Biology subjects

Grillova, L.

Publications and source records attributed to Grillova, L..

5 recordsLinked to original sources

bioRxiv2025

Solubilization of Membrane Proteins using designed protein WRAPS

The development of therapies and vaccines targeting integral membrane proteins has been complicated by their extensive hydrophobic surfaces, which can make production and structural characterization difficult. Here we describe a general deep learning-based design approach for solubilizing native membrane proteins while preserving their sequence, fold, and function using genetically encoded de novo protein WRAPs (Water-soluble RFdiffused Amphipathic Proteins) that surround the lipid-interacting hydrophobic surfaces, rendering them stable and water-soluble without the need for detergents. We design WRAPs for both beta-barrel outer membrane and helical multi-pass transmembrane proteins, and show that the solubilized proteins retain the binding and enzymatic functions of the native targets with enhanced stability. Syphilis vaccine development has been hindered by difficulties in characterizing and producing the outer membrane protein antigens; we generated soluble versions of four Treponema pallidum outer membrane beta barrels which are potential syphilis vaccine antigens. A 4.0 [A] cryo-EM map of WRAPed TP0698 is closely consistent with the design model. WRAPs should be broadly useful for facilitating biochemical and structural characterization of integral membrane proteins, enabling therapeutic discovery by screening against purified soluble targets, and generating antigenically intact immunogens for vaccine development.

biochemistry↗

bioRxiv2024

High-quality transcriptome profile of Treponema pallidum subsp. pallidum: confirmation of transcriptional landscape

Syphilis remains a critical global health challenge due to its potential for severe complications and the increase in its incidence rate over recent years. Until recently, the infectious agent of syphilis, Treponema pallidum subsp. pallidum (TPA), could not be cultured in vitro. Advances in co-culture techniques have finally allowed for effective long-term cultivation of TPA, providing a platform to study its biology. Limited transcriptional data from TPA have been reported so far and many genes in treponemal genomes are annotated based on in silico prediction of putative coding sequences without functional validation. To inform future syphilis vaccine development, experimental validation of in silico predicted genes coupled with functional annotation is necessary. In this study, strand-specific RNA-sequencing was used to reconstruct a high-quality transcriptome profile of TPA, confirming the active transcription of genes previously annotated as hypothetical, paving the way for more accurate identification of vaccine target candidates. Our transcriptomic data also revealed, for the first time, the organization of genes into transcription units, an abundance of anti-sense RNAs, and transcripts from intergenic regions, providing crucial insights for future functional genomics studies of TPA. Author SummaryIn our study, we explored the genetic activity of the bacteria responsible for syphilis, Treponema pallidum subsp. pallidum (TPA). Although syphilis has been a known disease for centuries, the bacterium causing it has remained difficult to study because it couldnt be easily grown in the lab. Recently, new techniques have allowed us to cultivate TPA successfully, enabling deeper investigation into its genetics. By employing directional RNA sequencing, we have mapped out which genes are actively transcribed, including those previously labeled as hypothetical. Our study has also revealed new insights into the gene organization and uncovered the presence of antisense RNA, which may regulate gene expression. These findings offer critical information that could inform future research and vaccine development efforts for syphilis.

microbiology↗

bioRxiv2024

Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted Green Fluorescent Protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes. Graphical abstractBy employing genetic engineering, T. pallidum was modified to express GFP, and Sf1Ep cells to express mCherry on the cytoplasmic membrane and BFP in the nucleus. These new resources for syphilis research will facilitate experimental designs to better define the complex interplay between T. pallidum and the host during infection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/596454v1_ufig1.gif" ALT="Figure 1"> View larger version (114K): org.highwire.dtl.DTLVardef@1cc5bcborg.highwire.dtl.DTLVardef@19e8a12org.highwire.dtl.DTLVardef@17aafdborg.highwire.dtl.DTLVardef@1af6823_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Core genome sequencing and genotyping of Leptospira interrogans in clinical samples by target capture sequencing

The life-threatening pathogen Leptospira interrogans is the most common agent of leptospirosis, an emerging zoonotic disease. However, little is known about the strains that are circulating worldwide due to the fastidious nature of the bacteria and its difficulty to be culture isolated. In addition, the paucity of bacteria in blood and other clinical samples has proven to be a considerable challenge for directly genotyping the agent of leptospirosis directly from patient material. Here, to elucidate the genomic diversity of Leptospira circulating strains, hybridization capture followed by Illumina sequencing of the core genome was performed directly from 20 biological samples that were PCR positive for pathogenic Leptospira. A set of samples subjected to capture with RNA probes covering the L. interrogans core genome resulted in 72 to 13,000-fold increase in pathogen reads when compared to standard sequencing without capture. A SNP analysis of the genomes sequenced from the biological samples using 273 Leptospira reference genome was then performed in order to determine the genotype of the infecting strain. For samples with sufficent coverage (19/20 samples with coverage >8X), we could unambigously identify L. interrogans sv Icterohaemorrhagiae (14 samples), L. kirschneri sv Grippotyphosa (4 samples) and L. interrogans sv Pyrogenes (1 sample) as the infecting strain. In conclusion, we obtained for most of our biological samples high quality genomic data at suitable coverage for confident core genome genotyping of the agent of leptospirosis. The ability to generate culture-free genomic data opens new opportunities to better understand the epidemiology and evolution of this fastidious pathogen.

microbiology↗

bioRxiv2021

Whole genome sequences of Treponema pallidum subsp. endemicum isolated from Cuban patients: the non-clonal character of isolates suggests a persistent human infection rather than a single outbreak

Bejel (endemic syphilis) is a neglected non-venereal disease caused by Treponema pallidum subsp. endemicum (TEN). Although it is mostly present in hot, dry climates, a few cases have been found outside of these areas. The aim of this work was the sequencing and analysis of TEN isolates obtained from "syphilis patients" in Cuba, which is not considered an endemic area for bejel. Genomes were obtained by pool segment genome sequencing or direct sequencing methods, and the bioinformatics analysis was performed according to an established pipeline. We obtained four genomes with 100%, 81.7%, 52.6%, and 21.1% of broad coverage, respectively. The sequenced genomes revealed a non-clonal character, with nucleotide variability ranging between 0.2-10.3 nucleotide substitutions per 100 kbp among the TEN isolates. Nucleotide changes affected 27 genes, and the analysis of the completely sequenced genome also showed a recombination event between tprC and tprI, in TP0488 as well as in the intergenic region between TP0127-TP0129. Despite limitations in the quality of samples affecting broad sequencing coverage, the determined non-clonal character of the isolates suggests a persistent infection in the Cuban population rather than a single outbreak caused by imported case. Author summaryThe incidence of venereal syphilis has greatly increased in the last years, however endemic syphilis (bejel) which have been considered as a disease restricted to dry arid areas such as the Sahel and the Middle East, remain as a neglected disease. In Cuba, which is a tropical country, several bejel cases were unexpectedly detected few years ago in "syphilis" patients with no records of travel abroad or sex with foreign partners. In this study, we explored the whole genome sequences from four of the Cuban Treponema pallidum subsp. endemicum (TEN) isolates and the substantial genetic diversity detected among them suggests a persistent infection of TEN within the human population rather than a single outbreak of a TEN isolate introduced from an area where it is typically endemic. This finding has significant implications on this neglected and also possibly tropical disease in terms of geographical/temporal distribution, and highlights the importance of keeping in mind neglected diseases in apparently non-endemic areas.

microbiology↗