Biology subjects

Lastovka, F.

Publications and source records attributed to Lastovka, F..

8 recordsLinked to original sources

bioRxiv2026

Changes in the translational landscape during red clover necrotic mosaic virus infection

Viruses alter host gene expression to create a proviral environment while the host simultaneously regulates gene expression to restrict the virus spread. Owing to RNA virus complete reliance on the host translational machinery, it is important to assess the translational control during virus infection. Therefore, we used ribosome profiling (ribo-seq) paired with RNAseq to observe how red clover necrotic mosaic virus (RCNMV) infection of Arabidopsis plants alters cellular gene expression at the levels of mRNA abundance and translation efficiency. We determined that at 5 days post-inoculation (dpi), the translational response to RCNMV infection is enriched in genes of the innate immune system. Expression of a tumor necrosis factor receptor-associated factor (TRAF)-like protein, a regulator of development and immune response, was translationally but not transcriptionally upregulated early in systemic infection. By 8 dpi, many pathways were regulated/dysregulated, and unfolded protein response (UPR) genes were transcriptionally upregulated but with reduced translation efficiency. Ribosome profiling of RCNMV RNAs revealed (i) -1 programmed ribosomal frameshifting at 7.5-8.0%, the first direct measurement of frameshift efficiency in infected cells for any plant virus; (ii) that coat protein is translated at extremely high efficiency, while the RNA-dependent RNA polymerase is translated least efficiently, and (iii) an unexpected extremely strong ribosomal pause site in the open reading frame that encodes the movement protein. To our knowledge, this is the first genome-wide study that assesses the translational control of gene expression in plants infected with a virus from the large and diverse Tombusviridae family. ImportancePositive strand RNA viruses usurp the hosts translation machinery to synthesize viral proteins. Moreover, translation of host mRNAs is altered by virus infection, both as part of the host immune response and by the virus to inhibit host defenses. To assess all these changes globally, we used ribosome profiling of plants infected with a member of the large and ubiquitous Tombusviridae family. We identified key host genes and pathways that were differentially altered in translation efficiency, giving us an understanding of host responses not detectable by conventional RNA sequencing. Moreover, ribosome profiling revealed (i) the most accurate calculation of efficiency of ribosomal frameshifting during infection for any plant virus, (ii) the extremely high level of translation of viral coat protein, and (iii) an unexpected strong ribosomal pause site in the movement protein gene. This work provides understanding of a new dimension of gene expression control in plant-virus interactions.

microbiology↗

bioRxiv2025

Toxoplasma strikes preemptively to swiftly suppress macrophage immune response during active infection

The apicomplexan parasite Toxoplasma gondii is known to manipulate its host in multiple ways, ranging from proteins secreted into the host cell to hormone balance disruption and behavioral changes. Host immune system is crucial in managing the outcome with macrophages as part of the first line of defense. However, the initial triggers that ultimately result in characteristic complex responses and, at times, health hazards, remain poorly understood. This study focuses on filling the gaps in our knowledge of acute transcriptomic changes taking place in a mouse macrophage T. gondii infection model. We performed time-resolved transcriptomic profiling to simultaneously capture host and parasite gene expression profiles during the course of infection, focusing on the initial time frame of fifteen to 120 minutes, a crucial window for the host to activate innate immunity but also for the parasite to establish within the host macrophage. Further, utilization of inactivated parasite stimulation enabled dissection of transcriptomic response to active parasite infection from innate immune responses. Here, we observed that macrophages upregulate transcripts encoding suppressors of cytokine signaling by 30 minutes, specific to live parasite infection. Additionally, both pro-growth and stress marker genes were dysregulated. Concurrently, transcriptional response of T. gondii was milder in magnitude, with initial changes pointing at increasing transcription and growth capacity, followed by a delayed transcriptional response pertaining to secreted proteins. Taken together, these results demonstrate that macrophages mount a rapid transcriptional response upon active invasion by T. gondii. In contrast, the delayed transcriptional activation in the invading Toxoplasma highlights its reliance on alternative regulatory mechanisms to establish its replicative niche within the host. Author summaryToxoplasma gondii, a eukaryotic intracellular parasite, is often regarded as one of the most globally successful parasites because it can infect virtually any warm-blooded animal. It uses a repertoire of secretory proteins to gain a foothold in a host cell, often resulting in a dormant infection in vivo due to sufficient immune suppression. However, the timing of the signaling events as Toxoplasma invades is not yet fully understood. In this work we implement our user-friendly transcriptomic method to simultaneously capture T. gondii and murine macrophage protein-coding RNA contents over a time course to track cellular responses during infection. In particular, we focus on the first 2 hours of infection, a time window where the initial transcriptomic changes within macrophage are generally expected to take place and potentially define further course of infection. Our analysis reveals a robust host macrophage immune response and a moderate more gradual T. gondii response. These findings complement the currently existing picture of all the cellular regulation layers involved in Toxoplasma-macrophage interaction.

