| 1. |
- Thorslund, Sara E, et al.
(författare)
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The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function
- 2011
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Ingår i: PLOS ONE. - San Francisco : Public Library of Science. - 1932-6203. ; 6:2, s. e16784-
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Tidskriftsartikel (refereegranskat)abstract
- Many Gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated beta1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.
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| 2. |
- Bakkeren, Erik, et al.
(författare)
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Salmonella persisters promote the spread of antibiotic resistance plasmids in the gut
- 2019
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Ingår i: Nature. - : NATURE PUBLISHING GROUP. - 0028-0836 .- 1476-4687. ; 573:7773, s. 276-280
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of antibiotic-resistant bacteria through mutations or the acquisition of genetic material such as resistance plasmids represents a major public health issue(1,2). Persisters are subpopulations of bacteria that survive antibiotics by reversibly adapting their physiology(3-10), and can promote the emergence of antibiotic-resistant mutants(11). We investigated whether persisters can also promote the spread of resistance plasmids. In contrast to mutations, the transfer of resistance plasmids requires the co-occurrence of both a donor and a recipient bacterial strain. For our experiments, we chose the facultative intracellular entero-pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and Escherichia coli, a common member of the microbiota(12). S. Typhimurium forms persisters that survive antibiotic therapy in several host tissues. Here we show that tissue-associated S. Typhimurium persisters represent long-lived reservoirs of plasmid donors or recipients. The formation of reservoirs of S. Typhimurium persisters requires Salmonella pathogenicity island (SPI)-1 and/or SPI-2 in gut-associated tissues, or SPI-2 at systemic sites. The re-seeding of these persister bacteria into the gut lumen enables the co-occurrence of donors with gut-resident recipients, and thereby favours plasmid transfer between various strains of Enterobacteriaceae. We observe up to 99% transconjugants within two to three days of re-seeding. Mathematical modelling shows that rare re-seeding events may suffice for a high frequency of conjugation. Vaccination reduces the formation of reservoirs of persisters after oral infection with S. Typhimurium, as well as subsequent plasmid transfer. We conclude that-even without selection for plasmid-encoded resistance genes-small reservoirs of pathogen persisters can foster the spread of promiscuous resistance plasmids in the gut.
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| 3. |
- Berggren, Sofia, et al.
(författare)
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ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis
- 2024
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Ingår i: mSphere. - : American Society for Microbiology. - 2379-5042. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Gastrointestinal disease caused by Salmonella enterica is associated with the pathogen's ability to replicate within epithelial cells and macrophages. Upon host cell entry, the bacteria express a type-three secretion system encoded within Salmonella pathogenicity island 2, through which host-manipulating effector proteins are secreted to establish a stable intracellular niche. Transcription of this intracellular virulence program is activated by the PhoPQ two-component system that senses the low pH and the reduced magnesium concentration of host cell vacuoles. In addition to transcriptional control, Salmonella commonly employ RNA-binding proteins (RBPs) and small regulatory RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. ProQ is a globally acting RBP in Salmonella that promotes expression of the intracellular virulence program, but its RNA repertoire has previously been characterized only under standard laboratory growth conditions. Here, we provide a high-resolution ProQ interactome during conditions mimicking the environment of the Salmonella-containing vacuole (SCV), revealing hundreds of previously unknown ProQ binding sites in sRNAs and mRNA 3 ' UTRs. ProQ positively affected both the levels and the stability of many sRNA ligands, some of which were previously shown to associate with the well-studied and infection-relevant RBP Hfq. We further show that ProQ activates the expression of PhoP at the post-transcriptional level, which, in turn, leads to upregulation of the intracellular virulence program.
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| 4. |
- Brännström, Kristoffer, et al.
(författare)
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A Generic Method for Design of Oligomer-Specific Antibodies
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:3, s. e90857-
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e. g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the A beta peptide and alpha-synuclein that are associated with Alzheimer's and Parkinson's diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies.
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| 5. |
- Brännström, Kristoffer, et al.
(författare)
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The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling
- 2009
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 77:3, s. 1144-1154
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Tidskriftsartikel (refereegranskat)abstract
- The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.
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| 6. |
- Di Martino, Maria Letizia, et al.
