Biology subjects

Luallen, R. J.

Publications and source records attributed to Luallen, R. J..

7 recordsLinked to original sources

bioRxiv2025

A type III secretion system is required for Bordetella atropi host cell invasion in vivo

Bordetella atropi is an intracellular bacterial pathogen that infects the intestinal epithelia of the nematode host Oscheius tipulae. We previously showed that the bacteria use filamentation as a novel cell-to-cell spreading mechanism once inside the intestinal cell. However, how the bacteria invade the host cells and what factors contribute to B. atropi infection process remain unknown. In this study, we investigate the roles of type III (T3SS) and type VI secretion systems (T6SS) in B. atropi pathogenesis, which are employed by many bacterial pathogens, both extracellular and intracellular, to deliver effectors that manipulate host physiology to their advantage. We found that the two T6SSs encoded in B. atropi genome played no obvious roles in the invasion or intracellular spreading. In contrast, a T3SS was required for intestinal cell invasion. T3SS mutants showed loss of host cell protrusions from the apical surface that normally engulf invading wild type bacteria, as seen by both electron microscopy and confocal fluorescent microscopy. These protrusions bear morphological similarities to membrane ruffles triggered by the T3SS-mediated invasion seen in other pathogens such as Salmonella and Shigella spp. Additionally, we conducted dual transcriptomics and saw upregulation of T3SS in vivo, along with several putative effectors and the virulence regulator BvgS of the genus Bordetellae. We knocked out these effector candidates and found that deletion of one of these genes, deiA (decreased invasion protein A), leads to a reduction in the number of invasion events and overall percentage of infected animals in the population. In addition, deletion of the virulence regulator bvgS resulted in a complete loss of B. atropi invasion, suggesting it may regulate T3SS for host cell invasion.

microbiology↗

bioRxiv2025

The virulence regulator bvgS is required for nutrient-induced filamentation in Bordetella avium

Bacteria can change morphology in response to stressors and changes in their environment, including infection of a host. We previously identified the bacterial species, Bordetella atropi, which uses nutrient-induced filamentation as a novel mechanism for cell-to-cell spreading in the intestinal epithelial cells of a nematode host. To further investigate the conservation of nutrient-induced filamentation in Bordetellae, we utilized the turkey-infecting species Bordetella avium which filaments in vitro when switched from a standard growth media to an enriched media. We conducted a selection-based filamentation screen with B. avium and isolated two independent non-filamentous mutants that failed to filament in highly enriched media. These mutants contained different alleles in bvgS, the sensor in the two-component master virulence regulator (BvgAS) conserved across the Bordetella genus. To investigate the role of bvgS in nutrient-induced filamentation, we conducted transcriptomics and found that bvgS mutation resulted in loss of responsiveness to highly-enriched media, especially in genes related to nutrient uptake and metabolism. The most dysregulated gene in the bvgS mutant encoded for succinyl-CoA:acetate CoA-transferase (SCACT) and we were able to regulate filamentation with exogenous metabolites up and downstream of this enzyme. These data suggest that bvgS regulates nutrient-induced filamentation by controlling metabolic capacity. Overall, we found that the virulence regulator bvgS is required for nutrient-induced filamentation in B. avium, suggesting there may be conservation in Bordetellae for utilizing this morphological change as a virulence phenotype.

microbiology↗

bioRxiv2022

Genomic and phenotypic evolution of nematode-infecting microsporidia

Microsporidia are a large phylum of intracellular parasites that can infect most types of animals. Species in the Nematocida genus can infect nematodes including Caenorhabditis elegans, which has become an important model to study mechanisms of microsporidia infection. To understand the genomic properties and evolution of nematode-infecting microsporidia, we sequenced the genomes of nine species of microsporidia, including two genera, Enteropsectra and Pancytospora, without any previously sequenced genomes. Core cellular processes, including metabolic pathways, are mostly conserved across genera of nematode-infecting microsporidia. Each species encodes unique proteins belonging to large gene families that are likely used to interact with host cells. Most strikingly, we observed one such family, NemLGF1, is present in both Nematocida and Pancytospora species, suggesting horizontal gene transfer between species from different genera. To understand how Nematocida phenotypic traits evolved, we measured the host range, tissue specificity, spore size, and polar tube length of several species in the genus. Our phylogenetic analysis shows that Nematocida is composed of two groups of species with distinct traits and that species with longer polar tubes infect multiple tissues. Together, our work details both genomic and trait evolution between related microsporidia species and provides a useful resource for further understanding microsporidia evolution and infection mechanisms. Author SummaryMicrosporidia are microbial parasites that can infect many animals. Nematodes have become a useful system to study microsporidia as these animals are commonly infected by microsporidia and these infections can be easily studied in a laboratory environment. To better understand how microsporidia evolve and change their properties as they infect different hosts, we sequenced the genomes of nine microsporidia species which infect nematodes. We found that metabolic pathways were mostly conserved between different clades of these species. Surprisingly, we found a family of proteins predicted to facilitate host interactions that is present in two distinct genera, suggesting that these genes were transferred between microsporidia species. We also determined the hosts and tissues that these microsporidia can infect as well as morphological properties of the microsporidia. We show that these properties are quite variable in related microsporidia species. Our results provide insight into the evolution of microsporidia and also provide a resource for studying microsporidia infections in nematodes.

