Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612422
Nicole Eisenhuth, Elisa Theres Rauh, Melina Mitnacht, Andrea Debus, Falk Butter, Ulrike Schleicher, Katerina Pruzinova, Petr Volf, Christian J Janzen
Conserved histone methyltransferases of the DOT1 family are involved in replication regulation, cell cycle progression, stage differentiation and gene regulation in trypanosomatids. However, the specific functions of these enzymes depend on the host evasion strategies of the parasites. In his study, we investigated the role of DOT1B in Leishmania mexicana, focusing on life cycle progression and infectivity. In contrast to Trypanosoma brucei, in which DOT1B is essential for the differentiation of mammal-infective bloodstream forms to insect procyclic forms, L. mexicana DOT1B (LmxDOT1B) is not critical for the differentiation of promastigotes to amastigotes in vitro. Additionally, there are no significant differences in the ability to infect or differentiate in macrophages or sand fly vectors between the LmxDOT1B-depleted and control strains. These findings highlight the divergency of the function of DOT1B in these related parasites, suggesting genus-specific adaptations in the use of histone modifications for life cycle progression and host adaptation processes.
{"title":"The histone methyltransferase DOT1B is dispensable for stage differentiation and macrophage infection in Leishmania mexicana","authors":"Nicole Eisenhuth, Elisa Theres Rauh, Melina Mitnacht, Andrea Debus, Falk Butter, Ulrike Schleicher, Katerina Pruzinova, Petr Volf, Christian J Janzen","doi":"10.1101/2024.09.11.612422","DOIUrl":"https://doi.org/10.1101/2024.09.11.612422","url":null,"abstract":"Conserved histone methyltransferases of the DOT1 family are involved in replication regulation, cell cycle progression, stage differentiation and gene regulation in trypanosomatids. However, the specific functions of these enzymes depend on the host evasion strategies of the parasites. In his study, we investigated the role of DOT1B in Leishmania mexicana, focusing on life cycle progression and infectivity. In contrast to Trypanosoma brucei, in which DOT1B is essential for the differentiation of mammal-infective bloodstream forms to insect procyclic forms, L. mexicana DOT1B (LmxDOT1B) is not critical for the differentiation of promastigotes to amastigotes in vitro. Additionally, there are no significant differences in the ability to infect or differentiate in macrophages or sand fly vectors between the LmxDOT1B-depleted and control strains. These findings highlight the divergency of the function of DOT1B in these related parasites, suggesting genus-specific adaptations in the use of histone modifications for life cycle progression and host adaptation processes.","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":"142214021","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 : 2024-09-10DOI: 10.1101/2024.09.10.612305
Hong Liu, Jianfeng Lin, Quynh T Phan, Vincent Michael Bruno, Scott G. Filler
The epidermal growth factor receptor (EGFR) has been identified as an epithelial cell receptor for Mucorales fungi and Candida albicans. Blocking EGFR with small molecule inhibitors reduces disease severity in mouse models of mucormycosis and oropharyngeal candidiasis. In contrast, cases of invasive aspergillosis have been reported in cancer patients who were treated with EGFR inhibitors, suggesting that EGFR signaling may play a protective role in the host defense against this infection. Here, we analyzed transcriptomic data from the lungs of mice with invasive aspergillosis and found evidence that Aspergillus fumigatus infection activates multiple genes that are predicted to function in the EGFR signaling pathway. We also found that A. fumigatus infection activates EGFR in both a human small airway epithelial (HSAE) cell line and in the lungs of immunosuppressed mice. EGFR signaling in HSAE cells is required for maximal endocytosis of A. fumigatus and for fungal-induced proinflammatory cytokine and chemokine production. In a corticosteroid immunosuppressed mouse model of invasive pulmonary aspergillosis, inhibition of EGFR with gefitinib decreased whole lung chemokine levels and reduced accumulation of phagocytes in the lung, leading to a decrease in fungal killing, an increase in pulmonary fungal burden, and accelerated mortality. Thus, EGFR signaling is required for pulmonary epithelial cells to orchestrate the host innate immune defense against invasive aspergillosis in immunosuppressed hosts.
