Molecular and cellular characterization of four putative nucleotide transporters from the shrimp microsporidian Enterocytozoon hepatopenaei (EHP).

Microsporidia are obligate intracellular parasites that lost several enzymes required in energy production. The expansion of transporter families in these organisms enables them to hijack ATP from hosts. In this study, nucleotide transporters of the microsporidian Enterocytozoon hepatopenaei (EHP), which causes slow growth in economically valuable Penaeus shrimp, were characterized. Analysis of the EHP genome suggested the presence of four putative nucleotide transporter genes, namely EhNTT1, EhNTT2, EhNTT3, and EhNTT4. Sequence alignment revealed four charged amino acids that are conserved in previously characterized nucleotide transporters. Phylogenetic analysis suggested that EhNTT1, 3, and 4 were derived from one horizontal gene transfer event, which was independent from that of EhNTT2. Localization of EhNTT1 and EhNTT2 using immunofluorescence analysis revealed positive signals within the envelope of developing plasmodia and on mature spores. Knockdown of EhNTT2 by double administration of sequence specific double-stranded RNA resulted in a significant reduction in EHP copy numbers, suggesting that EhNTT2 is crucial for EHP replication in shrimp. Taken together, the insight into the roles of NTTs in microsporidian proliferation can provide the biological basis for the development of alternative control strategies for microsporidian infection in shrimp.

Andrographolide Induces ROS-Mediated Cytotoxicity, Lipid Peroxidation, and Compromised Cell Integrity in Saccharomyces cerevisiae.

Andrographolide, a bioactive compound found in Andrographis paniculata, has gained significant attention for its potential therapeutic properties. Despite its promising benefits, the understanding of its side effects and underlying mechanisms remains limited. Here, we investigated the impact of andrographolide in Saccharomyces cerevisiae and observed that andrographolide induced cytotoxicity, particularly when oxidative phosphorylation was active. Furthermore, andrographolide affected various cellular processes, including vacuole fragmentation, endoplasmic reticulum stress, lipid droplet accumulation, reactive oxygen species levels, and compromised cell integrity. Moreover, we unexpectedly observed that andrographolide induced the precipitation of biomolecules secreted from yeast cells, adding an additional source of stress. Overall, this study provides insights into the cellular effects and potential mechanisms of andrographolide in yeast, shedding light on its side effects and underlying cytotoxicity pathways.

Bridging the Gap: Can COVID-19 Research Help Combat African Swine Fever?

African swine fever (ASF) is a highly contagious and economically devastating disease affecting domestic pigs and wild boar, caused by African swine fever virus (ASFV). Despite being harmless to humans, ASF poses significant challenges to the swine industry, due to sudden losses and trade restrictions. The ongoing COVID-19 pandemic has spurred an unparalleled global research effort, yielding remarkable advancements across scientific disciplines. In this review, we explore the potential technological spillover from COVID-19 research into ASF. Specifically, we assess the applicability of the diagnostic tools, vaccine development strategies, and biosecurity measures developed for COVID-19 for combating ASF. Additionally, we discuss the lessons learned from the pandemic in terms of surveillance systems and their implications for managing ASF. By bridging the gap between COVID-19 and ASF research, we highlight the potential for interdisciplinary collaboration and technological spillovers in the battle against ASF.

Transcription machinery of the minimalist: comparative genomic analysis provides insights into the (de)regulated transcription mechanism of microsporidia - fungal-relative parasites.

Microsporidia are eukaryotic obligate intracellular parasites closely related to fungi. Co-evolving with infected hosts, microsporidia have highly reduced their genomes and lacked several biological components. As it is beneficial for intracellular parasites like microsporidia to reduce their genome size, it is therefore reasonable to assume that genes encoding multifactorial complex machinery of transcription could be a potential target to be excluded from microsporidian genomes during the reductive evolution. In such a case, an evolutionary dilemma occurs because microsporidia cannot remove all transcription-machinery-encoding genes, products of which are essential for initialthe initial steps of gene expression. Here, I propose that while genes encoding core machinery are conserved, several genes known to function in fine-tune regulation of transcription are absent. This genome compaction strategy may come at the cost of loosely regulated or less controllable transcription. Alternatively, analogous to microsporidian polar tube, the parasites may have specialized factors to regulate their RNA synthesis.

