Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2×105 compared to that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and is marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.
{"title":"Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination","authors":"Go Kamoshida, Daiki Yamaguchi, Yuki Kaya, Toshiki Yamakado, Kenta Yamashita, Moe Aoyagi, Saaya Nagai, Noriteru Yamada, Yu Kawagishi, Mizuki Sugano, Yoshiaki Sakairi, Mikako Ueno, Norihiko Takemoto, Yuji Morita, Yukihito Ishizaka, Kinnosuke Yahiro","doi":"10.1093/pnasnexus/pgae328","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae328","url":null,"abstract":"Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2×105 compared to that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and is marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141968990","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 : 2024-08-07DOI: 10.1093/pnasnexus/pgae330
Junyue Wang, Thomas Schaefer, Aliaksandra Lisouskaya, Daniele S Firak, Xiaoyue Xin, Lingjun Meng, Hartmut Herrmann, Virender K Sharma, Ching-Hua Huang
Acetylperoxyl radical (CH3C(O)OO●) is among highly reactive organic radicals which are known to play crucial roles in atmospheric chemistry, aqueous chemistry and, most recently, peracetic acid (PAA)-based advanced oxidation processes. However, fundamental knowledge for its reactivity is scarce and severely hampers the understanding of relevant environmental processes. Herein, three independent experimental approaches were exploited for revelation and quantification of the reaction rates of acetylperoxyl radical. First, we developed and verified laser flash photolysis (LFP) of biacetyl, ultraviolet (UV) photolysis of biacetyl, and pulse radiolysis of acetaldehyde, each as a clean source of CH3C(O)OO●. Then, using competition kinetics and selection of suitable probe and competitor compounds, the rate constants between CH3C(O)OO● and compounds of diverse structures were determined. The three experimental approaches complemented in reaction time scale and ease of operation, and provided cross-validation of the rate constants. Moreover, formation of CH3C(O)OO● was verified by spin-trapped electron paramagnetic resonance (EPR), and potential influence of other reactive species in the systems was assessed. Overall, CH3C(O)OO● displays distinctively high reactivity and selectivity, reacting especially favorably with naphthyl and diene compounds (k ∼ 107-108 M-1s-1) but sluggishly with N- and S-containing groups. Significantly, we demonstrated that incorporating acetylperoxyl radical-oxidation reactions significantly improved the accuracy in modeling the degradation of environmental micropollutants by UV/PAA treatment. This study is among the most comprehensive investigation for peroxyl radical reactivity to date, and establishes a robust methodology for investigating organic radical chemistry. The determined rate constants strengthen kinetic databases and improve modeling accuracy for natural and engineered systems.
乙酰过氧自由基（CH3C(O)OO●）是一种高活性有机自由基，在大气化学、水化学以及最近基于过乙酸（PAA）的高级氧化过程中发挥着至关重要的作用。然而，有关其反应性的基础知识非常匮乏，严重阻碍了对相关环境过程的了解。在此，我们采用了三种独立的实验方法来揭示和量化乙酰过氧自由基的反应速率。首先，我们开发并验证了生物乙酰的激光闪烁光解（LFP）、生物乙酰的紫外线（UV）光解和乙醛的脉冲辐射分解，每种方法都是 CH3C(O)OO● 的清洁来源。然后，利用竞争动力学并选择合适的探针和竞争化合物，测定了 CH3C(O)OO● 与不同结构的化合物之间的速率常数。这三种实验方法在反应时间范围和操作简便性方面互为补充，并对速率常数进行了交叉验证。此外，还通过自旋俘获电子顺磁共振（EPR）验证了 CH3C(O)OO● 的形成，并评估了体系中其他反应物的潜在影响。总之，CH3C(O)OO● 显示出明显的高反应性和选择性，与萘基和二烯化合物的反应尤其有利（k ∼ 107-108 M-1s-1），但与含 N 和 S 的基团的反应则较为迟缓。重要的是，我们证明了加入乙酰过氧自由基氧化反应可显著提高紫外线/PAA 处理环境微污染物降解模型的准确性。这项研究是迄今为止对过氧自由基反应性进行的最全面的研究之一，为研究有机自由基化学建立了一种可靠的方法。确定的速率常数加强了动力学数据库，提高了自然和工程系统建模的准确性。
