events that maintain the transcriptional oscillation of pluripotency genes. Only a handful of transcription factors (TFs) are necessary to preserve pluripotency and reprogram differentiated cells into stem cells. Paradoxically, while the protein players are known, the challenge remains to decipher the series of steps that TFs undertake to modulate on and off fluctuations of gene transcription. Here, we use single-molecule tracking combined with the STREAMING-tag transcriptional reporter systems to reveal temporal clustering patterns of endogenous SOX2 occupancy at the Nanog locus in relation to its nascent mRNA synthesis in live embryonic stem cells. These patterns distinctively outline multifaceted regulatory behaviours of SOX2 associated with various stages of the Nanog transcription cycle. This study exposes that SOX2 clustering activity is out-of-phase with regulatory factors that engage with transcription burst at the Nanog gene locus.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212743","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612500
Hekmat A. Owaid, Mushtak T. S. Al-Ouqaili, Farah Al-Marzooq
Background: Gene editing techniques have been identified as potential tools to combat antimicrobial resistance (AMR). Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated sequences (Cas) can be key players in this process. Their presence in bacteria can impact the success of this technology in combating AMR. The aim of this study is to investigate whether CRISPR loci are associated by multidrug, extensive drug, or pan-drug resistance in Klebsiella pneumoniae from Iraq. Methods: Antibiotic susceptibility testing was performed for 100 isolates to detect patterns of resistance. PCR was used to investigate CRISPR/Cas systems. Whole genome sequencing (WGS) was performed on relevant isolates using a DNA nanoball sequencing platform. Results: Out of 81 K. pneumoniae isolates, 81% were resistant to antibiotics, with 71% producing ESBLs and 21% producing carbapenemases. Additionally, 53% were MDR, 19% XDR, and 9% PDR. Complete CRISPR/Cas systems were found in 38% of isolates, while 78% had incomplete systems. Furthermore, intact CRISPR-1, CRISPR2, and CRISPR3 types were found in 27.0%, 34%, and 18.0% of the isolates, respectively. An inverse correlation was found between antibiotic resistance levels and the presence of CRISPR/Cas systems. Two carbapenemase-producing K. pneumoniae (XDR and PDR) isolates were characterized by WGS. They were found to be carrying blaNDM-5 and blaOXA-181 genes with additional resistance genes against various antibiotic classes, in addition to CRISPR/Cas systems. Phylogenetic analysis indicated relationships with United Kingdom and Chinese strains. Furthermore, the entire genome revealed the presence of unique virulence and antibiotic resistance genes in Klebsiella pneumoniae. Conclusion: An inverse relationship was found between CRISPR/Cas systems and antimicrobial resistance in K. pneumoniae. The discovery of blaNDM-5 and blaOXA-181 genes in Iraqi strains is alarming as this can increase the risk of nosocomial outbreaks. This study elucidates the importance of monitoring CRISPR/Cas systems and antimicrobial resistance for more efficient control and prevention of infection in healthcare settings.