{"title":"Epidermal Growth Factor Receptor Signaling Governs the Host Inflammatory Response to Invasive Aspergillosis","authors":"Hong Liu, Jianfeng Lin, Quynh T Phan, Vincent Michael Bruno, Scott G. Filler","doi":"10.1101/2024.09.10.612305","DOIUrl":"https://doi.org/10.1101/2024.09.10.612305","url":null,"abstract":"The epidermal growth factor receptor (EGFR) has been identified as an epithelial cell receptor for Mucorales fungi and Candida albicans. Blocking EGFR with small molecule inhibitors reduces disease severity in mouse models of mucormycosis and oropharyngeal candidiasis. In contrast, cases of invasive aspergillosis have been reported in cancer patients who were treated with EGFR inhibitors, suggesting that EGFR signaling may play a protective role in the host defense against this infection. Here, we analyzed transcriptomic data from the lungs of mice with invasive aspergillosis and found evidence that Aspergillus fumigatus infection activates multiple genes that are predicted to function in the EGFR signaling pathway. We also found that A. fumigatus infection activates EGFR in both a human small airway epithelial (HSAE) cell line and in the lungs of immunosuppressed mice. EGFR signaling in HSAE cells is required for maximal endocytosis of A. fumigatus and for fungal-induced proinflammatory cytokine and chemokine production. In a corticosteroid immunosuppressed mouse model of invasive pulmonary aspergillosis, inhibition of EGFR with gefitinib decreased whole lung chemokine levels and reduced accumulation of phagocytes in the lung, leading to a decrease in fungal killing, an increase in pulmonary fungal burden, and accelerated mortality. Thus, EGFR signaling is required for pulmonary epithelial cells to orchestrate the host innate immune defense against invasive aspergillosis in immunosuppressed hosts.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214054","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 : 2024-09-10DOI: 10.1101/2024.09.09.612158
peng zhou, Bibek G C, Chenggang Wu
Fusobacterium nucleatum is an opportunistic pathogen with four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA), each with distinct disease potentials. Research on fusobacterial pathogenesis has mainly focused on the model strain ATCC 23726 from FNN. However, this narrow focus may overlook significant behaviors of other FNN strains and those from other subspecies, given the genetic and phenotypic diversity within F. nucleatum. While ATCC 23726 is highly transformable, most other Fusobacterium strains exhibit low transformation efficiency, complicating traditional gene deletion methods that rely on non-replicating plasmids. To address this, we developed a conditional plasmid system in which the RepA protein, essential for replication of a pCWU6-based shuttle plasmid, is controlled by an inducible system combining an fdx promoter with a theophylline-responsive riboswitch. This system allows plasmid replication in host cells upon induction and plasmid loss when the inducer is removed, forcing chromosomal integration via homologous recombination in the presence of the antibiotic thiamphenicol. We validated this approach by targeting the galK gene, successfully generating mutants in FNN (ATCC 23726, CTI-2), FNP (ATCC 10953), FNA (21_1A), and the closely related species Fusobacterium periodonticum. Incorporating a sacB counterselection marker in this conditional plasmid enabled the deletion of the radD gene in non-model strains. Interestingly, while radD deletion in 23726, 10953, and 21_1A abolished coaggregation with Actinomyces oris, the CTI-2 mutant retained this ability, suggesting the involvement of other unknown adhesins. This work significantly advances gene deletion in genetically recalcitrant F. nucleatum strains, enhancing our understanding of this pathogen.
