Pub Date : 2024-07-02DOI: 10.1021/acsinfecdis.4c00113
Valeria Francesconi, Marco Rizzo, Cecilia Pozzi, Lorenzo Tagliazucchi, Claude U Konchie Simo, Giulia Saporito, Giacomo Landi, Stefano Mangani, Anna Carbone, Silvia Schenone, Nuno Santarém, Joana Tavares, Anabela Cordeiro-da-Silva, Maria Paola Costi, Michele Tonelli
Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.
{"title":"Identification of Innovative Folate Inhibitors Leveraging the Amino Dihydrotriazine Motif from Cycloguanil for Their Potential as Anti-<i>Trypanosoma brucei</i> Agents.","authors":"Valeria Francesconi, Marco Rizzo, Cecilia Pozzi, Lorenzo Tagliazucchi, Claude U Konchie Simo, Giulia Saporito, Giacomo Landi, Stefano Mangani, Anna Carbone, Silvia Schenone, Nuno Santarém, Joana Tavares, Anabela Cordeiro-da-Silva, Maria Paola Costi, Michele Tonelli","doi":"10.1021/acsinfecdis.4c00113","DOIUrl":"https://doi.org/10.1021/acsinfecdis.4c00113","url":null,"abstract":"<p><p>Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (<b>1</b>) and 2-guanidino benzimidazoles (<b>2</b>), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of <i>Tb</i>DHFR and <i>Tb</i>PTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against <i>T. brucei</i> and <i>L. infantum</i> and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole <b>2g</b> resulted to be the most potent (<i>K</i><sub>i</sub> = 9 nM) and highly selective <i>Tb</i>DHFR inhibitor, 6000-fold over <i>Tb</i>PTR1 and 394-fold over <i>h</i>DHFR. The 5,6-dimethyl tricyclic analogue <b>1g</b>, despite showing a lower potency and selectivity profile than <b>2g</b>, shared a comparable antiparasitic activity against <i>T. brucei</i> in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles <b>2c</b> and <b>2d</b> revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of <i>T. brucei</i> and <i>L. infantum</i> parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489865","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01DOI: 10.1021/acsinfecdis.4c00280
Tingting Guo, Liying Yang, Na Zhou, Zichen Wang, Changchao Huan, Jun Zhou, Tao Lin, Guangyu Bao, Jian Hu, Guocai Li
Biofilm formation of Klebsiella pneumoniae can protect bacteria from antibiotics and is difficult to eradicate. Thus, the influence of subinhibitory concentrations of antibiotics on bacteria is becoming increasingly important. Our study showed that subminimum inhibitory concentrations (sub-MICs) of tetracycline antibiotics can increase biofilm formation in minocycline-resistant Klebsiella pneumoniae clinical strains. However, in the bacterial adhesion and invasion experiments, the adhesion and invasion ability decreased and the survival rate of Galleria mellonella increased. Under sub-MICs of tetracycline antibiotics treatment, abnormal stretching of bacteria was observed by scanning electron microscopy. Treatment with sub-MICs of tetracyclines leads to increased surface hydrophobicity and eDNA content and decreased outer membrane permeability. The expression levels of the fimA, luxS, qseB, and qseC genes decreased, the expression level of mrkA increased, and the expression level of acrA was inconsistent under different tetracycline antibiotics treatments. Together, our results suggested that the increase in Klebsiella pneumoniae biofilm formation caused by sub-MICs of tetracycline antibiotics may occur by affecting bacterial physical and chemical properties and associated genes expression.
