Expression Of Fimbriae Research Articles

The role of urease in the acid stress response and fimbriae expression in Klebsiella pneumoniae CG43.

Two urease operons were identified in Klebsiella pneumoniae CG43, ure-1 and ure-2. This study investigates whether a differential regulation of the expression of ure-1 and ure-2 exists and how urease activity influences the acid stress response and expression of type 1 and type 3 fimbriae. The ureA1 and ureA2 gene specific deletion mutants were constructed. Promoter activity was assessed using a LacZ reporter system. The sensitivity to acid stress was determined by assessing the survival after pH 2.5 treatment. The influence on type 1 and type 3 fimbriae expression was assessed using western blotting and mannose-sensitive yeast agglutination and biofilm formation assay, respectively. Bacterial growth analysis in mM9-U or modified Stuart broth revealed that ure-1 was the principal urease system, and ure-2 had a negative effect on ure-1 activity. Deletion of the fur or nac gene had no apparent effect on the activity of Pure1, Pure2-1, and Pure2-2. The Pure2-2 activity was enhanced by deletion of the hns gene. ureA1 deletion increased acid stress sensitivity, whereas the deleting effect of ureA2 was notable without hns. Deletion of ureA1 or ureA2 significantly induced the expression of type 1 fimbriae but decreased MrkA production and biofilm formation. ure-1 is the primary expression system in K. pneumoniae CG43, while ure-2 is active in the absence of hns. Impairment of urease activity increases the sensitivity to acid stress, and the accumulation of urea induces the expression of type 1 fimbriae but represses type 3 fimbriae expression.

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

Five nanometer size highly positive silver nanoparticles are bactericidal targeting cell wall and adherent fimbriae expression

To tackle growing antibiotic resistance (AR) and hospital-acquired infections (HAIs), novel antimicrobials are warranted that are effective against HAIs and safer for human use. We hypothesize that small 5 nm size positively charged nanoparticles could specifically target bacterial cell wall and adherent fimbriae expression, serving as the next generation antibacterial agent. Herein we show highly positively charged, 5 nm amino-functionalized silver nanoparticles (NH2–AgNPs) were bactericidal; highly negatively charged, 45 nm citrate-functionalized AgNPs (Citrate–AgNPs) were nontoxic; and Ag+ ions were bacteriostatic forming honeycomb-like potentially resistant phenotype, at 10 µg Ag/mL in E. coli. Further, adherent fimbriae were expressed with Citrate–AgNPs (0.5–10 µg/mL), whereas NH2–AgNPs (0.5–10 µg/mL) or Ag+ ions (only at 10 µg/mL) inhibited fimbriae expression. Our results also showed no lipid peroxidation in human lung epithelial and dermal fibroblast cells upon NH2–AgNPs treatments, suggesting NH2–AgNPs as a biocompatible antibacterial candidate. Potent bactericidal effects demonstrated by biocompatible NH2–AgNPs and the lack of toxicity of Citrate–AgNPs lend credence to the hypothesis that small size, positively charged AgNPs may serve as a next-generation antibacterial agent, potentially addressing the rising HAIs and patient health and safety.

Regulation of ECP fimbriae-related genes by the transcriptional regulator RcsAB in Klebsiella pneumoniae NTUH-K2044.

Klebsiella pneumoniae is one of the major pathogens causing nosocomial infections. The regulator of capsule synthesis (Rcs) system is a complex signal transduction pathway that is involved in the regulation of virulence factors of K. pneumoniae as an important transcriptional regulator. The RcsAB box-like sequence was found to be present in the promoter-proximal regions of ykgK, one of the ECP fimbriae-related genes, which suggested the expression of ECP fimbriae may be regulated by RcsAB. The ykgK gene in K. pneumoniae has 86% similarity to the ecpR gene in Escherichia coli. Nucleotide sequence alignment revealed a similar ECP fimbriae gene cluster including six genes in K. pneumoniae, which was proved to be on the same operon in this study. The electrophoretic mobility shift assay and DNase I assay, relative fluorescence expression, β-galactosidase activity, and relative gene expression of ykgK in the wild-type and mutant strains were performed to determine the transcriptional regulation mechanism of RcsAB on ECP fimbriae. The mutant ΔykgK and complementary strain ΔykgK/cΔykgK were constructed to complete theGalleria mellonella larvae infection experiment and biofilm formation assay. This study showed that RcsAB binds directly to the promoter region of the ykgK gene to positively regulate ECP fimbriae-related gene clusters, and then positively affect the biofilm formation.