{"title":"Unveiling the environmental significance of acetylperoxyl radical: reactivity quantification and kinetic modeling","authors":"Junyue Wang, Thomas Schaefer, Aliaksandra Lisouskaya, Daniele S Firak, Xiaoyue Xin, Lingjun Meng, Hartmut Herrmann, Virender K Sharma, Ching-Hua Huang","doi":"10.1093/pnasnexus/pgae330","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae330","url":null,"abstract":"Acetylperoxyl radical (CH3C(O)OO●) is among highly reactive organic radicals which are known to play crucial roles in atmospheric chemistry, aqueous chemistry and, most recently, peracetic acid (PAA)-based advanced oxidation processes. However, fundamental knowledge for its reactivity is scarce and severely hampers the understanding of relevant environmental processes. Herein, three independent experimental approaches were exploited for revelation and quantification of the reaction rates of acetylperoxyl radical. First, we developed and verified laser flash photolysis (LFP) of biacetyl, ultraviolet (UV) photolysis of biacetyl, and pulse radiolysis of acetaldehyde, each as a clean source of CH3C(O)OO●. Then, using competition kinetics and selection of suitable probe and competitor compounds, the rate constants between CH3C(O)OO● and compounds of diverse structures were determined. The three experimental approaches complemented in reaction time scale and ease of operation, and provided cross-validation of the rate constants. Moreover, formation of CH3C(O)OO● was verified by spin-trapped electron paramagnetic resonance (EPR), and potential influence of other reactive species in the systems was assessed. Overall, CH3C(O)OO● displays distinctively high reactivity and selectivity, reacting especially favorably with naphthyl and diene compounds (k ∼ 107-108 M-1s-1) but sluggishly with N- and S-containing groups. Significantly, we demonstrated that incorporating acetylperoxyl radical-oxidation reactions significantly improved the accuracy in modeling the degradation of environmental micropollutants by UV/PAA treatment. This study is among the most comprehensive investigation for peroxyl radical reactivity to date, and establishes a robust methodology for investigating organic radical chemistry. The determined rate constants strengthen kinetic databases and improve modeling accuracy for natural and engineered systems.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141938411","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 : 2024-08-07DOI: 10.1093/pnasnexus/pgae325
Jason Nideffer, Maureen Ty, Michele Donato, Rek John, Richard Kajubi, Xuhuai Ji, Felistas Nankya, Kenneth Musinguzi, Kathleen Dantzler Press, Nora Yang, Kylie Camanag, Bryan Greenhouse, Moses Kamya, Margaret E Feeney, Grant Dorsey, P J Utz, Bali Pulendran, Purvesh Khatri, Prasanna Jagannathan
The regulation of inflammation is a critical aspect of disease tolerance and naturally acquired clinical immunity to malaria. Here, we demonstrate using RNA sequencing and epigenetic landscape profiling by cytometry by Time-Of-Flight (EpiTOF), that the regulation of inflammatory pathways during asymptomatic parasitemia occurs downstream of pathogen sensing—at the epigenetic level. The abundance of certain epigenetic markers (methylation of H3K27 and dimethylation of arginine residues) and decreased prevalence of histone variant H3.3 correlated with suppressed cytokine responses among monocytes of Ugandan children. Such an epigenetic signature was observed across diverse immune cell populations and not only characterized active asymptomatic parasitemia but also correlated with future long-term disease tolerance and clinical immunity when observed in uninfected children. Pseudotime analyses revealed a potential trajectory of epigenetic change that correlated with a child’s age and recent parasite exposure and paralleled the acquisition of clinical immunity. Thus, our data support a model whereby exposure to Plasmodium falciparum induces epigenetic changes that regulate excessive inflammation and contribute to naturally acquired clinical immunity to malaria.
