Flagella, Type I Fimbriae and Curli of Uropathogenic Escherichia coli Promote the Release of Proinflammatory Cytokines in a Coculture System

Background. Urinary tract infections (UTIs) are a public health problem in Mexico, and uropathogenic Escherichia coli (UPEC) is one of the main etiological agents. Flagella, type I fimbriae, and curli promote the ability of these bacteria to successfully colonize its host. Aim. This study aimed to determine whether flagella-, type I fimbriae-, and curli-expressing UPEC induces the release of proinflammatory cytokines in an established coculture system. Methods. The fliC, fimH, and csgA genes by UPEC strain were disrupted by allelic replacement. Flagella, type I fimbriae, and curli were visualized by transmission electron microscopy (TEM). HTB-5 (upper chamber) and HMC-1 (lower chamber) cells cocultured in Transwell® plates were infected with these UPEC strains and purified proteins. There was adherence to HTB-5 cells treated with different UPEC strains and they were quantified as colony-forming units (CFU)/mL. Results. High concentrations of IL-6 and IL-8 were induced by the FimH and FliC proteins; however, these cytokines were detected in low concentrations in presence of CsgA. Compared with UPEC CFT073, CFT073ΔfimH, CFT073ΔfimHΔfliC, and CFT073ΔcsgAΔfimH strains significantly reduced the adherence to HTB-5 cells. Conclusion. The FimH and FliC proteins are involved in IL-6 and IL-8 release in a coculture model of HTB-5 and HMC-1 cells.

Insights into the structure of Escherichia coli outer membrane as the target for engineering microbial cell factories

Escherichia coli is generally used as model bacteria to define microbial cell factories for many products and to investigate regulation mechanisms. E. coli exhibits phospholipids, lipopolysaccharides, colanic acid, flagella and type I fimbriae on the outer membrane which is a self-protective barrier and closely related to cellular morphology, growth, phenotypes and stress adaptation. However, these outer membrane associated molecules could also lead to potential contamination and insecurity for fermentation products and consume lots of nutrients and energy sources. Therefore, understanding critical insights of these membrane associated molecules is necessary for building better microbial producers. Here the biosynthesis, function, influences, and current membrane engineering applications of these outer membrane associated molecules were reviewed from the perspective of synthetic biology, and the potential and effective engineering strategies on the outer membrane to improve fermentation features for microbial cell factories were suggested.

“Polar mutagenesis of bacterial transcriptional units using Cas12a”

Bacterial genes are often organized in functionally related transcriptional units or operons. One such example is the fimAICDFGH operon, which codes for type I fimbriae in Escherichia coli. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in the fim operon. Cas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 30%, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Our results showed that some of the obtained mutants, including one with a single base substitution at the fim locus, had decreased mRNA levels of fimA, suggesting that the regulation of the fim operon was disrupted. We corroborated the polar effect of these mutants by phenotypic assays and quantitative PCR, showing up to a 43 fold decrease in expression of genes downstream fimA. We believe this strategy could be useful in engineering the transcriptional shut-down of multiple genes in one single step. For bio-production in E. coli, this opens the possibility of inhibiting competing metabolic routes.

Escherichia colicultures maintain stable subpopulation structure during long-term evolution

How genetic variation is generated and maintained remains a central question in evolutionary biology. When presented with a complex environment, microbes can take advantage of genetic variation to exploit new niches. Here we present a massively parallel experiment where WT and repair-deficient (∆mutL)Escherichia colipopulations have evolved over 3 y in a spatially heterogeneous and nutritionally complex environment. Metagenomic sequencing revealed that these initially isogenic populations evolved and maintained stable subpopulation structure in just 10 mL of medium for up to 10,000 generations, consisting of up to five major haplotypes with many minor haplotypes. We characterized the genomic, transcriptomic, exometabolomic, and phenotypic differences between clonal isolates, revealing subpopulation structure driven primarily by spatial segregation followed by differential utilization of nutrients. In addition to genes regulating the import and catabolism of nutrients, major polymorphisms of note included insertion elements transposing intofimE(regulator of the type I fimbriae) and upstream ofhns(global regulator of environmental-change and stress-response genes), both known to regulate biofilm formation. Interestingly, these genes have also been identified as critical to colonization in uropathogenicE. coliinfections. Our findings illustrate the complexity that can arise and persist even in small cultures, raising the possibility that infections may often be promoted by an evolving and complex pathogen population.

Effective anti-adhesives of uropathogenic Escherichia coli

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are among the most common infectious diseases in humans. Due to their frequent occurrence in the community and nosocomial settings, as well as the development of resistance to the commonly prescribed antimicrobial agents, an enormous financial burden is placed on healthcare systems around the world. Therefore, novel approaches to the prevention and treatment of UTIs are needed. Although UPEC may harbour a plethora of virulence factors, type I fimbriae and P pili are two of the most studied adhesive organelles, since the attachment to host cells in the urinary tract is a crucial step towards infection. Design of receptor analogues that competitively bind to UPEC surface adhesins placed at the top of pili organelles led to the development of anti-adhesive drugs that are increasingly recognized as important and promising alternatives to antibiotic treatment of UTIs.

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.

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

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.

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

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.