Hypothetical Protein Research Articles (Page 1)

Duck enteritis virus (DEV), an epornitic pathogen, causes substantial economic losses in the commercial duck industry and poses persistent risks to wild and migratory waterfowl populations. However, due to the large genomic capacity of the DEV, the understanding of the virulence-associated genes of DEV is still limited. In previous studies, we developed an attenuated strain E74 by serial passage of a virulent strain E1 on primary chicken embryo fibroblasts (CEFs). The bird experiment showed that the mortality rate of E1 on ducks reached 100%, and high-titered viruses were detected in all tested tissue samples. In contrast, the E74 virus has lost its pathogenicity in ducks and can only be detected at a relatively low viral load in the spleen. Furthermore, the E74 stimulated a significant increase in antibodies in the ducks at 7 days post-inoculation. To further investigate the molecular basis of the attenuation of DEV in ducks, the complete genomes of E74 and E1 were sequenced and analyzed. Compared with E1, E74 had a 5152 bp deletion in the UL region, which resulted in the lack of the hypothetical protein, LORF5, UL55 and LORF4 genes. To test the influence of the deletion on the viral pathogenicity, a rescued virus rE1-Δ5152 with the 5152 bp deletion in the UL region was generated on the E1 backbone. Animal experiments showed that the lethality of rE1-Δ5152 in ducks had disappeared. Those findings suggest that the hypothetical protein, LORF5, UL55, and LORF4 genes of DEV are associated with virus virulence, and the flexibility of this region provided excellent insertion sites for exogenous genes when DEV is used as a recombinant vaccine vector.

Trichomonas vaginalis is a flagellated protozoan parasite and the causative agent of trichomoniasis, the most prevalent non-viral sexually transmitted infection, with significant impact on public health and economy. Here, we present a chromosome-level genome of T. vaginalis strain IR-78 (ATCC 50138), assembled using short and long-read sequencing technologies as well as chromatin conformation capture sequencing (Hi-C). The assembled genome is 173.3 Mb in size, with an N50 scaffold length of 26.4 Mb. The assembly is anchored in six super-scaffolds, corresponding to the six chromosomes of T. vaginalis, covering 93.5% of the genome. A total of 43,326 protein-coding genes were predicted, of which 95.4% were annotated using multiple databases, including Gene Ontology, eggNOG, and KEGG. Among these, 16,656 genes encoded hypothetical proteins. This high-quality genome assembly provides an additional resource for ongoing research on T. vaginalis pathogenesis, drug resistance, and host-parasite interactions. It furthermore enables comparative genomic studies to assess genomic variety across different Trichomonas species.

Immunoinformatics-driven construction of a next-generation epitope-based vaccine from conserved hypothetical proteins of M. tuberculosis for enhanced TB control.

Molecular mechanisms for exopolysaccharides synthesis in Lactobacillus helveticus: Relationship between structural characteristics and genomics.

Comprehensive whole-genome analysis of Streptococcus infantarius strains from Moroccan farmhouse dairy products: Genomic insights into dairy adaptation, safety, and biotechnological potential.

ABSTRACTBrucella abortus S19 is a widely used live attenuated vaccine strain for bovine brucellosis control; however, its long‐term efficacy is challenged by genomic plasticity and adaptive mechanisms. This study presents a comprehensive comparative genomic and immunoinformatics analysis of a field strain (B. abortus S19, BAS19) isolated from an aborted cattle placenta 3 years post‐vaccination in Erzurum, Turkey. Whole‐genome sequencing was performed using Oxford Nanopore Technology, followed by genome assembly, functional annotation and comparative analyses against the reference strain (B. abortus S19, BAR19). Genomic variations, including 1153 single nucleotide polymorphisms (SNPs), 120 insertions and 2501 deletions, were identified. Annotation revealed 772 hypothetical proteins in BAS19 compared to 604 in BAR19, with distinct differences in virulence‐associated genes. Immunoinformatics analysis of 95 outer membrane proteins (OMPs) indicated significant antigenic variation, with 47 proteins exhibiting epitope loss and 11 displaying novel epitope gains. Beta‐barrel structure prediction demonstrated a reduction in structural stability, with nine OMPs losing beta‐barrel motifs, potentially influencing host‐pathogen interactions. These findings highlight key genomic adaptations in BAS19 that may influence its immunogenic properties and vaccine efficacy. The results contribute to a deeper understanding of B. abortus genomic diversity, providing insights for the rational design of improved vaccines and therapeutics tailored to regional epidemiological needs.

