Biology subjects

Fahlgren, A.

Publications and source records attributed to Fahlgren, A..

6 recordsLinked to original sources

bioRxiv2025

6S RNA facilitates bacterial virulence and adaptation at the epithelial barrier

Enteropathogenic bacteria must adapt dynamically to the complex gastrointestinal environment to successfully colonize host tissue and evade immune defenses. Using Yersinia pseudotuberculosis as a model, we performed in vivo spatial transcriptomics to investigate bacterial gene expression as it translocates from cecal lumen to associated lymphoid tissue in the mouse intestine. By optimizing bacterial RNA enrichment, we achieved near-complete transcriptome coverage and identified compartment-specific transcriptional profiles. Bacteria in lymphoid tissue exhibited elevated expression of virulence-associated type III secretion system (T3SS) genes and markers of increased replication, alongside a higher plasmid copy number. Oxygen availability emerged as an environmental cue for T3SS induction. Importantly, we discovered a role for the non-coding 6S RNA in accelerating virulence gene expression. Tissue-localized bacteria had significantly upregulated 6S RNA levels, and deletion of the ssrS gene encoding 6S RNA impaired T3SS gene expression and effector secretion. Further, spatial analyses of bacterial gene expression in foci of infected lymphoid tissue revealed heterogeneous expression patterns with significantly elevated expression of T3SS and 6S RNA in bacteria located close to surrounding phagocytes. Together these findings demonstrate that Y. pseudotuberculosis undergoes rapid transcriptional reprogramming upon epithelial barrier crossing coordinated by environmental sensing, where 6S RNA accelerates tissue colonization by promoting efficient expression of T3SS.

microbiology↗

bioRxiv2025

Tartrate-resistant acid phosphatase (TRAP/ACP5) sex-specifically regulates bone maintenance in old mice, but the anabolic effects of mechanical loading is regulated in a sex-independent way

Tartrate-resistant acid phosphatase (TRAP/ACP5), primarily known as an osteoclast marker, has emerged as a critical regulator of skeletal integrity, regulating sex-specific bone growth, and bones response to mechanical load in young adult male mice. In this study, we investigated the sex-specific roles of TRAP in bone structure and response to mechanical stimuli in old (19-month-old) wild-type (WT) and TRAP-deficient (TRAP-/-) mice using micro-computed tomography, serum bone turnover markers, in vivo axial mechanical loading, and in vitro mechanotransduction assays. Our findings revealed that TRAP-/- mice of both sexes maintained shorter tibiae than WT mice independent of sex. Notably, male, but not female, TRAP-/- mice have increased trabecular bone volume fraction and cortical bone area compared to WT, indicative of disrupted bone remodelling processes in male mice. Interestingly, TRAP-deficiency substantially impaired the anabolic bone response to mechanical loading, affecting both trabecular and cortical compartments in both sexes, indicating that when challenged, TRAP is important for bone formation also in female mice. Mechanical stimulation in vitro of hematopoietic progenitor cells from WT and TRAP-/- mice revealed that the increased ATP-release in response to mechanical stimulation was only disrupted in male mice, while mechanically induced increase in osteoclast formation was inhibited in TRAP-/- mice of both sexes. These results highlight the importance of TRAP in maintaining trabecular architecture and cortical bone in male mice and underscore its critical function in mediating adaptive responses to mechanical loading of both sexes, during aging. Future investigations should focus on elucidation of TRAP-dependent pathways as potential therapeutic targets to counteract age-related deficits in bone adaptation and remodelling.

developmental biology↗

bioRxiv2023

A Two-Step Activation Mechanism Enables Mast Cells to Differentiate their Response between Extracellular and Invasive Enterobacterial Infection

Mast cells (MCs) localize to mucosal tissues and contribute to innate immune defenses against infection. How MCs sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other closely related enterobacteria, we here demonstrate that MCs can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and MCs colocalize in the Salmonella-infected mouse gut. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure LPS, causes a slow and modest induction of MC cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human MCs triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to promote the full-blown MC response. Supernatants from S.Tm-infected MCs boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that MCs can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

immunology↗

bioRxiv2022

In vitro and in vivo infection models reveal connection between periplasmic protease Prc and alternative peptidoglycan synthase PBP3SAL in Salmonella enterica serovar Typhimurium