A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

Receptor binding protein of prophage reversibly recognizes the low-molecular weight subunit of the surface-layer protein SlpA in Clostridioides difficile.

Receptor-binding proteins (RBPs) are located at the viral tail and mediate the initial recognition of phage to a specific bacterial host. Phage RBPs have co-evolved with numerous types of host receptors resulting in the formation of a diverse assortment of cognate pairs of RBP-receptors that function during the phage attachment step. Although several Clostridioides difficile bacteriophages have been discovered, their RBPs are poorly described. Using homology analysis, putative prophage-tail structure (pts) genes were identified from the prophage genome of the C. difficile HN10 strain. Competition and enzyme-linked immunosorbent assays, using recombinant PtsHN10M, demonstrated the interaction of this Pts to C. difficile cells, suggesting a role as a phage RBP. Gel filtration and cross-linking assay revealed the native form of this protein as a homotrimer. Moreover, truncated variants indicated that the C-terminal domain of PtsHN10M was important for binding to C. difficile cells. Interaction of PtsHN10M was also observed to the low-molecular weight subunit of surface-layer protein A (SlpA), located at the outermost surface of C. difficile cells. Altogether, our study highlights the function of PtsHN10M as an RBP and potentially paves the way toward phage engineering and phage therapy against C. difficile infection.

Insights into the Mechanism of Pre-mRNA Splicing of Tiny Introns from the Genome of a Giant Ciliate Stentor coeruleus.

Stentor coeruleus is a ciliate known for its regenerative ability. Recent genome sequencing reveals that its spliceosomal introns are exceptionally small. We wondered whether the multimegadalton spliceosome has any unique characteristics for removal of the tiny introns. First, we analyzed intron features and identified spliceosomal RNA/protein components. We found that all snRNAs are present, whereas many proteins are conserved but slightly reduced in size. Some regulators, such as Serine/Arginine-rich proteins, are noticeably undetected. Interestingly, while most parts of spliceosomal proteins, including Prp8's positively charged catalytic cavity, are conserved, regions of branching factors projecting to the active site are not. We conjecture that steric-clash avoidance between spliceosomal proteins and a sharply looped lariat might occur, and splicing regulation may differ from other species.

Potential Role of the Host-Derived Cell-Wall Binding Domain of Endolysin CD16/50L as a Molecular Anchor in Preservation of Uninfected Clostridioides difficile for New Rounds of Phage Infection.

Endolysin is a phage-encoded cell-wall hydrolase which degrades the peptidoglycan layer of the bacterial cell wall. The enzyme is often expressed at the late stage of the phage lytic cycle and is required for progeny escape. Endolysins of bacteriophage that infect Gram-positive bacteria often comprises two domains: a peptidoglycan hydrolase and a cell-wall binding domain (CBD). Although the catalytic domain of endolysin is relatively well-studied, the precise role of CBD is ambiguous and remains controversial. Here, we focus on the function of endolysin CBD from a recently isolated Clostridioides difficile phage. We found that the CBD is not required for lytic activity, which is strongly prevented by the surface layer of C. difficile. Intriguingly, hidden Markov model analysis suggested that the endolysin CBD is likely derived from the CWB2 motif of C. difficile cell-wall proteins but possesses a higher binding affinity to bacterial cell-wall polysaccharides. Moreover, the CBD forms a homodimer, formation of which is necessary for interaction with the surface saccharides. Importantly, endolysin diffusion and sequential cytolytic assays showed that CBD of endolysin is required for the enzyme to be anchored to post-lytic cell-wall remnants, suggesting its physiological roles in limiting diffusion of the enzyme, preserving neighboring host cells, and thereby enabling the phage progeny to initiate new rounds of infection. Taken together, this study provides an insight into regulation of endolysin through CBD and can potentially be applied for endolysin treatment against C. difficile infection. IMPORTANCE Endolysin is a peptidoglycan hydrolase encoded in a phage genome. The enzyme is attractive due to its potential use as antibacterial treatment. To utilize endolysin for the therapeutic propose, understanding of the fundamental role of endolysin becomes important. Here, we investigate the function of cell-wall binding domain (CBD) of an endolysin from a C. difficile phage. The domain is homologous to a cell-wall associating module of bacterial cell-wall proteins, likely acquired during phage-host coevolution. The interaction of CBD to bacterial cell walls reduces enzyme diffusion and thereby limits cell lysis of the neighboring bacteria. Our findings indicate that the endolysin is trapped to the cell-wall residuals through CBD and might serve as an advantage for phage replication. Thus, employing a CBD-less endolysin might be a feasible strategy for using endolysin for the treatment of C. difficile infection.

