In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.

The emergence of multidrug-resistant bacteria poses a significant threat to human health, necessitating a comprehensive understanding of their underlying mechanisms. Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, is frequently associated with multidrug resistance and recurrent infections. To elucidate the mechanism of resistance of UPEC to beta-lactam antibiotics, we generated ampicillin-resistant UPEC strains through continuous exposure to low and high levels of ampicillin in the laboratory, referred to as Low AmpR and High AmpR, respectively. Whole-genome sequencing revealed that both Low and High AmpR strains contained mutations in the marR, acrR, and envZ genes. The High AmpR strain exhibited a single additional mutation in the nlpD gene. Using protein modeling and qRT-PCR analyses, we validated the contributions of each mutation in the identified genes to antibiotic resistance in the AmpR strains, including a decrease in membrane permeability, increased expression of multidrug efflux pump, and inhibition of cell lysis. Furthermore, the AmpR strain does not decrease the bacterial burden in the mouse bladder even after continuous antibiotic treatment in vivo, implicating the increasing difficulty in treating host infections caused by the AmpR strain. Interestingly, ampicillin-induced mutations also result in multidrug resistance in UPEC, suggesting a common mechanism by which bacteria acquire cross-resistance to other classes of antibiotics.

Adherent and invasive Escherichia coli (AIEC) is a pathobiont that is involved in the onset and exacerbation of Crohn\'s disease. Although the inducible expression of virulence traits is a critical step for AIEC colonization in the host, the mechanism underlying AIEC colonization remains largely unclear. We here showed that the two-component signal transduction system CpxRA contributes to AIEC gut competitive colonization by activating type 1 fimbriae expression. CpxRA from AIEC strain LF82 functioned as a transcriptional regulator, as evidenced by our finding that an isogenic cpxRA mutant exhibits reduced expression of cpxP, a known regulon gene. Transcription levels of cpxP in LF82 increased in response to envelope stress, such as exposure to antimicrobials compromising the bacterial membrane, whereas the cpxRA mutant did not exhibit this response. Furthermore, we found that the cpxRA mutant exhibits less invasiveness into host cells than LF82, primarily due to reduced expression of the type 1 fimbriae. Finally, we found that the cpxRA mutant is impaired in gut competitive colonization in a mouse model. The colonization defects were reversed by the introduction of a plasmid encoding the cpxRA gene or expressing the type 1 fimbriae. Our findings indicate that modulating CpxRA activity could be a promising approach to regulating AIEC-involved Crohn\'s disease.

Currently, fresh, unprocessed food has become a relevant element of the chain of transmission of enteropathogenic infections. To survive on a plant surface and further spread the infections, pathogens like Salmonella have to attach stably to the leaf surface. Adhesion, driven by various virulence factors, including the most abundant fim operon encoding type 1 fimbriae, is usually an initial step of infection, preventing physical removal of the pathogen. Adhesion properties of Salmonella\'s type 1 fimbriae and its FimH adhesin were investigated intensively in the past. However, there is a lack of knowledge regarding its role in interaction with plant cells. Understanding the mechanisms and structures involved in such interaction may facilitate efforts to decrease the risk of contamination and increase fresh food safety. Here, we applied Salmonella genome site-directed mutagenesis, adhesion assays, protein-protein interactions, and biophysics methods based on surface plasmon resonance to unravel the role of FimH adhesin in interaction with spinach leaves. We show that FimH is at least partially responsible for Salmonella binding to spinach leaves, and this interaction occurs in a mannose-independent manner. Importantly, we identified a potential FimH receptor as endo-1,3-β-d-Glucanase and found that this interaction is strong and specific, with a dissociation constant in the nanomolar range. This research advances our comprehension of Salmonella\'s interactions with plant surfaces, offering insights that can aid in minimizing contamination risks and improving the safety of fresh, unprocessed foods.

Salmonella enterica is a food-borne pathogen able to cause a wide spectrum of diseases ranging from mild gastroenteritis to systemic infections. During almost all stages of the infection process Salmonella is likely to be exposed to a wide variety of host-derived antimicrobial peptides (AMPs). AMPs are important components of the innate immune response which integrate within the bacterial membrane, thus forming pores which lead ultimately to bacterial killing. In contrast to other AMPs Bactericidal/Permeability-increasing Protein (BPI) displayed only weak bacteriostatic or bactericidal effects towards Salmonella enterica sv. Typhimurium (STM) cultures. Surprisingly, we found that sub-antimicrobial concentrations of BPI fold-containing (BPIF) superfamily members mediated adhesion of STM depending on pre-formed type 1 fimbriae. BPIF proteins directly bind to type 1 fimbriae through mannose-containing oligosaccharide modifications. Fimbriae decorated with BPIF proteins exhibit extended binding specificity, allowing for bacterial adhesion on a greater variety of abiotic and biotic surfaces likely promoting host colonization. Further, fimbriae significantly contributed to the resistance against BPI, probably through sequestration of the AMP before membrane interaction. In conclusion, functional subversion of innate immune proteins of the BPIF family through binding to fimbriae promotes Salmonella virulence by survival of host defense and promotion of host colonization.

Bacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus. We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (∆fimE) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.IMPORTANCEType 6 secretion systems (T6SS) are molecular weapons employed by gram-negative bacteria to eliminate neighboring microbes. T6SS plays a pivotal role as a virulence factor, enabling pathogenic gram-negative bacteria to compete with the established communities to colonize hosts and induce infections. Gaining a deeper understanding of bacterial interactions will allow the development of strategies to control the action of systems such as the T6SS that can manipulate bacterial communities. In this context, we demonstrate that bacteria targeted by T6SS attacks from the enteric pathogen Cronobacter malonaticus, which poses a significant threat to infants, can develop a collective protective mechanism centered on the production of type I fimbriae. These adhesive structures promote the aggregation of bacterial preys and the formation of microcolonies, which protect the cells from T6SS attacks.