{"title":"Subminimum Inhibitory Concentrations Tetracycline Antibiotics Induce Biofilm Formation in Minocycline-Resistant <i>Klebsiella pneumonia</i> by Affecting Bacterial Physical and Chemical Properties and Associated Genes Expression.","authors":"Tingting Guo, Liying Yang, Na Zhou, Zichen Wang, Changchao Huan, Jun Zhou, Tao Lin, Guangyu Bao, Jian Hu, Guocai Li","doi":"10.1021/acsinfecdis.4c00280","DOIUrl":"https://doi.org/10.1021/acsinfecdis.4c00280","url":null,"abstract":"<p><p>Biofilm formation of <i>Klebsiella pneumoniae</i> can protect bacteria from antibiotics and is difficult to eradicate. Thus, the influence of subinhibitory concentrations of antibiotics on bacteria is becoming increasingly important. Our study showed that subminimum inhibitory concentrations (sub-MICs) of tetracycline antibiotics can increase biofilm formation in minocycline-resistant <i>Klebsiella pneumoniae</i> clinical strains. However, in the bacterial adhesion and invasion experiments, the adhesion and invasion ability decreased and the survival rate of <i>Galleria mellonella</i> increased. Under sub-MICs of tetracycline antibiotics treatment, abnormal stretching of bacteria was observed by scanning electron microscopy. Treatment with sub-MICs of tetracyclines leads to increased surface hydrophobicity and eDNA content and decreased outer membrane permeability. The expression levels of the <i>fimA</i>, <i>luxS</i>, <i>qseB</i>, and <i>qseC</i> genes decreased, the expression level of <i>mrkA</i> increased, and the expression level of <i>acrA</i> was inconsistent under different tetracycline antibiotics treatments. Together, our results suggested that the increase in <i>Klebsiella pneumoniae</i> biofilm formation caused by sub-MICs of tetracycline antibiotics may occur by affecting bacterial physical and chemical properties and associated genes expression.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141475355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01DOI: 10.1021/acsinfecdis.4c00089
Charmante Registre, Luciana Miranda Silva, Farah Registre, Rodrigo Dian de Oliveira Aguiar Soares, Karina Taciana Santos Rubio, Simone Pinto Carneiro, Orlando David Henrique Dos Santos
Millions of people worldwide are affected by leishmaniasis, caused by the Leishmania parasite. Effective treatment is challenging due to the biological complexity of the parasite, drug toxicity, and increasing resistance to conventional drugs. To combat this disease, the development of specific strategies to target and selectively eliminate the parasite is crucial. This Review highlights the importance of amino acids in the developmental stages of Leishmania as a factor determining whether the infection progresses or is suppressed. It also explores the use of peptides as alternatives in parasite control and the development of novel targeted treatments. While these strategies show promise for more effective and targeted treatment, further studies to address the remaining challenges are imperative.
{"title":"Targeting <i>Leishmania</i> Promastigotes and Amastigotes Forms through Amino Acids and Peptides: A Promising Therapeutic Strategy.","authors":"Charmante Registre, Luciana Miranda Silva, Farah Registre, Rodrigo Dian de Oliveira Aguiar Soares, Karina Taciana Santos Rubio, Simone Pinto Carneiro, Orlando David Henrique Dos Santos","doi":"10.1021/acsinfecdis.4c00089","DOIUrl":"https://doi.org/10.1021/acsinfecdis.4c00089","url":null,"abstract":"<p><p>Millions of people worldwide are affected by leishmaniasis, caused by the <i>Leishmania</i> parasite. Effective treatment is challenging due to the biological complexity of the parasite, drug toxicity, and increasing resistance to conventional drugs. To combat this disease, the development of specific strategies to target and selectively eliminate the parasite is crucial. This Review highlights the importance of amino acids in the developmental stages of <i>Leishmania</i> as a factor determining whether the infection progresses or is suppressed. It also explores the use of peptides as alternatives in parasite control and the development of novel targeted treatments. While these strategies show promise for more effective and targeted treatment, further studies to address the remaining challenges are imperative.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141475356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.
