Pub Date : 2024-09-11DOI: 10.1101/2024.09.10.612269
Louize Zheng, Alistair Raymond Legione
Recombinant koala retrovirus (recKoRV) is a recently discovered variant of koala retrovirus (KoRV), which likely emerged due to the insertion of another retrovirus (likely Phascolartus endogenous retrovirus) into the backbone of KoRV. KoRV endogenisation was thought to be ongoing in Victoria based on the low prevalence of the virus based on molecular detection of the pol gene, however recKoRV was not incorporated into the previous KoRV diagnostic test results. In this study, a new 5'-region-based PCR assay was developed, capable of detecting both intact KoRV and recKoRV. Using this assay, 319 archived DNA samples from 287 Victorian koalas were retested to investigate KoRV endogenisation. We found a 98.3% (282/287) of these samples were positive for the KoRV-5' fragment, the majority of which were KoRV-pol negative (222/287) on prior testing. Our findings demonstrate extensive KoRV integration into the Victorian koala populations, suggestive of a historic presence of KoRV in Victorian koalas. This finding makes biological sense relative to the translocation history of Victorian koalas, compared to the prior paradigm of ongoing endogenisation, and provides new epidemiological and practical management implications.
{"title":"A High Frequency of Detection of Recombinant Koala Retrovirus (recKoRV) in Victorian Koalas Suggests Historic Integration of KoRV","authors":"Louize Zheng, Alistair Raymond Legione","doi":"10.1101/2024.09.10.612269","DOIUrl":"https://doi.org/10.1101/2024.09.10.612269","url":null,"abstract":"Recombinant koala retrovirus (recKoRV) is a recently discovered variant of koala retrovirus (KoRV), which likely emerged due to the insertion of another retrovirus (likely Phascolartus endogenous retrovirus) into the backbone of KoRV. KoRV endogenisation was thought to be ongoing in Victoria based on the low prevalence of the virus based on molecular detection of the pol gene, however recKoRV was not incorporated into the previous KoRV diagnostic test results. In this study, a new 5'-region-based PCR assay was developed, capable of detecting both intact KoRV and recKoRV. Using this assay, 319 archived DNA samples from 287 Victorian koalas were retested to investigate KoRV endogenisation. We found a 98.3% (282/287) of these samples were positive for the KoRV-5' fragment, the majority of which were KoRV-pol negative (222/287) on prior testing. Our findings demonstrate extensive KoRV integration into the Victorian koala populations, suggestive of a historic presence of KoRV in Victorian koalas. This finding makes biological sense relative to the translocation history of Victorian koalas, compared to the prior paradigm of ongoing endogenisation, and provides new epidemiological and practical management implications.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214047","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: 10.1101/2024.09.11.612277
Ali Zahedi Amiri, Choudhary Ahmed, Subha Dahal, Filomena Grosso, Haomin Leng, Peter Stoilov, Maria Mangos, Johanne Toutant, Lulzim Shkreta, Liliana Attisano, Benoit Chabot, Martha Brown, Alan Cochrane
Viruses continue to pose a significant health burden in the human population and recent history has shown a concerning surge in viral threats.Treatment options for viral infections are limited and viruses have proven adept at evolving resistance to existing therapies, highlighting a significant vulnerability in our defences. In response to this challenge,we explored the modulation of cellular RNA metabolic processes as an alternative paradigm to antiviral development. Many viruses depend on the host cell's RNA splicing machinery and small alterations in this host process results in catastrophic changes in virakl protein production, ultimately inhibiting virus replication. Previously, the small molecule 5342191 was identified as a potent inhibitor of HIV-1 replication by altering viral RNA accumulation at doses that minimally affect host cell gene expression. In this report, we document 5342191 as a potent inhibitor of adenovirus, coronavirus, and influenza replication. In each case, 5342191's reduction in virus replication was associated with altered viral RNA accumulation and loss of viral structural protein expression. Interestingly, while resistant viruses were rapidly isolated for compounds targeting either virus-encoded proteases or polymerases, we have not yet isolated 5342191-resistant variants of coronavirus or influenza.Like HIV-1, 5342191's inhibition of cornavirus and influenza is mediated through the activation of specific cell signaling networks, including GPCR and/or MAPK signaling pathways that ultimately affect SR kinase expression. Together, these studies highlight the therapeutic potential of compounds that target cellular processes essential for replication of multiple viruses. Not only do these compounds hold promise as broad-spectrum antivirals, but they also offer the potential for greater durability in combating viral infections.