Initial stages of Salmonella Typhimurium infection involve a series of coordinated events aimed at reaching, attaching to, and invading host cells. Virulence factors such as flagella, fimbriae, and secretion systems play crucial roles in these events and are regulated in response to the host environment. The first point of contact between the pathogen and host is the intestinal epithelial layer, which normally serves as a barrier against invading pathogens, but can also be an entry site for pathogens. The integrity of this barrier can be modulated by the hypoxic environment of the intestines, created by the presence of trillions of microbes. Variable oxygen concentrations can strongly affect many functions of the gut, including secretion of cytokines and growth factors from the host site and affect the ability of Salmonella to persist, invade, and replicate. In this study, we investigated the first stages of Salmonella Typhimurium infection under hypoxic conditions in vitro and found that low oxygen levels significantly decreased bacterial adhesion. Using adhesion and motility assays, biofilm formation tests, as well as gene expression and cytokine secretion analysis, we identified a hypoxia-specific cross-talk between the expression of type 1 fimbriae and flagella, suggesting that altered flagellin expression levels affect the motility of bacteria and further impact their adhesion level, biofilm formation ability, and innate immune response. Overall, understanding how Salmonella interacts with its variable host environment provides insights into the virulence mechanisms of the bacterium and information regarding strategies for preventing or treating infections. Further research is required to fully understand the complex interplay between Salmonella and its host environment.

BACKGROUND: Avian Escherichia coli (E.coli) type 1 fimbriae adhere to avian tracheal epithelial cells through the FimH protein. However, the adhesion-related antigen is still unknown. The purpose of this study was to analyze the antigenicity of the type 1 fimbrial FimH protein of wild-type avian E. coli, screen antigen epitopes, and prepare monoclonal antibodies (mAbs) that can block the adhesion of avian E. coli.
RESULTS: In this study, the nucleic acid homologies of MG2 (O11), TS12 (O18), and YR5 (O78) with K12 were 97.7%, 99.6%, and 97.7%, respectively, and the amino acid sequence similarity reached 98.7%, 99.3%, and 98.0%, respectively. The epitopes and hydrophilicities of the FimH proteins of these three strains were similar. The more obvious lectin domain epitopes were located at FimH protein positions 111-124 and 154-162. The mAbs 7C2 and 7D8 against these two epitopes were prepared. An adhesion inhibition test showed that 7C2 and 7D8 blocked bacterial adhesion to avian tracheal epithelial cells. The mAb 7C2 against the 111-124 epitope inhibited O78 strain adhesion by 93%, and the mAb 7D8 against the 154-162 epitope inhibited O78 strain adhesion by 49%, indicating that these two epitopes are closely related to the adhesion of type 1 fimbriae. However, only the 111-124 epitope-recognizing mAb 7C2 inhibited bacterial agglutination of erythrocytes, indicating that host cell receptor binding and erythrocyte agglutination are not mediated by the same spatial locations within the FimH protein.
CONCLUSIONS: The results demonstrate that the mAbs 7C2 and 7D8 against FimH protein positions 111-124 and 154-162 could inhibit the adhesion of E.coli to the chicken trachea.

Bacterial interactions with surfaces rely on the coordinated expression of a vast repertoire of surface-exposed adhesins. However, how bacteria dynamically modulate their adhesion potential to achieve successful surface colonization is not yet well understood. Here, we investigated changes in adhesion capacity of an initially poorly adherent Escherichia coli strain using experimental evolution and positive selection for mutations improving adhesion and biofilm formation on abiotic surfaces. We showed that all identified evolved populations and clones acquired mutations located almost exclusively in the lectin domain of fimH, the gene coding for the α-d-mannose-specific tip adhesin of type 1 fimbriae, a key E. coli virulence factor. While most of these fimH mutants showed reduced mannose-binding ability, they all displayed enhanced binding to abiotic surfaces, indicating a trade-off between FimH-mediated specific and nonspecific adhesion properties. Several of the identified mutations were already reported in the FimH lectin domain of pathogenic and environmental E. coli, suggesting that, beyond pathoadaptation, FimH microevolution favoring nonspecific surface adhesion could constitute a selective advantage for natural E. coli isolates. Consistently, although E. coli deleted for the fim operon still evolves an increased adhesion capacity, mutants selected in the ∆fim background are outcompeted by fimH mutants revealing clonal interference for adhesion. Our study therefore provides insights into the plasticity of E. coli adhesion potential and shows that evolution of type 1 fimbriae is a major driver of the adaptation of natural E. coli to colonization.

The pathogenic Escherichia coli can be parsed into specific variants (pathovars) depending on their phenotypic behavior and/or expression of specific virulence factors. These pathogens are built around chromosomally-encoded core attributes and through acquisition of specific virulence genes that direct their interaction with the host. Engagement of E. coli pathovars with CEACAMs is determined both by core elements common to all E. coli as well as extrachromosomally-encoded pathovar-specific virulence traits, which target amino terminal immunoglobulin variable-like (IgV) regions of CEACAMs. Emerging data suggests that engagement of CEACAMs does not unilaterally benefit the pathogen and that these interactions may also provide an avenue for pathogen elimination.