{"title":"An Engineered N-Glycosylated Dengue Envelope Protein Domain III Facilitates Epitope-Directed Selection of Potently Neutralizing and Minimally Enhancing Antibodies.","authors":"Napon Nilchan, Romchat Kraivong, Prasit Luangaram, Anunyaporn Phungsom, Mongkhonphan Tantiwatcharakunthon, Somchoke Traewachiwiphak, Tanapan Prommool, Nuntaya Punyadee, Panisadee Avirutnan, Thaneeya Duangchinda, Prida Malasit, Chunya Puttikhunt","doi":"10.1021/acsinfecdis.4c00058","DOIUrl":"https://doi.org/10.1021/acsinfecdis.4c00058","url":null,"abstract":"<p><p>The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed \"EDIII mutant N\". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141464295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A newly discovered E3 ubiquitin ligase, UBR7, plays a crucial role in histone H2BK120 monoubiquitination. Here, we report a novel function of UBR7 in promoting hepatitis B virus (HBV) pathogenesis, which further leads to HBV-induced hepatocellular carcinoma (HCC). Transcriptomics analysis from HCC patients revealed the deregulation of UBR7 in cancer. Remarkably, targeting UBR7, particularly its catalytic function, led to a significant decrease in viral copy numbers. We also identified the speckled family protein Sp110 as an important substrate of UBR7. Notably, Sp110 has been previously shown to be a resident of promyelocytic leukemia nuclear bodies (PML-NBs), where it remains SUMOylated, and during HBV infection, it undergoes deSUMOylation and exits the PML body. We observed that UBR7 ubiquitinates Sp110 at critical residues within its SAND domain. Sp110 ubiquitination downregulates genes in the type I interferon response pathway. Comparative analysis of RNA-Seq from the UBR7/Sp110 knockdown data set confirmed that the IFN-β signaling pathway gets deregulated in HCC cells in the presence of HBV. Single-cell RNA-Seq analysis of patient samples further confirmed the inverse correlation between the expression of Sp110/UBR7 and the inflammation score. Notably, silencing of UBR7 induces IRF7 phosphorylation, thereby augmenting interferon (IFN)-β and the downstream interferon-stimulated genes (ISGs). Further, wild-type but not the ubiquitination-defective mutant of Sp110 could be recruited to the type I interferon response pathway genes. Our study establishes a new function of UBR7 in non-histone protein ubiquitination, promoting viral persistence, and has important implications for the development of therapeutic strategies targeting HBV-induced HCC.
{"title":"UBR7 E3 Ligase Suppresses Interferon-β Mediated Immune Signaling by Targeting Sp110 in Hepatitis B Virus-Induced Hepatocellular Carcinoma.","authors":"Vipin Singh, Atanu Mondal, Santanu Adhikary, Payel Mondal, Niranjan Shirgaonkar, Ramanuj DasGupta, Siddhartha Roy, Chandrima Das","doi":"10.1021/acsinfecdis.4c00213","DOIUrl":"https://doi.org/10.1021/acsinfecdis.4c00213","url":null,"abstract":"<p><p>A newly discovered E3 ubiquitin ligase, UBR7, plays a crucial role in histone H2BK120 monoubiquitination. Here, we report a novel function of UBR7 in promoting hepatitis B virus (HBV) pathogenesis, which further leads to HBV-induced hepatocellular carcinoma (HCC). Transcriptomics analysis from HCC patients revealed the deregulation of UBR7 in cancer. Remarkably, targeting UBR7, particularly its catalytic function, led to a significant decrease in viral copy numbers. We also identified the speckled family protein Sp110 as an important substrate of UBR7. Notably, Sp110 has been previously shown to be a resident of promyelocytic leukemia nuclear bodies (PML-NBs), where it remains SUMOylated, and during HBV infection, it undergoes deSUMOylation and exits the PML body. We observed that UBR7 ubiquitinates Sp110 at critical residues within its SAND domain. Sp110 ubiquitination downregulates genes in the type I interferon response pathway. Comparative analysis of RNA-Seq from the UBR7/Sp110 knockdown data set confirmed that the IFN-β signaling pathway gets deregulated in HCC cells in the presence of HBV. Single-cell RNA-Seq analysis of patient samples further confirmed the inverse correlation between the expression of Sp110/UBR7 and the inflammation score. Notably, silencing of UBR7 induces IRF7 phosphorylation, thereby augmenting interferon (IFN)-β and the downstream interferon-stimulated genes (ISGs). Further, wild-type but not the ubiquitination-defective mutant of Sp110 could be recruited to the type I interferon response pathway genes. Our study establishes a new function of UBR7 in non-histone protein ubiquitination, promoting viral persistence, and has important implications for the development of therapeutic strategies targeting HBV-induced HCC.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141464297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The ability of pathogenic bacteria to evade antibiotic treatment is an intricate and multifaceted phenomenon. Over the years, treatment failure among patients due to determinants of antimicrobial resistance (AMR) has been the focal point for the research and development of new therapeutic agents. However, the survival of bacteria by persisting under antibiotic stress has largely been overlooked. Bacterial persisters are a subpopulation of sensitive bacterial cells exhibiting a noninheritable drug-tolerant phenotype. They are linked to the recalcitrance of infections in healthcare settings, in turn giving rise to AMR variants. The importance of bacterial persistence in recurring infections has been firmly recognized. Fundamental work over the past decade has highlighted numerous unique tolerance factors contributing to the persister phenotype in many clinically relevant pathogens. This review summarizes contributing factors that could aid in developing new strategies against bacterial antibiotic persisters.