UBL5/Hub1: An Atypical Ubiquitin-Like Protein with a Typical Role as a Stress-Responsive Regulator.

Members of the ubiquitin-like protein family are known for their ability to modify substrates by covalent conjugation. The highly conserved ubiquitin relative UBL5/Hub1, however, is atypical because it lacks a carboxy-terminal di-glycine motif required for conjugation, and the whole E1-E2-E3 enzyme cascade is likely absent. Though the conjugation-mediated role of UBL5/Hub1 is controversial, it undoubtedly functions by interacting non-covalently with its partners. Several interactors of UBL5/Hub1 identified to date have suggested broad stress-responsive functions of the protein, for example, stress-induced control of pre-mRNA splicing, Fanconi anemia pathway of DNA damage repair, and mitochondrial unfolded protein response. While having an atypical mode of function, UBL5/Hub1 is still a stress protein that regulates feedback to various stimuli in a similar manner to other ubiquitin-like proteins. In this review, I discuss recent progress in understanding the functions of UBL5/Hub1 and the fundamental questions which remain to be answered.

Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA.

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.

Ambient pH regulates secretion of lipases in Malassezia furfur.

Malassezia is a lipophilic cutaneous commensal yeast and associated with various skin disorders. The yeast also causes bloodstream infection via intravascular catheters and can be detected even in human gut microbiota. Ambient pH is one of the major factors that affect the physiology and metabolism of several pathogenic microorganisms. Although dynamic changes of pH environment in different parts of the body is a great challenge for Malassezia to confront, the role that ambient pH plays in Malassezia is largely unknown. In this study, we investigated the impact of ambient pH on physiology and expression of lipases in M. furfur grown under different pH conditions. The yeast was able to grow in media ranging from pH 4 to 10 without morphological alteration. Elevation in pH value enhanced the extracellular lipase activity but decreased that of intracellular lipase. The qPCR results revealed that a set of functional lipase genes, LIP3-6, were constitutively expressed regardless of pH conditions or exposure time. Based on the data, we conclude that the external pH plays a promotional role in the secretion of lipases but exerts less effect on transcription of the genes and morphology in M. furfur.

Stress-induced upregulation of the ubiquitin-relative Hub1 modulates pre-mRNA splicing and facilitates cadmium tolerance in Saccharomyces cerevisiae.

Splicing is a fundamental RNA-processing step for eukaryotic gene expression involved in the removal of intronic sequences of pre-mRNA. As the process is utilized for quantitative and qualitative regulation of gene expression, uncontrolled splicing can result in potential cellular dysfunctions. Accumulating evidence suggests that fidelity of splicing is regulated by a family of DEAD/DExH-box RNA helicases. Recently, we have shown that the evolutionarily-conserved ubiquitin-relative Hub1 binds directly to the DEAD-box RNA helicase Prp5, a key regulator of splicing fidelity, and stimulates its ATPase activity. When overexpressed, Hub1 enhances splicing efficiency and relaxes the constraints on splice-site and branch-site usages; yet physiological relevance of cellular Hub1 overexpression remains unknown. Here we show that Hub1 is upregulated at the transcriptional level via the yeast-specific AP1 regulon upon oxidative and heavy metal stresses, and promotes efficient splicing of introns with non-canonical splice-sites. While nonessential for yeast viability, Hub1 becomes important for cadmium tolerance when metallothionein-mediated defense system is impaired. Moreover, mutant variants of other splicing factors also showed a similar cadmium sensitivity, suggesting the role of splicing in facilitating tolerance of heavy metal stress. Taken together, we propose that cells adjust gene expression landscape required for heavy metal detoxification by promoting intron-specific splicing through the stress-induced overexpression of Hub1.