(författare)
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Barcoded Consortium Infections Resolve Cell Type-Dependent Salmonella enterica Serovar Typhimurium Entry Mechanisms
- 2019
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Ingår i: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial host cell invasion mechanisms depend on the bacterium's virulence factors and the properties of the target cell. The enteropathogen Salmonella enterica serovar Typhimurium (S. Tm) invades epithelial cell types in the gut mucosa and a variety of immune cell types at later infection stages. The molecular mechanism(s) of host cell entry has, however, been studied predominantly in epithelial cell lines. S. Tm uses a type three secretion system (TTSS-1) to translocate effectors into the host cell cytosol, thereby sparking actin ruffle-dependent entry. The ruffles also fuel cooperative invasion by bystander bacteria. In addition, several TTSS-1-independent entry mechanisms exist, involving alternative S. Tm virulence factors, or the passive uptake of bacteria by phagocytosis. However, it remains ill-defined how S. Tm invasion mechanisms vary between host cells. Here, we developed an internally controlled and scalable method to map S. Tm invasion mechanisms across host cell types and conditions. The method relies on host cell infections with consortia of chromosomally tagged wild-type and mutant S. Tm strains, where the abundance of each strain can be quantified by qPCR or amplicon sequencing. Using this methodology, we quantified cooccurring TTSS-1-dependent, cooperative, and TTSS-1-independent invasion events in epithelial, monocyte, and macrophage cells. We found S. Tm invasion of epithelial cells and monocytes to proceed by a similar MOI-dependent mix of TTSS-1-dependent and cooperative mechanisms. TTSS-1-independent entry was more frequent in macrophages. Still, TTSS-1-dependent invasion dominated during the first minutes of interaction also with this cell type. Finally, the combined action of the SopB/SopE/SopE2 effectors was sufficient to explain TTSS-1-dependent invasion across both epithelial and phagocytic cells. IMPORTANCE Salmonella enterica serovar Typhimurium (S. Tm) is a widespread and broad-host-spectrum enteropathogen with the capacity to invade diverse cell types. Still, the molecular basis for the host cell invasion process has largely been inferred from studies of a few selected cell lines. Our work resolves the mechanisms that Salmonellae employ to invade prototypical host cell types, i.e., human epithelial, monocyte, and macrophage cells, at a previously unattainable level of temporal and quantitative precision. This highlights efficient bacterium-driven entry into innate immune cells and uncovers a type III secretion system effector module that dominates active bacterial invasion of not only epithelial cells but also monocytes and macrophages. The results are derived from a generalizable method, where we combine barcoding of the bacterial chromosome with mixed consortium infections of cultured host cells. The application of this methodology across bacterial species and infection models will provide a scalable means to address host-pathogen interactions in diverse contexts.
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| 7. |
- Diard, Médéric, et al.
(författare)
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Antibiotic treatment selects for cooperative virulence of Salmonella typhimurium.
- 2014
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Ingår i: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 24:17
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Tidskriftsartikel (refereegranskat)abstract
- Antibiotics are powerful therapeutics but are not equally effective against all cells in bacterial populations. Bacteria that express an antibiotic-tolerant phenotype ("persisters") can evade treatment [1]. Persisters can cause relapses of the infection after the end of the therapy [2]. It is still poorly understood whether persistence affects the evolution of bacterial virulence. During infections, persisters have been found preferentially at particular sites within the host [3, 4]. If bacterial virulence factors are required to reach such sites, treatment with antibiotics could impose selection on the expression of virulence genes, in addition to their well-established effects on bacterial resistance. Here, we report that treatment with antibiotics selects for virulence and fosters transmissibility of Salmonella Typhimurium. In a mouse model for Salmonella diarrhea, treatment with the broad-spectrum antibiotic ciprofloxacin reverses the outcome of competition between wild-type bacteria and avirulent mutants that can spontaneously arise during within-host evolution [5]. While avirulent mutants take over the gut lumen and abolish disease transmission in untreated mice, ciprofloxacin tilts the balance in favor of virulent, wild-type bacteria. This is explained by the need for virulence factors to invade gut tissues and form a persistent reservoir. Avirulent mutants remain in the gut lumen and are eradicated. Upon cessation of antibiotic treatment, tissue-lodged wild-type pathogens reseed the gut lumen and thereby facilitate disease transmissibility to new hosts. Our results suggest a general principle by which antibiotic treatment can promote cooperative virulence during within-host evolution, increase duration of transmissibility, and thereby enhance the spread of an infectious disease.
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| 8. |
- Diard, Mederic, et al.
(författare)
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Inflammation boosts bacteriophage transfer between Salmonella spp.
- 2017
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 355:6330, s. 1211-1215
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Tidskriftsartikel (refereegranskat)abstract
- Bacteriophage transfer (lysogenic conversion) promotes bacterial virulence evolution. There is limited understanding of the factors that determine lysogenic conversion dynamics within infected hosts. Amurine Salmonella Typhimurium (S.Tm) diarrhea model was used to study the transfer of SopEF, a prophage from S.Tm SL1344, to S.Tm ATCC14028S. Gut inflammation and enteric disease triggered >55% lysogenic conversion of ATCC14028S within 3 days. Without inflammation, SopEF transfer was reduced by up to 10(5)-fold. This was because inflammation (e.g., reactive oxygen species, reactive nitrogen species, hypochlorite) triggers the bacterial SOS response, boosts expression of the phage antirepressor Tum, and thereby promotes free phage production and subsequent transfer. Mucosal vaccination prevented a dense intestinal S.Tm population from inducing inflammation and consequently abolished SopEF transfer. Vaccination may be a general strategy for blocking pathogen evolution that requires disease-driven transfer of temperate bacteriophages.