病原菌逃避抗生素治疗的能力是一个错综复杂的多方面现象。多年来,由于抗菌药耐药性(AMR)的决定因素导致患者治疗失败一直是研究和开发新治疗药物的焦点。然而,细菌在抗生素压力下通过持久存活的现象却在很大程度上被忽视了。细菌持久体是敏感细菌细胞的一个亚群,表现出不可遗传的耐药表型。它们与医疗环境中的顽固性感染有关,反过来又会产生 AMR 变种。细菌持久性在反复感染中的重要性已得到充分认识。过去十年的基础研究工作强调了许多独特的耐受性因素,这些因素导致了许多临床相关病原体的持久性表型。本综述总结了这些因素,它们有助于开发新的策略来对付细菌抗生素耐药菌。
{"title":"Decoding Bacterial Persistence: Mechanisms and Strategies for Effective Eradication.","authors":"Abhiroop Sett, Vineet Dubey, Somok Bhowmik, Ranjana Pathania","doi":"10.1021/acsinfecdis.4c00270","DOIUrl":"https://doi.org/10.1021/acsinfecdis.4c00270","url":null,"abstract":"<p><p>The ability of pathogenic bacteria to evade antibiotic treatment is an intricate and multifaceted phenomenon. Over the years, treatment failure among patients due to determinants of antimicrobial resistance (AMR) has been the focal point for the research and development of new therapeutic agents. However, the survival of bacteria by persisting under antibiotic stress has largely been overlooked. Bacterial persisters are a subpopulation of sensitive bacterial cells exhibiting a noninheritable drug-tolerant phenotype. They are linked to the recalcitrance of infections in healthcare settings, in turn giving rise to AMR variants. The importance of bacterial persistence in recurring infections has been firmly recognized. Fundamental work over the past decade has highlighted numerous unique tolerance factors contributing to the persister phenotype in many clinically relevant pathogens. This review summarizes contributing factors that could aid in developing new strategies against bacterial antibiotic persisters.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141464296","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-26DOI: 10.1021/acsinfecdis.4c00289
Ayman Hemasa, Christina Spry, Matthias Mack, Kevin J Saliba
The riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilization. To determine their mechanism of action, we generated roseoflavin-resistant parasites by in vitro evolution. Relative to wild-type, these parasites were 4-fold resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Whole genome sequencing of the resistant parasites revealed a missense mutation leading to an amino acid change (L672H) in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene. The IC50 values for roseoflavin and 8-aminoriboflavin against the roseoflavin-resistant parasites created through in vitro evolution were indistinguishable from those against parasites in which the missense mutation was introduced into the native PfFK. We also generated two parasite lines episomally expressing GFP-tagged versions of either the wild-type or mutant forms of PfFK. We found that PfFK-GFP localizes to the parasite cytosol and that immunopurified PfFK-GFP phosphorylated riboflavin, roseoflavin, and 8-aminoriboflavin. The L672H mutation increased the KM for roseoflavin, explaining the resistance phenotype. Mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonized by increasing the extracellular concentration of riboflavin, consistent with it also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore, are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite proliferation by inhibiting riboflavin phosphorylation and via the generation of toxic flavin cofactor analogues.