Emerging Roles of Ubiquitin-like Proteins in Pre-mRNA Splicing.

Ubiquitin-like proteins (UBLs) belong to the protein family whose members share a globular beta-grasp fold structure. The archetypal member, ubiquitin, is known for its function in proteasome-mediated protein degradation. UBLs have been shown to play several crucial roles besides protein turnover, including DNA damage response, cell cycle control, cellular signaling, protein trafficking, and innate immunity activation. In the past few years, accumulating evidence illustrates that four UBLs, namely, ubiquitin, SUMO, Hub1, and Sde2, are involved in eukaryotic pre-mRNA splicing. They modify the spliceosomes and promote splicing by adding new surfaces for intermolecular interactions, thereby refining the outcome of gene expression. In this review article, we highlight recent discoveries with an emphasis on the emerging roles of UBLs in splicing regulation.

Error-Prone Splicing Controlled by the Ubiquitin Relative Hub1.

Accurate pre-mRNA splicing is needed for correct gene expression and relies on faithful splice site recognition. Here, we show that the ubiquitin-like protein Hub1 binds to the DEAD-box helicase Prp5, a key regulator of early spliceosome assembly, and stimulates its ATPase activity thereby enhancing splicing and relaxing fidelity. High Hub1 levels enhance splicing efficiency but also cause missplicing by tolerating suboptimal splice sites and branchpoint sequences. Notably, Prp5 itself is regulated by a Hub1-dependent negative feedback loop. Since Hub1-mediated splicing activation induces cryptic splicing of Prp5, it also represses Prp5 protein levels and thus curbs excessive missplicing. Our findings indicate that Hub1 mediates enhanced, but error-prone splicing, a mechanism that is tightly controlled by a feedback loop of PRP5 cryptic splicing activation.

Splicing and beyond: the many faces of the Prp19 complex.

The conserved Prp19 complex (Prp19C) - also known as NineTeen Complex (NTC) - functions in several processes of paramount importance for cellular homeostasis. NTC/Prp19C was discovered as a complex that functions in splicing and more specifically during the catalytic activation of the spliceosome. More recent work revealed that NTC/Prp19C plays a role in transcription elongation in Saccharomyces cerevisiae and in genome maintenance in higher eukaryotes. In addition, mouse PRP19 might ubiquity late proteins targeted for degradation and guide them to the proteasome. Furthermore, NTC/Prp19C has been implicated in lipid droplet biogenesis. In the future, the molecular function of NTC/Prp19C in all of these processes needs to be refined or elucidated. Most of NTC/Prp19C's functions have been shown in only one or few organisms. However, since this complex is highly conserved it is likely that it has the same functions across all species. Moreover, one NTC/Prp19C or different subcomplexes could function in the above-mentioned processes. Intriguingly, NTC/Prp19C might link these different processes to ensure an optimal coordination of cellular processes. Thus, many important questions about the functions of this interesting complex remain to be investigated. In this review we discuss the different functions of NTC/Prp19C focusing on the novel and emerging ones as well as open questions.

Prp19C and TREX: interacting to promote transcription elongationand mRNA export.

During transcription of protein coding genes by RNA Polymerase II the mRNA is processed and packaged into an mRNP. Among the proteins binding cotranscriptionally to the mRNP are mRNA export factors. One of the protein complexes thus coupling transcription to mRNA export is the TREX complex. However, despite the fact that TREX was identified and characterized about a decade ago, it had remained enigmatic how TREX is recruited to genes. The conserved Prp19 complex (Prp19C) has long been known for its function in splicing. We recently identified Prp19C to be essential for a second step in gene expression namely TREX occupancy at transcribed genes, answering this long-standing question but also raising new ones.

