Transcriptomic profiling of high-density Giardia foci encysting in the murine proximal intestine

AbstractGiardiais a highly prevalent, understudied protistan parasite causing significant diarrheal disease worldwide. Its life cycle consists of two stages: infectious cysts ingested from contaminated food or water sources, and motile trophozoites that colonize and attach to the gut epithelium, later encysting to form new cysts that are excreted into the environment. Current understanding of parasite physiology in the host is largely inferred from transcriptomic studies usingGiardiagrown axenically or in co-culture with mammalian cell lines. The dearth of information about the diversity of host-parasite interactions occurring within distinct regions of the gastrointestinal tract has been exacerbated by a lack of methods to directly and non-invasively interrogate disease progression and parasite physiology in live animal hosts. By visualizingGiardiainfections in the mouse gastrointestinal tract using bioluminescent imaging (BLI) of tagged parasites, we recently showed that parasites colonize the gut in high-density foci that may cause localized pathology to the epithelium. Encystation is also initiated in these foci throughout the entire course of infection, yet how the physiology of parasites within high-density foci in the host gut differs from that of cells in laboratory culture is unclear. Here we use BLI to precisely select parasite samples from high-density foci in the proximal intestine to interrogatein vivo Giardiagene expression in the host. Relative to axenic culture, we noted significantly higher expression (> 10-fold) of oxidative stress, membrane transporter, and metabolic and structural genes associated with encystation in the high-density foci. These differences in gene expression within parasite foci in the host may reflect physiological changes associated with high-density growth in localized regions of the gut. We also identified and verified six novel cyst-specific proteins, including new components of the cyst wall that were highly expressed in these foci. Ourin vivotranscriptome data support an emerging view that parasites encyst early in localized regions in the gut, possibly as a consequence of nutrient limitation, and also impact local metabolism and physiology.

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Giardia colonizes and encysts in high density foci in the murine small intestine

10.1101/080226 ◽

2016 ◽

Cited By ~ 2

Author(s):

NR Barash ◽

C Nosala ◽

JK Pham ◽

SG Mclnally ◽

S Gourguechon ◽

...

Keyword(s):

Small Intestine ◽

Gastrointestinal Tract ◽

Diarrheal Disease ◽

Laboratory Culture ◽

High Density ◽

Infection Dynamics ◽

Proximal Small Intestine ◽

Entire Duration ◽

Duration Of Infection

AbstractGiardiais a highly prevalent, yet understudied protistan parasite causing diarrheal disease worldwide. Hosts ingestGiardiacysts from contaminated sources. In the gastrointestinal tract, cysts excyst to become motile trophozoites, colonizing and attaching to the gut epithelium. Trophozoites later differentiate into infectious cysts that are excreted and contaminate the environment. Due to the limited accessibility of the gut, the temporospatial dynamics of giardiasis in the host is largely inferred from laboratory culture and thus may not mirrorGiardiaphysiology in the host. Here we have developed bioluminescent imaging (BLI) to directly interrogate and quantify thein vivotemporospatial dynamics of giardiasis, thereby providing an improved murine model to evaluate anti-Giardiadrugs. Using BLI, we determined that parasites primarily colonize the proximal small intestine non-uniformly in high-density foci. By imaging encystation-specific bioreporters, we show that encystation initiates shortly after inoculation and continues throughout the entire duration of infection. Encystation also initiates in high-density foci in the proximal small intestine, and high-density laboratory cultures of parasites are also stimulated to encyst. This work overturns the assumption that parasites encyst later during infection as they are dislodged and travel through the colon. We suggest that these high-density regions of parasite colonization likely result in localized pathology to the epithelium, and encystation occurs when trophozoites reach a threshold density due to local nutrient depletion. This more accurate visualization of giardiasis redefines the dynamics ofin vivo Giardialife cycle, paving the way for future mechanistic studies of density-dependent parasitic processes in the host.SignificanceGiardiais a single-celled parasite causing both acute and chronic diarrheal disease in over one billion people worldwide. Due to limited access to the site of infection in the gastrointestinal tract, our understanding of the dynamics ofGiardiainfections in the host has remained limited, and largely inferred from laboratory culture. To better understand giardiasis in the host, we developed imaging methods to quantifyGiardiaexpressing bioluminescent physiological reporters in live mice. We discovered that parasites primarily colonize and encyst in the proximal small intestine in discrete, high-density foci. Furthermore, this work provides evidence of a parasite density-based threshold for the differentiation ofGiardiainto cysts in the host. These findings overturn existing paradigms of giardiasis infection dynamics in the host.

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Giardia Colonizes and Encysts in High-Density Foci in the Murine Small Intestine

mSphere ◽

10.1128/msphere.00343-16 ◽

2017 ◽

Vol 2 (3) ◽

Cited By ~ 14

Author(s):

N. R. Barash ◽

C. Nosala ◽

J. K. Pham ◽

S. G. McInally ◽

S. Gourguechon ◽

...