{"title":"Genomic approach to studying the relationship between the CRISPR/Cas system and multidrug resistance in clinical isolates of Klebsiella pneumoniae","authors":"Hekmat A. Owaid, Mushtak T. S. Al-Ouqaili, Farah Al-Marzooq","doi":"10.1101/2024.09.11.612500","DOIUrl":"https://doi.org/10.1101/2024.09.11.612500","url":null,"abstract":"Background: Gene editing techniques have been identified as potential tools to combat antimicrobial resistance (AMR). Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated sequences (Cas) can be key players in this process. Their presence in bacteria can impact the success of this technology in combating AMR. The aim of this study is to investigate whether CRISPR loci are associated by multidrug, extensive drug, or pan-drug resistance in Klebsiella pneumoniae from Iraq.\u0000Methods: Antibiotic susceptibility testing was performed for 100 isolates to detect patterns of resistance. PCR was used to investigate CRISPR/Cas systems. Whole genome sequencing (WGS) was performed on relevant isolates using a DNA nanoball sequencing platform.\u0000Results: Out of 81 K. pneumoniae isolates, 81% were resistant to antibiotics, with 71% producing ESBLs and 21% producing carbapenemases. Additionally, 53% were MDR, 19% XDR, and 9% PDR. Complete CRISPR/Cas systems were found in 38% of isolates, while 78% had incomplete systems. Furthermore, intact CRISPR-1, CRISPR2, and CRISPR3 types were found in 27.0%, 34%, and 18.0% of the isolates, respectively. An inverse correlation was found between antibiotic resistance levels and the presence of CRISPR/Cas systems. Two carbapenemase-producing K. pneumoniae (XDR and PDR) isolates were characterized by WGS. They were found to be carrying blaNDM-5 and blaOXA-181 genes with additional resistance genes against various antibiotic classes, in addition to CRISPR/Cas systems. Phylogenetic analysis indicated relationships with United Kingdom and Chinese strains. Furthermore, the entire genome revealed the presence of unique virulence and antibiotic resistance genes in Klebsiella pneumoniae.\u0000Conclusion: An inverse relationship was found between CRISPR/Cas systems and antimicrobial resistance in K. pneumoniae. The discovery of blaNDM-5 and blaOXA-181 genes in Iraqi strains is alarming as this can increase the risk of nosocomial outbreaks. This study elucidates the importance of monitoring CRISPR/Cas systems and antimicrobial resistance for more efficient control and prevention of infection in healthcare settings.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612485
Cunren Liu, Rosa Serra
Previously, we showed that Papss2 expression is regulated through a Sox9-dependent pathway. Here we explore molecular mechanisms whereby Sox9 regulates mouse Papss2. A 509bp Sox9-responsive DNA element (Region C) was identified upstream of the Papss2 second start site using co-transfection and luciferase reporter assays. A Sox9 responsive element was narrowed down to 32bps within Region C (Sox9RE). Putative SoxE and C/EBPβ binding sites were identified within S9RE. C/EBPβ was identified as a repressor for Sox9-mediated activity. In cells transfected with expression vectors for C/EBPβ and Sox9, increasing amounts of C/EBPβ resulted in attenuation of Sox9-mediated activation of Region C while increasing amounts of Sox9 activated transcription in the presence of C/EBPβ. Using electromobility shift assays, three protein complexes were identified on S9RE after incubation with nuclear extracts from ATDC5 cells. Super shift assays indicated that under basal conditions C/EBPβ was present in the DNA-protein complexes observed. Unlabeled S9RE with point mutations in the predicted SoxE binding site competed with protein complex formation on the S9RE while excess oligo corresponding to the predicted SoxE binding site did not, suggesting that proteins do not bind to SoxE motiff under basal conditions. Under conditions of high Sox9 expression, the formation of protein-DNA complexes on S9RE was inhibited. We then showed by western blot that increasing Sox9 protein resulted in reduced C/EBPβ protein levels. Co-immunoprecipitation indicated interaction of Sox9 and C/EBPβ proteins. We propose that Sox9 acts to derepress C/EBPβ-inhibited transcription of Papss2 by first interacting with C/EBPβ to prevent it from binding DNA, then reducing C/EBPβ expression.
