Quantification of filamentation by uropathogenic Escherichia coli during experimental bladder cell infection by using semi-automated image analysis

Klein, Kasper; Palarasah, Yaseelan; Kolmos, Hans Jørn; Møller‐Jensen, Jakob; Andersen, Thomas Emil

doi:10.1016/j.mimet.2014.12.017

Cited by 28 publications

(30 citation statements)

References 28 publications

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“…Induction of UPEC filamentation via transient DamX overproduction from plasmid pSKdamX occurred only when seeded on a solid surface, thus implying a role for surface sensing and possibly fluid flow, irrespective of whether that surface is biotic (bladder epithelial cells) or abiotic (glass). This dependency of UPEC filamentation on surface growth has been confirmed in other recent studies of UTI89 grown in flow chambers ( 22 , 23 ), implying that the process of filamentation does not restrict itself exclusively to the UPEC infection cascade.…”

Section: Discussionsupporting

confidence: 79%

“…While some studies have aimed at identifying genes involved in bacterial filamentation ( 13 , 19 – 21 ), this study is the first to systematically analyze the UPEC gene expression profiles at each individual phase of its pathogenesis, with the aim of identifying genes that are critical to its morphological differentiation. Using microarray technology and a previously described ( 22 ) flow chamber-based bladder cell infection model, this study not only reaffirms recent reports regarding the importance of a contact surface and fluid flow in UPEC filamentation ( 23 ) but also identifies DamX as a key factor that controls reversible switching between normal rod-shaped and filamentous morphology during infection. While previous studies have linked UPEC filamentation with the SOS response and SulA ( 9 , 12 , 20 ), our present study identifies transient DamX overproduction as a key inducer of filamentation during this cycle.…”

Section: Introductionsupporting

confidence: 86%

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DamX Controls Reversible Cell Morphology Switching in Uropathogenic Escherichia coli

Khandige

Asferg

Rasmussen

et al. 2016

mBio

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The ability to change cell morphology is an advantageous characteristic adopted by multiple pathogenic bacteria in order to evade host immune detection and assault during infection. Uropathogenic Escherichia coli (UPEC) exhibits such cellular dynamics and has been shown to transition through a series of distinct morphological phenotypes during a urinary tract infection. Here, we report the first systematic spatio-temporal gene expression analysis of the UPEC transition through these phenotypes by using a flow chamber-based in vitro infection model that simulates conditions in the bladder. This analysis revealed a novel association between the cell division gene damX and reversible UPEC filamentation. We demonstrate a lack of reversible bacterial filamentation in a damX deletion mutant in vitro and absence of a filamentous response by this mutant in a murine model of cystitis. While deletion of damX abrogated UPEC filamentation and secondary surface colonization in tissue culture and in mouse infections, transient overexpression of damX resulted in reversible UPEC filamentation. In this study, we identify a hitherto-unknown damX-mediated mechanism underlying UPEC morphotypical switching. Murine infection studies showed that DamX is essential for establishment of a robust urinary tract infection, thus emphasizing its role as a mediator of virulence. Our study demonstrates the value of an in vitro methodology, in which uroepithelium infection is closely simulated, when undertaking targeted investigations that are challenging to perform in animal infection models.

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Section: Discussionsupporting

confidence: 79%

Section: Introductionsupporting

confidence: 86%

DamX Controls Reversible Cell Morphology Switching in Uropathogenic Escherichia coli

Khandige

Asferg

Rasmussen

et al. 2016

mBio

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“…The physiological role of this chromosomally encoded toxin family remains unknown, as do the molecular interactions with its target. A change in cell morphology has been previously characterized as a survival mechanism of E. coli present in urinary tract infections (French, Cote, Stokes, Truant, & Brown, ; Justice, Hunstad, Cegelski, & Hultgren, ; Klein, Palarasah, Kolmos, Moller‐Jensen, & Andersen, ), and CIP‐induced filaments were found to give rise to antibiotic‐resistant populations (Bos et al, ). This survival mechanism is linked to pathway activation in response to DNA breaks, thus promoting error‐prone repair and driving evolution (Recacha et al, ; Torres‐Barcelo et al, ; Valencia, Esposito, Spira, Blazquez, & Galhardo, ).…”

