bluuebunny/crossref_metadata_2025_split huggingface dataset:
A growth-based screening strategy for engineering the catalytic activity of an oxygen-sensitive formate dehydrogenase
Authors: Li Feilong , Scheller Silvan , Lienemann Michael 
Published: August 2024

ABSTRACT

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of
Escherichia coli
. This system relies on the complementation of the
E. coli
formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (
Ec
FDH-H). Using an
Ec
FDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active
Ec
FDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using
Ec
FDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032
Ec
FDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling
E. coli
FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.

IMPORTANCE

Oxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO
2
and N
2
at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications.
Nationwide upsurge in invasive disease in the context of longitudinal surveillance of carriage and invasive
Streptococcus pyogenes
2009–2023, the Netherlands: a molecular epidemiological study
Authors: Rümke Lidewij W. , Davies Matthew A. , Vestjens Stefan M. T. , van der Putten Boas C. L. , Bril-Keijzers Wendy C. M. , van Houten Marlies A. , Rots Nynke Y. , Wijmenga-Monsuur Alienke J. , van der Ende Arie , de Gier Brechje , Vlaminckx Bart J. M. , van Sorge Nina M. 
Published: August 2024

ABSTRACT

Since 2022, many countries have reported an upsurge in invasive group A streptococcal (iGAS) infections. We explored whether changes in
Streptococcus pyogenes
carriage rates or emergence of strains with potentially altered virulence, such as
emm
1 variants M1
UK
and M1
DK
, contributed to the 2022/2023 surge in the Netherlands. We determined
emm
(sub)type distribution for 2,698 invasive and 351
S
.
pyogenes
carriage isolates collected between January 2009 and March 2023. Genetic evolution of
emm
1 was analyzed by whole-genome sequencing of 497
emm
1 isolates. The nationwide iGAS upsurge coincided with a sharp increase of
emm
1.0 from 18% (18/100) of invasive isolates in Q1 2022 to 58% (388/670) in Q1 2023 (Fisher’s exact test,
P
< 0.0001). M1
UK
became dominant among invasive
emm
1 isolates in 2016 and further expanded from 72% in Q1 2022 to 96% in Q1 2023. Phylogenetic comparison revealed evolution and clonal expansion of four new M1
UK
clades in 2022/2023. DNase Spd1 and superantigen SpeC were acquired in 9% (46/497) of
emm
1 isolates.
S. pyogenes
carriage rates and
emm
1 proportions in carriage isolates remained stable during this surge, and the expansion of M1
UK
in iGAS was not reflected in carriage isolates. During the 2022/2023 iGAS surge in the Netherlands, expansion of four new M1
UK
clades was observed among invasive isolates, but not carriage isolates, suggesting increased virulence and fitness of M1
UK
compared to contemporary M1 strains. The emergence of more virulent clades has important implications for public health strategies such as antibiotic prophylaxis for close contacts of iGAS patients.

IMPORTANCE

This study describes the molecular epidemiology of invasive group A streptococcal (iGAS) infections in the Netherlands based on >3,000
Streptococcus pyogenes
isolates from both asymptomatic carriers and iGAS patients collected before, during, and after the COVID-19 pandemic period (2009–2023) and is the first to assess whether changes in carriage rates or carried
emm
types contributed to the alarming post-COVID-19 upsurge in iGAS infections. We show that the 2022/2023 iGAS surge coincided with a sharp increase of
emm
1, particularly the toxicogenic M1
UK
variant, in invasive isolates, but not in carriage isolates. These findings suggest that increased virulence and fitness of M1
UK
likely contributes to an increased dissemination between hosts. The emergence of a more virulent and fit lineage has important implications for iGAS control interventions such as antibiotic prophylaxis for close contacts of iGAS patients and calls for a reappraisal of iGAS control interventions and guidelines.
ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis
Authors: Zhu Junji , Liu GuanQun , Sayyad Zuberwasim , Goins Christopher M. , Stauffer Shaun R. , Gack Michaela U. 
Published: August 2024

ABSTRACT

Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host immune proteins such as MDA5 and IRF3 in a process called ISGylation, thereby promoting type I IFN induction to limit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through deISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387, and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.