{"title":"SRY-Box Transcription Factor 9 regulates 3'-Phosphoadenosine 5'-Phosphosulfate Synthase 2 mRNA expression through derepression of the transcriptional repressor, CCAAT/enhancer-binding protein beta","authors":"Cunren Liu, Rosa Serra","doi":"10.1101/2024.09.11.612485","DOIUrl":"https://doi.org/10.1101/2024.09.11.612485","url":null,"abstract":"Previously, we showed that Papss2 expression is regulated through a Sox9-dependent pathway. Here we explore molecular mechanisms whereby Sox9 regulates mouse Papss2. A 509bp Sox9-responsive DNA element (Region C) was identified upstream of the Papss2 second start site using co-transfection and luciferase reporter assays. A Sox9 responsive element was narrowed down to 32bps within Region C (Sox9RE). Putative SoxE and C/EBPβ binding sites were identified within S9RE. C/EBPβ was identified as a repressor for Sox9-mediated activity. In cells transfected with expression vectors for C/EBPβ and Sox9, increasing amounts of C/EBPβ resulted in attenuation of Sox9-mediated activation of Region C while increasing amounts of Sox9 activated transcription in the presence of C/EBPβ. Using electromobility shift assays, three protein complexes were identified on S9RE after incubation with nuclear extracts from ATDC5 cells. Super shift assays indicated that under basal conditions C/EBPβ was present in the DNA-protein complexes observed. Unlabeled S9RE with point mutations in the predicted SoxE binding site competed with protein complex formation on the S9RE while excess oligo corresponding to the predicted SoxE binding site did not, suggesting that proteins do not bind to SoxE motiff under basal conditions. Under conditions of high Sox9 expression, the formation of protein-DNA complexes on S9RE was inhibited. We then showed by western blot that increasing Sox9 protein resulted in reduced C/EBPβ protein levels. Co-immunoprecipitation indicated interaction of Sox9 and C/EBPβ proteins. We propose that Sox9 acts to derepress C/EBPβ-inhibited transcription of Papss2 by first interacting with C/EBPβ to prevent it from binding DNA, then reducing C/EBPβ expression.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"100 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612461
Annabelle Campbell, Hanna F. Esser, A. Maxwell Burroughs, Otto Berninghausen, L. Aravind, Thomas Becker, Rachel Green, Roland Beckmann, Allen R. Buskirk
Although many antibiotics inhibit bacterial ribosomes, loss of known factors that rescue stalled ribosomes does not lead to robust antibiotic sensitivity in E. coli, suggesting the existence of additional mechanisms. Here, we show that the RNA helicase HrpA rescues stalled ribosomes in E. coli. Acting selectively on ribosomes that have collided, HrpA uses ATP hydrolysis to split stalled ribosomes into subunits. Cryo-EM structures reveal how HrpA simultaneously binds to two collided ribosomes, explaining its selectivity, and how its helicase module engages downstream mRNA, such that by exerting a pulling force on the mRNA, it would destabilize the stalled ribosome. These studies show that ribosome splitting is a conserved mechanism that allows proteobacteria to tolerate ribosome-targeting antibiotics.
{"title":"The RNA helicase HrpA rescues collided ribosomes in E. coli","authors":"Annabelle Campbell, Hanna F. Esser, A. Maxwell Burroughs, Otto Berninghausen, L. Aravind, Thomas Becker, Rachel Green, Roland Beckmann, Allen R. Buskirk","doi":"10.1101/2024.09.11.612461","DOIUrl":"https://doi.org/10.1101/2024.09.11.612461","url":null,"abstract":"Although many antibiotics inhibit bacterial ribosomes, loss of known factors that rescue stalled ribosomes does not lead to robust antibiotic sensitivity in <em>E. coli</em>, suggesting the existence of additional mechanisms. Here, we show that the RNA helicase HrpA rescues stalled ribosomes in <em>E. coli</em>. Acting selectively on ribosomes that have collided, HrpA uses ATP hydrolysis to split stalled ribosomes into subunits. Cryo-EM structures reveal how HrpA simultaneously binds to two collided ribosomes, explaining its selectivity, and how its helicase module engages downstream mRNA, such that by exerting a pulling force on the mRNA, it would destabilize the stalled ribosome. These studies show that ribosome splitting is a conserved mechanism that allows proteobacteria to tolerate ribosome-targeting antibiotics.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212747","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612387