Inhibitory effect of modified atmosphere packaging on Escherichia coli O157:H7 in fresh-cut cucumbers (Cucumis sativus L.) and effectively maintain quality during storage

Modified atmosphere packaging (MAP) can inhibit microbial growth and prolong shelf life of fresh-cut cucumbers. This study compared the effects of different packaging gases on the growth of E. coli O157:H7 and sensory characteristics of fresh-cut cucumbers. Changes in key movement, adhesion, and oxidative stress genes expression of strain under optimal MAP and air were determined. Cell population density, the extracellular carbohydrate complex content and expression of curli fimbriae were evaluated. Results revealed that the growth of E. coli O157:H7 in fresh-cut cucumbers could be effectively inhibited under MAP (atmosphere = 2% O2, 7% CO2, 91% N2), and better maintained the sensory characteristics. Furthermore, the inhibition mechanism was revealed by inhibiting the expression of movement (fliC), adhesion (eaeA) and oxidative stress (rpoS and sodB) genes in E. coli O157:H7, reducing biofilm formation, extracellular carbohydrate production and curli fimbriae expression. Proper MAP can maintain the quality and safety of fresh-cut cucumbers.

Sef fimbria operon construction, expression, and function for direct rapid detection of Salmonella Enteritidis.

Salmonella Enteritidis (SE) causes both horizontal and vertical transmission of diseases in poultry industry and is also one of the main causes of human food poisoning. Sequence analysis of the sef operon of poultry-derived Salmonella serotypes showed the presence of an entire sef operon in SE, whereas only sef pseudogenes were found in Salmonella Gallinarum and Salmonella Pullorum. Subsequently, the sef operon of SE was cloned into the pBR322 plasmid and expressed in a modified Escherichia coli strain SE5000. sef operon expression was demonstrated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, agglutination assay, and transmission electron microscopy. The results showed that SE5000+Sef, but not SE5000+pBR322, could specifically react with SE-positive chicken serum in an agglutination assay, which could be clearly visualized by the naked eye within less than 2 min. In contrast, SE5000+Sef could not be recognized in Salmonella Gallinarum- and Salmonella Pullorum-positive chicken sera. Next, taking advantage of the exclusive presence of an entire sef operon in SE, we set up an agglutination-based detection system to monitor the dynamics of Sef-targeted antibody from SE-infected chicks for 47 days. Using the proposed detection method, SE was readily detectable starting from 2 weeks post-infection. Finally, we compared the proposed SE5000+Sef-based detection system with commercially available agglutination antigen using the classical bacterial isolation and identification procedure as reference. The results showed that the SE5000+Sef system was more consistent with the results of bacterial isolation and identification with almost 100% accuracy. We established a simple, sensitive, and cheap agglutination method for rapid and specific detection of SE-infected chickens, which can facilitate epidemiological investigation and eradication of SE infections. KEY POINTS: • Only the Salmonella Enteritidis serotype expressed Sef fimbriae in chicken infected with SE. • A rapid, large-scale method of detection by the naked eye of detection of SE-infected chicken is presented.