{"title":"Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals","authors":"Ali Zahedi Amiri, Choudhary Ahmed, Subha Dahal, Filomena Grosso, Haomin Leng, Peter Stoilov, Maria Mangos, Johanne Toutant, Lulzim Shkreta, Liliana Attisano, Benoit Chabot, Martha Brown, Alan Cochrane","doi":"10.1101/2024.09.11.612277","DOIUrl":"https://doi.org/10.1101/2024.09.11.612277","url":null,"abstract":"Viruses continue to pose a significant health burden in the human population and recent history has shown a concerning surge in viral threats.Treatment options for viral infections are limited and viruses have proven adept at evolving resistance to existing therapies, highlighting a significant vulnerability in our defences. In response to this challenge,we explored the modulation of cellular RNA metabolic processes as an alternative paradigm to antiviral development. Many viruses depend on the host cell's RNA splicing machinery and small alterations in this host process results in catastrophic changes in virakl protein production, ultimately inhibiting virus replication. Previously, the small molecule 5342191 was identified as a potent inhibitor of HIV-1 replication by altering viral RNA accumulation at doses that minimally affect host cell gene expression. In this report, we document 5342191 as a potent inhibitor of adenovirus, coronavirus, and influenza replication. In each case, 5342191's reduction in virus replication was associated with altered viral RNA accumulation and loss of viral structural protein expression. Interestingly, while resistant viruses were rapidly isolated for compounds targeting either virus-encoded proteases or polymerases, we have not yet isolated 5342191-resistant variants of coronavirus or influenza.Like HIV-1, 5342191's inhibition of cornavirus and influenza is mediated through the activation of specific cell signaling networks, including GPCR and/or MAPK signaling pathways that ultimately affect SR kinase expression. Together, these studies highlight the therapeutic potential of compounds that target cellular processes essential for replication of multiple viruses. Not only do these compounds hold promise as broad-spectrum antivirals, but they also offer the potential for greater durability in combating viral infections.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214037","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: 10.1101/2024.09.11.612390
Aiswarya Prasad, Asha Devi Pallujam, Rajath Siddaganga, Ashwin Suryanarayanan, Florent Mazel, Axel Brockmann, Sze Huei Yek, Philipp Engel
Studying gut microbiota evolution across animals is crucial for understanding symbiotic interactions but is hampered by the lack of high-resolution genomic data. Honeybees, with their specialized gut microbiota and well-known ecology, offer an ideal system to study this evolution. Using shotgun metagenomics on 200 honeybee workers from five species, we recovered thousands of metagenome-assembled genomes, identifying several novel bacterial species. While microbial communities were mostly host-specific, we found both specialist and generalist bacteria, even among closely related species, with notable variation between host species. Some generalists emerged host-specific only at the strain level, suggesting recent host switches. Unexpectedly, we found no evidence of codiversification between hosts and symbionts. Instead, symbiont gains, losses, and replacements led to functional differences, such as the ability to degrade pollen-derived pectin. Our results provide new insights into gut microbiota evolution and uncover the functional potential of the previously underexplored gut microbiota of these important pollinators.
{"title":"Symbiont loss and gain, rather than co-diversification shapes honeybee gut microbiota diversity and function","authors":"Aiswarya Prasad, Asha Devi Pallujam, Rajath Siddaganga, Ashwin Suryanarayanan, Florent Mazel, Axel Brockmann, Sze Huei Yek, Philipp Engel","doi":"10.1101/2024.09.11.612390","DOIUrl":"https://doi.org/10.1101/2024.09.11.612390","url":null,"abstract":"Studying gut microbiota evolution across animals is crucial for understanding symbiotic interactions but is hampered by the lack of high-resolution genomic data. Honeybees, with their specialized gut microbiota and well-known ecology, offer an ideal system to study this evolution. Using shotgun metagenomics on 200 honeybee workers from five species, we recovered thousands of metagenome-assembled genomes, identifying several novel bacterial species. While microbial communities were mostly host-specific, we found both specialist and generalist bacteria, even among closely related species, with notable variation between host species. Some generalists emerged host-specific only at the strain level, suggesting recent host switches. Unexpectedly, we found no evidence of codiversification between hosts and symbionts. Instead, symbiont gains, losses, and replacements led to functional differences, such as the ability to degrade pollen-derived pectin. Our results provide new insights into gut microbiota evolution and uncover the functional potential of the previously underexplored gut microbiota of these important pollinators.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214039","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: 10.1101/2024.09.11.612460