IMPORTANCE
The role of protein ISGylation in regulating host cellular processes has been studied extensively; however, how ISG15 conjugation influences the activity of viral proteins, particularly coronaviral proteins, is largely unknown. Our study uncovered that the nucleocapsid (N) protein of SARS-CoV-2 is ISGylated by the HERC5 ISGylation machinery and that this modification impedes the functional assembly of N into oligomers ultimately inhibiting viral RNA synthesis. This antiviral restriction mechanism is antagonized by the PLpro deISGylation activity of SARS-CoV-2 NSP3. This study deepens our understanding of SARS-CoV-2 protein regulation by posttranslational modifications and may open new avenues for designing antiviral strategies for COVID-19.
Nectin1 is a pivotal host factor involved in attachment and entry of red-spotted grouper nervous necrosis virus in the early stages of the viral life cycle
Authors: Zhang Zhiqi , Xing Jing , Tang Xiaoqian , Sheng Xiuzhen , Chi Heng , Zhan Wenbin 
Published: August 2024

ABSTRACT

Nervous necrosis virus (NNV) is a highly neurotropic virus that poses a persistent threat to the survival of multiple fish species. However, its inimitable neuropathogenesis remains largely elusive. To rummage potential partners germane to the nervous system, we investigated the interaction between red-spotted grouper NNV (RGNNV) and grouper brain by immunoprecipitation coupled with mass spectrometry and discerned Nectin1 as a novel host factor subtly involved in viral early invasion events. Nectin1 was abundant in neural tissues and implicated in the inception of tunnel nanotubes triggered by RGNNV. Its overexpression not only dramatically potentiated the replication dynamics of RGNNV in susceptible cells, but also empowered non-sensitive cells to expeditiously capture free virions within 2 min. This potency was impervious to low temperatures but was dose-dependently suppressed by soluble protein or specific antibody of Nectin1 ectodomain, indicating Nectin1 as an attachment receptor for RGNNV. Mechanistically, efficient hijacking of virions by Nectin1 strictly depended on intricate linkages to different modules of viral capsid protein, especially the direct binding between the IgC1 loop and P-domain. More strikingly, despite abortive proliferation in Nectin1-reconstructed CHSE-214 cells, a non-sensitive cell, RGNNV could gain access to the intracellular compartment by capitalizing on Nectin1, thereby inducing canonical cytoplasmic vacuolation. Altogether, our findings delineate a candidate entrance for RGNNV infiltration into the nervous system, which may shed unprecedented insights into the exploration and elucidation of RGNNV pathogenesis.

IMPORTANCE
Nervous necrosis virus (NNV) is one of the most virulent pathogens in the aquaculture industry, which inflicts catastrophic damage to ecology, environment, and economy annually around the world. Nevertheless, its idiosyncratic invasion and latency mechanisms pose enormous hardships to epidemic prevention and control. In this study, deploying grouper brain as a natural screening library, a single-transmembrane glycoprotein, Nectin1, was first identified as an emergent functional receptor for red-spotted grouper NNV (RGNNV) that widely allocated in nervous tissues and directly interacted with viral capsid protein through distinct Ig-like loops to bridge virus-host crosstalk, apprehend free virions, and concomitantly propel viral entry. Our findings illuminate the critical role of Nectin1 in RGNNV attachment and entry and provide a potential target for future clinical intervention strategies in the therapeutic race against RGNNV.
Characterization of viroplasm-like structures by co-expression of NSP5 and NSP2 across rotavirus species A to J
Authors: Lee Melissa , Cosic Ariana , Tobler Kurt , Aguilar Claudio , Fraefel Cornel , Eichwald Catherine 
Published: August 2024

ABSTRACT

Rotaviruses (RVs) are classified into nine species, A–D and F–J, with species A being the most studied. In rotavirus of species A (RVA), replication occurs in viroplasms, which are cytosolic globular inclusions composed of main building block proteins NSP5, NSP2, and VP2. The co-expression of NSP5 with either NSP2 or VP2 in uninfected cells leads to the formation of viroplasm-like structures (VLSs). Although morphologically identical to viroplasms, VLSs do not produce viral progeny but serve as excellent tools for studying complex viroplasms. A knowledge gap exists regarding non-RVA viroplasms due to the lack of specific antibodies and suitable cell culture systems. In this study, we explored the ability of NSP5 and NSP2 from non-RVA species to form VLSs. The co-expression of these two proteins led to globular VLSs in RV species A, B, D, F, G, and I, while RVC formed filamentous VLSs. The co-expression of NSP5 and NSP2 of RV species H and J did not result in VLS formation. Interestingly, NSP5 of all RV species self-oligomerizes, with the ordered C-terminal region, termed the tail, being necessary for self-oligomerization of RV species A–C and G–J. Except for NSP5 from RVJ, all NSP5 interacted with their cognate NSP2. We also found that interspecies VLS are formed between closely related RV species B with G and D with F. Additionally, VLS from RVH and RVJ formed when the tail of NSP5 RVH and RVJ was replaced by the tail of NSP5 from RVA and co-expressed with their respective NSP2.