{"title":"Mutation of the <i>Plasmodium falciparum</i> Flavokinase Confers Resistance to Roseoflavin and 8-Aminoriboflavin.","authors":"Ayman Hemasa, Christina Spry, Matthias Mack, Kevin J Saliba","doi":"10.1021/acsinfecdis.4c00289","DOIUrl":"https://doi.org/10.1021/acsinfecdis.4c00289","url":null,"abstract":"<p><p>The riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilization. To determine their mechanism of action, we generated roseoflavin-resistant parasites by in vitro evolution. Relative to wild-type, these parasites were 4-fold resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Whole genome sequencing of the resistant parasites revealed a missense mutation leading to an amino acid change (L672H) in the gene coding for a putative flavokinase (<i>Pf</i>FK), the enzyme responsible for converting riboflavin into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the phenotype, we generated parasites with the missense mutation incorporated into the <i>Pf</i>FK gene. The IC<sub>50</sub> values for roseoflavin and 8-aminoriboflavin against the roseoflavin-resistant parasites created through in vitro evolution were indistinguishable from those against parasites in which the missense mutation was introduced into the native <i>Pf</i>FK. We also generated two parasite lines episomally expressing GFP-tagged versions of either the wild-type or mutant forms of <i>Pf</i>FK. We found that <i>Pf</i>FK-GFP localizes to the parasite cytosol and that immunopurified <i>Pf</i>FK-GFP phosphorylated riboflavin, roseoflavin, and 8-aminoriboflavin. The L672H mutation increased the <i>K</i><sub>M</sub> for roseoflavin, explaining the resistance phenotype. Mutant <i>Pf</i>FK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonized by increasing the extracellular concentration of riboflavin, consistent with it also inhibiting parasite growth through competitive inhibition of <i>Pf</i>FK. Our findings, therefore, are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite proliferation by inhibiting riboflavin phosphorylation and via the generation of toxic flavin cofactor analogues.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Antimicrobial peptides (AMPs) are becoming next-generation alternative antibacterial agents because of the rapid increase in resistance in bacteria against existing antibiotics, which can also be attributed to the formation of resilient biofilms. However, their widespread use is limited because of their poor absorption, higher dosage requirements, and delayed onset of the bioactivity to elicit a desired response. Here we developed a short AMP that specifically targeted Fusobacterium nucleatum. We conjugated 23R to a statherin-derived peptide (SDP) through rational design; this conjugate binds to FomA, a major porin protein of F. nucleatum. The SDP-tagged 23R exhibited rapid and highly specific bactericidal efficacy against F. nucleatum. Further, IC50 values were in the nanomolar range, and they were 100-fold lower than those obtained with unconjugated 23R. In a human gut microbiota model, 0.1 nM SDP-23R achieved 99% clearance of F. nucleatum ATCC 25586 without markedly altering resident microbiota. Here we demonstrated that binding-peptide-coupled AMPs show increased killing efficacy and specificity for the target pathogen without affecting the resident microbiota.