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| 9. |
- Ek, Viktor, et al.
(författare)
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A motile doublet form of Salmonella Typhimurium diversifies target search behavior at the epithelial surface
- 2022
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Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 117:5, s. 1156-1172
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Tidskriftsartikel (refereegranskat)abstract
- The behaviors of infectious bacteria are commonly studied in bulk. This is effective to define the general properties of a given isolate, but insufficient to resolve subpopulations and unique single-microbe behaviors within the bacterial pool. We here employ microscopy to study single-bacterium characteristics among Salmonella enterica serovar Typhimurium (S.Tm), as they prepare for and launch invasion of epithelial host cells. We find that during the bacterial growth cycle, S.Tm populations switch gradually from fast planktonic growth to a host cell-invasive phenotype, characterized by flagellar motility and expression of the Type-three-secretion-system-1. The indistinct nature of this shift leads to the establishment of a transient subpopulation of S.Tm "doublets"-waist-bearing bacteria anticipating cell division-which simultaneously express host cell invasion machinery. In epithelial cell culture infections, these S.Tm doublets outperform their "singlet" brethren and represent a hyperinvasive subpopulation. Atop both glass and enteroid-derived monolayers, doublets swim along markedly straighter trajectories than singlets, thereby diversifying search patterns and improving the surface exploration capacity of the total bacterial population. The straighter swimming, combined with an enhanced cell-adhesion propensity, suffices to account for the hyperinvasive doublet phenotype. This work highlights bacterial cell length heterogeneity as a key determinant of target search patterns atop epithelia.
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| 10. |
- Ek, Viktor
(författare)
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It Takes Two to Tango : Bacterial heterogeneity and host cell features govern Salmonella infection
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Salmonella enterica serovar Typhimurium (S.Tm) causes enterocolitis with significant worldwide morbidity and mortality. The general aim of this thesis is to investigate variation in host cell invasion mechanisms used by S.Tm across different host cell contexts, as well as the influence of bacterial cell-cell heterogeneity on invasion-relevant S.Tm behaviours. The thesis is divided into four sub-projects, each a section in the presented work.First, a genetic barcoding technique for tracking bacteria in mixed consortium infections was developed and applied to evaluate the dependency on the type three secretion system 1 (T3SS-1) and its effectors for host cell entry. It was found that S.Tm invasion of cultured epithelial cells and monocytes is mainly mediated by T3SS-1, or by cooperative uptake of bystander bacteria. T3SS-1-independent entry was possible in cultured macrophages, although T3SS-1-dependent entry was predominant also there. In fact, active invasion was promoted by the same T3SS-1 effectors in all three cell types. Second, an in-depth comparison of S.Tm infections in cell line cultures and in the mouse gut mucosa in vivo highlighted a “discreet-invasion” modality in vivo, in sharp contrast to the prevailing “ruffle” model for host cell invasion. While ruffle-mediated entry into epithelial cell lines was driven by the T3SS-1 effectors SopBEE2, discreet-invasion into the murine gut absorptive epithelium is driven predominantly by the SipA effector, as well as the SiiE adhesin. Furthermore, discreet-invasion targeted apicolateral “hot spots” near cell-cell junctions, dependent on the local cell neighbourhood, which was further charted in the final two sub-projects. Third, single-bacterium characteristics among S.Tm populations were studied using time-lapse microscopy. The indistinct nature of the shift from growth to virulence induction spawned a transient subpopulation of S.Tm “doublets”, cell division intermediates also exhibiting pronounced swimming and host cell invasion aptitude. The longer doublets also displayed a different search pattern during near-surface swimming, highlighting bacterial cell length heterogeneity as a key determinant of target search atop epithelia. Fourth, the morphogenic impact of clinically relevant antibiotics were explored, in context of the previous data. Even S.Tm bacteria with the most extreme morphological abnormalities (e.g. highly filamentous or coccoid individuals), induced by chloramphenicol, ciprofloxacin, nitrofurantoin, and meropenem, could robustly swim and invade epithelial host cells. While high concentrations of these antibiotics were effective at suppressing growth and virulence, a range of low, sub-inhibitory concentrations even enhanced host cell invasion capacity and affected the near-surface swimming behaviour among surviving bacteria. In summary, the present investigation highlights the pivotal importance of taking both host cell features and bacterial heterogeneity into account when studying infection processes.
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