microbiology↗

bioRxiv2024

Controlling heterologous protein synthesis through a plant RNA ThermoSwitch

Plants have evolved sophisticated mechanisms to adapt to temperature fluctuations, including transcriptional, post-transcriptional, and post-translational processes. Recent discoveries highlight RNA ThermoSwitches, cis-acting elements in several plant mRNAs that regulate protein synthesis based on temperature changes. These mechanisms, first identified in Arabidopsis thaliana, offer a promising tool for biotechnology by enabling temperature-sensitive control of protein expression. This study demonstrates, for the first time, the feasibility the application of plant RNA ThermoSwitches in Agrobacterium-mediated transient expression systems, presenting a novel method for controlled gene expression in plants. This system is particularly advantageous due to its homogeneous nature and independence from chemical inducers or suppressors.

plant biology↗

bioRxiv2024

Activity of Salmonella SPI-1 inhibits the TLR4-dependent transcriptional but not translational response during macrophage infection

Changes in gene expression during bacterial infection are the combined result of altered transcription and translation, with the latter comparatively understudied. Gram-negative bacteria rapidly trigger cytokine gene transcription in macrophages through the activation of pathogen associated molecular pattern receptors, for example detection of Salmonella lipopolysaccharide (LPS) from the bacterial cell envelope by Toll-like receptor 4 (TLR4). Here, through time-resolved parallel translatomic and transcriptomic profiling, we now show temporal TLR4-specific translational upregulation of cell signalling proteins in macrophages induced by Salmonella. While transcriptional upregulation of these genes is dampened through the activity of the Salmonella SPI-1 type three secretion system, a robust translational response remains. These data reveal an important host-pathogen translational regulatory network that modifies the innate immune response of macrophages to infection.

molecular biology↗

bioRxiv2023

Pervasive translational control of photosynthesis genes during photomorphogenesis is acquired by C4 genes

C4 photosynthesis allows increased efficiency and has evolved in more than sixty-six plant lineages. Underpinning this repeated appearance of the C4 pathway is a major transcriptional reprogramming of photosynthesis genes. Here we investigated whether evolution has also significantly modified translational control by defining the translational dynamics of C3 rice and C4 sorghum during photomorphogenesis. In the dark rice photosynthesis transcripts are low abundance but highly translated. After exposure to light translational efficiency declines. The same phenomena occur in sorghum but in addition C4 cycle genes show this response. We propose a model in which translational control of photosynthesis genes permits a rapid response to light and that this translational regulation is gained by C4 genes during the evolution of the C4 pathway.

molecular biology↗

bioRxiv2023

Salmonella impairs macrophage immunity through effector-independent rapid translational induction in response to membrane puncture by the SPI-1 injectisome

