Biology subjects

Rusniok, C.

Publications and source records attributed to Rusniok, C..

8 recordsLinked to original sources

bioRxiv2026

Bacterial targeting of host paraspeckles uncovers a new SFPQ-based regulation

Nuclear functions are key in protecting cells against infections, yet intracellular pathogens like Legionella pneumophila can exploit these mechanisms to survive. We characterized a L. pneumophila protein, LpDot1, which shares sequence similarity with the eukaryotic catalytic domain of the histone methyltransferase DOT1. Structure determination, together with biochemical and biophysical analyses, revealed that LpDot1 methylates non-histone nuclear proteins, notably the splicing factor proline-glutamine rich protein (SFPQ). Importantly, LpDot1 targets the previously uncharacterized K518, located on an important structural motif of SFPQ, therefore impairing its dimerization in vitro. During infection, L. pneumophila modulates SFPQ abundance and activities in a LpDot1-dependent manner, thereby hijacking paraspeckle organization and the host cell splicing machinery, leading to alternative splice variants of infection related genes such as NF-kB2 and CD45. To our knowledge, this is the first report of a bacterial effector directly modifying paraspeckle dynamics, providing new insight into previously uncharacterized eukaryotic regulatory pathways.

microbiology↗

bioRxiv2025

Avirulent Pseudomonas aeruginosa T3SS-negative strains belonging to Clade 5 produce variable quantities of secondary metabolites

Pseudomonas species are ubiquitous in the environment and serve as valuable source of enzymes and secondary metabolites for industrial applications. P. aeruginosa secretes metalloproteases such as elastase LasB and produces bioactive small molecules, including pyocyanin, rhamnolipids, and pyoverdine, with potential biotechnological applications. However, the interest in P. aeruginosa for industrial use has been limited due to the virulence-associated Type III Secretion System (T3SS), a key factor in host-pathogen interactions. In this study, we genotypically and phenotypically characterized a collection of P. aeruginosa strains naturally lacking T3SS-encoding genes. Phylogenetic analysis revealed that these strains belong to two distinct clades. Several strains exhibited low or no cytotoxicity on epithelial cell lines and were avirulent in the Galleria infection model. The level of LasB and the three metabolites -- pyocyanin, rhamnolipids, and pyoverdine -- varied independently of virulence profiles. Notably, we identified avirulent strains capable of producing at least two secondary metabolites, including monorhamnolipids, highlighting their potential for biotechnological applications.

microbiology↗

bioRxiv2023

The unique Legionella longbeachae capsule favors intracellular replication and immune evasion

Legionella longbeachae and Legionella pneumophila cause Legionnaires disease despite species-specific differences in environmental niches, disease epidemiology, and genomic content. Here, we characterized a new L. longbeachae virulence factor, a capsule that is expressed in post-exponential growth phase as shown by electron microscopy. Analysis of the capsule composition via HLPC revealed the presence of a highly anionic polysaccharide absent in a capsule mutant. The capsule is crucial for replication and virulence in vivo in a mouse model of infection and in the natural host Acanthamoeba castellanii. It has anti-phagocytic function when encountering innate immune cells, it is involved in a low cytokine responses in mice and in human monocyte derived macrophages and helps to dampen the innate immune response. The L. longbeachae capsule is a novel virulence factor, unique among known Legionella species, that aids L. longbeachae to survive in specific niches and partly confers L. longbeachae its unique infection characteristics.

microbiology↗

bioRxiv2022

A Legionella pneumophila effector impedes host gene silencing to promote virulence

RNA silencing is a gene silencing mechanism directed by small RNAs. Human miRNAs act as central regulators of host-bacteria interactions. However, it is unknown whether human pathogenic bacteria could impede RNA silencing to promote virulence. Here, we show that the Legionella pneumophila type IV-secreted effector LegK1 efficiently suppresses siRNA and miRNA activities in human cells. This effect depends on its known kinase activity, but also on its novel capacity, found here, to bind Argonaute (Ago) proteins. We further demonstrate that the ability of LegK1 to activate NF-{kappa}B signaling is required for RNA silencing suppression, establishing a link between effector-mediated NF-{kappa}B signaling and RNA silencing suppression. LegK1 also promotes L. pneumophila growth in both amoeba and human macrophages, supporting a role for this effector in virulence. Finally, we show that, in infected-macrophages, the latter activity relies, in part, on the genetic targeting of human Ago4. These findings indicate that a L. pneumophila effector has evolved to suppress RNA silencing to promote virulence. Significance StatementIt is now well established that mammalian viruses suppress RNAi to promote their replication in host cells. However, whether mammalian pathogenic bacteria use a similar virulence strategy remains unknown. Here, we show that the LegK1 effector from Legionella pneumophia, the causal agent of Legionnaires disease, efficiently suppresses RNAi in human cells. This effect depends on its ability to interact with Argonaute (Ago) proteins and to activate NF-{kappa}B signaling. In addition, LegK1 promotes virulence in infected-macrophages through the genetic targeting of human Ago4. Based on the lack of NF-{kappa}B-related factors in amoebae, and on the presence of canonical Ago proteins in these natural L. pneumophila hosts, we propose that the RNAi suppression activity of LegK1 represents its primary virulence function.