SdiA, a Quorum-Sensing Regulator, Suppresses Fimbriae Expression, Biofilm Formation, and Quorum-Sensing Signaling Molecules Production in Klebsiella pneumoniae

Klebsiella pneumoniae is a Gram-negative pathogen that has become a worldwide concern due to the emergence of multidrug-resistant isolates responsible for various invasive infectious diseases. Biofilm formation constitutes a major virulence factor for K. pneumoniae and relies on the expression of fimbrial adhesins and aggregation of bacterial cells on biotic or abiotic surfaces in a coordinated manner. During biofilm aggregation, bacterial cells communicate with each other through inter- or intra-species interactions mediated by signallng molecules, called autoinducers, in a mechanism known as quorum sensing (QS). In most Gram-negative bacteria, intra-species communication typically involves the LuxI/LuxR system: LuxI synthase produces N-acyl homoserine lactones (AHLs) as autoinducers and the LuxR transcription factor is their cognate receptor. However, K. pneumoniae does not produce AHL but encodes SdiA, an orphan LuxR-type receptor that responds to exogenous AHL molecules produced by other bacterial species. While SdiA regulates several cellular processes and the expression of virulence factors in many pathogens, the role of this regulator in K. pneumoniae remains unknown. In this study, we describe the characterization of sdiA mutant strain of K. pneumoniae. The sdiA mutant strain has increased biofilm formation, which correlates with the increased expression of type 1 fimbriae, thus revealing a repressive role of SdiA in fimbriae expression and bacterial cell adherence and aggregation. On the other hand, SdiA acts as a transcriptional activator of cell division machinery assembly in the septum, since cells lacking SdiA regulator exhibited a filamentary shape rather than the typical rod shape. We also show that K. pneumoniae cells lacking SdiA regulator present constant production of QS autoinducers at maximum levels, suggesting a putative role for SdiA in the regulation of AI-2 production. Taken together, our results demonstrate that SdiA regulates cell division and the expression of virulence factors such as fimbriae expression, biofilm formation, and production of QS autoinducers in K. pneumoniae.

Exacerbation of AMD Phenotype in Lasered CNV Murine Model by Dysbiotic Oral Pathogens.

Emerging evidence underscores an association between age-related macular degeneration (AMD) and periodontal disease (PD), yet the biological basis of this linkage and the specific role of oral dysbiosis caused by PD in AMD pathophysiology remains unclear. Furthermore, a simple reproducible model that emulates characteristics of both AMD and PD has been lacking. Hence, we established a novel AMD+PD murine model to decipher the potential role of oral infection (ligature-enhanced) with the keystone periodontal pathogen Porphyromonas gingivalis, in the progression of neovasculogenesis in a laser-induced choroidal-neovascularization (Li-CNV) mouse retina. By a combination of fundus photography, optical coherence tomography, and fluorescein angiography, we documented inflammatory drusen-like lesions, reduced retinal thickness, and increased vascular leakage in AMD+PD mice retinae. H&E further confirmed a significant reduction of retinal thickness and subretinal drusen-like deposits. Immunofluorescence microscopy revealed significant induction of choroidal/retinal vasculogenesis in AMD+PD mice. qPCR identified increased expression of oxidative-stress, angiogenesis, pro-inflammatory mediators, whereas antioxidants and anti-inflammatory genes in AMD+PD mice retinae were notably decreased. Through qPCR, we detected Pg and its fimbrial 16s-RrNA gene expression in the AMD+PD mice retinae. To sum-up, this is the first in vivo study signifying a role of periodontal infection in augmentation of AMD phenotype, with the aid of a pioneering AMD+PD murine model established in our laboratory.

An Attenuated Escherichia coli K88ac LT(S63K)ΔSTb Efficiently Provides Protection Against Enterotoxigenic Escherichia coli in the Mouse Model.

To develop an attenuated vaccine candidate against K88ac enterotoxigenic Escherichia coli (ETEC), a novel Escherichia coli (E. coli) K88ac LT(S63K)ΔSTb with LT(S63K) mutation and ST1 deletion was generated using site mutagenesis and λ-Red homologous recombination based on wild paternal ETEC strain C83902. E. coli K88ac LT(S63K)ΔSTb showed very similar fimbriae expression and growth kinetics to the wild strain C83902, but it was significantly attenuated according to the results of a rabbit ligated ileal loop assay and mouse infection study. Oral inoculation with E. coli K88ac LT(S63K)ΔSTb stimulated the mucosa immune response and induced the secretion of IgA to K88ac in the intestines in mice. A challenge experiment revealed that the attenuated strain provided efficient protection against C83902 in the following 7 days and at the 24th day post-inoculation, suggesting that the attenuated isolate could act as an ecological protectant and vaccine in preventing K88ac ETEC.