{"title":"Development of a Conditional Plasmid for Gene Deletion in Non-Model Fusobacterium nucleatum strains","authors":"peng zhou, Bibek G C, Chenggang Wu","doi":"10.1101/2024.09.09.612158","DOIUrl":"https://doi.org/10.1101/2024.09.09.612158","url":null,"abstract":"Fusobacterium nucleatum is an opportunistic pathogen with four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA), each with distinct disease potentials. Research on fusobacterial pathogenesis has mainly focused on the model strain ATCC 23726 from FNN. However, this narrow focus may overlook significant behaviors of other FNN strains and those from other subspecies, given the genetic and phenotypic diversity within F. nucleatum. While ATCC 23726 is highly transformable, most other Fusobacterium strains exhibit low transformation efficiency, complicating traditional gene deletion methods that rely on non-replicating plasmids. To address this, we developed a conditional plasmid system in which the RepA protein, essential for replication of a pCWU6-based shuttle plasmid, is controlled by an inducible system combining an fdx promoter with a theophylline-responsive riboswitch. This system allows plasmid replication in host cells upon induction and plasmid loss when the inducer is removed, forcing chromosomal integration via homologous recombination in the presence of the antibiotic thiamphenicol. We validated this approach by targeting the galK gene, successfully generating mutants in FNN (ATCC 23726, CTI-2), FNP (ATCC 10953), FNA (21_1A), and the closely related species Fusobacterium periodonticum. Incorporating a sacB counterselection marker in this conditional plasmid enabled the deletion of the radD gene in non-model strains. Interestingly, while radD deletion in 23726, 10953, and 21_1A abolished coaggregation with Actinomyces oris, the CTI-2 mutant retained this ability, suggesting the involvement of other unknown adhesins. This work significantly advances gene deletion in genetically recalcitrant F. nucleatum strains, enhancing our understanding of this pathogen.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214086","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 : 2024-09-10DOI: 10.1101/2024.09.10.612332
Margaret Mars Brisbin, McCaela Acord, Rachel Davitt, Shavonna Bent, Benjamin A.S. Van Mooy, Eliott Flaum, Andreas Norlin, Jessica Turner, Arianna Krinos, Harriet Alexander, Mak Saito
Interactions between phytoplankton and bacteria play critical roles in shaping marine ecosystems. However, the intricate relationships within these communities - particularly in extreme and rapidly changing environments like the coastal Southern Ocean - remain poorly understood. Here, we apply targeted methods to directly characterize the microbiomes of individual colonies of Phaeocystis antarctica, a keystone phytoplankton species in the Southern Ocean, for the first time. We show that colony microbiomes are consistent in distinct geographic locations at approximately the same time, but shift significantly after a year of laboratory culture. The bacterial orders Alteromonadales, Oceanospirillales, and Sphingomonadales dominated the microbiomes of all field-collected colonies, whereas Caulobacterales, Cellvibrionales, and Rhodobacterales dominated colony microbiomes after culturing. Notably, the most abundant genera in field-collected colony microbiomes, the psychrophiles Paraglaciecola and Colwellia, were lost in culture. The shift in microbiome structure emphasizes the importance of field-based studies to capture the complexity of microbial interactions, especially for species from polar environments that are difficult to replicate in laboratory conditions. Furthermore, the relative abundances of bacterial taxa comprising the majority of field-collected colony microbiome - e.g., Paraglaciecola sp. (Alteromonadales) and Nitrincolaceae (Oceanospirillales) - were strongly associated with Phaeocystis abundance in surface waters, highlighting their potential roles in bloom dynamics and carbon cycling. This research provides valuable insights into the ecological significance of prokaryotic interactions with a key phytoplankton species and underscores the necessity of considering these dynamics in the context of climate-driven shifts in marine ecosystems.
{"title":"Exploring the Phaeosphere: characterizing the microbiomes of Phaeocystis antarctica colonies from the coastal Southern Ocean and laboratory culture","authors":"Margaret Mars Brisbin, McCaela Acord, Rachel Davitt, Shavonna Bent, Benjamin A.S. Van Mooy, Eliott Flaum, Andreas Norlin, Jessica Turner, Arianna Krinos, Harriet Alexander, Mak Saito","doi":"10.1101/2024.09.10.612332","DOIUrl":"https://doi.org/10.1101/2024.09.10.612332","url":null,"abstract":"Interactions between phytoplankton and bacteria play critical roles in shaping marine ecosystems. However, the intricate relationships within these communities - particularly in extreme and rapidly changing environments like the coastal Southern Ocean - remain poorly understood. Here, we apply targeted methods to directly characterize the microbiomes of individual colonies of <em>Phaeocystis antarctica</em>, a keystone phytoplankton species in the Southern Ocean, for the first time. We show that colony microbiomes are consistent in distinct geographic locations at approximately the same time, but shift significantly after a year of laboratory culture. The bacterial orders Alteromonadales, Oceanospirillales, and Sphingomonadales dominated the microbiomes of all field-collected colonies, whereas Caulobacterales, Cellvibrionales, and Rhodobacterales dominated colony microbiomes after culturing. Notably, the most abundant genera in field-collected colony microbiomes, the psychrophiles <em>Paraglaciecola</em> and <em>Colwellia</em>, were lost in culture. The shift in microbiome structure emphasizes the importance of field-based studies to capture the complexity of microbial interactions, especially for species from polar environments that are difficult to replicate in laboratory conditions. Furthermore, the relative abundances of bacterial taxa comprising the majority of field-collected colony microbiome - e.g., <em>Paraglaciecola sp.</em> (Alteromonadales) and Nitrincolaceae (Oceanospirillales) - were strongly associated with <em>Phaeocystis</em> abundance in surface waters, highlighting their potential roles in bloom dynamics and carbon cycling. This research provides valuable insights into the ecological significance of prokaryotic interactions with a key phytoplankton species and underscores the necessity of considering these dynamics in the context of climate-driven shifts in marine ecosystems.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214056","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 : 2024-09-10DOI: 10.1101/2024.09.10.612348