Detection of Neorickettsia risticii in antemortem fecal and postmortem fetal samples, with genomic insights from complete genome sequencing of a strain recovered from an aborted equine fetus.

Controlling agricultural insect pests primarily depends on chemical insecticides, which pose environmental and health risks. This study comprehensively characterizes Bacillus thuringiensis (Bt) strain T419 and explores its potential as an eco-friendly biopesticide. Morphological analysis revealed diverse crystal structures including bipyramidal, spherical, and cuboidal shapes, indicative of broad insecticidal activity. SDS-PAGE confirmed the presence of key proteins corresponding to Cry1 (~ 130kDa), Cry2 (~ 65kDa), and Vip3 (~ 88kDa), known for their efficacy against lepidopteran pests. PCR screening confirmed the presence of genes encoding these insecticidal proteins, such as cry1, cry1Ac, cry1Ab, cry2, cry2Aa, cry2Ab, and vip3A. Whole genome sequencing yielded a 6.4Mb draft genome with 6,962 protein-coding sequences, including 2,327 hypothetical proteins, suggesting significant potential for novel gene discovery. Notably, 14 plasmids were identified which likely harboring key insecticidal genes. Bioassays demonstrated T419's effectiveness against economically important lepidopteran pests such as Spodoptera frugiperda (LC50 value of 2.821µg/mL), Spodoptera litura (LC50 value of 1.569µg/mL), and Plutella xylostella (LC50 value of 0.363µg/mL). Genome annotation revealed Bt genome T419 possesses many insecticidal toxin proteins such as Cry1Aa18, Cry1Ab11, Cry1Ab14, Spp1Aa1, Mpp46Ab1, Cry1Ac5, Cry1Ia42, Cry2Aa9, Cry2Ab41, and Vip3Aa86. In addition, it also possesses various virulence factors, including InhA metalloproteases, phospholipase C, sphingomyelinase, and chitinase, elucidating T419's sophisticated pathogenic mechanisms. These findings suggest that the Bt strain T419 is a promising candidate for developing new biopesticide formulations, offering a potent combination of broad-spectrum insecticidal activity and genetic diversity.

Phylloplane-associated bacteria contribute significantly to sustainable plant disease management by modulating host defense mechanisms and directly antagonizing pathogens. In this study, Pseudomonas oryzihabitans Pg-Slp82, a dominant phylloplane isolate from pomegranate, was evaluated for its potential as a biocontrol agent against Xanthomonas axonopodis pv. punicae, the causal agent of bacterial blight. Whole-genome sequencing revealed a 5.0Mb genome comprising 7,193 coding sequences, including 1,029 hypothetical proteins, 15 tRNAs, 15 rRNAs, and 60 repetitive elements, indicating a genomically diverse strain with potential biocontrol traits. Transcriptomic analysis of P. oryzihabitans Pg-Slp82-treated pomegranate leaves generated 403million reads (GC content 49-50%) and showed peak host gene expression at 24h post-inoculation (hpi), with activation of 28.3% of unique transcripts. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations revealed enrichment in pathways related to stress response, secondary metabolism, and defense signaling, notably phenylpropanoid and shikimate biosynthesis. Antioxidant assays indicated early activation of SOD at 24 hpi, with maximum activity of POX, PAL, and PPO at 72 hpi. qPCR validation confirmed early upregulation of defense-related genes (SIRK1, CAT1, RLP47) at 72 hpi, followed by a decline in expression of immune regulatory genes (EMS1, PR1, PR5, POX) at 120 hpi, suggesting a transition toward immune modulation. These findings demonstrate the ability of P. oryzihabitans Pg-Slp82 to induce a transient but effective defense response, followed by successful epiphytic adaptation. The integrated genomic and transcriptomic data support its potential as a biocontrol agent for managing bacterial blight in pomegranate.

Encounters between bacteria and bacteriophages have led to the evolution of phage and counter-defense arsenals, respectively. Multicopy phage resistance protein, Mpr, in mycobacteria is one such molecule providing resistance against D29 mycobacteriophage, probably by viral DNA degradation. Here, we demonstrate that the DUF4352 domain of Mpr (MprDUF4352) has viral origin, exhibits A/GTP hydrolysis, and synthesizes an unusual guanosine nucleotide, ppGp. The sequence and structure of MprDUF4352 are found to be conserved, and it shows compensatory effects when replaced in Mpr with phage-encoded hypothetical proteins harboring the DUF4352 domain. MprDUF4352 provides a structural framework and facilitates Mpr oligomerization, which is sensitive to GDP concentration. Oligomerized Mpr in the cell membrane cleaves mycobacteriophage genomic DNA, rendering the phage non-viable. As a counter-defense, D29 mutates Gp32 (minor tail protein), and escapes bacterial immunity. In conclusion, we present evidence that the host bacterium has repurposed a phage-encoded DUF4352 to orchestrate a non-abortive host phage defense.