{"title":"Engineering Short Antimicrobial Peptides to Specifically Target <i>Fusobacterium nucleatum</i> in the Mixed Microbial Population.","authors":"Zhao Liu, Yijie Wang, Chen Zhang, Yongshuai Yang, Junfeng Zhang","doi":"10.1021/acsinfecdis.4c00387","DOIUrl":"10.1021/acsinfecdis.4c00387","url":null,"abstract":"<p><p>Antimicrobial peptides (AMPs) are becoming next-generation alternative antibacterial agents because of the rapid increase in resistance in bacteria against existing antibiotics, which can also be attributed to the formation of resilient biofilms. However, their widespread use is limited because of their poor absorption, higher dosage requirements, and delayed onset of the bioactivity to elicit a desired response. Here we developed a short AMP that specifically targeted <i>Fusobacterium nucleatum</i>. We conjugated 23R to a statherin-derived peptide (SDP) through rational design; this conjugate binds to FomA, a major porin protein of <i>F. nucleatum</i>. The SDP-tagged 23R exhibited rapid and highly specific bactericidal efficacy against <i>F. nucleatum</i>. Further, IC<sub>50</sub> values were in the nanomolar range, and they were 100-fold lower than those obtained with unconjugated 23R. In a human gut microbiota model, 0.1 nM SDP-23R achieved 99% clearance of <i>F. nucleatum</i> ATCC 25586 without markedly altering resident microbiota. Here we demonstrated that binding-peptide-coupled AMPs show increased killing efficacy and specificity for the target pathogen without affecting the resident microbiota.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141449020","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-25DOI: 10.1021/acsinfecdis.4c00251
Zaret G Denieva, Peter I Kuzmin, Timur R Galimzyanov, Siddhartha A K Datta, Alan Rein, Oleg V Batishchev
Human immunodeficiency virus (HIV) assembly at an infected cell's plasma membrane requires membrane deformation to organize the near-spherical shape of an immature virus. While the cellular expression of HIV Gag is sufficient to initiate budding of virus-like particles, how Gag generates membrane curvature is not fully understood. Using highly curved lipid nanotubes, we have investigated the physicochemical basis of the membrane activity of recombinant nonmyristoylated Gag-Δp6. Gag protein, upon adsorption onto the membrane, resulted in the shape changes of both charged and uncharged nanotubes. This shape change was more pronounced in the presence of charged lipids, especially phosphatidylinositol bisphosphate (PI(4,5)P2). We found that Gag modified the interfacial tension of phospholipid bilayer membranes, as judged by comparison with the effects of amphipathic peptides and nonionic detergent. Bioinformatic analysis demonstrated that a region of the capsid and SP1 domains junction of Gag is structurally similar to the amphipathic peptide magainin-1. This region accounts for integral changes in the physical properties of the membrane upon Gag adsorption, as we showed with the synthetic CA-SP1 junction peptide. Phenomenologically, membrane-adsorbed Gag could diminish the energetic cost of increasing the membrane area in a way similar to foam formation. We propose that Gag acts as a surface-active substance at the HIV budding site that softens the membrane at the place of Gag adsorption, lowering the energy for membrane bending. Finally, our experimental data and theoretical considerations give a lipid-centric view and common mechanism by which proteins could bend membranes, despite not having intrinsic curvature in their molecular surfaces or assemblies.
{"title":"Human Immunodeficiency Virus Type 1 Gag Polyprotein Modulates Membrane Physical Properties like a Surfactant: Potential Implications for Virus Assembly.","authors":"Zaret G Denieva, Peter I Kuzmin, Timur R Galimzyanov, Siddhartha A K Datta, Alan Rein, Oleg V Batishchev","doi":"10.1021/acsinfecdis.4c00251","DOIUrl":"https://doi.org/10.1021/acsinfecdis.4c00251","url":null,"abstract":"<p><p>Human immunodeficiency virus (HIV) assembly at an infected cell's plasma membrane requires membrane deformation to organize the near-spherical shape of an immature virus. While the cellular expression of HIV Gag is sufficient to initiate budding of virus-like particles, how Gag generates membrane curvature is not fully understood. Using highly curved lipid nanotubes, we have investigated the physicochemical basis of the membrane activity of recombinant nonmyristoylated Gag-Δp6. Gag protein, upon adsorption onto the membrane, resulted in the shape changes of both charged and uncharged nanotubes. This shape change was more pronounced in the presence of charged lipids, especially phosphatidylinositol bisphosphate (PI(4,5)P<sub>2</sub>). We found that Gag modified the interfacial tension of phospholipid bilayer membranes, as judged by comparison with the effects of amphipathic peptides and nonionic detergent. Bioinformatic