{"title":"Clinical immunity to malaria involves epigenetic reprogramming of innate immune cells","authors":"Jason Nideffer, Maureen Ty, Michele Donato, Rek John, Richard Kajubi, Xuhuai Ji, Felistas Nankya, Kenneth Musinguzi, Kathleen Dantzler Press, Nora Yang, Kylie Camanag, Bryan Greenhouse, Moses Kamya, Margaret E Feeney, Grant Dorsey, P J Utz, Bali Pulendran, Purvesh Khatri, Prasanna Jagannathan","doi":"10.1093/pnasnexus/pgae325","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae325","url":null,"abstract":"The regulation of inflammation is a critical aspect of disease tolerance and naturally acquired clinical immunity to malaria. Here, we demonstrate using RNA sequencing and epigenetic landscape profiling by cytometry by Time-Of-Flight (EpiTOF), that the regulation of inflammatory pathways during asymptomatic parasitemia occurs downstream of pathogen sensing—at the epigenetic level. The abundance of certain epigenetic markers (methylation of H3K27 and dimethylation of arginine residues) and decreased prevalence of histone variant H3.3 correlated with suppressed cytokine responses among monocytes of Ugandan children. Such an epigenetic signature was observed across diverse immune cell populations and not only characterized active asymptomatic parasitemia but also correlated with future long-term disease tolerance and clinical immunity when observed in uninfected children. Pseudotime analyses revealed a potential trajectory of epigenetic change that correlated with a child’s age and recent parasite exposure and paralleled the acquisition of clinical immunity. Thus, our data support a model whereby exposure to Plasmodium falciparum induces epigenetic changes that regulate excessive inflammation and contribute to naturally acquired clinical immunity to malaria.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969040","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 : 2024-08-07DOI: 10.1093/pnasnexus/pgae322
Liping Zhang, Kelly G Ten Hagen
Glycoproteins are abundant within the human reproductive system and alterations in glycosylation lead to reproductive disorders, suggesting that glycans play important roles in reproductive function. In this study, we used the Drosophila reproductive system as a model to investigate the biological functions of O-glycosylation. We found that O-glycosylation in the male accessory glands (MAGs), an organ responsible for secreting seminal fluid proteins, plays important roles in female post-mating behavior. Loss of one O-glycosyltransferase, PGANT9, in the male reproductive system resulted in decreased egg production in mated females. We identified one substrate of PGANT9, the lectin-46Ca (CG1656), which is known to affect female post-mating responses. We further show that the loss of lectin-46Ca O-glycosylation affects its ability to associate with sperm tails, resulting in reduced transfer within the female reproductive system. Our results provide the first example that O-glycosylation of a seminal fluid protein affects its ability to associate with sperm in vivo. These studies may shed light on the biological function of O-glycans in mammalian reproduction.
糖蛋白在人类生殖系统中含量丰富，糖基化的改变会导致生殖系统疾病，这表明聚糖在生殖功能中发挥着重要作用。在这项研究中，我们以果蝇生殖系统为模型，研究了O-糖基化的生物学功能。我们发现，雄性附属腺体（MAGs）中的O-糖基化在雌性交配后的行为中起着重要作用，雄性附属腺体是负责分泌精液蛋白的器官。雄性生殖系统中一种O-糖基转移酶PGANT9的缺失会导致交配雌性产卵量减少。我们发现了 PGANT9 的一种底物--凝集素-46Ca（CG1656），已知它能影响雌性交配后的反应。我们进一步发现，凝集素-46Ca O-糖基化的缺失会影响其与精子尾部结合的能力，导致其在雌性生殖系统内的转移能力降低。我们的研究结果首次提供了精液蛋白的O-糖基化影响其在体内与精子结合能力的实例。这些研究可能会揭示 O 型糖在哺乳动物生殖过程中的生物学功能。
{"title":"O-Glycosylation of a male seminal fluid protein influences sperm binding and female post-mating behavior","authors":"Liping Zhang, Kelly G Ten Hagen","doi":"10.1093/pnasnexus/pgae322","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae322","url":null,"abstract":"Glycoproteins are abundant within the human reproductive system and alterations in glycosylation lead to reproductive disorders, suggesting that glycans play important roles in reproductive function. In this study, we used the Drosophila reproductive system as a model to investigate the biological functions of O-glycosylation. We found that O-glycosylation in the male accessory glands (MAGs), an organ responsible for secreting seminal fluid proteins, plays important roles in female post-mating behavior. Loss of one O-glycosyltransferase, PGANT9, in the male reproductive system resulted in decreased egg production in mated females. We identified one substrate of PGANT9, the lectin-46Ca (CG1656), which is known to affect female post-mating responses. We further show that the loss of lectin-46Ca O-glycosylation affects its ability to associate with sperm tails, resulting in reduced transfer within the female reproductive system. Our results provide the first example that O-glycosylation of a seminal fluid protein affects its ability to associate with sperm in vivo. These studies may shed light on the biological function of O-glycans in mammalian reproduction.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141968952","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 : 2024-08-07DOI: 10.1093/pnasnexus/pgae331