Keyword(s):

Small Intestine ◽

Gastrointestinal Tract ◽

Animal Models ◽

Diarrheal Disease ◽

Laboratory Culture ◽

High Density ◽

Proximal Small Intestine ◽

High Parasite Density ◽

High Parasite

ABSTRACT Giardia is a single-celled parasite causing significant diarrheal disease in several hundred million people worldwide. Due to limited access to the site of infection in the gastrointestinal tract, our understanding of the dynamics of Giardia infections in the host has remained limited and largely inferred from laboratory culture. To better understand Giardia physiology and colonization in the host, we developed imaging methods to quantify Giardia expressing bioluminescent physiological reporters in two relevant animal models. We discovered that parasites primarily colonize and encyst in the proximal small intestine in discrete, high-density foci. We also show that high parasite density contributes to encystation initiation. Giardia lamblia is a highly prevalent yet understudied protistan parasite causing significant diarrheal disease worldwide. Hosts ingest Giardia cysts from contaminated sources. In the gastrointestinal tract, cysts excyst to become motile trophozoites, colonizing and attaching to the gut epithelium. Trophozoites later differentiate into infectious cysts that are excreted and contaminate the environment. Due to the limited accessibility of the gut, the temporospatial dynamics of giardiasis in the host are largely inferred from laboratory culture and thus may not mirror Giardia physiology in the host. Here, we have developed bioluminescent imaging (BLI) to directly interrogate and quantify the in vivo temporospatial dynamics of Giardia infection, thereby providing an improved murine model to evaluate anti-Giardia drugs. Using BLI, we determined that parasites primarily colonize the proximal small intestine nonuniformly in high-density foci. By imaging encystation-specific bioreporters, we show that encystation initiates shortly after inoculation and continues throughout the duration of infection. Encystation also initiates in high-density foci in the proximal small intestine, and high density contributes to the initiation of encystation in laboratory culture. We suggest that these high-density in vivo foci of colonizing and encysting Giardia likely result in localized disruption to the epithelium. This more accurate visualization of giardiasis redefines the dynamics of the in vivo Giardia life cycle, paving the way for future mechanistic studies of density-dependent parasitic processes in the host. IMPORTANCE Giardia is a single-celled parasite causing significant diarrheal disease in several hundred million people worldwide. Due to limited access to the site of infection in the gastrointestinal tract, our understanding of the dynamics of Giardia infections in the host has remained limited and largely inferred from laboratory culture. To better understand Giardia physiology and colonization in the host, we developed imaging methods to quantify Giardia expressing bioluminescent physiological reporters in two relevant animal models. We discovered that parasites primarily colonize and encyst in the proximal small intestine in discrete, high-density foci. We also show that high parasite density contributes to encystation initiation.

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Multiplexed biosensors for precision bacteria tropism in vivo

10.1101/851311 ◽

2019 ◽

Cited By ~ 2

Author(s):

Tiffany Chien ◽

Tetsuhiro Harimoto ◽

Benjamin Kepecs ◽

Kelsey Gray ◽

Courtney Coker ◽

...

Keyword(s):

Gene Expression ◽

Gastrointestinal Tract ◽

Bacterial Growth ◽

Essential Gene ◽

Logic Gate ◽

Control Mechanisms ◽

Lower Gastrointestinal Tract

AbstractThe engineering of microbes spurs biotechnological innovations, but requires control mechanisms to confine growth within defined environments for translation. Here we engineer bacterial growth tropism to sense and grow in response to specified oxygen, pH, and lactate signatures. Coupling biosensors to drive essential gene expression reveals engineered bacterial localization within upper or lower gastrointestinal tract. Multiplexing biosensors in an AND logic-gate architecture reduced bacterial off-target colonization in vivo.

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Fermented vegetable and fruit extract (OM-X®) stimulates murine gastrointestinal tract cells and RAW264.7 cells in vitro and regulates liver gene expression in vivo

Integrative Molecular Medicine ◽

10.15761/imm.1000266 ◽

2016 ◽

Vol 4 (1) ◽

Author(s):

Koji Wakame ◽

Akifumi Nakata ◽

Keisuke Sato ◽

Yoshihiro Mihara ◽

Muneaki Takahata ◽

...