analysis demonstrated that a region of the capsid and SP1 domains junction of Gag is structurally similar to the amphipathic peptide magainin-1. This region accounts for integral changes in the physical properties of the membrane upon Gag adsorption, as we showed with the synthetic CA-SP1 junction peptide. Phenomenologically, membrane-adsorbed Gag could diminish the energetic cost of increasing the membrane area in a way similar to foam formation. We propose that Gag acts as a surface-active substance at the HIV budding site that softens the membrane at the place of Gag adsorption, lowering the energy for membrane bending. Finally, our experimental data and theoretical considerations give a lipid-centric view and common mechanism by which proteins could bend membranes, despite not having intrinsic curvature in their molecular surfaces or assemblies.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448945","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-24DOI: 10.1021/acsinfecdis.3c00728
Cheng Wang, Shaobo Wang, Xiangyu Ma, Xiaohong Yao, Kegang Zhan, Zai Wang, Di He, Wenting Zuo, Songling Han, Gaomei Zhao, Bin Cao, Jinghong Zhao, Xiuwu Bian, Junping Wang
SARS-CoV-2 infection starts from the association of its spike 1 (S1) subunit with sensitive cells. Vesicular endothelial cells and platelets are among the cell types that bind SARS-CoV-2, but the effectors that mediate viral attachment on the cell membrane have not been fully elucidated. Herein, we show that P-selectin (SELP), a biomarker for endothelial dysfunction and platelet activation, can facilitate the attachment of SARS-CoV-2 S1. Since we observe colocalization of SELP with S1 in the lung tissues of COVID-19 patients, we perform molecular biology experiments on human umbilical vein endothelial cells (HUVECs) to confirm the intermolecular interaction between SELP and S1. SELP overexpression increases S1 recruitment to HUVECs and enhances SARS-CoV-2 spike pseudovirion infection. The opposite results are determined after SELP downregulation. As S1 causes endothelial inflammatory responses in a dose-dependent manner, by activating the interleukin (IL)-17 signaling pathway, SELP-induced S1 recruitment may contribute to the development of a "cytokine storm" after viral infection. Furthermore, SELP also promotes the attachment of S1 to the platelet membrane. Employment of PSI-697, a small inhibitor of SELP, markedly decreases S1 adhesion to both HUVECs and platelets. In addition to the role of membrane SELP in facilitating S1 attachment, we also discover that soluble SELP is a prognostic factor for severe COVID-19 through a meta-analysis. In this study, we identify SELP as an adhesive site for the SARS-CoV-2 S1, thus providing a potential drug target for COVID-19 treatment.
{"title":"P-selectin Facilitates SARS-CoV-2 Spike 1 Subunit Attachment to Vesicular Endothelium and Platelets.","authors":"Cheng Wang, Shaobo Wang, Xiangyu Ma, Xiaohong Yao, Kegang Zhan, Zai Wang, Di He, Wenting Zuo, Songling Han, Gaomei Zhao, Bin Cao, Jinghong Zhao, Xiuwu Bian, Junping Wang","doi":"10.1021/acsinfecdis.3c00728","DOIUrl":"https://doi.org/10.1021/acsinfecdis.3c00728","url":null,"abstract":"<p><p>SARS-CoV-2 infection starts from the association of its spike 1 (S1) subunit with sensitive cells. Vesicular endothelial cells and platelets are among the cell types that bind SARS-CoV-2, but the effectors that mediate viral attachment on the cell membrane have not been fully elucidated. Herein, we show that P-selectin (SELP), a biomarker for endothelial dysfunction and platelet activation, can facilitate the attachment of SARS-CoV-2 S1. Since we observe colocalization of SELP with S1 in the lung tissues of COVID-19 patients, we perform molecular biology experiments on human umbilical vein endothelial cells (HUVECs) to confirm the intermolecular interaction between SELP and S1. SELP overexpression increases S1 recruitment to HUVECs and enhances SARS-CoV-2 spike pseudovirion infection. The opposite results are determined after SELP downregulation. As S1 causes endothelial inflammatory responses in a dose-dependent manner, by activating the interleukin (IL)-17 signaling pathway, SELP-induced S1 recruitment may contribute to the development of a \"cytokine storm\" after viral infection. Furthermore, SELP also promotes the attachment of S1 to the platelet membrane. Employment of PSI-697, a small inhibitor of SELP, markedly decreases S1 adhesion to both HUVECs and platelets. In addition to the role of membrane SELP in facilitating S1 attachment, we also discover that soluble SELP is a prognostic factor for severe COVID-19 through a meta-analysis. In this study, we identify SELP as an adhesive site for the SARS-CoV-2 S1, thus providing a potential drug target for COVID-19 treatment.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141441699","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}