Brent W Simpson, Amanda B McLean, M. Stephen Trent
Gram-negative bacteria produce a multilayered cell envelope in which their peptidoglycan is sandwiched between two membranes, an inner membrane made of glycerophospholipids and an asymmetric outer membrane with glycerophospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. The Acinetobacter baumannii outer membrane contains lipooligosaccharide (LOS), a variant of LPS lacking O-antigen. LPS/LOS is typically essential, but A. baumannii can survive without LOS. Previously, we found that the peptidoglycan biogenesis protein NlpD becomes essential during LOS-deficiency. NlpD is typically redundant and is one of the cell's amidase activators for regulating peptidoglycan degradation, a process critical for cell division. We found that NlpD is essential under these conditions because a second putative amidase activator, termed WthA (cell wall turnover hub protein A), no longer functions in LOS-deficient cells. Mutants lacking WthA had severe cell division defects and were synthetically sick with loss of NlpD. Both Acinetobacter WthA and NlpD were found to activate an amidase activity of Oxa51, a chromosomally encoded beta-lactamase. Further, WthA is homologous to Pseudomonas LbcA that impacts two other classes of peptidoglycan degradation enzymes, endopeptidases and lytic transglycosylases. WthA/LbcA homologs were identified across Proteobacteria, Bacteroidota, and Chlorobiota, suggesting they belong to a conserved family involved in regulation of peptidoglycan turnover. While Acinetobacter WthA may share functions of Pseudomonas LbcA, we found no evidence that LbcA is an amidase activator. Altogether, we have identified a missing player in Acinetobacter peptidoglycan biogenesis, a conserved hub protein that regulates multiple peptidoglycan turnover enzymes including cell division amidases.
革兰氏阴性细菌产生多层细胞包膜，其肽聚糖夹在两层膜之间，一层是由甘油磷脂组成的内膜，另一层是不对称的外膜，内叶为甘油磷脂，外叶为脂多糖（LPS）。鲍曼不动杆菌外膜含有脂寡糖（LOS），这是一种缺乏 O 抗原的 LPS 变体。LPS/LOS 通常是必不可少的，但鲍曼不动杆菌在没有 LOS 的情况下也能存活。此前，我们发现肽聚糖生物生成蛋白 NlpD 在 LOS 缺乏时变得至关重要。NlpD 通常是冗余的，它是细胞的酰胺酶激活剂之一，用于调节肽聚糖降解，这是细胞分裂的关键过程。我们发现 NlpD 在这些条件下是必不可少的，因为第二个假定的酰胺酶激活剂 WthA（细胞壁周转枢纽蛋白 A）在 LOS 缺乏的细胞中不再起作用。缺乏 WthA 的突变体有严重的细胞分裂缺陷，并且由于 NlpD 的缺失而出现合成疾病。研究发现，WthA 和 NlpD 都能激活 Oxa51（一种染色体编码的 beta-内酰胺酶）的酰胺酶活性。此外，WthA 与假单胞菌的 LbcA 同源，而 LbcA 会影响另外两类肽聚糖降解酶，即内肽酶和溶解性转糖基酶。WthA/LbcA的同源物在变形菌群、类杆菌群和氯生物群中都被发现，表明它们属于参与调节肽聚糖周转的保守家族。虽然不动杆菌 WthA 可能与假单胞菌 LbcA 具有相同的功能，但我们没有发现 LbcA 是酰胺酶激活剂的证据。总之，我们发现了肽聚糖生物发生过程中一个缺失的角色，它是一个保守的中枢蛋白，可调控多种肽聚糖周转酶，包括细胞分裂酰胺酶。
{"title":"A conserved hub protein for coordinating peptidoglycan turnover that activates cell division amidases in Acinetobacter baumannii","authors":"Brent W Simpson, Amanda B McLean, M. Stephen Trent","doi":"10.1101/2024.09.11.612460","DOIUrl":"https://doi.org/10.1101/2024.09.11.612460","url":null,"abstract":"Gram-negative bacteria produce a multilayered cell envelope in which their peptidoglycan is sandwiched between two membranes, an inner membrane made of glycerophospholipids and an asymmetric outer membrane with glycerophospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. The Acinetobacter baumannii outer membrane contains lipooligosaccharide (LOS), a variant of LPS lacking O-antigen. LPS/LOS is typically essential, but A. baumannii can survive without LOS. Previously, we found that the peptidoglycan biogenesis protein NlpD becomes essential during LOS-deficiency. NlpD is typically redundant and is one of the cell's amidase activators for regulating peptidoglycan degradation, a process critical for cell division. We found that NlpD is essential under these conditions because a second putative amidase activator, termed WthA (cell wall turnover hub protein A), no longer functions in LOS-deficient cells. Mutants lacking WthA had severe cell division defects and were synthetically sick with loss of NlpD. Both Acinetobacter WthA and NlpD were found to activate an amidase activity of Oxa51, a chromosomally encoded beta-lactamase. Further, WthA is homologous to Pseudomonas LbcA that impacts two other classes of peptidoglycan degradation enzymes, endopeptidases and lytic transglycosylases. WthA/LbcA homologs were identified across Proteobacteria, Bacteroidota, and Chlorobiota, suggesting they belong to a conserved family involved in regulation of peptidoglycan turnover. While Acinetobacter WthA may share functions of Pseudomonas LbcA, we found no evidence that LbcA is an amidase activator. Altogether, we have identified a missing player in Acinetobacter peptidoglycan biogenesis, a conserved hub protein that regulates multiple peptidoglycan turnover enzymes including cell division amidases.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214042","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: 10.1101/2024.09.11.612557
Chantal Emade Nkwelle, Unique Stephens, Kimberly Liang, Joel Cassel, Joseph Salvino, Luis J. Montaner, Roland N Ndip, Seraphine N Esemu, Fidele Ntie-Kang, Ian Tietjen