Keyword(s):

Gene Expression ◽

Gastrointestinal Tract ◽

Raw264.7 Cells ◽

Fruit Extract ◽

Liver Gene

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Gene expression profiles of vitrified in vivo derived 8-cell stage mouse embryos detected by high density oligonucleotide microarrays

Molecular Reproduction and Development ◽

10.1002/mrd.20588 ◽

2006 ◽

Vol 73 (11) ◽

pp. 1380-1392 ◽

Cited By ~ 31

Author(s):

Solomon Mamo ◽

Szilard Bodo ◽

Julianna Kobolak ◽

Zsuzsanna Polgar ◽

Gergely Tolgyesi ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Mouse Embryos ◽

High Density ◽

Oligonucleotide Microarrays ◽

Cell Stage

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cDNA Array Analysis of cag Pathogenicity Island-Associated Helicobacter pylori Epithelial Cell Response Genes

Infection and Immunity ◽

10.1128/iai.69.11.6970-6980.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6970-6980 ◽

Cited By ~ 70

Author(s):

Joanne M. Cox ◽

Christopher L. Clayton ◽

Toshihiko Tomita ◽

Don M. Wallace ◽

Philip A. Robinson ◽

...

Keyword(s):

Gene Expression ◽

Helicobacter Pylori ◽

Epithelial Cells ◽

Cdna Array ◽

Pathogenicity Island ◽

High Density ◽

Differentially Expressed ◽

Gastric Epithelial Cells ◽

H Pylori

ABSTRACT Helicobacter pylori strains containing thecag pathogenicity island (PAI) induce NF-κB activation and interleukin-8 secretion in gastric epithelial cells. The aim of this study was to investigate changes in epithelial gene expression induced by cag PAI-positive and -negative strains ofH. pylori using high-density cDNA array hybridization technology. Radio-labeled cDNA prepared from H. pylori-infected Kato 3 gastric epithelial cells was hybridized to high-density cDNA arrays to identify changes in epithelial gene expression compared to noninfected controls. In vivo expression of selected, differentially expressed genes was examined by reverse transcription-PCR analysis of H. pylori-positive and -negative gastric mucosa. Screening of ca. 57,800 cDNAs identified 208 known genes and 48 novel genes and/or expressed sequence tags of unknown function to be differentially expressed in Kato 3 cells following H. pylori infection. Marked differences in gene expression profiles were observed following cagPAI-positive and cag PAI-negative infection with 15 novel cDNAs and 92 known genes being differentially expressed. H. pylori was found to change the expression of genes encoding growth factors and cytokine/chemokines and their receptors, apoptosis proteins, transcription factors and metalloprotease-disintegrin proteins (ADAMs), and tissue inhibitors of metalloproteinases. Gastric differential expression of selected known genes (amphiregulin and ADAM 10) and a novel gene (HPYR1) was confirmed in vivo in patients with H. pylori infection. Confirmation of the in vivo expression of selected genes demonstrates the usefulness of this approach for investigating pathogen-induced changes in host gene expression.

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RstA Regulation ofClostridioides difficileToxin Production and Sporulation in Phenotypically Diverse Strains

10.1101/774125 ◽

2019 ◽

Author(s):

Adrianne N. Edwards ◽

Ellen G. Krall ◽

Shonna M. McBride

Keyword(s):

Gene Expression ◽

Gastrointestinal Tract ◽

Diarrheal Disease ◽

Toxin Production ◽

Toxin Gene ◽

Disease Symptoms ◽

Clostridioides Difficile ◽

Sequence Dependent ◽

Toxin Gene Expression ◽

Strain Dependent

ABSTRACTThe anaerobic spore-former,Clostridioides difficile, causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores which subsequently germinate within the host gastrointestinal tract. Here, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical forC. difficilepathogenesis, the regulatory links between these two physiological processes are not well understood and are strain-dependent. Previously, we identified a conservedC. difficileregulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin gene transcription in the historical isolate, 630Δerm. To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created anrstAmutant in the epidemic 027 ribotype strain, R20291. RstA affects sporulation and toxin gene expression similarly in R20291, although more robust regulatory effects are observed in this strain than in 630Δerm. Reporter assays measuring transcriptional regulation oftcdR, the sigma factor essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverseC. difficilestrains. We provide evidence that RstA contributes totcdRbistability in R20291 by biasing cells to a toxin-OFF state. Finally, sequence-dependent and strain-dependent differences were evident in RstA negative autoregulation ofrstAtranscription. Our data establish RstA as an important regulator ofC. difficilevirulence traits, and implicate RstA as a contributor to the variety of sporulation and toxin phenotypes observed in distinct isolates.IMPORTANCETwo critical traits ofClostridioides difficilepathogenesis are the production of toxins, which cause disease symptoms, and the formation of spores, which permit survival outside of the gastrointestinal tract. The multifunctional regulator, RstA, promotes sporulation and prevents toxin production in the historical strain, 630Δerm. Here, we show that RstA functions similarly in an epidemic isolate, R20291, although strain-specific effects on toxin andrstAexpression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator, TcdR, contribute to regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates, and may allow strains to differentially control toxin production in response to a variety of signals.

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