7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

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Prof.

Development of a genetic system for Haloferax gibbonsii LR2-5, model host for haloarchaeal viruses

Phages and microbial defense systems 

Like organisms from the other domains of life, archaea are predated upon by viruses. These viruses can have strong effects on host populations and even entire ecosystems. However, the infection mechanisms of archaeal viruses remain poorly understood. While there have been several new discoveries in viruses infecting thermoproteota (formerly crenarchaeota) in recent years, euryarchaeal viruses remain particularly understudied. To improve our understanding of these viruses, model virus-host systems are required.The halophilic euryarchaeon Haloferax gibbonsii LR2-5 was isolated along with the virus HFTV1 from the hypersaline lake Retba in Senegal [1]. The strain grows well in established media used for other Haloferax species and is highly motile. Its genome has been fully sequenced and annotated. Additionally, it is well suited for light and electron microscopy [2]. Besides the virus it was isolated with, Hfx. gibbonsii LR2-5 has been shown to be infected by nine other viruses [3]. This makes it an attractive model organism for the study of virus-host interactions.To better research the molecular mechanisms underpinning archaeal virus-host interactions, we have established a genetic system in Hfx. gibbonsii LR2-5 by adapting the well-established system of Hfx. volcanii. This system can be used for plasmid-based protein expression as well as gene deletions. Both expression- and deletion plasmids designed for Hfx. volcanii have been demonstrated to function in Hfx. gibbonsii. This opens up a versatile genetic toolbox for the study of archaeal virus-host interactions.ReferencesMizuno, C.M., et al., Novel haloarchaeal viruses from Lake Retba infecting Haloferax and Halorubrum species. Environ Microbiol, 2019. 21(6): p. 2129-2147.Tittes, C., et al., Cellular and Genomic Properties of Haloferax gibbonsii LR2-5, the Host of Euryarchaeal Virus HFTV1. Front Microbiol, 2021. 12: p. 625599.Aguirre Sourrouille, Z., et al., The Viral Susceptibility of the Haloferax Species. Viruses, 2022. 14(6).

Oral Presentation

Prof. Dr.

Screening anti-viral compounds against a diverse set of Bacteriophages

Under the constant threat of predation by bacteriophages (or phages), bacteria evolved a huge arsenal of anti-phage defense mechanism. The majority of antiphage defense systems reported so far revolve around proteins or RNA complexes that function at the cellular level. Recently, the DNA-intercalating anthracyclines1 as well as several aminoglycoside antibiotics2, produced by Streptomyces, were shown to specifically block DNA replication of various dsDNA phages. Despite this discovery, the precise mechanism by which these compounds exert their effects and the specific phage characteristics that render them susceptible to these substances remain unknown.Most antiphage molecules reported so far are produced by environmental bacteria of the genus Streptomyces. This study centers on antiphage compounds produced by Streptomyces and seeks to evaluate the specific phage characteristics that render them susceptible to chemical defense mechanisms1,2. For this purpose, the BASEL phage collection, comprising 69 diverse Escherichia coli phages, as well as several Streptomyces phages from our own collection were screened to investigate sensitivity determinants on the phage genome towards these anti-viral compounds3. This screening revealed distinct and taxonomically-related clusters of phages which are sensitive to specific compounds and emphasize an important impact of phage epigenomic modifications.References1 Aël Hardy, Larissa Kever, and Julia Frunzke* "Antiphage small molecules produced by bacteria – beyond protein-mediated defences" (2022), doi: 10.1016/j.tim.2022.08.0012 Kever, L. et al. "Aminoglycoside Antibiotics Inhibit Phage Infection by Blocking an Early Step of the Infection Cycle" (2022), doi: 10.1128/mbio.00783-223 Maffei, E. et al. "Systematic exploration of Escherichia coli phage-host interactions with the BASEL phage collection" (2021), doi: 10.1371/journal.pbio.3001424