J-Lat cells are derivatives of the Jurkat CD4+ T cell line that contain a non-infectious, inducible HIV provirus with a GFP tag. While these cells have substantially advanced our understanding of HIV latency, their use by many laboratories in low and middle-income countries is restricted by limited access to flow cytometry. To overcome this barrier, we describe a modified J-Lat assay using a standard microplate reader that detects HIV-GFP expression following treatment with latency-reversing agents (LRAs). We show that HIV reactivation by control LRAs like prostratin and romidepsin is readily detected with dose dependence and with significant correlation and sensitivity to standard flow cytometry. For example, 10 micromol prostratin induced a 20.1 +/- 3.3-fold increase in GFP fluorescence in the microplate reader assay, which corresponded to 64.2 +/- 5.0% GFP-positive cells detected by flow cytometery. Similarly, 0.3 micromol prostratin induced a 1.7 +/- 1.2-fold increase compared to 8.7 +/- 5.7% GFP-positive cells detected. Using this method, we screen 79 epigenetic modifiers and identify molibresib, quisinostat, and CUDC-101 as novel LRAs. This microplate reader-based method offers accessibility to researchers in resource-limited regions to work with J-Lat cells and more actively participate in global HIV cure research efforts.
{"title":"A high-throughput, microplate reader-based method to monitor in vitro HIV latency reversal in the absence of flow cytometry","authors":"Chantal Emade Nkwelle, Unique Stephens, Kimberly Liang, Joel Cassel, Joseph Salvino, Luis J. Montaner, Roland N Ndip, Seraphine N Esemu, Fidele Ntie-Kang, Ian Tietjen","doi":"10.1101/2024.09.11.612557","DOIUrl":"https://doi.org/10.1101/2024.09.11.612557","url":null,"abstract":"J-Lat cells are derivatives of the Jurkat CD4+ T cell line that contain a non-infectious, inducible HIV provirus with a GFP tag. While these cells have substantially advanced our understanding of HIV latency, their use by many laboratories in low and middle-income countries is restricted by limited access to flow cytometry. To overcome this barrier, we describe a modified J-Lat assay using a standard microplate reader that detects HIV-GFP expression following treatment with latency-reversing agents (LRAs). We show that HIV reactivation by control LRAs like prostratin and romidepsin is readily detected with dose dependence and with significant correlation and sensitivity to standard flow cytometry. For example, 10 micromol prostratin induced a 20.1 +/- 3.3-fold increase in GFP fluorescence in the microplate reader assay, which corresponded to 64.2 +/- 5.0% GFP-positive cells detected by flow cytometery. Similarly, 0.3 micromol prostratin induced a 1.7 +/- 1.2-fold increase compared to 8.7 +/- 5.7% GFP-positive cells detected. Using this method, we screen 79 epigenetic modifiers and identify molibresib, quisinostat, and CUDC-101 as novel LRAs. This microplate reader-based method offers accessibility to researchers in resource-limited regions to work with J-Lat cells and more actively participate in global HIV cure research efforts.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214043","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: 10.1101/2024.09.11.612426
Julian Bulssico, Swapnesh Panigrahi, Nicolas Ginet, Mireille Ansaldi
A distinctive manifestation of phage infection in solid media is the appearance of lysis plaques, corresponding to the circular thinning of a bacterial lawn. During plaque formation, successive cycles of phage replication take place from a single point of infection and spread radially in a matrix of immobilized bacterial hosts. Many factors affect plaque size, such as the composition and the reticulation of the propagation matrix, the characteristics of the phage, but also parameters related to the physiology of the bacterial host. As the combined administration of antibiotics and phages is a common practice in compassionate treatments, our research focuses on the effects of antibiotics on phage predation, which may be of crucial importance for phage therapeutic applications. Sublethal concentrations can drastically affect bacterial physiology, allowing phages to spread more rapidly and resulting in better bacterial eradication. Previous experimental work has focused on the phage characteristics. However, as plaque formation is strongly influenced by host growth dynamics, a comprehensive model integrating both the host growth and phage infection parameters is required. We suggest that plaque enlargement is linked to morphological changes of the host that have an impact on the rate of epidemic propagation and on phage diffusion into the matrix. To support this hypothesis, we characterized the growth parameters of two different phages and bacteria in semi-solid media in the presence of various antibiotics. By combining these data, we have produced a mathematical model that accounts for these observations and explains the increase in plaque size when the host morphology is affected.