Sharmada Swaminath, Marisa Mendes, Yipeng Zhang, Kaleigh A Remick, Isabel Mejia, Melissa Guereca, Aartjan J.W. te Velthuis, Alistair B Russell
Influenza A virus encodes promoters in both the sense and antisense orientations. These support the generation of new genomes, antigenomes, and mRNA transcripts. Using minimal replication assays—transfections with viral polymerase, nucleoprotein, and a genomic template—the influenza promoter sequences were identified as 13nt at the 5' end of the viral genomic RNA (U13) and 12nt at the 3' end (U12). Other than the fourth 3' nucleotide, the U12 and U13 sequences are identical between all eight RNA molecules that comprise the segmented influenza genome. Despite possessing identical promoters, individual segments can exhibit different transcriptional dynamics during infection. However flu promoter sequences were defined in experiments without influenza NS2, a protein which modulates transcription and replication differentially between genomic segments. This suggests that the identity of the "complete" promoter may depend on NS2. Here we assess how internal sequences of two genomic segments, HA and PB1, may contribute to NS2-dependent replication as well as map such interactions down to individual nucleotides in PB1. We find that the expression of NS2 significantly alters sequence requirements for efficient replication beyond the identical U12 and U13 sequence, providing a mechanism for the divergent replication and transcription dynamics across the influenza A virus genome.
{"title":"Efficient genome replication in influenza A virus requires NS2 and sequence beyond the canonical promoter","authors":"Sharmada Swaminath, Marisa Mendes, Yipeng Zhang, Kaleigh A Remick, Isabel Mejia, Melissa Guereca, Aartjan J.W. te Velthuis, Alistair B Russell","doi":"10.1101/2024.09.10.612348","DOIUrl":"https://doi.org/10.1101/2024.09.10.612348","url":null,"abstract":"Influenza A virus encodes promoters in both the sense and antisense orientations. These support the generation of new genomes, antigenomes, and mRNA transcripts. Using minimal replication assays—transfections with viral polymerase, nucleoprotein, and a genomic template—the influenza promoter sequences were identified as 13nt at the 5' end of the viral genomic RNA (U13) and 12nt at the 3' end (U12). Other than the fourth 3' nucleotide, the U12 and U13 sequences are identical between all eight RNA molecules that comprise the segmented influenza genome. Despite possessing identical promoters, individual segments can exhibit different transcriptional dynamics during infection. However flu promoter sequences were defined in experiments without influenza NS2, a protein which modulates transcription and replication differentially between genomic segments. This suggests that the identity of the \"complete\" promoter may depend on NS2. Here we assess how internal sequences of two genomic segments, HA and PB1, may contribute to NS2-dependent replication as well as map such interactions down to individual nucleotides in PB1. We find that the expression of NS2 significantly alters sequence requirements for efficient replication beyond the identical U12 and U13 sequence, providing a mechanism for the divergent replication and transcription dynamics across the influenza A virus genome.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214064","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 : 2024-09-10DOI: 10.1101/2024.09.10.612303
Collin Pete Pickens, Dongyu Wang, Chongle Pan, Kara B De Leon
Ubiquitous in nature, biofilms provide stability in a fluctuating environment and provide protection from stressors. Biofilms formed in industrial processes are exceedingly problematic and costly. While biofilms of sulfate-reducing bacteria in the environment are often beneficial because of their capacity to remove toxic metals from water, in industrial pipelines, these biofilms cause a major economic impact due to their involvement in metal and concrete corrosion. The mechanisms by which biofilms of sulfate-reducing bacteria form, however, is not well understood. Our previous work identified two proteins, named by their gene loci DVU1012 and DVU1545, as adhesins in the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough. Both proteins are localized to the cell surface and the presence of at least one of the proteins, with either being sufficient, is necessary for biofilm formation to occur. In this study, differences in cell attachment and early biofilm formation in single deletion mutants of these adhesins were identified. Cells lacking DVU1012 had a different attachment strategy from wild-type and ΔDVU1545 cells, more often attaching as single cells than aggregates, which indicated that DVU1012 was more important for cell-to-cell attachment. ΔDVU1545 cells had increased cell attachment compared to wild-type cells when grown in static cultures. To date, comparisons of the D. vulgaris Hildenborough have been made to the large adhesion protein (Lap) system in environmental pseudomonads. Yet, we and others have shown distinct mechanistic differences in the systems. We propose to name these proteins in D. vulgaris Hildenborough biofilm formation system (Bfs) to facilitate comparisons.
