Influence of Type I Fimbriae and Fluid Shear Stress on Bacterial Behavior and Multicellular Architecture of Early
                    Escherichia coli
                    Biofilms at Single-Cell Resolution

ABSTRACT Biofilm formation on abiotic surfaces in the food and medical industry can cause severe contamination and infection, yet how biological and physical factors determine the cellular architecture of early biofilms and the bacterial behavior of the constituent cells remains largely unknown. In this study, we examined the specific role of type I fimbriae in nascent stages of biofilm formation and the response of microcolonies to environmental flow shear at the single-cell resolution. The results show that type I fimbriae are not required for reversible adhesion from plankton, but they are critical for the irreversible adhesion of Escherichia coli strain MG1655 cells that form biofilms on polyethylene terephthalate (PET) surfaces. Besides establishing firm cell surface contact, the irreversible adhesion seems necessary to initiate the proliferation of E. coli on the surface. After the application of shear stress, bacterial retention is dominated by the three-dimensional architecture of colonies, independent of the population size, and the multilayered structure could protect the embedded cells from being insulted by fluid shear, while the cell membrane permeability mainly depends on the biofilm population size and the duration of the shear stress. IMPORTANCE Bacterial biofilms could lead to severe contamination problems in medical devices and food processing equipment. However, biofilms are usually studied at a rough macroscopic level; thus, little is known about how individual bacterium behavior within biofilms and the multicellular architecture are influenced by bacterial appendages (e.g., pili/fimbriae) and environmental factors during early biofilm formation. We applied confocal laser scanning microscopy (CLSM) to visualize Escherichia coli microcolonies at a single-cell resolution. Our findings suggest that type I fimbriae are vital to the initiation of bacterial proliferation on surfaces. We also found that the fluid shear stress affects the biofilm architecture and cell membrane permeability of the constituent bacteria in a different way: the onset of the biofilm is linked with the three-dimensional morphology, while membranes are regulated by the overall population of microcolonies.

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Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.00369-16 ◽

2016 ◽

Vol 84 (11) ◽

pp. 3220-3231 ◽

Cited By ~ 9

Author(s):

Kumiko Kurabayashi ◽

Tomohiro Agata ◽

Hirofumi Asano ◽

Haruyoshi Tomita ◽

Hidetada Hirakawa

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Antimicrobial Agents ◽

Host Cells ◽

Type I ◽

Wild Type ◽

Content Type ◽

Type I Fimbriae ◽

Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD , which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs.

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IscR Controls Iron-Dependent Biofilm Formation in Escherichia coli by Regulating Type I Fimbria Expression

Journal of Bacteriology ◽

10.1128/jb.01086-08 ◽

2008 ◽

Vol 191 (4) ◽

pp. 1248-1257 ◽

Cited By ~ 92

Author(s):

Yun Wu ◽

F. Wayne Outten

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Developmental Process ◽

Bacterial Species ◽

Type I ◽

Surface Attachment ◽

Environmental Signals ◽

Regulatory Pathways ◽

E Coli ◽

Type I Fimbriae

ABSTRACT Biofilm formation is a complex developmental process regulated by multiple environmental signals. In addition to other nutrients, the transition metal iron can also regulate biofilm formation. Iron-dependent regulation of biofilm formation varies by bacterial species, and the exact regulatory pathways that control iron-dependent biofilm formation are often unknown or only partially characterized. To address this gap in our knowledge, we examined the role of iron availability in regulating biofilm formation in Escherichia coli. The results indicate that biofilm formation is repressed under low-iron conditions in E. coli. Furthermore, a key iron regulator, IscR, controls biofilm formation in response to changes in cellular Fe-S homeostasis. IscR regulates the FimE recombinase to control expression of type I fimbriae in E. coli. We propose that iron-dependent regulation of FimE via IscR leads to decreased surface attachment and biofilm dispersal under iron-limiting conditions.