During bacterial infection both the host cell and its invader must divert intracellular resources to synthesise specific proteins in a timely manner. For the host, these factors may be needed for innate immune responses, including programmed cell death, and in the bacteria newly synthesized proteins may be survival factors needed to counteract host responses. Salmonella is an important food-borne bacterial pathogen that invades and multiplies within host cells. It is well established that invasion of epithelial cells is dependent upon the SPI-1 Type III injectisome, a biological needle that penetrates and secretes effectors into host cells to promote internalization. However, the importance of the SPI-1 injectisome in infection of professional phagocytes such as macrophages, which are the predominant host cell type during systemic infection, is less clear. Through time resolved parallel transcriptomic and translatomic studies of macrophage infection, we revealed that SPI-1 injectisome-dependent infection of macrophages triggers rapid translation of transcription factor mRNAs, including Early Growth Response 1 (Egr1). Despite the short half-life of EGR1 protein, its swift synthesis within the initial hour of infection is sufficient to inhibit transcription of pro-inflammatory genes and thereby restrain inflammatory responses and programmed cell death within the first hour of during early infection. This transient period of inflammatory suppression in macrophages is exploited by Salmonella to establish infection and sheds new insight on the importance of translational activation in host-pathogen dynamics during Salmonella infection.

molecular biology↗

bioRxiv2023

The distinct translational landscapes of Gram-positive and Gram-negative bacteria

Translational control in pathogenic bacteria is fundamental to gene expression and affects virulence and other infection phenotypes. We used an enhanced ribosome profiling protocol coupled with parallel transcriptomics to capture accurately the global translatome of two evolutionarily distant pathogenic bacteria - the Gram-negative bacterium Salmonella and the Gram positive bacterium Listeria We find that the two bacteria use different mechanisms to translationally regulate protein synthesis. In Salmonella, in addition to the expected correlation between translational efficiency and cis-regulatory features such as Shine-Dalgarno (SD) strength and RNA secondary structure around the initiation codon, our data reveal an effect of the 2nd and 3rd codons, where the presence of tandem lysine codons (AAA-AAA) enhances translation in both Salmonella and E. coli. Strikingly, none of these features are seen in efficiently translated Listeria transcripts. Instead, approximately 15% of efficiently translated Listeria genes exhibit 70S footprints seven nt upstream of the authentic start codon, suggesting that these genes may be subject to a novel translational initiation mechanism. Our results show that SD strength is not a direct hallmark of translational efficiency in all bacteria. Instead, Listeria has evolved additional mechanisms to control gene expression level that are distinct from those utilised by Salmonella and E. coli. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising

microbiology↗

bioRxiv2020

Resistance to Ceftazidime/Avibactam Plus Meropenem/Vaborbactam When Both are Used Together Achieved in Four Steps From Metallo-β-Lactamase Negative Klebsiella pneumoniae.

Serine cephalosporinases and carbapenemases are dominant causes of critically important {beta}-lactam resistance in Klebsiella pneumoniae. This has led to the recent clinical deployment of new serine {beta}-lactamase inhibitors used in combination with {beta}-lactams. Starting with clinical K. pneumoniae isolates and adding plasmids carrying the OXA-48-like class D carbapenemase, OXA-232, the class A carbapenemase KPC-3, the class A cephalosporinase CTX-M-14 and mutant derivatives of these enzymes, we set out to identify the steps required to give resistance to the recently approved {beta}-lactam/{beta}-lactamase inhibitor pairs ceftazidime/avibactam and meropenem/vaborbactam when both are used together. We show that four steps: ompK36 and ramR loss-of-function plus carriage of OXA-232 and KPC-3-D178Y, all of which have been observed in clinical isolates, allow K. pneumoniae to resist the combined use of both {beta}-lactam/{beta}-lactamase inhibitor pairs. These findings have implications for decision making about sequential and combinatorial use of {beta}-lactam/{beta}-lactamase inhibitor pairs to treat K. pneumoniae infections, and suggest simple surveillance activities that might identify intermediate stages in resistance acquisition and therefore guide therapy to reduce the emergence of dual resistant strains.

microbiology↗