Weiyue Xin, Maria M Santore
Biological or biomimetic membranes are examples within the larger material class of flexible ultrathin lamellae and contoured fluid sheets that require work or energy to impose bending deformations. Bending elasticity also dictates the interactions and assembly of integrated phases or molecular clusters within fluid lamellae, for instance enabling critical cell functions in biomembranes. More broadly, lamella and other thin fluids that integrate dispersed objects, inclusions, and phases behave as contoured 2D colloidal suspensions governed by elastic interactions. To elucidate the breadth of interactions and assembled patterns accessible through elastic interactions, we consider the bending elasticity-driven assembly of 1-10 μm solid plate-shaped Brownian domains (the 2D colloids), integrated into a fluid phospholipid membrane (the 2D fluid). Here the fluid membranes of giant unilamellar vesicles, 20-50 μm in diameter, each contain 4-100 monodisperse plate-domains at an overall solid area fraction of 17±3%. Three types of reversible plate arrangements are found: persistent vesicle-encompassing quasi-hexagonal lattices, persistent closely associated chains or concentrated lattices, and a dynamic disordered state. The interdomain distances evidence combined attractive and repulsive elastic interactions up to 10 μm, far exceeding the ranges of physio-chemical interactions. Bending contributions are controlled through membrane slack (excess area) producing, for a fixed composition, a sharp cooperative multi-body transition in plate arrangement, while domain size and number contribute intricacy.
{"title":"Bending-Driven patterning of solid inclusions in lipid membranes: Colloidal assembly and transitions in elastic 2D fluids","authors":"Weiyue Xin, Maria M Santore","doi":"10.1093/pnasnexus/pgae331","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae331","url":null,"abstract":"Biological or biomimetic membranes are examples within the larger material class of flexible ultrathin lamellae and contoured fluid sheets that require work or energy to impose bending deformations. Bending elasticity also dictates the interactions and assembly of integrated phases or molecular clusters within fluid lamellae, for instance enabling critical cell functions in biomembranes. More broadly, lamella and other thin fluids that integrate dispersed objects, inclusions, and phases behave as contoured 2D colloidal suspensions governed by elastic interactions. To elucidate the breadth of interactions and assembled patterns accessible through elastic interactions, we consider the bending elasticity-driven assembly of 1-10 μm solid plate-shaped Brownian domains (the 2D colloids), integrated into a fluid phospholipid membrane (the 2D fluid). Here the fluid membranes of giant unilamellar vesicles, 20-50 μm in diameter, each contain 4-100 monodisperse plate-domains at an overall solid area fraction of 17±3%. Three types of reversible plate arrangements are found: persistent vesicle-encompassing quasi-hexagonal lattices, persistent closely associated chains or concentrated lattices, and a dynamic disordered state. The interdomain distances evidence combined attractive and repulsive elastic interactions up to 10 μm, far exceeding the ranges of physio-chemical interactions. Bending contributions are controlled through membrane slack (excess area) producing, for a fixed composition, a sharp cooperative multi-body transition in plate arrangement, while domain size and number contribute intricacy.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141938276","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 : 2024-08-01DOI: 10.1093/pnasnexus/pgae316
Anshu Saran, Hey-Min Kim, Ireland Manning, Mark A Hancock, Claus Schmitz, Mariusz Madej, Jan Potempa, Maria Sola, Jean-François Trempe, Yongtao Zhu, Mary Ellen Davey, Natalie Zeytuni
The Type-IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo. First, our structural studies revealed PorX harbours a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorX’s effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorX’s dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the non-pathogenic Flavobacterium johnsoniae, but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.