{"title":"Antibiotic-Induced Morphological Changes Enhance Phage Predation: A Mathematical Model of Plaque Formation in Structured Environments","authors":"Julian Bulssico, Swapnesh Panigrahi, Nicolas Ginet, Mireille Ansaldi","doi":"10.1101/2024.09.11.612426","DOIUrl":"https://doi.org/10.1101/2024.09.11.612426","url":null,"abstract":"A distinctive manifestation of phage infection in solid media is the appearance of lysis plaques, corresponding to the circular thinning of a bacterial lawn. During plaque formation, successive cycles of phage replication take place from a single point of infection and spread radially in a matrix of immobilized bacterial hosts. Many factors affect plaque size, such as the composition and the reticulation of the propagation matrix, the characteristics of the phage, but also parameters related to the physiology of the bacterial host. As the combined administration of antibiotics and phages is a common practice in compassionate treatments, our research focuses on the effects of antibiotics on phage predation, which may be of crucial importance for phage therapeutic applications. Sublethal concentrations can drastically affect bacterial physiology, allowing phages to spread more rapidly and resulting in better bacterial eradication. Previous experimental work has focused on the phage characteristics. However, as plaque formation is strongly influenced by host growth dynamics, a comprehensive model integrating both the host growth and phage infection parameters is required. We suggest that plaque enlargement is linked to morphological changes of the host that have an impact on the rate of epidemic propagation and on phage diffusion into the matrix. To support this hypothesis, we characterized the growth parameters of two different phages and bacteria in semi-solid media in the presence of various antibiotics. By combining these data, we have produced a mathematical model that accounts for these observations and explains the increase in plaque size when the host morphology is affected.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214041","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: 10.1101/2024.09.11.612086
Leo Song, Amanda T.P. Alker, Agnes Oromi-Bosch, Sophia E. Swartz, Jonathan N.V. Martinson, Jigyasa Arora, Abby M. Wang, Rachel Rovinsky, Sara J. Smith, Emily C. Pierce, Adam M. Deutschbauer, Jennifer A. Doudna, Brady F. Cress, Benjamin E. Rubin
CRISPR-Associated Transposases (CASTs) hold tremendous potential for microbial genome editing due to their ability to integrate large DNA cargos in a programmable and site-specific manner. However, the widespread application of CASTs has been hindered by their low efficiency in diverse, non-model bacteria. In an effort to address this shortcoming, we conducted the first genome-wide screen for host factors impacting Vibrio cholerae CAST (VchCAST) activity and used the findings to increase VchCAST editing efficiency. A genome-wide loss-of-function mutant library in E. coli was screened to identify 15 genes that impact type VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity and two were found to be inhibitory. Informed by homologous recombination involved effectors, RecD and RecA, we tested the λ-Red recombineering system in our VchCAST editing vectors, which increased its insertion meditated-editing efficiency by 25.7-fold in E. coli while maintaining high target specificity and similar insertion arrangements. Furthermore, λ-Red-enhanced VchCAST achieved increased editing efficiency in the industrially important bacteria Pseudomonas putida and the emerging pathogen Klebsiella michiganensis. This study improves understanding of factors impacting VchCAST activity and enhances its efficiency as a bacterial genome editor.