Molecular Characterization and Biofilm Formation of Escherichia coli from Vegetables

Escherichia coli can cause diarrheal and extraintestinal illnesses in humans. Diarrheagenic E. coli can be transmit to human through consumption of contaminated food, including vegetables. Biofilm produced by E. coli during food processing plays a role in development of foodborne illnesses. Vegetables have often been involved in diarrheal E. coli infections. A total of 40 E. coli isolates from vegetables were tested to determine biofilm formation at 12°C, 25°C and 37°C by the crystal violet and MTT assays. All isolates were performed for the production of curli fimbriae and cellulose associated with biofilm formation on Congo red agar. Biofilm formation at 37°C, 25°C and 12°C was detected in 87.5%, 70% and 70% of the isolates, respectively. Biofilm formation among the E. coli isolates using the crystal violet and MTT assays showed a statistically significant difference between 12°C and 25°C as well as 12°C and 37°C (p < 0.05). However, no significant difference between 25 and 37°C (p > 0.05) was obtained. Three different morhotypes (bdar, pdar and saw) were identified based on the expression of curli fimbriae and cellulose. The incidence of the bdar morhotype was 27.5% and 50% at 25°C and 37°C, respectively. Prevalence of the pdar morphotype was 50% and 70% at 25°C and 37°C, respectively. At 25°C, only one isolate (2.5%) showed the saw morphotype. All isolates tested expressed curli fimbriae or cellulose, only three of which were non-biofilm producer using the crystal violet assay. This study demonstrated that the presence of biofilm forming E.coli isolates in vegetables may cause a risk to human health and food safety.

Functional Insights From KpfR, a New Transcriptional Regulator of Fimbrial Expression That Is Crucial for Klebsiella pneumoniae Pathogenicity

Although originally known as an opportunistic pathogen, Klebsiella pneumoniae has been considered a worldwide health threat nowadays due to the emergence of hypervirulent and antibiotic-resistant strains capable of causing severe infections not only on immunocompromised patients but also on healthy individuals. Fimbriae is an essential virulence factor for K. pneumoniae, especially in urinary tract infections (UTIs), because it allows the pathogen to adhere and invade urothelial cells and to form biofilms on biotic and abiotic surfaces. The importance of fimbriae for K. pneumoniae pathogenicity is highlighted by the large number of fimbrial gene clusters on the bacterium genome, which requires a coordinated and finely adjusted system to control the synthesis of these structures. In this work, we describe KpfR as a new transcriptional repressor of fimbrial expression in K. pneumoniae and discuss its role in the bacterium pathogenicity. K. pneumoniae with disrupted kpfR gene exhibited a hyperfimbriated phenotype with enhanced biofilm formation and greater adhesion to and replication within epithelial host cells. Nonetheless, the mutant strain was attenuated for colonization of the bladder in a murine model of urinary tract infection. These results indicate that KpfR is an important transcriptional repressor that, by negatively controlling the expression of fimbriae, prevents K. pneumoniae from having a hyperfimbriated phenotype and from being recognized and eliminated by the host immune system.

Identification of Crp as a novel regulator of the Std fimbrial expression in Salmonella.

The Salmonella enterica serovar Typhi genome contains 14 putative fimbrial systems. The Std fimbriae belong to the chaperone-usher family and its regulation has not been investigated in S. Typhi. Several regulators of Std were previously identified in the closely related serovar Typhimurium. We hypothesize that regulators of S. Typhimurium may be shared with S. Typhi, but that several other regulators remain to be discovered. Here, we describe the role of more than 50 different candidate regulators on std expression. Three types of regulators were investigated: known regulators in S. Typhimurium, in silico predicted regulators and virulence/metabolic regulators. Expression of std was determined in the regulator mutants and compared with the wild-type strain. Overall, 21 regulator mutations affect std promoter expression. The role of Crp, a newly identified factor for std expression, was further investigated. Crp acted as an activator of std expression on a distal region of the std promoter region. Together, our results demonstrate the major influence of Crp as a novel transcriptional factor on std promoter expression and later production of Std fimbriae in Salmonella.