{"title":"Unveiling the molecular mechanisms of the Type-IX secretion system's response regulator: Structural and functional insights","authors":"Anshu Saran, Hey-Min Kim, Ireland Manning, Mark A Hancock, Claus Schmitz, Mariusz Madej, Jan Potempa, Maria Sola, Jean-François Trempe, Yongtao Zhu, Mary Ellen Davey, Natalie Zeytuni","doi":"10.1093/pnasnexus/pgae316","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae316","url":null,"abstract":"The Type-IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo. First, our structural studies revealed PorX harbours a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorX’s effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorX’s dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the non-pathogenic Flavobacterium johnsoniae, but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141886761","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 : 2024-08-01DOI: 10.1093/pnasnexus/pgae317
João Felipe M Salgado, Balakrishnan N V Premkrishnan, Elaine L Oliveira, Vineeth Kodengil Vettath, Feng Guang Goh, Xinjun Hou, Daniela I Drautz-Moses, Yu Cai, Stephan C Schuster, Ana Carolina M Junqueira
Blood feeding is crucial for the reproductive cycle of the mosquito Aedes aegypti, as well as for the transmission of arboviruses to hosts. It is postulated that blood meals may influence the mosquito microbiome but shifts in microbial diversity and function during digestion remain elusive. We used whole-genome shotgun metagenomics to monitor the midgut microbiome in 60 individual females of A. aegypti throughout digestion, after 12, 24, and 48 hours following blood or sugar meals. Additionally, ten individual larvae were sequenced, showing microbiomes dominated by Microbacterium sp. The high metagenomic coverage allowed for microbial assignments at the species taxonomic level, also providing functional profiling. Females in the post-digestive period and larvae displayed low microbiome diversities. A striking proliferation of Enterobacterales was observed during digestion in blood-fed mosquitoes. The compositional shift was concomitant with enrichment in genes associated with carbohydrate and protein metabolism, as well as virulence factors for antimicrobial resistance and scavenging. The bacterium Elizabethkingia anophelis (Flavobacteriales), a known human pathogen, was the dominant species at the end of blood digestion. Phylogenomics suggests that its association with hematophagous mosquitoes occurred several times. We consider evidence of mutually beneficial host-microbe interactions raised from this association, potentially pivotal for the mosquito’s resistance to arbovirus infection. After digestion, the observed shifts in blood-fed females’ midguts shifted to a sugar-fed-like microbial profile. This study provides insights into how the microbiome of A. aegypti is modulated to fulfill digestive roles following blood meals, emphasizing proliferation of potential symbionts in response to the dynamic midgut environment.
{"title":"The dynamics of the midgut microbiome in aedes aegypti during digestion reveal putative symbionts","authors":"João Felipe M Salgado, Balakrishnan N V Premkrishnan, Elaine L Oliveira, Vineeth Kodengil Vettath, Feng Guang Goh, Xinjun Hou, Daniela I Drautz-Moses, Yu Cai, Stephan C Schuster, Ana Carolina M Junqueira","doi":"10.1093/pnasnexus/pgae317","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae317","url":null,"abstract":"Blood feeding is crucial for the reproductive cycle of the mosquito Aedes aegypti, as well as for the transmission of arboviruses to hosts. It is postulated that blood meals may influence the mosquito microbiome but shifts in microbial diversity and function during digestion remain elusive. We used whole-genome shotgun metagenomics to monitor the midgut microbiome in 60 individual females of A. aegypti throughout digestion, after 12, 24, and 48 hours following blood or sugar meals. Additionally, ten individual larvae were sequenced, showing microbiomes dominated by Microbacterium sp. The high metagenomic coverage allowed for microbial assignments at the species taxonomic level, also providing functional profiling. Females in the post-digestive period and larvae displayed low microbiome diversities. A striking proliferation of Enterobacterales was observed during digestion in blood-fed mosquitoes. The compositional shift was concomitant with enrichment in genes associated with carbohydrate and protein metabolism, as well as virulence factors for antimicrobial resistance and scavenging. The bacterium Elizabethkingia anophelis (Flavobacteriales), a known human pathogen, was the dominant species at the end of blood digestion. Phylogenomics suggests that its association with hematophagous mosquitoes occurred several times. We consider evidence of mutually beneficial host-microbe interactions raised from this association, potentially pivotal for the mosquito’s resistance to arbovirus infection. After digestion, the observed shifts in blood-fed females’ midguts shifted to a sugar-fed-like microbial profile. This study provides insights into how the microbiome of A. aegypti is modulated to fulfill digestive roles following blood meals, emphasizing proliferation of potential symbionts in response to the dynamic midgut environment.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141886762","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 : 2024-08-01DOI: 10.1093/pnasnexus/pgae288
Mohammad Bagher Khamechian, Mohammad Reza Daliri, Stefan Treue, Moein Esghaei
Performing visually-guided behavior involves flexible routing of sensory information towards associative areas. We hypothesize that in visual cortical areas, this routing is shaped by a gating influence of the local neuronal population on the activity of the same population’s single neurons. We analyzed beta frequencies (representing local population activity), high-gamma frequencies (representative of the activity of local clusters of neurons), and the firing of single neurons in cortical area MT of behaving rhesus monkeys. Our results show an influence of beta activity on single neurons, predictive of behavioral performance. Similarly, the temporal dependence of high-gamma on beta predicts behavioral performance. These demonstrate a unidirectional influence of network-level neural dynamics on single neuron activity, preferentially routing relevant information. This demonstration of a local top-down influence offers a new perspective onto a core feature of cortical information processing: the selective transmission of sensory information to downstream areas based on behavioral relevance.