{"title":"Absence of biofilm adhesin proteins changes surface attachment and cell strategy for Desulfovibrio vulgaris Hildenborough","authors":"Collin Pete Pickens, Dongyu Wang, Chongle Pan, Kara B De Leon","doi":"10.1101/2024.09.10.612303","DOIUrl":"https://doi.org/10.1101/2024.09.10.612303","url":null,"abstract":"Ubiquitous in nature, biofilms provide stability in a fluctuating environment and provide protection from stressors. Biofilms formed in industrial processes are exceedingly problematic and costly. While biofilms of sulfate-reducing bacteria in the environment are often beneficial because of their capacity to remove toxic metals from water, in industrial pipelines, these biofilms cause a major economic impact due to their involvement in metal and concrete corrosion. The mechanisms by which biofilms of sulfate-reducing bacteria form, however, is not well understood. Our previous work identified two proteins, named by their gene loci DVU1012 and DVU1545, as adhesins in the model sulfate-reducing bacterium, <em>Desulfovibrio vulgaris</em> Hildenborough. Both proteins are localized to the cell surface and the presence of at least one of the proteins, with either being sufficient, is necessary for biofilm formation to occur. In this study, differences in cell attachment and early biofilm formation in single deletion mutants of these adhesins were identified. Cells lacking DVU1012 had a different attachment strategy from wild-type and ΔDVU1545 cells, more often attaching as single cells than aggregates, which indicated that DVU1012 was more important for cell-to-cell attachment. ΔDVU1545 cells had increased cell attachment compared to wild-type cells when grown in static cultures. To date, comparisons of the <em>D. vulgaris</em> Hildenborough have been made to the large adhesion protein (Lap) system in environmental pseudomonads. Yet, we and others have shown distinct mechanistic differences in the systems. We propose to name these proteins in <em>D. vulgaris</em> Hildenborough biofilm formation system (Bfs) to facilitate comparisons.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214062","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 : 2024-09-10DOI: 10.1101/2024.09.09.612141
Ian Tietjen, Daniel C Kwan, Annett Petrich, Roland Zell, Ivi Theodosia Antoniadou, Agni Gavriilidou, Christina Tzitzoglaki, Michail Rallis, David Fedida, Francesc X Sureda, Cato Mestdagh, Lieve MJ Naesens, Salvatore Chiantia, F Brent Johnson, Antonios Kolocouris
To better manage seasonal and pandemic influenza infections, new drugs are needed with enhanced activity against contemporary amantadine- and rimantadine-resistant influenza A virus (IAV) strains containing the S31N variant of the viral M2 ion channel (M2S31N). Here we tested 36 amantadine analogs against a panel of viruses containing either M2S31N or the parental, M2 S31 wild-type variant (M2WT). We found that several analogs, primarily those with sizeable lipophilic adducts, inhibited up to three M2S31N-containing viruses with activities at least 5-fold lower than rimantadine, without inhibiting M2S31N proton currents or modulating endosomal pH. While M2WT viruses in passaging studies rapidly gained resistance to these analogs through the established M2 mutations V27A and/or A30T, resistance development was markedly slower for M2S31N viruses and did not associate with additional M2 mutations. Instead, a subset of analogs, exemplified by 2-propyl-2-adamantanamine (38), but not 2-(1-adamantyl)piperidine (26), spiro[adamantane-2,2-pyrrolidine] (49), or spiro[adamantane-2,2-piperidine] (60), inhibited cellular entry of infectious IAV following pre-treatment and/or H1N1 pseudovirus entry. Conversely, an overlapping subset of the most lipophilic analogs including compounds 26, 49, 60, and others, disrupted viral M2-M1 protein colocalization required for intracellular viral assembly and budding. Finally, a pilot toxicity study in mice demonstrated that 38 and 49 were tolerated at doses approaching those of amantadine. Together, these results indicate that amantadine analogs act on multiple, complementary mechanisms to inhibit replication of M2S31N viruses.
