Here, there, and everywhere: How pathogenic<i>Escherichia coli</i>sense and respond to gastrointestinal biogeography

Woodward, Sarah E.; Krekhno, Zakhar

doi:10.1111/cmi.13107

Cited by 31 publications

(20 citation statements)

References 136 publications

(173 reference statements)

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“…The molecular obstacle(s) that make polarized non-transformed IECs more challenging to invade than typical cell lines constitutes an intriguing area for further enquiry. The prolonged bacterial attachment also suggests an additional biologically relevant epithelial colonization stage in the infection cycle of Salmonella , in apparent analogy to attaching-effacing enteropathogens that use the epithelial surface as a primary colonization niche (60).…”

Section: Discussionmentioning

confidence: 99%

High definition DIC imaging uncovers transient stages of pathogen infection cycles on the surface of human adult stem cell-derived intestinal epithelium

Rijn

Eriksson

Grüttner

et al. 2021

Preprint

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Interactions between individual pathogenic microbes and host tissues involve fast and dynamic processes that ultimately impact the outcome of infection. Using live-cell microscopy, these dynamics can be visualized to study e.g. microbe motility, binding and invasion of host cells, and intra-host-cell survival. Such methodology typically employs confocal imaging of fluorescent tags in tumor-derived cell line infections on glass. This allows high-definition imaging, but poorly reflects the host tissues physiological architecture and may result in artifacts. We developed a method for live-cell imaging of microbial infection dynamics on human adult stem cell-derived intestinal epithelial cell (IEC) layers. These IEC monolayers are grown in alumina membrane chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows sub-second visualization of both microbial and epithelial surface ultrastructure at high resolution without using fluorescent reporters. We employed this technology to probe the behavior of two model pathogens, Salmonella enterica Typhimurium (Salmonella) and Giardia intestinalis (Giardia), at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium, showing that Salmonella adheres to the IEC surface for prolonged periods before host-cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. This method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. Thereby, this work describes a powerful, generalizable, and relatively inexpensive approach to study dynamic pathogen interactions with IEC surfaces at high resolution and under near-native conditions.

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Section: Discussionmentioning

confidence: 99%

High definition DIC imaging uncovers transient stages of pathogen infection cycles on the surface of human adult stem cell-derived intestinal epithelium

Rijn

Eriksson

Grüttner

et al. 2021

Preprint

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“…Oligonucleotide primers used in this study. 1 All primers were designed in this study except for pNLP10_F/R (21) 2 Restriction sites are underlined, enzymes indicated in primer name.…”

Section: Table S3 Average Cps/od600 Of Three Biological Replicates 1-hour Post-resuspension In Lbmentioning

confidence: 99%

“…and high-glucose DMEM with MOPS for cpxP-, yebE-, ygiB-, bssR-, and htpX-lux reporter plasmids. 1 Values (CPS/OD600) are the same as those represented in Figure 1A 2 P-value indicates significant difference between wild-type and ΔcpxRA mutant cells grown in the same condition (LB or HG-DMEM), Student's t-test Reporter activity was plotted relative to OD600, and data represents the mean and standard deviation of three biological replicate cultures.…”

Section: Table S3 Average Cps/od600 Of Three Biological Replicates 1-hour Post-resuspension In Lbmentioning

confidence: 99%

Simulated colonic fluid replicates the in vivo growth capabilities of Citrobacter rodentium mutants and highlights a critical role for the Cpx envelope stress response in mediating stressors encountered in the gastrointestinal tract