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Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms

Applied and Environmental Microbiology ◽

10.1128/aem.01569-16 ◽

2016 ◽

Vol 82 (17) ◽

pp. 5320-5331 ◽

Cited By ~ 16

Author(s):

Cathy L. Abberton ◽

Ludmila Bereschenko ◽

Paul W. J. J. van der Wielen ◽

Cindy J. Smith

Keyword(s):

Escherichia Coli ◽

Shear Stress ◽

Drinking Water ◽

Biofilm Formation ◽

Pipe Wall ◽

Health Concern ◽

Wall Material ◽

Content Type ◽

E Coli ◽

Contamination Events

ABSTRACTEscherichia coliis the most commonly used indicator for fecal contamination in drinking water distribution systems (WDS). The assumption is thatE. colibacteria are of enteric origin and cannot persist for long outside their host and therefore act as indicators of recent contamination events. This study investigates the fate ofE. coliin drinking water, specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of threeE. colistrains (two enteric isolates and one soil isolate) in sterile and nonsterile drinking water microcosms at 8 and 17°C, withT90(time taken for a reduction in cell number of 1 log10unit) values ranging from 17.4 ± 1.8 to 149 ± 67.7 days, using standard plate counts and a series of (reverse transcription-)quantitative PCR [(RT-)Q-PCR] assays targeting 16S rRNA,tuf,uidA, androdAgenes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dynes [dyn] cm−2; BioFlux system; Fluxion); however, cell numbers did not increase when drinking water flowed over the biofilm (P> 0.05 byttest). Finally,E. coliregrowth within drinking water microcosms containing polyethylene PE-100 pipe wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods forE. coli. The results of this work highlight that whenE. colienters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely.IMPORTANCEThe provision of clean, safe drinking water is fundamental to society. WDS deliver water to consumers via a vast network of pipes. E. coliis used as an indicator organism for recent contamination events based on the premise that it cannot survive for long outside its host. A key public health concern therefore arises around the fate ofE. colion entering a WDS; its survival, ability to form a biofilm, and potential for regrowth. In particular, ifE. colibacteria have the ability to incorporate and regrow within the pipe wall biofilm of a WDS, they could reinoculate the water at a later stage. This study sheds light on the fate of environmental and enteric strains ofE. coliin drinking water showing extended survival, the potential for biofilm formation under shear stress, and importantly, that regrowth in the presence of an indigenous microbial community is unlikely.

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Cellular chain formation in Escherichia coli biofilms

Microbiology ◽

10.1099/mic.0.026419-0 ◽

2009 ◽

Vol 155 (5) ◽

pp. 1407-1417 ◽

Cited By ~ 23

Author(s):

Rebecca Munk Vejborg ◽

Per Klemm

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Immunofluorescence Microscopy ◽

Asymptomatic Bacteriuria ◽

Type I ◽

Chain Formation ◽

Biofilm Growth ◽

E Coli ◽

Type I Fimbriae ◽

K 12

In this study we report on a novel structural phenotype in Escherichia coli biofilms: cellular chain formation. Biofilm chaining in E. coli K-12 was found to occur primarily by clonal expansion, but was not due to filamentous growth. Rather, chain formation was the result of intercellular interactions facilitated by antigen 43 (Ag43), a self-associating autotransporter (SAAT) protein, which has previously been implicated in auto-aggregation and biofilm formation. Immunofluorescence microscopy suggested that Ag43 was concentrated at or near the cell poles, although when the antigen was highly overexpressed, a much more uniform distribution was seen. Immunofluorescence microscopy also indicated that other parameters, including dimensional constraints (flow, growth alongside a surface), may also affect the final biofilm architecture. Moreover, chain formation was affected by other surface structures; type I fimbriae expression significantly reduced cellular chain formation, presumably by steric hindrance. Cellular chain formation did not appear to be specific to E. coli K-12. Although many urinary tract infection (UTI) isolates were found to form rather homogeneous, flat biofilms, three isolates, including the prototypic asymptomatic bacteriuria strain, 83972, formed highly elaborate cellular chains during biofilm growth in human urine. Combined, these results illustrate the diversity of biofilm architectures that can be observed even within a single microbial species.