In vitro type 1 fimbriae expression and antibiotic resistance by uro-pathogenic bacterial isolates from Human Immunodeficiency Virus infected patients

Background: Somebacteria possess fimbriae that support their adherence to host cell surface, colonization of human epithelial cells and resistance to antibiotics. Aim: The expression of type 1 fimbriae and antibiotic resistance by isolates from HIV infected patients. Methods: Bacteriological analysis of mid-stream urine (MSU) samples of human immunodeficiency virus infected patients (n=61) was determined using microbiological technique. In vitro type 1 fimbriae expression and antibiotic susceptibility of bacteria was determined using human erythrocytes with D-mannose and disc diffusion technique. Results: Of the 61 MSU samples from subjects, 31.1% had positive bacterial growth ≥ 105 CFU/mL. Forty-nine (49) bacterial isolates, belonging to genera Staphylococcus, Escherichia, Pseudomonas, Streptococcus, Klebsiella, Enterococcus and Proteus were recovered from the MSU samples. Escherichia coli was predominant isolate with 26.5%, followed by S. aureus (18.4%), while E. faecalis had the lowest occurrence (4.1%). Of the 29 isolates exhibiting hemagglutinating activity, type 1 fimbriae was expressed by 16 isolates, while 13 isolates showed P-fimbriae. The results showed 18.4 % isolates exhibiting resistance to Gentamycin; between 30.6 % and 38.8% isolates were Tetracycline and Nitrofurantoin resistant, while ≤ 44.9% isolates were resistant to Chloramphenicol and Ampicillin. Proteus sp had a low level of resistance to Ciprofloxacin, while ≥ 46.2 % E. coli were resistant to Nitrofurantoin. There was significant relationship (p < 0.05) between isolates expressing type 1 fimbriae and resistance to Gentamycin, Nalidixic Acid and Ampicillin. Conclusion: This study has shown the phenotypic expression of type 1 fimbriae and antibiotic resistance by isolates from HIV infected patients.

Enzyme-assisted extraction of flavanones from citrus pomace: Obtention of natural compounds with anti-virulence and anti-adhesive effect against Salmonella enterica subsp. enterica serovar Typhimurium

Food poisoning has been recognized as a global challenge, causing illness in millions of people. Salmonella enterica subsp. enterica serovar Typhimurium is a major foodborne pathogen causing disease in humans by expressing virulence genes to invade the host epithelial cells. Thus, the search for natural compounds with anti-virulence and anti-adhesive effect could help to mitigate Salmonella infection. This study aimed to determine the effect of flavanones on S. Typhimurium virulence and adhesion to Caco-2 cells. Flavanones were obtained from citrus pomace (CP) by enzyme-assisted (CP-E) and conventional hydroalcoholic (CP–H) extraction methods, which produced extracts rich in aglycones and glycosylated flavanones, respectively. CP-E and aglycone standards (naringenin and hesperetin) decreased bacterial growth (25–90%), motility (50–90%), and adhesion to Caco-2 cells (25–50%). CP-H and glycosylated flavanones (naringin and hesperidin) affected only bacterial adhesion (decreased 20–75%). Agglutination assays demonstrated the binding of extracts and pure flavanones to S. Typhimurium adhesion structures. Fimbrial gene expression (fimA and fimZ) was downregulated in the presence of CP-H, CP-E, and aglycones. Both CP extracts decreased the expression of genes associated with cell invasion (hilA, ssrB and sopD). These results suggest that CP extracts and flavanones inhibit S. Typhimurium adhesion by interacting with fimbriae and flagella structures and downregulating fimbrial and virulence genes. CP-E and aglycone flavanones, rarely found in nature, exhibited superior inhibitory effects on bacterial viability, motility, adhesion and virulence gene expression than glycosylated flavanones. The outcomes of this research advance the understanding of how aglycone and glycosylated flavanones impact S. Typhimurium virulence activity and adhesion, contributing to their application as functional food ingredients for the prevention of adhesion, invasion, and the development of this pathogen in the gut.