microbiology↗

bioRxiv2022

High-throughput phenotyping of C. elegans wild isolates reveals specific resistance and susceptibility traits to infection by distinct microsporidia species

Animals are under constant selective pressure from a myriad of diverse pathogens. Microsporidia are ubiquitous animal parasites, but the influence they exert on shaping animal genomes is mostly unknown. Using multiplexed competition assays, we measured the impact of four different species of microsporidia on 22 wild isolates of Caenorhabditis elegans. This resulted in the identification and confirmation of 13 strains with significantly altered population fitness profiles under infection conditions. One of these identified strains, JU1400, is sensitive to an epidermal-infecting species by lacking tolerance to infection. JU1400 is also resistant to an intestinal-infecting species and can specifically recognize and destroy this pathogen. Genetic mapping of JU1400 demonstrates that these two opposing phenotypes are caused by separate loci. Transcriptional analysis reveals the JU1400 sensitivity to epidermal microsporidia infection results in a response pattern that shares similarity to toxin-induced responses. In contrast, we do not observe JU1400 intestinal resistance being regulated at the transcriptional level. The transcriptional response to these four microsporidia species is conserved, with C. elegans strain-specific differences in potential immune genes. Together, our results show that phenotypic differences to microsporidia infection amongst C. elegans are common and that animals can evolve species-specific genetic interactions.

genomics↗

bioRxiv2022

RNA fluorescence in situ hybridization (FISH) to visualize microbial colonization and infection in the Caenorhabditis elegans intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. This protocol introduces RNA fluorescence in situ hybridization (FISH) as a tool used for the identification, visualization, and quantification of the microbes within the intestines of C. elegans. FISH probes that label the highly abundant small subunit ribosomal RNA can produce a bright signal for bacteria and microsporidian cells, and similar probes can be used to label viral RNA. FISH probes can be ordered from a commercial source as single-stranded DNA end-labeled with fluorophores. One limitation is that FISH may not provide robust signal against low copy targets, although signal can be boosted by using multiple probes (so-called single-molecule FISH). FISH staining involves collecting colonized or infected animals, washing to eliminate external contamination, followed by fixation in either paraformaldehyde or acetone. After fixation, FISH probes are incubated with samples to allow for the hybridization of probes to the desired target. To remove excess background, the animals are washed again, and then examined on microscope slides or using automated approaches. Overall, this protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available. SUMMARYGut microbiome bacteria and intestinal intracellular pathogens, like the Orsay virus and microsporidia, are often found associated with wild Caenorhabditis nematodes. This protocol presents RNA FISH as a method for the detection, quantification, and identification of colonizing or infectious microbes within the context of intact C. elegans nematodes.

microbiology↗

bioRxiv2021

Bacterial filamentation is an in vivo mechanism for cell-to-cell spreading

Intracellular pathogens are challenged with limited space and resources while replicating in a single host cell. Mechanisms for direct invasion of neighboring host cells have been discovered in cell culture, but we lack an understanding of how bacteria directly spread from cell-to-cell in vivo. Here, we describe the discovery of a bacterial species that uses filamentation as an in vivo mechanism for intracellular spreading between the intestinal epithelial cells of its host, the rhabditid nematode Oscheius tipulae. In vitro and in vivo filamentation by this bacterium, Bordetella atropi, requires a highly conserved nutrient-sensing pathway used by divergent bacteria to detect rich conditions and inhibit the divisome. Thus, B. atropi uses a distinct mechanism for cell-to-cell spreading by coopting a pathway that normally regulates bacterial cell size to trigger filamentation inside host cells.

microbiology↗

bioRxiv2019

A newly defined cullin-RING ubiquitin ligase promotes thermotolerance as part of the Intracellular Pathogen Response in C. elegans

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode C. elegans called the Intracellular Pathogen Response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other ubiquitin ligase components. Here we use co-immunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4 and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that co-immunoprecipitate with CUL-6 and find that FBXA-158 promotes thermotolerance. In summary, we have defined the three core components and an F-box adaptor of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.\n\nSignificance StatementIntracellular pathogen infection in the nematode Caenorhabditis elegans induces a robust transcriptional response as the host copes with infection. This response program includes several ubiquitin ligase components that are predicted to function in protein quality control. In this study, we show that these infection-induced ubiquitin ligase components form a protein complex that promotes increased tolerance of acute heat stress, an indicator of improved protein homeostasis capacity. These findings show that maintaining protein homeostasis may be a critical component of a multifaceted approach allowing the host to deal with stress caused by intracellular infection.

biochemistry↗