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The Activity of Escherichia coli Chaperone SurA Is Regulated by Conformational Changes Involving a Parvulin Domain

Journal of Bacteriology ◽

10.1128/jb.00889-15 ◽

2016 ◽

Vol 198 (6) ◽

pp. 921-929 ◽

Cited By ~ 22

Author(s):

Garner R. Soltes ◽

Jaclyn Schwalm ◽

Dante P. Ricci ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Conformational Changes ◽

Outer Membrane Proteins ◽

Membrane Integrity ◽

Minor Importance ◽

Type I ◽

Peptidyl Prolyl Isomerase ◽

Content Type ◽

Type I Fimbriae

ABSTRACTThe periplasmic chaperone SurA is critical for the biogenesis of outer membrane proteins (OMPs) and, thus, the maintenance of membrane integrity inEscherichia coli. The activity of this modular chaperone has been attributed to a core chaperone module, with only minor importance assigned to the two SurA peptidyl-prolyl isomerase (PPIase) domains. In this work, we used synthetic phenotypes and covalent tethering to demonstrate that the activity of SurA is regulated by its PPIase domains and, furthermore, that its activity is correlated with the conformational state of the chaperone. When combined with mutations in the β-barrel assembly machine (BAM), SurA mutations resulting in deletion of the second parvulin domain (P2) inhibit OMP assembly, suggesting that P2 is involved in the regulation of SurA. The first parvulin domain (P1) potentiates this autoinhibition, as mutations that covalently tether the P1 domain to the core chaperone module severely impair OMP assembly. Furthermore, these inhibitory mutations negate the suppression of and biochemically stabilize the protein specified by a well-characterized gain-of-function mutation in P1, demonstrating that SurA cycles between distinct conformational and functional states during the OMP assembly process.IMPORTANCEThis work reveals the reversible autoinhibition of the SurA chaperone imposed by a heretofore underappreciated parvulin domain. Many β-barrel-associated outer membrane (OM) virulence factors, including the P-pilus and type I fimbriae, rely on SurA for proper assembly; thus, a mechanistic understanding of SurA function and inhibition may facilitate antibiotic intervention against Gram-negative pathogens, such as uropathogenicEscherichia coli,E. coliO157:H7,Shigella, andSalmonella. In addition, SurA is important for the assembly of critical OM biogenesis factors, such as the lipopolysaccharide (LPS) transport machine, suggesting that specific targeting of SurA may provide a useful means to subvert the OM barrier.

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Microfluidic chip of concentration gradient and fluid shear stress on a single cell level

Chinese Chemical Letters ◽

10.1016/j.cclet.2021.10.026 ◽

2021 ◽

Author(s):

Xuexia Lin ◽

Jianlong Su ◽

Shufeng Zhou

Keyword(s):

Shear Stress ◽

Single Cell ◽

Microfluidic Chip ◽

Concentration Gradient ◽

Fluid Shear Stress ◽

Single Cell Level ◽

Fluid Shear ◽

Cell Level

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Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence

Journal of Experimental Medicine ◽

10.1084/jem.20141514 ◽

2015 ◽

Vol 212 (5) ◽

pp. 633-648 ◽

Cited By ~ 35

Author(s):

Peter Geon Kim ◽

Haruko Nakano ◽

Partha P. Das ◽

Michael J. Chen ◽

R. Grant Rowe ◽

...

Keyword(s):

Shear Stress ◽

Protein Kinase ◽

Protein Kinase A ◽

Fluid Shear Stress ◽

Camp Response Element Binding ◽

Bmp Signaling ◽

Type I ◽

Fluid Shear ◽

Hematopoietic Stem ◽

Kinase A

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta–gonad–mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)–cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA–CREB and BMP pathways in isolated AGM VE-cadherin+ cells from mid-gestation embryos, we demonstrate that PKA–CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA–CREB–BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.

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Activation of Smad2/3 signaling by low fluid shear stress mediates artery inward remodeling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2105339118 ◽

2021 ◽

Vol 118 (37) ◽

pp. e2105339118

Author(s):

Hanqiang Deng ◽

Elizabeth Min ◽

Nicolas Baeyens ◽

Brian G. Coon ◽

Rui Hu ◽

...

Keyword(s):

Shear Stress ◽

Fluid Shear Stress ◽

Transforming Growth Factor ◽

Nuclear Translocation ◽

Transmembrane Protein ◽

Type I ◽

Fluid Shear ◽

Neuropilin 1 ◽

Morphogenetic Protein ◽

Artery Remodeling

Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-β family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-β signaling.

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