BackgroundAmong fatalities due to parasitic diseases, deaths due to Leishmaniases rank second only to those caused by malaria. Endemic to 90 countries, more than a billion people are at risk of Leishmania infection. While there are no vaccines against Leishmaniases, treatments are available. However, the complexities of the treatment regimens, their high costs and toxic side effects, and most importantly the emerging problem of drug resistance, have continued to thrust forward research into this parasite’s cellular processes. The parasite is digenetic, reproducing by binary fission in both hosts, with DNA replication being central to the process. The absence of many conserved DNA replication proteins in Leishmania suggests the possibility of parasite-specific proteins being involved in the process. This investigation aimed to identify such proteins.MethodsUsing Leishmania donovani Cdc45, a component of the eukaryotic replicative helicase, as bait in immunoprecipitation reactions coupled to mass spectrometry, we looked for proteins that are a part of the Leishmania replisome. One of the promising candidates, annotated “hypothetical protein” in the Leishmania genome database, shared partial structural homology with Timeless of human, C. elegans and Drosophila, and hence we named it “LdTIM-like”. A second candidate, annotated “PIF1-like” in the genome database, was named “LdPIF6” based on sequence homology with T.brucei PIF6. Their possible involvement in DNA replication was examined by creating genomic knockouts and analyzing resultant phenotypes.ResultsLdTIM-like and LdPIF6 were constitutively nuclear, in keeping with a possible role in DNA replication. We found tim-like and pif6 were essential for parasite survival. Single allele knockouts lowered tim-like/pif6 expression significantly, and resulted in slower parasite growth. The mutant parasites took considerably longer to navigate S phase, and pulse-labelling with EdU revealed that the time taken by the mutant cell populations to complete DNA replication was significantly extended. The mutant parasites survived poorly in host macrophages as well.ConclusionsConsidering that these two proteins: LdTIM-like and LdPIF6, are essential to the parasite, coupled to their limited homology with human proteins, they could be investigated as potential sites for therapeutic intervention in future in-depth studies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-025-04377-7.

Leishmania major is a unicellular protozoan that causes cutaneous leishmaniasis in mammals and is mainly transmitted by the sand fly Phlebotomus papatasi. However, the contribution of microRNAs (miRNAs) and protein-coding genes to its pathogenic mechanisms remains largely unexplored. In this study, we systematically analyzed miRNAs and protein-coding genes in L. major, its insect vector, and mammalian hosts. Comparative genomic analysis revealed 2963 conserved proteins shared among the three groups, highlighting a core set of proteins across protozoa, vectors, and hosts. Among mammals, human proteins exhibited the highest homology with L. major, while P. papatasi displayed the lowest proportion of homologs. Functional annotation of 94 hypothetical proteins identified 27 infection-related proteins, including 24 protein kinases and three tyrosine phosphatases, which may represent novel therapeutic targets. In addition, an EST-based approach identified 29 novel miRNAs in L. major. Phylogenetic analysis indicated that these miRNAs diverged into two distinct evolutionary branches, and homology analysis revealed that seven miRNAs were absent in all mammalian species. For example, miR-10117-3p was detected only in nematode Heligosmoides polygyrus. Furthermore, miRNA-gene interaction network analysis highlighted four key genes potentially involved in L. major infection. Collectively, our findings expand current knowledge of protozoan virulence by identifying novel miRNAs and infection-related proteins and provide promising candidates for future drug development against leishmaniasis.