IMPORTANCE
Rotaviruses (RVs) are classified into nine species, A–D and F–J, infecting mammals and birds. Due to the lack of research tools, all cumulative knowledge on RV replication is based on RV species A (RVA). The RV replication compartments are globular cytosolic structures named viroplasms, which have only been identified in RV species A. In this study, we examined the formation of viroplasm-like structures (VLSs) by the co-expression of NSP5 with NSP2 across RV species A to J. Globular VLSs formed for RV species A, B, D, F, G, and I, while RV species C formed filamentous structures. The RV species H and J did not form VLS with their cognates NSP5 and NSP2. Similar to RVA, NSP5 self-oligomerizes in all RV species, which is required for VLS formation. This study provides basic knowledge of the non-RVA replication mechanisms, which could help develop strategies to halt virus infection across RV species.
Foot-and-mouth disease virus (FMDV) negatively regulates ZFP36 protein expression to alleviate its antiviral activity
Authors: Yin Mengge , Qian Ping , Wang Haoyuan , Zhao Qiongqiong , Zhang Huiyan , Zheng Zixuan , Zhang Min , Lu Zengjun , Li Xiangmin 
Published: August 2024

ABSTRACT

Zinc finger protein 36 (ZFP36) is a key regulator of inflammatory and cytokine production. However, the interplay between swine zinc-finger protein 36 (sZFP36) and foot-and-mouth disease virus (FMDV) has not yet been reported. Here, we demonstrate that overexpression of sZFP36 restricted FMDV replication, while the knockdown of sZFP36 facilitated FMDV replication. To subvert the antagonism of sZFP36, FMDV decreased sZFP36 protein expression through its non-structural protein 3C protease (3C
pro
). Our results also suggested that 3C
pro
-mediated sZFP36 degradation was dependent on its protease activity. Further investigation revealed that both N-terminal and C-terminal-sZFP36 could be degraded by FMDV and FMDV 3C
pro
. In addition, both N-terminal and C-terminal-sZFP36 decreased FMDV replication. Moreover, sZFP36 promotes the degradation of FMDV structural proteins VP3 and VP4 via the CCCH-type zinc finger and NES domains of sZFP36. Together, our results confirm that sZFP36 is a host restriction factor that negatively regulates FMDV replication.

IMPORTANCE

Foot-and-mouth disease (FMD) is an infectious disease of animals caused by the pathogen foot-and-mouth disease virus (FMDV). FMD is difficult to prevent and control because there is no cross-protection between its serotypes. Thus, we designed this study to investigate virus-host interactions. We first demonstrate that swine zinc-finger protein 36 (sZFP36) impaired FMDV structural proteins VP3 and VP4 to suppress viral replication. To subvert the antagonism of sZFP36, FMDV and FMDV 3C
pro
downregulate sZFP36 expression to facilitate FMDV replication. Taken together, the present study reveals a previously unrecognized antiviral mechanism for ZFP36 and elucidates the role of FMDV in counteracting host antiviral activity.
Single-cell transcriptome sequencing reveals that
Wolbachia
induces gene expression changes in
Drosophila
ovary cells to favor its own maternal transmission
Authors: Miao Yun-heng , Dou Wei-hao , Liu Jing , Huang Da-wei , Xiao Jin-hua 
Published: August 2024