{"title":"Identification of Proteins Influencing CRISPR-Associated Transposases for Enhanced Genome Editing","authors":"Leo Song, Amanda T.P. Alker, Agnes Oromi-Bosch, Sophia E. Swartz, Jonathan N.V. Martinson, Jigyasa Arora, Abby M. Wang, Rachel Rovinsky, Sara J. Smith, Emily C. Pierce, Adam M. Deutschbauer, Jennifer A. Doudna, Brady F. Cress, Benjamin E. Rubin","doi":"10.1101/2024.09.11.612086","DOIUrl":"https://doi.org/10.1101/2024.09.11.612086","url":null,"abstract":"CRISPR-Associated Transposases (CASTs) hold tremendous potential for microbial genome editing due to their ability to integrate large DNA cargos in a programmable and site-specific manner. However, the widespread application of CASTs has been hindered by their low efficiency in diverse, non-model bacteria. In an effort to address this shortcoming, we conducted the first genome-wide screen for host factors impacting Vibrio cholerae CAST (VchCAST) activity and used the findings to increase VchCAST editing efficiency. A genome-wide loss-of-function mutant library in E. coli was screened to identify 15 genes that impact type VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity and two were found to be inhibitory. Informed by homologous recombination involved effectors, RecD and RecA, we tested the λ-Red recombineering system in our VchCAST editing vectors, which increased its insertion meditated-editing efficiency by 25.7-fold in E. coli while maintaining high target specificity and similar insertion arrangements. Furthermore, λ-Red-enhanced VchCAST achieved increased editing efficiency in the industrially important bacteria Pseudomonas putida and the emerging pathogen Klebsiella michiganensis. This study improves understanding of factors impacting VchCAST activity and enhances its efficiency as a bacterial genome editor.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214044","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: 10.1101/2024.09.11.612551
Elissa G. Currie, Olga Rojas, Isaac S. Lee, Khashayar Khaleghi, Alberto Martin, Jen Gommerman, Scott Gray-Owen
Neisseria meningitidis is a human-restricted pathogen that can cause a rapidly progressing invasive meningococcal disease, yet it is also a regular inhabitant of the human nasopharynx. Vaccines that target N. meningitidis aim to prevent invasive disease, but their ability to interfere with nasal colonization could effectively eradicate this bacteria in a population, and so is an important target for meningococcal vaccine design. While protection against invasive meningococcal disease is classically attributed to IgG-dependent complement activation and bacterial killing, there remains no indication of what confers protection against nasopharyngeal colonization, making it impossible to deliberately target this stage during vaccine development. Moreover, without understanding what confers protection in this tissue site, it is impossible to understand the level of susceptibility within a population. To address this, we have taken advantage of the CEACAM1-humanized mouse model to characterize immune effectors that protect against nasal carriage of N. meningitidis. Protection against nasal colonization could be induced by live mucosal infection or by parenteral immunization with heat-killed bacteria. Mice possessing genetic deficiencies in B cells were used to evaluate the role of B cells and a specific antibody response, while neutrophil and complement depletion were used to evaluate their respective contributions to immunization-induced protection against meningococcal nasal carriage. Despite the essential role for complement killing in preventing invasive meningococcal disease, complement was not required for protection against nasal colonization. Instead, N. meningitidis-specific antibodies and neutrophils were both required to protect mice against the nasal infection. Combined, these data suggest that phagocytic bacterial killing is necessary for protection against mucosal colonization by N. meningitidis, indicating that nasal immunoglobulin with the ability to promote opsonophagocytosis must be considered as a correlate of protection against meningococcal carriage.