{"title":"Antiviral Mechanisms and Preclinical Evaluation of Amantadine Analogs that Continue to Inhibit Influenza A Viruses with M2 S31N-Based Drug Resistance","authors":"Ian Tietjen, Daniel C Kwan, Annett Petrich, Roland Zell, Ivi Theodosia Antoniadou, Agni Gavriilidou, Christina Tzitzoglaki, Michail Rallis, David Fedida, Francesc X Sureda, Cato Mestdagh, Lieve MJ Naesens, Salvatore Chiantia, F Brent Johnson, Antonios Kolocouris","doi":"10.1101/2024.09.09.612141","DOIUrl":"https://doi.org/10.1101/2024.09.09.612141","url":null,"abstract":"To better manage seasonal and pandemic influenza infections, new drugs are needed with enhanced activity against contemporary amantadine- and rimantadine-resistant influenza A virus (IAV) strains containing the S31N variant of the viral M2 ion channel (M2S31N). Here we tested 36 amantadine analogs against a panel of viruses containing either M2S31N or the parental, M2 S31 wild-type variant (M2WT). We found that several analogs, primarily those with sizeable lipophilic adducts, inhibited up to three M2S31N-containing viruses with activities at least 5-fold lower than rimantadine, without inhibiting M2S31N proton currents or modulating endosomal pH. While M2WT viruses in passaging studies rapidly gained resistance to these analogs through the established M2 mutations V27A and/or A30T, resistance development was markedly slower for M2S31N viruses and did not associate with additional M2 mutations. Instead, a subset of analogs, exemplified by 2-propyl-2-adamantanamine (38), but not 2-(1-adamantyl)piperidine (26), spiro[adamantane-2,2-pyrrolidine] (49), or spiro[adamantane-2,2-piperidine] (60), inhibited cellular entry of infectious IAV following pre-treatment and/or H1N1 pseudovirus entry. Conversely, an overlapping subset of the most lipophilic analogs including compounds 26, 49, 60, and others, disrupted viral M2-M1 protein colocalization required for intracellular viral assembly and budding. Finally, a pilot toxicity study in mice demonstrated that 38 and 49 were tolerated at doses approaching those of amantadine. Together, these results indicate that amantadine analogs act on multiple, complementary mechanisms to inhibit replication of M2S31N viruses.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214058","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}
Multigene family (MGF) 360 genes, which are African swine fever virus (ASFV) virulence genes, primarily target key host immune molecules to suppress host interferon (IFN) production and interferon-stimulated gene (ISG) transcription, impairing host innate immune responses for efficient viral replication. However, the interactions between MGF 360 virulence genes and host molecules, as well as the mechanisms through which MGF 360 genes regulate host immune responses and interferon signaling, require further elucidation. In this study, we discovered that ASFV MGF_360-4L interacts with MDA5 and recruits the mitochondrial selective autophagy receptor SQSTM1 to degrade MDA5, thus impairing interferon signaling and compromising host innate immune responses. Furthermore, MGF_360-4L inhibits the interaction between MDA5 and MAVS, blocking ISG15-mediated ISGylation of MDA5. MGF_360-4L deficiencysignificantly attenuated virus-induced mitochondrial autophagy in vitro. Additionally, OAS1 ubiquitinates MGF_360-4L at residues K290, K295 and K327. Finally, a recombinant ASFV lacking the MGF_360-4L gene (ASFV-?MGF_360-4L) was generated using ASFV-CN/SC/2019 as the backbone, which demonstrated that the replication kinetics of ASFV-ΔMGF_360-4L in PAM cells were like those of the highly virulent parental ASFV-WT in vitro. Domestic pigs infected with ASFV-ΔMGF_360-4L exhibited milder symptoms than those infected with parental ASFV-WT, and ASFV-ΔMGF_360-4L-infected pigs presented with enhanced host innate antiviral immune response, confirming that the deletion of the MGF_360-4L gene from the ASFV genome highly attenuated virulence in pigs and provided effective protection against parental ASFV challenge. In conclusion, we identified a novel ASFV virulence gene, MGF_360-4L, further elucidating ASFV infection mechanisms and providing a new candidate for vaccine development.