The sialidase inhibitor, DANA, reduces Porphyromonas gingivalis pathogenicity and exerts anti-inflammatory effects: An in vitro and in vivo experiment.

Sialidase has an important role in the pathogenesis of periodontitis and Porphyromonas gingivalis is a sialidase-producing organism implicated in periodontitis development. The aim of this study was to evaluate the anti-virulence and anti-inflammatory properties of the sialidase inhibitor, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA), in vitro and in vivo. The effects of DANA on P. gingivalis sialidase and cell viability were determined, and the effects of DANA on P. gingivalis virulence were evaluated by assessment of growth curves, cell morphology, biofilm formation, fimbriae gene expression, and gingipains and lipopolysaccharide (LPS) activity. Anti-inflammatory effects of DANA on LPS-induced macrophages were assessed by measurement of tumor necrosis factor-alpha (TNF-α), interleukin (IL-1β), inducible nitric oxide synthase (iNOS) secretions. The effect of DANA on P. gingivalis-induced periodontitis in rats was analyzed by radiography, stereoscopic microscopy, histopathology, and immunohistochemistry. Sialidase inhibition rate of 1mM DANA was 72.01%. Compared with untreated controls, treatment with DANA inhibited P. gingivalis growth and biofilm formation, and significantly decreased expression of the fimA, fimR, and fimS genes, as well as gingipains activity. DANA did not influence macrophage viability, but significantly inhibited TNF-α, IL-1β, and iNOS production in LPS-stimulated macrophages. In the periodontitis rat model, DANA prevented alveolar bone absorption and inhibited TNF-α and IL-1β production. DANA can reduce the growth, the biofilm formation and the virulence of P. gingivalis and exhibits anti-inflammatory effects, as well as effects against rat periodontitis, suggesting that DANA should be considered for development as a new adjunctive treatment for periodontitis.

Can Effector Molecules of Innate Immunity Target Microbial Motility?

Abstract Antimicrobial peptides are key effector molecules throughout nature responsible in part for the defense against microbial pathogens and influencing the composition of colonizing microbes. Defensins are the major antimicrobial peptides produced by Paneth cells in the mammalian small intestine and are essential for maintaining homeostasis between host and microbe. While mature defensin peptides typically have microbicidal activity, human alpha defensin 6 (HD6) lacks such activity yet provides protection from invasion by bacterial pathogens. Using a transgenic mouse model expressing HD6 in Paneth cells, we reported the importance of HD6 in preventing the translocation of pathogenic Salmonella Typhimurium (STM) across the small intestinal epithelium (doi:10.1126/science.1218831); however, the mechanism underlying the inhibition of invasion is not fully understood. We hypothesize HD6 binds to extracellular bacterial protein structures, such as flagella, and inhibits swimming motility through a process of direct binding and self-assembly. Using an agar based motility assay, we show STM radially swim at a slower rate when in the presence of HD6. In ongoing studies using live-microscopy to visualize GFP-expressing STM in the presence of HD6, we observe a decrease in STM motility in a concentration dependent manner with some STM appearing to be completely immobilized, but without changes in bacterial viability. Unexpectedly, it appears the immobilization observed is influenced more by the expression of fimbriae than by the expression of flagella. With these results in mind, we plan to expand our investigation to elucidate key molecular determinants and include other microbes to test the broader relevance of this activity of HD6.

Expression and Functional Characterization of Various Chaperon-Usher Fimbriae, Curli Fimbriae, and Type 4 Pili of Enterohemorrhagic Escherichia coli O157:H7 Sakai.