ABSTRACT

Wolbachia
is an obligate endosymbiont that is maternally inherited and widely distributed in arthropods and nematodes. It remains in the mature eggs of female hosts over generations through multiple strategies and manipulates the reproduction system of the host to enhance its spreading efficiency. However, the transmission of
Wolbachia
within the host’s ovaries and its effects on ovarian cells during oogenesis, have not been extensively studied. We used single-cell RNA sequencing to comparatively analyze cell-typing and gene expression in
Drosophila
ovaries infected and uninfected with
Wolbachia
. Our findings indicate that
Wolbachia
significantly affects the transcription of host genes involved in the extracellular matrix, cytoskeleton organization, and cytomembrane mobility in multiple cell types, which may make host ovarian cells more conducive for the transmission of
Wolbachia
from extracellular to intracellular. Moreover, the genes
nos
and
orb
, which are related to the synthesis of ribonucleoprotein complexes, are specifically upregulated in early germline cells of ovaries infected with
Wolbachia
, revealing that
Wolbachia
can increase the possibility of its localization to the host oocytes by enhancing the binding with host ribonucleoprotein-complex processing bodies (P-bodies). All these findings provide novel insights into the maternal transmission of
Wolbachia
between host ovarian cells.

IMPORTANCE

Wolbachia
, an obligate endosymbiont in arthropods, can manipulate the reproduction system of the host to enhance its maternal transmission and reside in the host’s eggs for generations. Herein, we performed single-cell RNA sequencing of ovaries from
Drosophila melanogaster
and observed the effects of
Wolbachia
(strain
w
Mel) infection on different cell types to discuss the potential mechanism associated with the transmission and retention of
Wolbachia
within the ovaries of female hosts. It was found that the transcriptions of multiple genes in the ovary samples infected with
Wolbachia
are significantly altered, which possibly favors the maternal transmission of
Wolbachia
. Meanwhile, we also discovered that
Wolbachia
may flexibly regulate the expression level of specific host genes according to their needs rather than rigidly changing the expression level in one direction to achieve a more suitable living environment in the host’s ovarian cells. Our findings contribute to a further understanding of the maternal transmission and possible universal effects of
Wolbachia
within the host.
The impact of small intestinal bacterial overgrowth on the efficacy of fecal microbiota transplantation in patients with chronic constipation
Authors: Wang Le , Xu Yue , Li Long , Yang Bo , Zhao Di , Ye Chen , Lin Zhiliang , Cui Jiaqu , Liu Yunkun , Zhu Wanyong , Li Ning , Tian Hongliang , Chen Qiyi 
Published: August 2024

ABSTRACT

To investigate the impact of Small Intestinal Bacterial Overgrowth (SIBO) on the efficacy of Fecal Microbiota Transplantation (FMT) in patients with chronic constipation, our research team included 218 patients with chronic constipation treated with FMT. Based on the results of the SIBO breath test, the patients were divided into two groups: the constipation with SIBO group (SIBO) and the constipation without SIBO group (non-SIBO). The efficacy of the two groups was evaluated using constipation-related scoring scales. At the same time, feces and small intestinal fluid samples were collected from both groups before and after FMT to compare the changes in the intestinal microbiota through 16S rRNA sequencing. In this study, it was found that the clinical efficacy of FMT in the SIBO group was superior to that in the non-SIBO group. After FMT treatment, both groups showed a significant increase in bowel frequency and improvement in stool characteristics. Abdominal symptoms, rectal symptoms, and defecation symptoms were significantly alleviated (
P
< 0.05), and patients’ quality of life was significantly enhanced (
P
< 0.05). After FMT, except for the Constipation Assessment Scale scores, other scale scores showed significant differences between the two groups, the SIBO group scoring significantly better than the non-SIBO group (
P
< 0.05). After FMT, there were minor changes in the colonic microbiota but more substantial changes in the small intestinal microbiota. At baseline, the SIBO group had a higher abundance of
Veillonella
, and lower abundances of
Escherichia-Shigella
and
Acinetobacter
compared to the non-SIBO group. Chronic constipation patients with SIBO have a better response to FMT than those without SIBO.

IMPORTANCE
Existing studies have rarely considered the impact of the small intestine’s microbial state on the efficacy of fecal microbiota transplantation (FMT), nor have they extensively explored the effect of the small intestine’s microbial state on the recovery of colonic motility. Therefore, this study investigates the influence of small intestinal bacterial overgrowth (SIBO) on the efficacy of FMT in treating constipation, specifically the impact of the microbial state of the small intestine on the restoration of colonic homeostasis, and consequently on the recovery of colonic motility.