Marie-Katherin Zuehlke, Alexandra Bahr, Daniel Bartosik, Vipul Solanki, Michelle Teune, Frank Unfried, Tristan Barbeyron, Elizabeth Ficko-Blean, Lionel Cladiere, Alexandra Jeudy, Anne Susemihl, Fabian Hartmann, Diane Jouanneau, Murielle Jam, Matthias Hoehne, Mihaela Delcea, Uwe Bornscheuer, Doerte Becher, Jan-Hendrik Hehemann, Mirjam Czjzek, Thomas Schweder
SusC/D-like proteins are essential components of glycan utilization machineries in Bacteroidota, but remain unknown in other bacterial phyla. The glycan-binding SusD-like protein forms a lid on top of the SusC-like TonB-dependent transporter (TBDT) and both are structurally designed to function as a complex in sugar uptake. In comparison, Gammaproteobacteria import glycans using classical TBDTs without an accessory SusD-like protein. We have now identified a SusD-like protein and a SusC-like TBDT in a fructan polysaccharide utilization locus (PUL) of the marine gammaproteobacterium Pseudoalteromonas distincta, which are tandemly organized as in Bacteroidota. Proteome analysis revealed an increased production of PUL-encoded proteins during growth on inulin- and levan-type fructans. However, P. distincta preferred inulin over plant-derived levan and hardly grew on bacterial levan. Further analysis showed that the SusD-like protein has a weak affinity (Ka 43 M-1) for oligosaccharides from inulin. The PUL-encoded glycoside hydrolase from family 32 (GH32) hydrolyzes inulin and plant-derived levan, but also has a low activity on bacterial levan, which confirms the growth experiments. Comparative genomics identified further SusC/D-like proteins in Gammaproteobacteria genomes, most of which (83%) were encoded in fructan PULs.
{"title":"SusC/D-like proteins in Gammaproteobacteria that utilize fructans","authors":"Marie-Katherin Zuehlke, Alexandra Bahr, Daniel Bartosik, Vipul Solanki, Michelle Teune, Frank Unfried, Tristan Barbeyron, Elizabeth Ficko-Blean, Lionel Cladiere, Alexandra Jeudy, Anne Susemihl, Fabian Hartmann, Diane Jouanneau, Murielle Jam, Matthias Hoehne, Mihaela Delcea, Uwe Bornscheuer, Doerte Becher, Jan-Hendrik Hehemann, Mirjam Czjzek, Thomas Schweder","doi":"10.1101/2024.09.11.612387","DOIUrl":"https://doi.org/10.1101/2024.09.11.612387","url":null,"abstract":"SusC/D-like proteins are essential components of glycan utilization machineries in <em>Bacteroidota</em>, but remain unknown in other bacterial phyla. The glycan-binding SusD-like protein forms a lid on top of the SusC-like TonB-dependent transporter (TBDT) and both are structurally designed to function as a complex in sugar uptake. In comparison, <em>Gammaproteobacteria</em> import glycans using classical TBDTs without an accessory SusD-like protein. We have now identified a SusD-like protein and a SusC-like TBDT in a fructan polysaccharide utilization locus (PUL) of the marine gammaproteobacterium <em>Pseudoalteromonas distincta</em>, which are tandemly organized as in <em>Bacteroidota</em>. Proteome analysis revealed an increased production of PUL-encoded proteins during growth on inulin- and levan-type fructans. However, <em>P. distincta</em> preferred inulin over plant-derived levan and hardly grew on bacterial levan. Further analysis showed that the SusD-like protein has a weak affinity (Ka 43 M<sup>-1</sup>) for oligosaccharides from inulin. The PUL-encoded glycoside hydrolase from family 32 (GH32) hydrolyzes inulin and plant-derived levan, but also has a low activity on bacterial levan, which confirms the growth experiments. Comparative genomics identified further SusC/D-like proteins in <em>Gammaproteobacteria</em> genomes, most of which (83%) were encoded in fructan PULs.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612449
Aurel Holzschuh, Anita Lerch, Christian Nsanzabana