Bacteriophages in ancient human gut metagenomes

Bacteriophages (phages), viruses that infect bacteria, are highly diverse and abundant everywhere on Earth. Recent advances in metagenomic sequencing and computational analysis have made it possible to explore the vast genomic diversity of phages and study their evolution and ecology. However, most studies only sample present-day phages, so they lack a long-term perspective on their evolution. In this study, we present the de novo assembly of 298 ancient phage genomes from publicly available ancient human gut metagenomes dated up to 5300 years before present. We authenticated their ancestral origin based on DNA deamination patterns. Next, we analyzed their relationships to modern viruses from the gastrointestinal tract and predicted hosts with cutting-edge tools. Our analysis revealed ancient representatives of known virus families and genera, as well as ancient phages that were unclassified or only distantly related to known viruses. We also discovered a genome of Mushuvirus mushu, a phage that infects Faecalibacterium prausnitzii, and other commensal bacteria in the human gut ecosystem. Despite 1300 years of evolution on different continents, modern and ancient Mushuvirus genomes share 97.7% nucleotide identity and suggest a long-term mutualistic relationship between the prophage and its host. Overall, our results demonstrate the feasibility of reconstructing high-quality ancient phage genomes and offer new insights into phage-bacteria interactions, expanding our understanding of the virosphere.

Wall teichoic acid substitution with glucose governs phage susceptibility of Staphylococcus epidermidis

The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits inter-species horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and of horizontal gene transfer and it will help in the design of anti-staphylococcal phage therapies.

Molecular cross-talk between Sa3int phages and their Staphylococcus aureus host

As a major opportunistic pathogen of human and animals Staphylococcus aureus asymptomatically colonizes the nasal cavity, but is also a leading cause of life-threatening acute and chronic infections. More than 90% of the human nasal isolates of S. aureus were found to carry Sa3int phages, which integrate as prophages into the bacterial hlb gene disrupting the expression of an important virulence factor. The virulence factor-encoding genes carried by the Sa3-phages are all highly human-specific and probably essential for bacterial survival in the human host. However, how the S. aureus host modulates the life cycle of those temperate phages remains largely unknown (1). Our data suggest that this regulation is strain specific, with certain S. aureus strains being more prone than others to support either a lysogenic or a lytic life cycle (2). We constructed S. aureus single lysogens with integrated Sa3int prophages and found significant differences in phage transfer rates between different strains. Based on this finding, strains were grouped into low and high transfer strains. To get a more precise picture of the regulatory circuits we constructed replication deficient mutants, performed differential RNAseq and analysed a set of mutant strains. Comparison of different mutants in phenotypic assays revealed roles of known global regulators of S. aureus, in phage infection and replication regulation. By transcriptional start site prediction we identified promoter-regions that are differentially active in high and low transfer strains and identified putative transcriptional regulators on phage and host site by pull down assays. Overall, our results suggest a more prominent role of the host background regulatory processes in different growth phases and strains than previously assumed.1. Rohmer C, Wolz C. The Role of hlb-Converting Bacteriophages in Staphylococcus aureus Host Adaption. Microb Physiol. 2021;31(2):109-122. doi:10.1159/0005166452. Rohmer C, Dobritz R, Tuncbilek-Dere D, et al. Influence of Staphylococcus aureus Strain Background on Sa3int Phage Life Cycle Switches. Viruses. 2022;14(11):2471. Published 2022 Nov 8. doi:10.3390/v14112471

Translation at the Phage Nucles in ΦKZ

Bacteriophages must seize control of the host gene expression machinery to promote their own protein synthesis. Since the bacterial hosts are armed with numerous anti-phage defence systems, it is essential that mechanisms of host takeover act immediately upon infection. Although individual proteins that modulate components of the bacterial gene expression apparatus have been described in several different phages, systematic approaches which capture the phage&quot;s arsenal for immediate targeting of host transcription and translation processes have been lacking. In particular, there are no known phage factors that associate directly with host ribosomes to modulate protein synthesis. Here, we take an integrative high-throughput approach to uncover numerous new proteins encoded by the jumbo phage ΦKZ that target the gene expression machinery of the Gram-negative human pathogen Pseudomonas aeruginosa immediately upon infection. By integrating biochemical and structural analyses, we identify a conserved phage factor that associates with the large ribosomal subunit by binding the 5S ribosomal RNA. This highly abundant factor is amongst the earliest ΦKZ proteins expressed after infection and stays bound to ribosomes during the entire translation cycle. Our study provides a general strategy to decipher molecular components of phage-mediated host take-over and argues that phage genomes represent a large discovery space for proteins that modulate the host gene expression machinery. Gerovac et al. 2023 bioRxiv

Oral Session
Biology of Microbial Viruses

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Oral SessionBiology of Microbial Viruses

Appointment

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Topic

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Oral Session
Biology of Microbial Viruses