脑膜炎奈瑟菌是一种人类限制性病原体，可引起进展迅速的侵袭性脑膜炎球菌疾病，但它也是人类鼻咽部的常住菌。针对脑膜炎球菌的疫苗旨在预防侵袭性疾病，但其干扰鼻腔定植的能力可有效消灭人群中的这种细菌，因此是脑膜炎球菌疫苗设计的一个重要目标。虽然对侵袭性脑膜炎球菌疾病的保护作用通常归因于依赖 IgG 的补体激活和细菌杀灭，但目前仍没有迹象表明是什么赋予了对鼻咽定植的保护作用，因此在疫苗开发过程中不可能刻意针对这一阶段。此外，如果不了解是什么在这一组织部位产生保护作用，就不可能了解人群的易感性水平。为了解决这个问题，我们利用 CEACAM1 人源化小鼠模型来描述保护鼻腔不被脑膜炎奈瑟菌携带的免疫效应因子。鼻腔定植的保护可通过活体粘膜感染或用热杀死的细菌进行肠外免疫诱导。利用遗传性 B 细胞缺陷的小鼠来评估 B 细胞和特异性抗体反应的作用，同时利用中性粒细胞和补体耗竭来评估它们各自对免疫诱导的脑膜炎球菌鼻腔携带保护作用的贡献。尽管补体杀伤在预防侵袭性脑膜炎球菌疾病中发挥着重要作用，但鼻腔定植却不需要补体来提供保护。相反，脑膜炎球菌特异性抗体和中性粒细胞都是保护小鼠免受鼻腔感染的必要条件。这些数据综合起来表明，要防止脑膜炎球菌在粘膜上定植，必须要有吞噬细菌的能力，这表明必须将具有促进嗜蛋白吞噬能力的鼻腔免疫球蛋白视为防止脑膜炎球菌携带的相关因素。
{"title":"Protection against Neisseria meningitidis nasopharyngeal colonization relies on antibody opsonization and phagocytosis by neutrophils","authors":"Elissa G. Currie, Olga Rojas, Isaac S. Lee, Khashayar Khaleghi, Alberto Martin, Jen Gommerman, Scott Gray-Owen","doi":"10.1101/2024.09.11.612551","DOIUrl":"https://doi.org/10.1101/2024.09.11.612551","url":null,"abstract":"Neisseria meningitidis is a human-restricted pathogen that can cause a rapidly progressing invasive meningococcal disease, yet it is also a regular inhabitant of the human nasopharynx. Vaccines that target N. meningitidis aim to prevent invasive disease, but their ability to interfere with nasal colonization could effectively eradicate this bacteria in a population, and so is an important target for meningococcal vaccine design. While protection against invasive meningococcal disease is classically attributed to IgG-dependent complement activation and bacterial killing, there remains no indication of what confers protection against nasopharyngeal colonization, making it impossible to deliberately target this stage during vaccine development. Moreover, without understanding what confers protection in this tissue site, it is impossible to understand the level of susceptibility within a population. To address this, we have taken advantage of the CEACAM1-humanized mouse model to characterize immune effectors that protect against nasal carriage of N. meningitidis. Protection against nasal colonization could be induced by live mucosal infection or by parenteral immunization with heat-killed bacteria. Mice possessing genetic deficiencies in B cells were used to evaluate the role of B cells and a specific antibody response, while neutrophil and complement depletion were used to evaluate their respective contributions to immunization-induced protection against meningococcal nasal carriage. Despite the essential role for complement killing in preventing invasive meningococcal disease, complement was not required for protection against nasal colonization. Instead, N. meningitidis-specific antibodies and neutrophils were both required to protect mice against the nasal infection. Combined, these data suggest that phagocytic bacterial killing is necessary for protection against mucosal colonization by N. meningitidis, indicating that nasal immunoglobulin with the ability to promote opsonophagocytosis must be considered as a correlate of protection against meningococcal carriage.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214040","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: 10.1101/2024.09.11.612453
Seungwoo Baek, Yong-Joon Cho, Eunna Choi, Soomin Choi, Eun-Jin Lee
Ribosomes translate mRNAs by matching every 3-nucleotide sequence in mRNA, producing the corresponding proteins. As the amino acid sequence directly dictates the activity of the protein, frameshifts often lead to unexpected effects. Here, ribosome profiling reveals that the intracellular pathogen Salmonella Typhimurium suppresses frameshift in the ugtL antimicrobial resistance gene during translation. This suppression of frameshift is mediated by a ribosome pause occurring in a newly-identified overlapping gene, serving as a non-slip bump. Given that the pause site contains a poly-proline motif and can be resolved by elongation factor P, the removal of the ribosome pause by substituting the motif induces ribosome slippage in ugtL, resulting in UgtL frameshifted protein production. This renders Salmonella sensitive to antimicrobial peptides but, in turn, protects the MgtC virulence factor from the FtsH-mediated proteolysis, indicating that elongation factor P-dependent ribosome pause is required for controlling both full antimicrobial resistance and mouse virulence. These findings reveal a new regulatory mechanism in which ribosome pause controls the production of two different protein isoforms by suppressing ribosome slippage-mediated frameshift.