molecular biology↗

bioRxiv2022

Legionella para-effectors target chromatin and promote bacterial replication

Legionella pneumophila replicates intracellularly by secreting effectors via a type IV secretion system. One of these effectors is a eukaryotic methyltransferase (RomA) that methylates K14 of histone H3 (H3K14me3) to counteract host immune responses. However, it is not known how L. pneumophila infection catalyses H3K14 methylation as this residue is usually acetylated. Here we show that L. pneumophila secretes a eukaryotic-like histone deacetylase (LphD) that specifically targets H3K14ac and works in synergy with RomA. Both effectors target host chromatin and bind the HBO1 histone acetyltransferase complex that acetylates H3K14. Full activity of RomA is dependent on the presence of LphD as H3K14 methylation levels are significantly decreased in a {Delta}lphD mutant. The dependency of these two chromatin-modifying effectors on each other is further substantiated by mutational and virulence assays revealing that the presence of only one of these two effectors impairs intracellular replication, while a double knockout ({Delta}lphD{Delta}romA) can restore intracellular replication. Uniquely, we present evidence for "para-effectors", an effector pair, that actively and coordinately modify host histones to hijack the host response. The identification of epigenetic marks modulated by pathogens opens new vistas for the development of innovative therapeutic strategies to counteract bacterial infection and strengthening host defences.

microbiology↗

bioRxiv2021

Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche

Legionella pneumophila, the causative agent of Legionnaires disease, a severe pneumonia, injects via a type-IV-secretion-system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here we show that despite reduced OXPHOS, the mitochondrial membrane potential ({Delta}{psi}m) is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the {Delta}{psi}m, preserves mitochondrial polarization and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the {Delta}{psi}m, but not LncP and MitF. The inhibition of the L. pneumophila-induced "reverse mode" of the FOF1-ATPase collapsed the {Delta}{psi}m and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the {Delta}{psi}m and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

microbiology↗

bioRxiv2019

Legionella pneumophila infection and antibiotic treatment engenders a highly disturbed pulmonary microbiome with decreased microbial diversity

BackgroundLung microbiome analyses have shown that the healthy lung is not sterile but it is colonized like other body sites by bacteria, fungi and viruses. However, little is known about the microbial composition of the lung microbiome during infectious diseases such as pneumonia and how it evolves during antibiotic therapy. To better understand the impact of the composition of the pulmonary microbiome on severity and outcome of pneumonia we analysed the composition and evolution of the human lung microbiome during pneumonia caused by the bacterium Legionella pneumophila.\n\nResultsWe collected 10 bronchoalveolar lavage (BAL) samples from three patients during long-term hospitalisation due to severe pneumonia and performed a longitudinal in-depth study of the composition of their lung microbiome by high-throughput Illumina sequencing of the 16S rRNA gene (bacteria and archaea), ITS region (fungi) and 18S rRNA gene (eukaryotes). We found that the composition of the bacterial lung microbiome during pneumonia is hugely disturbed containing a very high percentage of the pathogen, a very low bacterial diversity, and an increased presence of opportunistic microorganisms such as species belonging to Staphylococcaceae and Streptococcaceae. The microbiome of antibiotic treated patients cured from pneumonia represented a different perturbation state with a higher abundance of resistant bacteria (mainly Firmicutes) and a significantly different bacterial composition as that found in healthy individuals. In contrast, the mycobiome remains more stable during pneumonia and antimicrobial therapy. Interestingly we identified possible cooperation within and between both communities. Furthermore, archaea (Methanobrevibacter) and protozoa (Acanthamoeba and Trichomonas) were detected.\n\nConclusionsBacterial pneumonia leads to a collapse of the healthy microbiome and a strongly disturbed bacterial composition of the pulmonary microbiome that is dominated by the pathogen. Antibiotic treatment allows some bacteria to regrow or recolonize the lungs but the restoration of a healthy lung microbiome composition is only regained a certain time after the antibiotic treatment. Archaea and protozoa should also be considered, as they might be important but yet overseen members of the lung microbiome. Interactions between the micro- and the mycobiome might play a role in the restoration of the microbiome and the clinical evolution of the disease.

microbiology↗

bioRxiv2018

More than 16,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells

SignificanceLegionella pneumophila is a bacterial pathogen causing outbreaks of a lethal pneumonia. The genus Legionella comprises 65 species for which aquatic amoebae are the natural reservoirs. Using functional and comparative genomics to deconstruct the entire bacterial genus we reveal the surprising parallel evolutionary trajectories that have led to the emergence of human pathogenic Legionella. An unexpectedly large and unique repository of secreted proteins (>16,000) containing eukaryotic-like proteins acquired from all domains of life (plant, animal, fungal, archaea) is contrasting with a highly conserved type 4 secretion system. This study reveals an unprecedented environmental reservoir of bacterial virulence factors, and provides a new understanding of how reshuffling and gene-acquisition from environmental eukaryotic hosts, may allow for the emergence of human pathogens.\n\nAbstractThe bacterial genus Legionella comprises 65 species among, which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 16,000 secreted proteins encoding 137 different eukaryotic-like domains and more than 200 eukaryotic-like proteins is paired with a highly conserved T4SS. Specifically, we show that eukaryotic Rho and Rab GTPase domains are found nearly exclusively in eukaryotes and Legionella species. Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F/U-box and SET domains were present in >70% of all species suggesting that manipulation of host signal transduction, protein turnover and chromatin modification pathways, respectively are fundamental intracellular replication strategies for Legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by inter-domain gene transfer. Furthermore we revealed the surprising extent to which legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts and provides a new understanding of how dynamic reshuffling and gene-acquisition has led to the emergence of major human pathogens.

genomics↗