A hallmark in salmonellosis is the ability of the bacteria to proliferate within host cells. Most notably, Salmonella proliferates within professional phagocytes in a vacuolar compartment. During proliferation Salmonella has to build new cell wall, but how this is regulated within the intraphagosomal niche is not known. Here we show that genetically inactivating the periplasmic protease Prc, involved in cleaving peptidoglycan-processing enzymes, results in decreased fitness in macrophage-like RAW264.7 cells and in BALB/c mice, and in a decreased tolerance to redox stress. All these prc mutant phenotypes were conditional depending on pbp3sal, a recently defined paralogue for ftsl coding for the essential penicillin binding protein 3. These phenotypic connections between Prc and PBP3SAL adds to the phenotypes governed by Prc, and possibly adds PBP3SAL to the pool of target proteins involved in cell wall homeostasis that are regulated by Prc.

microbiology↗

bioRxiv2020

Spatiotemporal Variations in Growth Rate and Virulence Plasmid Copy Number during Yersinia pseudotuberculosis Infection

Pathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Y. pseudotuberculosis up-regulates the virulence plasmid copy number (PCN) during infection and the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the current study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyers Patches and caecum during the clonal invasive phase of the infection, while the fastest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues, and will be readily applicable to other infection models. ImportanceStudying pathogenic bacteria proliferating inside infected hosts is challenging using traditional methods, especially the transit and reversible genetic events. The bacteria are effectively diluted by the overwhelming number of host cells present in infected tissues. Using an innovative droplet digital PCR (ddPCR) approach, we have determined the virulence plasmid copy number (PCN) variations and growth rates of Yersinia during the course of infection in a mouse model. Here, we show that both the virulence plasmid copy number and bacterial growth rates display spatiotemporal variations in mice during infection. We demonstrate that the peak-to-trough ratio can be used as a proxy for determining the growth rate of invasive bacterial pathogen during infection, and ddPCR as the method of choice for quantifying DNA in host-pathogen interaction context. This proof-of-concept ddPCR approach can be easily applied for any bacterial pathogens and any infection models, for analysis of PCN, growth dynamics and bacterial loads.

microbiology↗

bioRxiv2020

Genome-scale mapping reveals complex regulatory activities of RpoN in Yersinia pseudotuberculosis

RpoN, an alternative sigma factor commonly known as sigma 54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function in this pathogen. RpoN was found essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping genome-wide associations of Y. pseudotuberculosis RpoN using chromatin immunoprecipitation coupled with next-generation sequencing identified an RpoN-binding motif located at 103 inter- and intragenic sites on both sense and anti-sense strands. Deletion of rpoN had a large impact on gene expression, including down-regulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with locations of the binding sites implicated around 130 genes or operons potentially activated or repressed by RpoN. Mutagenesis of selected intergenic binding sites confirmed both positive and negative regulatory effects of RpoN binding. Corresponding mutations of intragenic sense sites had less impact on associated gene expression. Surprisingly, mutating intragenic sites on the anti-sense strand commonly reduced expression of genes encoded by the corresponding sense strand. IMPORTANCEThe alternative sigma factor, RpoN ({sigma} 54), which is widely distributed in eubacteria have been implicated to control gene expression of importance for numerous functions including virulence. Proper responses to host environments are crucial for bacteria to establish infection and regulatory mechanisms involved are therefore of high interest for development of future therapeutics. Little is known about the function of RpoN in the intestinal pathogen Y. pseudotuberculosis and we therefore investigated its regulatory role in this pathogen. This regulator was indeed found to be critical for establishment of infection in mice, likely involving its requirement for motility and biofilm formation. The RpoN regulon involved both activating and suppressive effects on gene expression which could be confirmed with mutagenesis of identified binding sites. This is the first of its kind study of RpoN in Y. pseudotuberculosis revealing complex regulation of gene expression involving both productive and silent effects of its binding to DNA providing important information about RpoN regulation in enterobacteria.

microbiology↗