Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.08.05.668709

Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis

Ortiz-Severin, J.·Geoffroy, P.·Aravena, P.·Hödar, C.·Palma, D. E.·Gonzalez, M.·Cambiazo, V.
View original source ↗Export metadata ↓

Abstract

Piscirickettsia salmonis is the causative agent of Salmonid Rickettsial Septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an IPTG-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous gene encoding green fluorescent protein (sfGFP), and the endogenous homolog of the fur gene, whose gene product regulates intracellular iron homeostasis in bacteria. The inducible expression of dcas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains, when the expression of the fur gRNA was induced with IPTG. By RNA-seq we detected a significant increase in the expression of genes encoding the Fe+2 and Fe+3 acquisition systems and iron mobilization in the fur1 knockdown, after IPTG induction. All the genes with over two-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The successful implementation of the Mobile-CRISPRi system in P. salmonis paves the way for systematic analysis of gene function in this pathogen We anticipate that these analyses will be very valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis. ImportanceSalmon Rickettsial Septicemia (SRS) is an infectious disease caused by the marine bacterium Piscirickettsia salmonis. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen P. salmonis, inserting exogenous genes into the bacteriums chromosome to ensure their constitutive and inducible expression, and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Furs relevance to the iron metabolism in the pathogen.

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Ortiz-Severin, J., Geoffroy, P., Aravena, P., Hödar, C., Palma, D. E., Gonzalez, M., Cambiazo, V.. 2025-08-06. Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis. https://doi.org/10.1101/2025.08.05.668709

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Matrix-controlled emergence of biofilm architecture shapes antimicrobial survival

Biofilms are structured microbial communities whose extracellular matrix is widely regarded as a basis of their protection against antimicrobial compounds. Yet how matrix production by individual bacteria gives rise to collective architecture and antimicrobial protection remains poorly understood. Here, we systematically varied expression of the master biofilm regulator csgD in Salmonella enterica and found that increasing matrix production reorganizes biofilms from dense, isotropic packings into sparse, nematically aligned communities by altering cell-cell interactions. By combining experimentally measured biofilm architectures with reaction-diffusion modeling, we show that these structural changes produce distinct patterns of antimicrobial killing, ranging from preferential killing near the liquid-biofilm interface to more uniform killing throughout the community. Consequently, increasing matrix production unexpectedly reduces antimicrobial survival by shifting the biofilm into different transport regimes, while strain-specific physiological differences further modulate antimicrobial depletion. Rather than acting as a passive barrier, EPS therefore shapes antimicrobial susceptibility by reorganizing biofilm architecture and its transport properties. EPS thus provides a physical link between molecular regulation, collective architecture and antimicrobial survival, providing a quantitative framework for understanding how cellular matrix production generates emergent biofilm function.

microbiology↗
bioRxiv2026

Mapping virulence-associated protein interaction networks reveals regulators of thermotolerance in Cryptococcus neoformans

Protein-protein interactions (PPIs) influence critical biological processes in pathogenic microorganisms, such as the human fungal pathogen, Cryptococcus neoformans. Fungal thermotolerance and stress response pathways are key virulence determinants that directly impact pathogen adaptation and survival and the infection process. To establish a comprehensive baseline of PPIs in C. neoformans and explore these interactions to infer functional roles for uncharacterized proteins, we applied size exclusion chromatography coupled with mass spectrometry to the secreted and cellular proteomes of the fungi. As a result, 216 and 1699 unique proteins were identified across 24 secretome and proteome fractions, respectively. The predicted secretome networks included expected proteins associated with vesicles and virulence, indicating a role in extracellular defense. Whereas the cryptococcal proteome highlighted interactions among proteins with defined roles in fungal virulence for protein stability and thermotolerance, including two previously uncharacterized proteins, CNAG_00287 and CNAG_05199, putatively involved in complex formation with heat-shock proteins (HSP). Based on sequence and structure homology, we propose that CNAG_00287 is a tetratricopeptide repeat-containing co-chaperone that modulates Hsp 70 activity and CNAG_05199 functions as a Hsp70. We validated the thermotolerance role of CNAG_00287 in heat-related stress, as its absence significantly impaired fungal growth in nutrient-limited media at 37 {degrees}C. Together, this work resolves virulence-associated PPIs within C. neoformans and reveals new molecular regulators of thermotolerance that underpin fungal pathogenicity.

microbiology↗
bioRxiv2026

Environmental filtering and host identity collectively shape root-associated microbiomes of Ericaceae and ectomycorrhizal plants in fumarole fields

Background Symbiosis with microbes is a key strategy that has enabled plants to colonize extreme environments. Since the benefits conferred by root-associated microbes depend on both environmental conditions and host-microbe combinations, plant adaptation to harsh environments is closely linked to the assembly of root microbial communities. Understanding how environmental and host filtering jointly shape these communities is therefore fundamental to elucidating the mechanisms underlying plant adaptation to extreme environments. Results In this study, we investigated the differentiation of root-associated prokaryotic and fungal communities and individual operational taxonomic units (OTUs) across two contrasting habitats surrounding fumaroles, solfatara-field and forest-edge habitats, and six dominant Ericaceae and ectomycorrhizal plant taxa. Prokaryotic and fungal OTUs rarely exhibited strong preferences for both habitat and host identity. Instead, many of prokaryotic and fungal OTUs specialized to one of these niches, collectively generating root microbial communities differentiated by both factors. Nonetheless, striking specializations in habitat and host niches were observed in the fungal family Hyaloscyphaceae (Helotiales). To gain insight into the evolutionary basis of microbial specialization, we examined phylogenetic signals in preference phenotypes. The resulting weak phylogenetic signals in these preference phenotypes further suggest that this fungal clade has undergone substantial ecological divergence. Conclusion Overall, our findings indicate that root-associated microbial communities in extreme environments are assembled through the accumulation of microbial taxa specialized to either habitat or host, and that strong ecological specialization in fungi can arise with little phylogenetic constraint.

microbiology↗