Section: Discussionmentioning

confidence: 99%

Expression of different ParE toxins results in conserved phenotypes with distinguishable classes of toxicity

et al. 2019

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Toxin-antitoxin (TA) systems are found on both chromosomes and plasmids. These systems are unique in that they can confer both fatal and protective effects on bacterial cells-a quality that could potentially be harnessed given further understanding of these TA mechanisms. The current work focuses on the ParE subfamily, which is found throughout proteobacteria and has a sequence identity on average of approximately 12% (similarity at 30%-80%). Our aim is to evaluate the equivalency of chromosomally derived ParE toxin activity depending on its bacterial species of origin. Nine ParE toxins were analyzed, originating from six different bacterial species. Based on the resulting toxicity, three categories can be established: ParE toxins that do not exert toxicity under the experimental conditions, toxins that exert toxicity within the first four hours, and those that exert toxicity only after 10-12 hr of exposure. All tested ParE toxins produce a cellular morphologic change from rods to filaments, consistent with disruption of DNA topology. Analysis of the distribution of filamented cells within a population reveals a correlation between the extent of filamentation and toxicity. No membrane septation is visible along the length of the cell filaments, whereas aberrant lipid blebs are evident. Potent ParE-mediated toxicity is also correlated with a hallmark signature of abortive DNA replication, consistent with the inhibition of DNA gyrase. K E Y W O R D SDNA gyrase, DNA topology, filamented morphology, ParE toxin, toxin-antitoxin systems

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“…The drawback is that HTB9 is relatively fragile under high flow conditions and thus requires fixation to be used in the current assay. If live cells are needed to further study UPEC-uroepithelium interactions, the PD07i cell line (Klumpp et al, 2001 ) can be used in flow assays without fixation, as it is more flow resistant (Andersen et al, 2012 ) and stays confluent after prolonged infection experiments in urine flow (Klein et al, 2015 ). PD07i is however a relatively undifferentiated cell more resembling the underlying urothelial cells in the stratified bladder mucosa and, probably for this reason, is less supportive of UPEC adhesion/invasion (own unpublished data).…”

Section: Discussionmentioning

confidence: 99%

“…However, this requires termination of a rather laborious experiment, leaving only a single data point measured per chamber. Furthermore, CFU enumeration of bacteria exposed to in vivo -like stress conditions can be tricky, since bacteria may change morphology, i.e., become filamentous (Klein et al, 2015 ; Khandige et al, 2016 ), or aggregate (Loof et al, 2015 ; Grønnemose et al, 2017 ; Sønderholm et al, 2017 ), or slow down growth speed (Proctor et al, 2006 ), which may affect the accuracy of bacterial quantification by traditional plating techniques.…”

Section: Introductionmentioning

confidence: 99%

A Method for Quantification of Epithelium Colonization Capacity by Pathogenic Bacteria

Pedersen

Grønnemose

Stærk

et al. 2018

Front. Cell. Infect. Microbiol.

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Most bacterial infections initiate at the mucosal epithelium lining the gastrointestinal, respiratory, and urogenital tracts. At these sites, bacterial pathogens must adhere and increase in numbers to effectively breach the outer barrier and invade the host. If the bacterium succeeds in reaching the bloodstream, effective dissemination again requires that bacteria in the blood, reestablish contact to distant endothelium sites and form secondary site foci. The infectious potential of bacteria is therefore closely linked to their ability to adhere to, colonize, and invade epithelial and endothelial surfaces. Measurement of bacterial adhesion to epithelial cells is therefore standard procedure in studies of bacterial virulence. Traditionally, such measurements have been conducted with microtiter plate cell cultures to which bacteria are added, followed by washing procedures and final quantification of retained bacteria by agar plating. This approach is fast and straightforward, but yields only a rough estimate of the adhesive properties of the bacteria upon contact, and little information on the ability of the bacterium to colonize these surfaces under relevant physiological conditions. Here, we present a method in which epithelia/endothelia are simulated by flow chamber-grown human cell layers, and infection is induced by seeding of pathogenic bacteria on these surfaces under conditions that simulate the physiological microenvironment. Quantification of bacterial adhesion and colonization of the cell layers is then performed by in situ time-lapse fluorescence microscopy and automatic detection of bacterial surface coverage. The method is demonstrated in three different infection models, simulating Staphylococcus aureus endothelial infection and Escherichia coli intestinal- and uroepithelial infection. The approach yields valuable information on the fitness of the bacterium to successfully adhere to and colonize epithelial surfaces and can be used to evaluate the influence of specific virulence genes, growth conditions, and antimicrobial treatment on this process.

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Quantification of filamentation by uropathogenic Escherichia coli during experimental bladder cell infection by using semi-automated image analysis

Cited by 28 publications

References 28 publications

DamX Controls Reversible Cell Morphology Switching in Uropathogenic Escherichia coli

DamX Controls Reversible Cell Morphology Switching in Uropathogenic Escherichia coli

Expression of different ParE toxins results in conserved phenotypes with distinguishable classes of toxicity

A Method for Quantification of Epithelium Colonization Capacity by Pathogenic Bacteria

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