Discovering Yersinia–Host Interactions by Tissue Dual RNA-Seq

Kusmierek, Maria; Heroven, Ann Kathrin; Beckstette, Michael; Nuss, Aaron M.; Dersch, Petra

doi:10.1007/978-1-4939-9541-7_8

Cited by 9 publications

(6 citation statements)

References 21 publications

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“…The csrA gene was amplified (primers listed in Table S2 under “CsrA-his tag construct”), digested with NcoI/XhoI, and cloned into matching sites in pET28A (Novagen) to generate pET28:: csrA-his 6 , which was transformed into the E. coli strain, Bl21λDE3 pLysS. Cultures were induced as previously described ( 75 ). Cell pellets were resuspended in protein buffer (100 mM Tris‐HCl, 300 mM NaCl, pH 7.5, 20 mM imidazole), 26 U/ml Benzonase (Sigma), and one tablet of cOmplete Mini EDTA-free protease inhibitor cocktail (Roche) per 10 ml buffer and then lysed by sonication.…”

Section: Methodsmentioning

confidence: 99%

“…Cell pellets were resuspended in protein buffer (100 mM Tris‐HCl, 300 mM NaCl, pH 7.5, 20 mM imidazole), 26 U/ml Benzonase (Sigma), and one tablet of cOmplete Mini EDTA-free protease inhibitor cocktail (Roche) per 10 ml buffer and then lysed by sonication. Protein was purified from the supernatant using affinity chromatography as previously described ( 75 ). Relevant fractions were dialyzed with a 10,000 molecular weight cutoff (MWCO) Slide-A-Lyzer G2 dialysis cassette (Thermo Fisher Scientific) in dialysis buffer (protein buffer without imidazole).…”

Section: Methodsmentioning

confidence: 99%

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CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA

Silva-Rohwer

Held

Sagawa

et al. 2021

mBio

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Yersinia pestis , the bacterial agent of bubonic plague, produces a c-di-GMP-dependent biofilm-mediated blockage of the flea vector foregut to facilitate its transmission by flea bite. However, the intricate molecular regulatory processes that underlie c-di-GMP-dependent biofilm formation and, thus, biofilm-mediated blockage in response to the nutritional environment of the flea are largely undefined.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA

Silva-Rohwer

Held

Sagawa

et al. 2021

mBio

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“…This regulatory pathway could allow the bacteria to control T6SS4 expression in response to various different carbon sources/nutrients as well as antimicrobial peptides, low pH, and ions such as Mg 2+ which are sensed by the two-component systems. Moreover, CsrA seems to promote reciprocal regulation of the Yersinia T6SS4 and Ysc-T3SS; the latter was recently shown to be activated by CsrA [59].…”

Section: Discussionmentioning

confidence: 98%

RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression

et al. 2020

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Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.

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“…We show that in this model system, in contrast to small-animal models, S. Typhimurium infection is restricted to the epithelial layer and does not spread into the vascular compartment, thereby mimicking human disease. Dual transcriptome sequencing (dual RNA-seq), which comprehensively profiles host and pathogen gene expression during bacterial infections, has been successfully applied to infected cell line-based, two-dimensional (2D) monocultures (reviewed in reference 24) and mouse models of infection (25)(26)(27). For the first time, we here applied dual RNA-seq to a 3D tissue model to chart mRNA and noncoding RNA expression changes in the communicating, purified host cell types (intestinal epithelial cells [IECs], endothelial cells, monocytes, NK cells) and in Salmonella.…”

mentioning

confidence: 99%

An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection

Schulte

Schweinlin

Westermann

et al. 2020

mBio

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A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens. IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen’s infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.

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Discovering Yersinia–Host Interactions by Tissue Dual RNA-Seq

Cited by 9 publications

References 21 publications

CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA

CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA

RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression

An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection

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