{"title":"Elongation factor P controls ribosomal frameshift of a Salmonella antimicrobial resistance gene","authors":"Seungwoo Baek, Yong-Joon Cho, Eunna Choi, Soomin Choi, Eun-Jin Lee","doi":"10.1101/2024.09.11.612453","DOIUrl":"https://doi.org/10.1101/2024.09.11.612453","url":null,"abstract":"Ribosomes translate mRNAs by matching every 3-nucleotide sequence in mRNA, producing the corresponding proteins. As the amino acid sequence directly dictates the activity of the protein, frameshifts often lead to unexpected effects. Here, ribosome profiling reveals that the intracellular pathogen Salmonella Typhimurium suppresses frameshift in the ugtL antimicrobial resistance gene during translation. This suppression of frameshift is mediated by a ribosome pause occurring in a newly-identified overlapping gene, serving as a non-slip bump. Given that the pause site contains a poly-proline motif and can be resolved by elongation factor P, the removal of the ribosome pause by substituting the motif induces ribosome slippage in ugtL, resulting in UgtL frameshifted protein production. This renders Salmonella sensitive to antimicrobial peptides but, in turn, protects the MgtC virulence factor from the FtsH-mediated proteolysis, indicating that elongation factor P-dependent ribosome pause is required for controlling both full antimicrobial resistance and mouse virulence. These findings reveal a new regulatory mechanism in which ribosome pause controls the production of two different protein isoforms by suppressing ribosome slippage-mediated frameshift.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142227023","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: 10.1101/2024.09.11.612488
Marie-Therese Fischer, Katherine Shaohua Xue, Elizabeth Kate Costello, Mai Dvorak, Anna Robaczewska, Gaelle Raboisson, Stephanie Caty, David A. Relman, Lauren A O'Connell
Parent-offspring interactions constitute the first contact of many newborns with their environment, priming community assembly of microbes through priority effects. Early exposure to microbes can have lasting influences on the assembly and functionality of the host's microbiota, leaving a life-long imprint on host health and disease. Studies of the role played by parental care in microbial acquisition have primarily focused on humans and hosts with agricultural relevance. Anuran vertebrates offer the opportunity to examine microbial community composition across life stages as a function of parental investment. In this study, we investigate vertical transmission of microbiota during parental care in a poison frog (Family Dendrobatidae), where fathers transport their offspring piggyback-style from terrestrial clutches to aquatic nurseries. We found that substantial bacterial colonization of the embryo begins after hatching from the vitelline envelope, emphasizing its potential role as microbial barrier during early development. Using a laboratory cross-foster experiment, we demonstrated that poison frogs performing tadpole transport serve as a source of skin microbes for tadpoles on their back. To study how transport impacts the microbial skin communities of tadpoles in an ecologically relevant setting, we sampled frogs and tadpoles of sympatric species that do or do not exhibit tadpole transport in their natural habitat. We found more diverse microbial communities associated with tadpoles of transporting species compared to a non-transporting frog. However, we detected no difference in the degree of similarity between adult and tadpole skin microbiotas, based on whether the frog species exhibits transporting behavior or not. Using a field experiment, we confirmed that tadpole transport can result in the persistent colonization of tadpoles by isolated microbial taxa associated with the caregiver's skin, albeit often at low abundance. This is the first study to describe vertical transmission of skin microbes in anuran amphibians, showing that offspring transport may serve as a mechanism for transmission of parental skin microbes. Overall, these findings provide a foundation for further research on how vertical transmission in this order impacts host-associated microbiota and physiology.
{"title":"Effects of parental care on skin microbial community composition in poison frogs","authors":"Marie-Therese Fischer, Katherine Shaohua Xue, Elizabeth Kate Costello, Mai Dvorak, Anna Robaczewska, Gaelle Raboisson, Stephanie Caty, David A. Relman, Lauren A O'Connell","doi":"10.1101/2024.09.11.612488","DOIUrl":"https://doi.org/10.1101/2024.09.11.612488","url":null,"abstract":"Parent-offspring interactions constitute the first contact of many newborns with their environment, priming community assembly of microbes through priority effects. Early exposure to microbes can have lasting influences on the assembly and functionality of the host's microbiota, leaving a life-long imprint on host health and disease. Studies of the role played by parental care in microbial acquisition have primarily focused on humans and hosts with agricultural relevance. Anuran vertebrates offer the opportunity to examine microbial community composition across life stages as a function of parental investment. In this study, we investigate vertical transmission of microbiota during parental care in a poison frog (Family Dendrobatidae), where fathers transport their offspring piggyback-style from terrestrial clutches to aquatic nurseries. We found that substantial bacterial colonization of the embryo begins after hatching from the vitelline envelope, emphasizing its potential role as microbial barrier during early development. Using a laboratory cross-foster experiment, we demonstrated that poison frogs performing tadpole transport serve as a source of skin microbes for tadpoles on their back. To study how transport impacts the microbial skin communities of tadpoles in an ecologically relevant setting, we sampled frogs and tadpoles of sympatric species that do or do not exhibit tadpole transport in their natural habitat. We found more diverse microbial communities associated with tadpoles of transporting species compared to a non-transporting frog. However, we detected no difference in the degree of similarity between adult and tadpole skin microbiotas, based on whether the frog species exhibits transporting behavior or not. Using a field experiment, we confirmed that tadpole transport can result in the persistent colonization of tadpoles by isolated microbial taxa associated with the caregiver's skin, albeit often at low abundance. This is the first study to describe vertical transmission of skin microbes in anuran amphibians, showing that offspring transport may serve as a mechanism for transmission of parental skin microbes. Overall, these findings provide a foundation for further research on how vertical transmission in this order impacts host-associated microbiota and physiology.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214036","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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