Lukas Hafner

Institut Pasteur

Microbial pathogenesis is mediated by the expression of virulence genes. However, as microbes with identical virulence gene content can differ in their pathogenic potential, other virulence determinants must be involved. Here, by combining comparative genomics and transcriptomics of a large collection of isolates of the model pathogen Listeria mono...

We report a case of fulminant fatal neonatal listeriosis due to horizontal transmission of Listeria monocytogenes (Lm) in a neonatal double room. Genomic analyses reveal a close genetic relationship between clinical isolates, supporting cross-contamination. Oral inoculation experiments in adult and neonatal mice show that neonates are susceptible t...

Genome-wide Association Studies (GWAS) have been central to studying the genetics of complex human outcomes, and there is now tremendous interest in implementing GWAS-like approaches to study pathogenic bacteria. A variety of methods have been proposed to address the complex linkage structure of bacterial genomes, however, some questions remain abo...

Background Encephalitis is a worldwide public health issue, with a substantially high burden among children in southeast Asia. We aimed to determine the causes of encephalitis in children admitted to hospitals across the Greater Mekong region by implementing a comprehensive state-of-the-art diagnostic procedure harmonised across all centres, and id...

Listeria monocytogenes (Lm) is a foodborne pathogen and the etiological agent of listeriosis. This facultative intracellular Gram-positive bacterium has the ability to colonize the intestinal lumen, cross the intestinal, blood-brain and placental barriers, leading to bacteremia, neurolisteriosis and maternal-fetal listeriosis. Lm is a model microor...

Listeria genus comprises two pathogenic species, L. monocytogenes (Lm) and L. ivanovii, and non-pathogenic species. All can thrive as saprophytes, whereas only pathogenic species cause systemic infections. Identifying Listeria species’ respective biotopes is critical to understand the ecological contribution of Listeria virulence. In order to inves...

Neonates are highly susceptible to bacterial meningitis as compared to children and adults. Group B streptococcus (GBS) is a major cause of neonatal meningitis. Neonatal meningitis can result from GBS intestinal colonization and translocation across the intestinal barrier (IB). Here, we show that the immaturity of the neonatal intestinal microbiota...

Mittal et al. (2021; first brought to our attention in May 2019) have raised concerns regarding the Chromatin Endogenous Cleavage-sequencing (ChEC-seq) technique (Zentner et al., 2015) that may create a false impression that this method has fundamental flaws which prevent one from distinguishing between signal and noise. Although Mittal et al. focu...

Listeria genus comprises two opportunistic pathogenic species, L. monocytogenes ( Lm ) and L. ivanovii , and several non-pathogenic species. All can thrive as saprophytes, whereas only pathogenic species cause systemic infections in human and cattle. Identifying Listeria species′ respective biotopes is critical to understand the ecological contribu...

We thank Dmitry Ershov from the Bioinformatics and Biostatistics Hub (Institut Pasteur, USR 3756CNRS) and the Image Analysis Hub for their help with the image and/or data analysis. We thank George M. Haustant and Thomas Cokelaer (Biomics Platform, C2RT, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09-09) and IBISA) fo...

Listeria monocytogenes is a Gram-positive bacterium that can be found in a broad range of environments, including soil, food, animals, and humans. L. monocytogenes can cause a foodborne disease manifesting as sepsis and meningo-encephalitis. To evaluate signals of selection within the core genome of neuroinvasive L. monocytogenes strains, we sequen...

In buddying yeast, like all eukaryotes examined so far, DNA replication is under temporal control, such that some origins fire early and some late during S phase. This replication timing program is established in G1 phase, where chromatin states are thought to prevent binding of key-limiting initiation factors at late-firing origins. Although many...

The yeast Sfp1 protein regulates both cell division and growth but how it coordinates these processes is poorly understood. We demonstrate that Sfp1 directly controls genes required for ribosome production and many other growth-promoting processes. Remarkably, the complete set of Sfp1 target genes is revealed only by a combination of ChIP (chromati...

The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the re...

In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved ∼125-...

The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is simila...

FOB1 deletion rescues rDNA instability, reverses ERC accumulation but does not affect rif1Δ -promoted rARS activation. (A) The chromosomes (chr) from indicated strains were resolved using pulsed-field gel electrophoresis (PFGE) and stained with ethidium bromide (top panel). The same gel was transferred by Southern blot and hybridized with an rDNA p...

Synthetic sickness of rif1Δ with mre1Δ is due to rDNA RFBs. Part I. (A–B) Exponentially growing cultures of the indicated genotypes were serially diluted 1:10 and spotted onto solid YPAD media at the indicated temperature supplemented or not with indicated chemicals (PHL, MMS). (C) Tetrad dissection plates of the heterozygous diploids MRE11/mre11Δ...

The increase in rDNA instability in rif1Δ is not due to DNA damage response or Sir2 re-localization. (A-B) Representative pictures of plates with colonies of strains with indicated genotypes for the rDNA instability assays (ADE2 marker-loss assay) at Fig 3A–3C. The red sectors on the white colonies are marked with white arrowheads. (C) Exponentiall...

Increase in replication initiation in rif1Δ leads to elevated DRC. (A) Rad53 phosphorylation upon HU treatment in asynchronous cultures in S288C and JC482 backgrounds (upper panel) and additional rapamycin treatment of WT and RIF1-FRB anchor-away strains (both W303, tor1-1 fpr1Δ RPL13A-2xFKB12). (B) Rad53 phosphorylation upon HU treatment detected...

Rif1 inhibits DNA replication initiation at rARS and late origins. (A) Mcm4-13xMyc ChIP at indicated loci in G1-arrested WT and rif1Δ cells. (B) Representative FACS profiles from the G2/M arrest and 0.2M HU release experiments. (C) BrdU incorporation at the indicated loci in WT, rif1Δ mutant and negative control strains (neg ctrl; a WT strain that...

Telomere length phenotype of fob1Δ background and rDNA instability in rad9Δ background. (A) Telomere length assayed by Southern blot of XhoI-digested genomic DNA in the indicated strains. (B) rDNA instability measured by ADE2 loss in the indicated strains. (TIF)

Synthetic sickness of rif1Δ with mre1Δ is due to rDNA RFBs. Part II. (A) Tetrad dissection plate of a diploid strain heterozygous for 4 gene deletions: RIF1/rif1Δ, RRM3/rrm3Δ, MRE11/mre11Δ, and FOB1/fob1Δ (left panel) and serial dilution spot assays with some of the derived strains (right panel). (B) Serial dilution spot assay of strains harboring...

List of yeast strains used in this study. (DOCX)

Rap1-interacting factor 1 (Rif1) was originally identified in the budding yeast Saccharomyces cerevisiae as a telomere-binding protein that negatively regulates telomerase-mediated telomere elongation. Although this function is conserved in the distantly related fission yeast Schizosaccharomyces pombe, recent studies, both in yeasts and in metazoan...