Showing papers by "Iain G. Duggin published in 2023"

The parasitic lifestyle of an archaeal symbiont

Abstract: DPANN Archaea are a diverse group of organisms typically characterised by small cells and reduced genomes. To date, all cultivated DPANN Archaea are ectosymbionts that require direct cell contact with an archaeal host species for proliferation. However, the dynamics of DPANN – host interactions and the impacts of these interactions on the host species are poorly understood. Here, we show that one DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. Our data also suggest that these interactions involve invasion of the host cell by the nanohaloarchaeon. This is the first reported instance of such a predatory-like lifestyle amongst Archaea and indicates that some DPANN Archaea may interact with host populations in a manner similar to viruses.

Diversity and Potential Multifunctionality of Archaeal CetZ Tubulin-like Cytoskeletal Proteins

Abstract: Tubulin superfamily (TSF) proteins are widespread, and are known for their multifaceted roles as cytoskeletal proteins underpinning many basic cellular functions, including morphogenesis, division, and motility. In eukaryotes, tubulin assembles into microtubules, a major component of the dynamic cytoskeletal network of fibres, whereas the bacterial homolog FtsZ assembles the division ring at midcell. The functions of the lesser-known archaeal TSF proteins are beginning to be identified and show surprising diversity, including homologs of tubulin and FtsZ as well as a third archaea-specific family, CetZ, implicated in the regulation of cell shape and possibly other unknown functions. In this study, we define sequence and structural characteristics of the CetZ family and CetZ1 and CetZ2 subfamilies, identify CetZ groups and diversity amongst archaea, and identify potential functional relationships through analysis of the genomic neighbourhoods of cetZ genes. We identified at least three subfamilies of orthologous CetZ proteins in the archaeal class Halobacteria, including CetZ1 and CetZ2 as well as a novel uncharacterized subfamily. CetZ1 and CetZ2 were correlated to one another as well as to cell shape and motility phenotypes across diverse Halobacteria. Among other known CetZ clusters in orders Archaeoglobales, Methanomicrobiales, Methanosarcinales, and Thermococcales, an additional uncharacterized group from Archaeoglobales and Methanomicrobiales is affiliated strongly with Halobacteria CetZs, suggesting that they originated via horizontal transfer. Subgroups of Halobacteria CetZ2 and Thermococcales CetZ genes were found adjacent to different type IV pili regulons, suggesting potential utilization of CetZs by type IV systems. More broadly conserved cetZ gene neighbourhoods include nucleotide and cofactor biosynthesis (e.g., F420) and predicted cell surface sugar epimerase genes. These findings imply that CetZ subfamilies are involved in multiple functions linked to the cell surface, biosynthesis, and motility.

Extracellular vesicles of Euryarchaeida: precursor to eukaryotic membrane trafficking

Abstract: Since their discovery, extracellular vesicles (EVs) have changed our view on how organisms interact with their extracellular world. EVs are able to traffic a diverse array of molecules across different species and even domains, facilitating numerous functions. In this study, we investigate EV production in Haloferax volcanii, as representative for Euryarchaeida. We uncover that EVs enclose RNA, with specific transcripts preferentially enriched, including those with regulatory potential, and conclude that EVs can act as an RNA communication system between haloarchaea. We demonstrate the key role of an EV-associated Ras superfamily GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. Ras superfamily GTPases are key players in eukaryotic intracellular vesicle formation and trafficking mechanisms that have been crucial for the emergence of Eukaryotes. Therefore, we propose that archaeal EV formation could reveal insights into the origin of the compartmentalized eukaryotic cell.

Improving the genetic system for Halorubrum lacusprofundi to allow in-frame deletions

Abstract: Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, Antarctica. Hrr. lacusprofundi is commonly used to study adaptation to cold environments and thereby a potential source for biotechnological products. Additionally, in contrast to other haloarchaeal model organisms, Hrr. lacusprofundi is also susceptible to a range of different viruses and virus-like elements, making it a great model to study virus-host interactions in a cold-adapted organism. A genetic system has previously been reported for Hrr. lacusprofundi; however, it does not allow in-frame deletions and multiple gene knockouts. Here, we report the successful generation of uracil auxotrophic (pyrE2) mutants of two strains of Hrr. lacusprofundi. Subsequently, we attempted to generate knockout mutants using the auxotrophic marker for selection. However, surprisingly, only the combination of the auxotrophic marker and antibiotic selection allowed the timely and clean in-frame deletion of a target gene. Finally, we show that vectors established for the model organism Haloferax volcanii are deployable for genetic manipulation of Hrr. lacusprofundi, allowing the use of the portfolio of genetic tools available for H. volcanii in Hrr. lacusprofundi.

Direct visualization of bacterial multispecies infection and symbiotic interactions within human bladder epithelial cells

Abstract: Urinary tract infections (UTIs) are a worsening global health problem. Multispecies bacterial UTIs are common but are poorly recognized and understood. Here we developed host cell co-infection models combined with high resolution imaging to compare single- and multi-species infections with common uropathogens, uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis. We demonstrate cooperation and direct interactions specifically between UPEC and E. faecalis within host cells that corresponded to an increased frequency of host cell infection. 3D imaging showed UPEC rods orientated perpendicular to the major host cell axis within single-species intracellular bacterial communities (IBCs), whereas UPEC cells often enveloped E. faecalis in multispecies IBCs. Furthermore, during dispersal from host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF) in multispecies infections. These first insights into intra-species synergies during UTIs at a single cell level should provide research pathways towards better diagnosis and treatment.