Background The assessment of antimalarial drug efficacy against Plasmodium falciparum requires PCR correction to distinguish recrudescence from new infections by comparing parasite genotypes before treatment and in recurrent infections. Nanopore sequencing offers a low-cost, portable, scalable, and rapid alternative to traditional methods, supporting the expansion and decentralization of sequencing in endemic, resource-limited settings, potentially providing rapid PCR-corrected drug failure estimates. Methods We optimized a multiplexed AmpSeq panel targeting six microhaplotypes for high and uniform coverage. We assessed sensitivity and specificity for detecting minority clones in polyclonal infections and evaluated genetic diversity across the microhaplotype markers. We used mixtures of four P. falciparum laboratory strains at different ratios and 20 paired patient samples from a clinical trial. A custom bioinformatics workflow was used to infer haplotypes from polyclonal infections, including minority clones, with defined cut-off criteria for accurate haplotype calling. Findings The nanopore AmpSeq assay achieved uniform and high read coverage across all six microhaplotype markers (median coverage: 12,989x to 15,440x for laboratory strain mixtures and 7,011x to 11,600x for patients' samples, respectively). We found high sensitivity in detecting minority clones (up to 50:1:1:1 in the 3D7:K1:HB3:FCB1 laboratory strain mixtures) and high specificity with less than 0.01% of all reads being false-positive haplotypes. Genetic diversity in the markers used was high (HE ≥ 0.98 and up to 31 unique haplotypes in 20 paired samples with cpmp), and concordant results in classifying new infections and recrudescence across all markers used were observed in 18 (90%) of 20 paired samples. Interpretation Our study demonstrates the feasibility of nanopore AmpSeq for distinguishing recrudescence from new infections in clinical trials.
{"title":"Multiplexed nanopore amplicon sequencing to distinguish recrudescence from new infection in antimalarial drug trials","authors":"Aurel Holzschuh, Anita Lerch, Christian Nsanzabana","doi":"10.1101/2024.09.11.612449","DOIUrl":"https://doi.org/10.1101/2024.09.11.612449","url":null,"abstract":"Background\u0000The assessment of antimalarial drug efficacy against <em>Plasmodium falciparum</em> requires PCR correction to distinguish recrudescence from new infections by comparing parasite genotypes before treatment and in recurrent infections. Nanopore sequencing offers a low-cost, portable, scalable, and rapid alternative to traditional methods, supporting the expansion and decentralization of sequencing in endemic, resource-limited settings, potentially providing rapid PCR-corrected drug failure estimates.\u0000Methods\u0000We optimized a multiplexed AmpSeq panel targeting six microhaplotypes for high and uniform coverage. We assessed sensitivity and specificity for detecting minority clones in polyclonal infections and evaluated genetic diversity across the microhaplotype markers. We used mixtures of four <em>P. falciparum</em> laboratory strains at different ratios and 20 paired patient samples from a clinical trial. A custom bioinformatics workflow was used to infer haplotypes from polyclonal infections, including minority clones, with defined cut-off criteria for accurate haplotype calling.\u0000Findings\u0000The nanopore AmpSeq assay achieved uniform and high read coverage across all six microhaplotype markers (median coverage: 12,989x to 15,440x for laboratory strain mixtures and 7,011x to 11,600x for patients' samples, respectively). We found high sensitivity in detecting minority clones (up to 50:1:1:1 in the 3D7:K1:HB3:FCB1 laboratory strain mixtures) and high specificity with less than 0.01% of all reads being false-positive haplotypes. Genetic diversity in the markers used was high (H<sub>E</sub> ≥ 0.98 and up to 31 unique haplotypes in 20 paired samples with <em>cpmp</em>), and concordant results in classifying new infections and recrudescence across all markers used were observed in 18 (90%) of 20 paired samples. Interpretation\u0000Our study demonstrates the feasibility of nanopore AmpSeq for distinguishing recrudescence from new infections in clinical trials.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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