Nocardia seriolae is the primary cause of fish nocardiosis, and it is urgent to develop an effective vaccine for controlling N. seriolae infection in aquaculture. In our previous study, two potential antigenic proteins, glutamate-cysteine ligase EgtA (EgtA) and hypothetical protein 6747 (hp6747), were determined via the invivo-induced antigen technique (IVIAT). A DNA vaccine was aimed to be developed using these two invivo-induced antigens as candidates for ligation with the pcDNA3.1-myc-his-A plasmid, and their immunological effects were evaluated in hybrid snakehead (Channa maculata ♀ × Channa argus ♂). Multiple epitopes of the EgtA and hp6747 proteins from N. seriolae were predicted on both T cells and B cells, and the transcription of the EgtA and hp6747 genes was found in the spleen, trunk kidney, muscle and liver in the fish after immunisation with pcDNA-EgtA and pcDNA-hp6747 DNA vaccines, respectively. Notably, the serum specific antibody (IgM) titres, as well as LYZ, ACP, AKP and SOD activities, were elevated in the vaccinated fish. Meanwhile, quantitative real-time PCR analysis demonstrated that the expression levels of several immune-related genes (TNFα, CD4, CD8α, IL-1β, MHCIIα and MHCIα) were significantly elevated across the aforementioned tissues in the vaccinated fish. Furthermore, they could provide the relative percent survival (RPS) at 61.04% and 43.73% against artificial challenge with N. seriolae in the fish after immunisation with pcDNA-EgtA and pcDNA-hp6747 DNA vaccines, respectively. Taken together, using these two invivo-induced antigens of EgtA and hp6747 for vaccine development was the ideal strategy for controlling fish nocardiosis in aquaculture.

Insect innate immunity has been well studied in Drosophila melanogaster. However, the mechanisms of immune invasion and host adaptation mediated by entomopathogens remain understudied. Here, it is reported that the Drosophila immune cytokine Spätzle (Spz, a Toll receptor ligand) can be targeted by two divergent virulence effectors (ETSs) of Metarhizium robertsii, a fungus that infects a wide range of invertebrates. Mechanistically, the M28-family aminopeptidase ETS1 degrades Spz and its mature ligand form C106, while the hypothetical protein ETS6 only binds C106. Both effectors, particularly ETS6, attenuate or disable Spz interaction with its processing enzyme, the formation of the C106 dimer, and ligand-receptor interaction. Mutagenesis of ETS6 revealed its structural uniqueness in hijacking C106. While mutant Drosophila lacking functional Spz are similarly killed by wild-type and mutant strains of M. robertsii, transgenesis with either ETS1 or ETS6 reduced fly resistance to fungal colonization. Both effectors can target the sequence-divergent yet structurally similar orthologous ligands of other invertebrates, unveiling a fungal mechanism for infecting and killing diverse host species. These findings reveal a rare instance of multiple effectors targeting a single immune factor in fungus-animal interactions, and offer a mechanistic insight into the manipulation of parasite host range.

Bovine mastitis, a multi-etiological disease, is driven by complex microbial consortia; however, the transcriptional activity of pathogens and their underlying molecular mechanisms remains insufficiently explored. To the best of our knowledge, no metatranscriptome study on bovine mastitis is available in the public domain that identifies transcriptionally active pathogens and their associated molecular signatures. In this study, an in silico metatranscriptomics approach is employed on publicly available bovine mastitis RNA sequencing (RNA-Seq) datasets to identify transcriptionally active pathogens and their gene expression signatures. The analysis of unmapped reads (those not mapped to the bovine genome) identified 25 transcriptionally active pathogenic genera, accounting for 8,995 sequences, approximately from 500 bacterial strains of different species. Major findings of the study includes: (I) list of emerging pathogens “Pseudomonas, Stenotrophomonas, Comamonas, and Sphingomonas” actively contributing to disease development alongside well-known pathogens; (II) expression profiling of 4,121 virulence proteins, 484 peptidases, 432 secretory proteins, and 74 antimicrobial resistance genes; (III) identification of numerous hypothetical proteins in Staphylococcus (112), Mycoplasma (69), and Escherichia (32), representing potential source for diagnostics and multi-epitope vaccine candidates; and (IV) negative correlations between beneficial bacteria (Blautia, Bacillus, Lactobacillus) and pathogenic species in microbial co-occurrence interaction networks, suggesting opportunities for microbiome-based therapeutic strategies to treat subclinical mastitis. This study demonstrated the advantages of the metatranscriptomics approach and publicly available dual RNA-Seq datasets in unraveling the complexity of polymicrobial infectious diseases.