The Prp19 complex is a novel transcription elongation factor required for TREX occupancy at transcribed genes.

Different steps in gene expression are intimately linked. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to nuclear messenger RNA (mRNA) export. However, it is unknown how TREX is recruited to actively transcribed genes. Here, we show that the Prp19 splicing complex functions in transcription elongation. The Prp19 complex is recruited to transcribed genes, interacts with RNA polymerase II (RNAPII) and TREX, and is absolutely required for TREX occupancy at transcribed genes. Importantly, the Prp19 complex is necessary for full transcriptional activity. Taken together, we identify the Prp19 splicing complex as a novel transcription elongation factor that is essential for TREX occupancy at transcribed genes and that thus provides a novel link between transcription and messenger ribonucleoprotein (mRNP) formation.

The transcription elongation factor Bur1-Bur2 interacts with replication protein A and maintains genome stability during replication stress.

Multiple DNA-associated processes such as DNA repair, replication, and recombination are crucial for the maintenance of genome integrity. Here, we show a novel interaction between the transcription elongation factor Bur1-Bur2 and replication protein A (RPA), the eukaryotic single-stranded DNA-binding protein with functions in DNA repair, recombination, and replication. Bur1 interacted via its C-terminal domain with RPA, and bur1-ΔC mutants showed a deregulated DNA damage response accompanied by increased sensitivity to DNA damage and replication stress as well as increased levels of persisting Rad52 foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant was suppressed by bur1 mutation, further underscoring a functional link between these two protein complexes. The transcription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress.

Transgenic overexpression of the alpha-synuclein interacting protein synphilin-1 leads to behavioral and neuropathological alterations in mice.

Synphilin-1 has been identified as an interacting protein of alpha-synuclein, Parkin, and LRRK2, proteins which are mutated in familial forms of Parkinson disease (PD). Subsequently, synphilin-1 has also been shown to be an intrinsic component of Lewy bodies in sporadic PD. In order to elucidate the role of synphilin-1 in the pathogenesis of PD, we generated transgenic mice overexpressing wild-type and mutant (R621C) synphilin-1 driven by a mouse prion protein promoter. Transgenic expression of both wild-type and the R621C variant synphilin-1 resulted in increased dopamine levels of the nigrostriatal system in 3-month-old mice. Furthermore, we found pathological ubiquitin-positive inclusions in cerebellar sections and dark-cell degeneration of Purkinje cells. Both transgenic mouse lines showed significant reduction of motor skill learning and motor performance. These findings suggest a pathological role of overexpressed synphilin-1 in vivo and will help to further elucidate the mechanisms of protein aggregation and neuronal cell death.

Hepatitis delta antigen binds to the clamp of RNA polymerase II and affects transcriptional fidelity.

Hepatitis delta virus (HDV) is an RNA virus whose replication and transcription are considered to proceed via RNA-dependent RNA synthesis by RNA polymerase II (Pol II), and the viral protein called hepatitis delta antigen (HDAg) is essential for these processes. HDAg was previously shown to stimulate Pol II elongation on both DNA and RNA templates in vitro. Here, the mechanism of elongation control by HDAg was investigated because it serves as a prototype of cellular transcription elongation factors and also plays an interesting role in HDV proliferation. With site-specific photocrosslinking and transcription using reconstituted elongation complexes, evidence is presented that HDAg functionally interacts with the clamp of Pol II, a mobile structure that holds DNA and RNA in place. Strikingly, HDAg not only increases the rate of elongation but also affects the decision of which nucleotide is incorporated. These and our previous findings lead us to propose a model in which HDAg interacts with and loosens the clamp, and thereby accelerates forward translocation of Pol II at the cost of fidelity. By reducing transcriptional fidelity in terms of not only discrimination of incoming nucleotides but also recognition of templates, HDAg may facilitate the unusual RNA-dependent RNA synthesis by Pol II.