{"title":"Coupled oscillations orchestrate selective information transmission in visual cortex","authors":"Mohammad Bagher Khamechian, Mohammad Reza Daliri, Stefan Treue, Moein Esghaei","doi":"10.1093/pnasnexus/pgae288","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae288","url":null,"abstract":"Performing visually-guided behavior involves flexible routing of sensory information towards associative areas. We hypothesize that in visual cortical areas, this routing is shaped by a gating influence of the local neuronal population on the activity of the same population’s single neurons. We analyzed beta frequencies (representing local population activity), high-gamma frequencies (representative of the activity of local clusters of neurons), and the firing of single neurons in cortical area MT of behaving rhesus monkeys. Our results show an influence of beta activity on single neurons, predictive of behavioral performance. Similarly, the temporal dependence of high-gamma on beta predicts behavioral performance. These demonstrate a unidirectional influence of network-level neural dynamics on single neuron activity, preferentially routing relevant information. This demonstration of a local top-down influence offers a new perspective onto a core feature of cortical information processing: the selective transmission of sensory information to downstream areas based on behavioral relevance.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141886808","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 : 2024-08-01DOI: 10.1093/pnasnexus/pgae306
Brennan Klein, Harrison Hartle, Munik Shrestha, Ana Cecilia Zenteno, David Barros Sierra Cordera, José R Nicolás-Carlock, Ana I Bento, Benjamin M Althouse, Bernardo Gutierrez, Marina Escalera-Zamudio, Arturo Reyes-Sandoval, Oliver G Pybus, Alessandro Vespignani, José Alberto Díaz Quiñonez, Samuel V Scarpino, Moritz U G Kraemer
During outbreaks of emerging infectious diseases, internationally connected cities often experience large and early outbreaks, while rural regions follow after some delay. This hierarchical structure of disease spread is influenced primarily by the multiscale structure of human mobility. However, during the COVID-19 epidemic, public health responses typically did not take into consideration the explicit spatial structure of human mobility when designing non-pharmaceutical interventions (NPIs). NPIs were applied primarily at national or regional scales. Here we use weekly anonymized and aggregated human mobility data and spatially highly resolved data on COVID-19 cases at the municipality level in Mexico to investigate how behavioural changes in response to the pandemic have altered the spatial scales of transmission and interventions during its first wave (March - June 2020). We find that the epidemic dynamics in Mexico were initially driven by SARS-CoV-2 exports from Mexico State and Mexico City, where early outbreaks occurred. The mobility network shifted after the implementation of interventions in late March 2020, and the mobility network communities became more disjointed while epidemics in these communities became increasingly synchronised. Our results provide dynamic insights into how to use network science and epidemiological modelling to inform the spatial scale at which interventions are most impactful in mitigating the spread of COVID-19 and infectious diseases in general.