The increasing worldwide trend of antibiotic-resistant Neisseria gonorrhoeae strains highlights the urgent need for new therapeutic strategies against this sexually transmitted pathogen, including a gonococcal vaccine. We previously designed a bioinformatics-based candidate selection pipeline (CASS) and identified potential novel gonococcal vaccine targets among hypothetical proteins expressed during natural human infection. One of these candidates, NGO1701, is a predicted periplasmic four-helix bundle protein with amino acid sequence homology to the copper storage protein 1 (Csp1) from Methylosinus trichosporium OB3b. In this study, we confirmed that purified NGO1701 binds 15 Cu(I) ions per monomer in vitro, supporting its function as Csp in N. gonorrhoeae. Using a ngo1701 deletion mutant generated in N. gonorrhoeae F62, we investigated its role in bacteria physiology. We showed that ablation of Csp was not limiting for bacterial growth and fitness in vitro, but the Δcsp strain became significantly more susceptible to copper mediated toxicity. This phenotype was rescued by csp gene complementation, indicating a role in protection against copper toxicity. Our results indicate that Csp participates in periplasmic copper homeostasis in N. gonorrhoeae, buffering excess copper to reduce toxicity and playing a putative role in copper delivery to important copper-enzymes. Csp does not appear to be involved in bacterial host cell interaction and activation in vitro, since no difference in the ability of N. gonorrhoeae to adhere/invade epithelial cells or induce IL-8 secretion was reported among wild type, csp deletion mutant and complemented strains. Furthermore, sera from mice immunized with NGO1701 failed to recognize Δcsp by dot blot and ELISA, and the sera’s ability to kill N. gonorrhoeae was abrogated against Δcsp. However, both functions were restored after gene complementation, supporting the relevance of Csp as a potential vaccine target. Allelic analysis of Neisseria species revealed that this gene is absent in N. meningitidis, thus making it a gonococcal-specific target.

Guanidine-responsive riboswitches control genes that enable either detoxification or assimilation of guanidino compounds. In Vreelandella boliviensis and other halophilic bacteria, genes encoding the guanidine carboxylase pathway are found in a guanidine riboswitch-regulated operon, along with two uncharacterized genes annotated as 2-oxoglutarate (2-OG/Fe(II))-dependent dioxygenase family protein and hypothetical protein, respectively. Here we show that the 2-OG/Fe(II)-dependent dioxygenase efficiently hydroxylates methylguanidine. The resulting N-(hydroxymethyl)guanidine constitutes an unexpectedly stable hemiaminal that slowly decays to guanidine and formaldehyde. The second protein strongly accelerates the fragmentation of N-(hydroxymethyl)guanidine into guanidine and formaldehyde, thus acting as N-(hydroxymethyl)guanidine lyase. Interestingly, the class II guanidine riboswitch in front of the guanidine carboxylase gene does not discriminate between guanidine and methylguanidine, whereas the guanidine class I riboswitch at the start of the entire operon is specific for guanidine. V. boliviensis exhibits growth in minimal media with either guanidine or methylguanidine as sole nitrogen source. Comparative proteome analysis revealed that the entire guanidine carboxylase operon is strongly expressed under these conditions. The presented study broadens our understanding of guanidine metabolism by describing two enzymatic activities that jointly catalyze the demethylation of methylguanidine.

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, harbors numerous uncharacterized proteins whose functions remain unexplored yet may be central to its survival and pathogenicity. Among its specialized organelles, food vacuole plays pivotal role in hemoglobin-catabolism, heme-detoxification, nutrient-assimilation and pharmacodynamic-interactions, thereby representing critical therapeutic target. However, numerous food vacuole-associated proteins remain uncharacterized. In this study, multiple bioinformatics tools were employed to comprehensively characterize a hypothetical food vacuole-associated protein, PF11_0364 (designated PfHDDCP). Conserved domain analysis identified HotDog fold, hallmark of acyl-CoA thioesterases, suggesting its possible role in lipid metabolism. 3D structural model of PfHDDCP was generated using I-TASSER and evaluated with PROCHECK and ProSA. Over 90% of residues were located in favored regions of Ramachandran plot, and ProSA Z-score fell within the range typical of native protein structures, indicating good model quality. Domain analysis via NCBI-CDD identified two putative ligand-binding sites in PfHDDCP. Molecular docking using HDOCK and AutoDock predicted that Acetyl-CoA and Acyl Carrier Protein, canonical substrates of thioesterases, bind at Binding-Site 1, which corresponds to the predicted catalytic site. In contrast, antimalarial compounds were predicted to bind at Binding-Site 2, distinct secondary pocket, suggesting possible allosteric site that may interfere with substrate binding. Molecular dynamics simulations performed with Desmond indicated stable PfHDDCP-ligand complexes and ligand-induced conformational changes, supporting model of ligand-mediated functional modulation. Although these results offer preliminary computational insights into structure, function, and druggability of PfHDDCP, they remain predictive and require experimental validation to confirm the proposed enzymatic activity and therapeutic relevance.

Exploration of hypothetical proteins and reverse vaccinology approach for novel multi-epitope vaccine design against multidrug-resistant clinical isolate Pseudomonas aeruginosa JJPA01.

Computational exploration of natural inhibitors against toxin-associated proteins in Naegleria fowleri Karachi strain.