Gilliland¹,

Gavino

Gruenheid

et al. 2021

Preprint

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Citrobacter rodentium is an attaching and effacing (A/E) pathogen used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. While in the host, C. rodentium must adapt to stresses in the gastrointestinal tract such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression and is necessary for C. rodentium pathogenesis in mice. To investigate genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, RNA-Seq, SILAC, and microarray data from previous research was mined and the genes yebE, ygiB, bssR, and htpX were confirmed to be strongly upregulated by the presence of CpxRA using lux reporter constructs. To determine the function of these genes in vivo, knockout mutants were tested in C57Bl/6J and C3H/HeJ mice. Although none of the mutants exhibited marked virulence phenotypes, the ΔcpxRA mutant had reduced colonization and attenuated virulence, as previously determined. We also found that the absence of the Cpx ESR resulted in higher expression of the LEE master regulator, ler. In addition, we determined that the ΔcpxRA mutant had a growth defect in medium simulating the colon, as did several of the mutants bearing deletions in Cpx-upregulated genes. Overall, these results indicate that the ΔcpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.

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“…Importantly, the transcription of LEEassociated genes is known to be extensively regulated by distinct transcriptional regulators such as Ler and specific environmental conditions (temperature, pH, iron, ammonium, calcium, bicarbonate, quorum-sensing signaling etc.) [5,6,8]. The other major EHEC virulence factor is the Shiga toxin Stx.…”

Section: Introductionmentioning

confidence: 99%

Transcriptional analysis reveals specific niche factors and response to environmental stresses of enterohemorrhagic Escherichia coli O157:H7 in bovine digestive contents

et al. 2021

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Background Enterohemorrhagic Escherichia coli (EHEC) are responsible for severe diseases in humans, and the ruminant digestive tract is considered as their main reservoir. Their excretion in bovine feces leads to the contamination of foods and the environment. Thus, providing knowledge of processes used by EHEC to survive and/or develop all along the bovine gut represents a major step for strategies implementation. Results We compared the transcriptome of the reference EHEC strain EDL933 incubated in vitro in triplicate samples in sterile bovine rumen, small intestine and rectum contents with that of the strain grown in an artificial medium using RNA-sequencing (RNA-seq), focusing on genes involved in stress response, adhesion systems including the LEE, iron uptake, motility and chemotaxis. We also compared expression of these genes in one digestive content relative to the others. In addition, we quantified short chain fatty acids and metal ions present in the three digestive contents. RNA-seq data first highlighted response of EHEC EDL933 to unfavorable physiochemical conditions encountered during its transit through the bovine gut lumen. Seventy-eight genes involved in stress responses including drug export, oxidative stress and acid resistance/pH adaptation were over-expressed in all the digestive contents compared with artificial medium. However, differences in stress fitness gene expression were observed depending on the digestive segment, suggesting that these differences were due to distinct physiochemical conditions in the bovine digestive contents. EHEC activated genes encoding three toxin/antitoxin systems in rumen content and many gene clusters involved in motility and chemotaxis in rectum contents. Genes involved in iron uptake and utilization were mostly down-regulated in all digestive contents compared with artificial medium, but feo genes were over-expressed in rumen and small intestine compared with rectum. The five LEE operons were more expressed in rectum than in rumen content, and LEE1 was also more expressed in rectum than in small intestine content. Conclusion Our results highlight various strategies that EHEC may implement to survive in the gastrointestinal environment of cattle. These data could also help defining new targets to limit EHEC O157:H7 carriage and shedding by cattle.

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Here, there, and everywhere: How pathogenicEscherichia colisense and respond to gastrointestinal biogeography

Cited by 31 publications

References 136 publications

High definition DIC imaging uncovers transient stages of pathogen infection cycles on the surface of human adult stem cell-derived intestinal epithelium

High definition DIC imaging uncovers transient stages of pathogen infection cycles on the surface of human adult stem cell-derived intestinal epithelium

Simulated colonic fluid replicates the in vivo growth capabilities of Citrobacter rodentium mutants and highlights a critical role for the Cpx envelope stress response in mediating stressors encountered in the gastrointestinal tract

Transcriptional analysis reveals specific niche factors and response to environmental stresses of enterohemorrhagic Escherichia coli O157:H7 in bovine digestive contents

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