{"title":"Spatial scales of COVID-19 transmission in Mexico","authors":"Brennan Klein, Harrison Hartle, Munik Shrestha, Ana Cecilia Zenteno, David Barros Sierra Cordera, José R Nicolás-Carlock, Ana I Bento, Benjamin M Althouse, Bernardo Gutierrez, Marina Escalera-Zamudio, Arturo Reyes-Sandoval, Oliver G Pybus, Alessandro Vespignani, José Alberto Díaz Quiñonez, Samuel V Scarpino, Moritz U G Kraemer","doi":"10.1093/pnasnexus/pgae306","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae306","url":null,"abstract":"During outbreaks of emerging infectious diseases, internationally connected cities often experience large and early outbreaks, while rural regions follow after some delay. This hierarchical structure of disease spread is influenced primarily by the multiscale structure of human mobility. However, during the COVID-19 epidemic, public health responses typically did not take into consideration the explicit spatial structure of human mobility when designing non-pharmaceutical interventions (NPIs). NPIs were applied primarily at national or regional scales. Here we use weekly anonymized and aggregated human mobility data and spatially highly resolved data on COVID-19 cases at the municipality level in Mexico to investigate how behavioural changes in response to the pandemic have altered the spatial scales of transmission and interventions during its first wave (March - June 2020). We find that the epidemic dynamics in Mexico were initially driven by SARS-CoV-2 exports from Mexico State and Mexico City, where early outbreaks occurred. The mobility network shifted after the implementation of interventions in late March 2020, and the mobility network communities became more disjointed while epidemics in these communities became increasingly synchronised. Our results provide dynamic insights into how to use network science and epidemiological modelling to inform the spatial scale at which interventions are most impactful in mitigating the spread of COVID-19 and infectious diseases in general.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141887388","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 : 2024-08-01DOI: 10.1093/pnasnexus/pgae296
Didem Pehlivanoglu, Alayna Shoenfelt, Ziad Hakim, Amber Heemskerk, Jialong Zhen, Mario Mosqueda, Robert C Wilson, Matthew Huentelman, Matthew D Grilli, Gary Turner, R Nathan Spreng, Natalie C Ebner
With technological advancements, financial exploitation tactics have expanded into the online realm. Older adults may be particularly susceptible to online scams due to age- and Alzheimer’s Disease-related changes in cognition. In this study, 182 adults ranging from 18-90 years underwent cognitive assessment, genotyping for apolipoprotein E e4 (APOE4), and completed the lab-based Short Phishing Email Suspicion Test (S-PEST) as well as the real-life PHishing Internet Task (PHIT). Across both paradigms, older age predicted heightened susceptibility to phishing, with this enhanced susceptibility pronounced among older APOE4 allele carriers with lower working memory. Additionally, performance in both phishing tasks was correlated, in that reduced ability to discriminate between phishing and safe emails in S-PEST predicted greater phishing susceptibility in PHIT. The current study identifies older age, APOE4, and lower cognition as risk factors of phishing vulnerability and introduces S-PEST as an easy-to-administer, ecologically valid tool for assessing phishing susceptibility.
{"title":"Phishing vulnerability compounded by older age, APOE4 genotype, and lower cognition","authors":"Didem Pehlivanoglu, Alayna Shoenfelt, Ziad Hakim, Amber Heemskerk, Jialong Zhen, Mario Mosqueda, Robert C Wilson, Matthew Huentelman, Matthew D Grilli, Gary Turner, R Nathan Spreng, Natalie C Ebner","doi":"10.1093/pnasnexus/pgae296","DOIUrl":"https://doi.org/10.1093/pnasnexus/pgae296","url":null,"abstract":"With technological advancements, financial exploitation tactics have expanded into the online realm. Older adults may be particularly susceptible to online scams due to age- and Alzheimer’s Disease-related changes in cognition. In this study, 182 adults ranging from 18-90 years underwent cognitive assessment, genotyping for apolipoprotein E e4 (APOE4), and completed the lab-based Short Phishing Email Suspicion Test (S-PEST) as well as the real-life PHishing Internet Task (PHIT). Across both paradigms, older age predicted heightened susceptibility to phishing, with this enhanced susceptibility pronounced among older APOE4 allele carriers with lower working memory. Additionally, performance in both phishing tasks was correlated, in that reduced ability to discriminate between phishing and safe emails in S-PEST predicted greater phishing susceptibility in PHIT. The current study identifies older age, APOE4, and lower cognition as risk factors of phishing vulnerability and introduces S-PEST as an easy-to-administer, ecologically valid tool for assessing phishing susceptibility.","PeriodicalId":516525,"journal":{"name":"PNAS Nexus","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141880492","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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