{"title":"The African Swine Fever Virus gene MGF_360-4L inhibits interferon signaling by recruiting mitochondrial selective autophagy receptor SQSTM1 degrading MDA5 antagonizing innate immune responses","authors":"Qingli Niu, lin hua Sun, Jifei Yang, Zhonghui Zhang, Mengli Wu, Zhancheng Tian, Ying Liu, Xiaoqiang Zhang, Jianhao Zhong, Songlin Yang, Yikang Chen, Jianxun Luo, Guiquan Guan, Hong Yin","doi":"10.1101/2024.09.09.612163","DOIUrl":"https://doi.org/10.1101/2024.09.09.612163","url":null,"abstract":"Multigene family (MGF) 360 genes, which are African swine fever virus (ASFV) virulence genes, primarily target key host immune molecules to suppress host interferon (IFN) production and interferon-stimulated gene (ISG) transcription, impairing host innate immune responses for efficient viral replication. However, the interactions between MGF 360 virulence genes and host molecules, as well as the mechanisms through which MGF 360 genes regulate host immune responses and interferon signaling, require further elucidation. In this study, we discovered that ASFV MGF_360-4L interacts with MDA5 and recruits the mitochondrial selective autophagy receptor SQSTM1 to degrade MDA5, thus impairing interferon signaling and compromising host innate immune responses. Furthermore, MGF_360-4L inhibits the interaction between MDA5 and MAVS, blocking ISG15-mediated ISGylation of MDA5. MGF_360-4L deficiencysignificantly attenuated virus-induced mitochondrial autophagy in vitro. Additionally, OAS1 ubiquitinates MGF_360-4L at residues K290, K295 and K327. Finally, a recombinant ASFV lacking the MGF_360-4L gene (ASFV-?MGF_360-4L) was generated using ASFV-CN/SC/2019 as the backbone, which demonstrated that the replication kinetics of ASFV-ΔMGF_360-4L in PAM cells were like those of the highly virulent parental ASFV-WT in vitro. Domestic pigs infected with ASFV-ΔMGF_360-4L exhibited milder symptoms than those infected with parental ASFV-WT, and ASFV-ΔMGF_360-4L-infected pigs presented with enhanced host innate antiviral immune response, confirming that the deletion of the MGF_360-4L gene from the ASFV genome highly attenuated virulence in pigs and provided effective protection against parental ASFV challenge. In conclusion, we identified a novel ASFV virulence gene, MGF_360-4L, further elucidating ASFV infection mechanisms and providing a new candidate for vaccine development.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214051","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 : 2024-09-10DOI: 10.1101/2024.09.10.612322
Glenn James Rapsinski, Alecia Rokes, Daria Van Tyne, Vaughn S Cooper
Identifying resistance mechanisms to novel antimicrobials informs treatment and antimicrobial development, but frequently identifies multiple candidate resistance mutations without resolving the driver mutation. Using whole genome sequencing of longitudinal Pseudomonas aeruginosa that developed imipenem/cilastatin/relebactam and ceftolozane/tazobactam resistance during ceftazidime/avibactam treatment, we determined mutations resulting in cross-resistance. Penicillin-binding protein ftsI, transcriptional repressor bepR, and virulence regulator pvdS were found in resistant isolates. We conclude that peptidoglycan synthesis gene mutations can alter the efficacy of multiple antimicrobials.
{"title":"Mutations Leading to Ceftolozane/Tazobactam and Imipenem/Cilastatin/Relebactam Resistance During in vivo exposure to Ceftazidime/Avibactam in Pseudomonas aeruginosa","authors":"Glenn James Rapsinski, Alecia Rokes, Daria Van Tyne, Vaughn S Cooper","doi":"10.1101/2024.09.10.612322","DOIUrl":"https://doi.org/10.1101/2024.09.10.612322","url":null,"abstract":"Identifying resistance mechanisms to novel antimicrobials informs treatment and antimicrobial development, but frequently identifies multiple candidate resistance mutations without resolving the driver mutation. Using whole genome sequencing of longitudinal Pseudomonas aeruginosa that developed imipenem/cilastatin/relebactam and ceftolozane/tazobactam resistance during ceftazidime/avibactam treatment, we determined mutations resulting in cross-resistance. Penicillin-binding protein ftsI, transcriptional repressor bepR, and virulence regulator pvdS were found in resistant isolates. We conclude that peptidoglycan synthesis gene mutations can alter the efficacy of multiple antimicrobials.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214060","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}
Background Comparing the in vitro fitness of dengue virus (DENV) isolates is a pivotal approach to assess the contribution of DENV strains replicative fitness to epidemiological contexts, including serotype replacements. Competition assays are the gold standard to compare the in vitro replicative fitness of viral strains. Implementing competition assays between DENV serotypes requires an experimental setup and an appropriate read-out to quantify the viral progeny of strains belonging to different serotypes. Results In the current study, we optimized an existing serotyping qRT-PCR by adapting primer/probe design and multiplexing the serotype-specific qRT-PCR reactions, allowing to accurately detect and quantify all four DENV serotypes. The qRT-PCR was specific, had a limit of detection of at least 5.08x10^1, 5.16x10^1, 7.14x10^1 and 1.36 x10^1 genome copies/uL, an efficiency of 1.993, 1.975, 1.902, 1.898 and a linearity (R^2) of 0.99975, 0.99975, 0.9985, 0.99965 for DENV-1, -2, -3 and -4 respectively. Challenge of this multiplex serotype-specific qRT-PCR on mixes of viral supernatants containing known concentrations of strains from two serotypes evidenced an accurate quantification of the amount of genome copies of each serotype. We next developed an in vitro assay to compare the replicative fitness of two DENV serotypes in the human hepatic cell line HuH7: quantification of the viral progeny of each serotype in the inoculum and the supernatant using the serotype-specific multiplex qRT-PCR unveiled an enrichment of the supernatant in DENV-1 genome copies, uncovering the enhanced replicative fitness of this DENV-1 isolate. Conclusions This optimized qRT-PCR combined to a relevant cellular model allowed to accurately quantify the viral progeny of two DENV strains belonging to two different serotypes in a competition assay, allowing to determine which strain had a replicative advantage. This reliable experimental setup is adaptable to the comparative study of the replicative fitness of any DENV serotypes.
{"title":"Development of a competition assay to assess the in vitro fitness of dengue virus serotypes using an optimized serotype-specific qRT-PCR","authors":"Anne-Fleur Griffon, Loeiza Rault, Etienne Simon-Loriere, Myrielle Dupont-Rouzeyrol, Catherine Inizan","doi":"10.1101/2024.09.10.611934","DOIUrl":"https://doi.org/10.1101/2024.09.10.611934","url":null,"abstract":"Background\u0000Comparing the in vitro fitness of dengue virus (DENV) isolates is a pivotal approach to assess the contribution of DENV strains replicative fitness to epidemiological contexts, including serotype replacements. Competition assays are the gold standard to compare the in vitro replicative fitness of viral strains. Implementing competition assays between DENV serotypes requires an experimental setup and an appropriate read-out to quantify the viral progeny of strains belonging to different serotypes.\u0000Results\u0000In the current study, we optimized an existing serotyping qRT-PCR by adapting primer/probe design and multiplexing the serotype-specific qRT-PCR reactions, allowing to accurately detect and quantify all four DENV serotypes. The qRT-PCR was specific, had a limit of detection of at least 5.08x10^1, 5.16x10^1, 7.14x10^1 and 1.36 x10^1 genome copies/uL, an efficiency of 1.993, 1.975, 1.902, 1.898 and a linearity (R^2) of 0.99975, 0.99975, 0.9985, 0.99965 for DENV-1, -2, -3 and -4 respectively. Challenge of this multiplex serotype-specific qRT-PCR on mixes of viral supernatants containing known concentrations of strains from two serotypes evidenced an accurate quantification of the amount of genome copies of each serotype. We next developed an in vitro assay to compare the replicative fitness of two DENV serotypes in the human hepatic cell line HuH7: quantification of the viral progeny of each serotype in the inoculum and the supernatant using the serotype-specific multiplex qRT-PCR unveiled an enrichment of the supernatant in DENV-1 genome copies, uncovering the enhanced replicative fitness of this DENV-1 isolate.\u0000Conclusions\u0000This optimized qRT-PCR combined to a relevant cellular model allowed to accurately quantify the viral progeny of two DENV strains belonging to two different serotypes in a competition assay, allowing to determine which strain had a replicative advantage. This reliable experimental setup is adaptable to the comparative study of the replicative fitness of any DENV serotypes.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142214055","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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