Enterohemorrhagic Escherichia coli (EHEC) is a highly pathogenic strain leading to hemorrhagic colitis and to the hemolytic-uremic syndrome (HUS) in humans. The mechanisms by which pathogenic E. coli infect and colonize humans leading to the typical disease pattern are in focus of many investigations. The adhesion of EHEC to epithelial cells by the coordinated translocation of receptor Tir and surface expression of corresponding adhesin intimin is a key event in host–pathogen-interaction. However, less is known about other adhesins encoded by EHEC, especially about the complex set of fimbrial adhesins varying among various serotypes. Here, we investigate EHEC serotype O157:H7 strain Sakai possessing at least 16 putative fimbrial gene clusters. Using a synthetic heterologous expression system in a non-pathogenic E. coli strain, a subset of 6 gene clusters for fimbrial adhesins was analyzed. We were able to visualize surface expression of two γ1 class fimbriae (Fim and Ycb), two γ4 class fimbriae (Yad and Yeh), and two fimbrial adhesins which are assembled by the nucleation/precipitation pathway (Curli fimbriae), and by a type 2 secretion system (type 4 pili). Further, we elucidated the impact of these fimbrial adhesins in adhesion to various epithelial cells lines (HeLa, MDCK, and CaCo2), and the contribution on biofilm formation. We demonstrate the ultrastructure of Fim fimbriae and Yad fimbriae of EHEC Sakai, and Yeh fimbriae of E. coli in general. The involvement of Fim fimbriae of EHEC Sakai to adhesion to various epithelial cell lines, and contribution to biofilm formation is reported here. Our approach provides first ultrastructural and functional data for novel EHEC adhesins, and enables further understanding of the involvement of fimbrial adhesins in pathogenesis of EHEC Sakai.

Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

Carbon Starvation Induces the Expression of PprB-Regulated Genes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa can cause severe infections in humans. This bacterium often adopts a biofilm lifestyle that is hard to treat. In several previous studies, the PprA-PprB two-component system (TCS), which controls the expression of type IVb pili, BapA adhesin, and CupE fimbriae, was shown to be involved in biofilm formation (M. Romero, H. Silistre, L. Lovelock, V. J. Wright, K.-G. Chan, et al., Nucleic Acids Res 46:6823-6840, 2018, https://doi.org/10.1093/nar/gky324; S. de Bentzmann, C. Giraud, C. S. Bernard, V. Calderon, F. Ewald F, et al., PLoS Pathog 8:e1003052, 2012, https://doi.org/10.1371/journal.ppat.1003052). However, signals or environmental conditions that can trigger the PprA-PprB TCS are still unknown, and the molecular mechanisms of PprB-mediated biofilm formation are poorly characterized. Here, we report that carbon starvation stress (CSS) can induce the expression of pprB and genes in the PprB regulon. CSS-induced pprB transcription is mediated by the stress response sigma factor RpoS rather than the two-component sensor PprA. We also observed a strong negative regulation of PprB on the transcription of itself. Further experiments showed that PprB overexpression greatly enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Specifically, under the background of PprB overexpression, both the BapA adhesin and CupE fimbriae displayed positive effects on CCA and CSA, while the type IVb pili showed an unexpected negative effect on CCA and no effect on CSA. In addition, expression of the PprB regulon genes were significantly increased in 3-day colony biofilms, indicating a possible carbon limitation state. The CSS-RpoS-PprB-Bap/Flp/CupE pathway identified in this study provides a new perspective on the process of biofilm formation in carbon-limited environments.IMPORTANCE Typically, the determination of the external signals that can trigger a regulatory system is crucial to understand the regulatory logic and inward function of that system. The PprA-PprB two-component system was reported to be involved in biofilm formation in Pseudomonas aeruginosa, but the signals triggering this system are unknown. In this study, we found that carbon starvation stress (CSS) induces transcription of pprB and genes in the PprB regulon through an RpoS-dependent pathway. Increased PprB expression leads to enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Both CCA and CSA are largely dependent on the Bap secretion system and are moderately dependent on the CupE fimbriae. Our findings suggest that PprB reinforces the structure of biofilms under carbon-limited conditions, and the Bap secretion system and CupE fimbriae are two potential targets for biofilm treatment.

The Role of the AggR Regulon in the Virulence of the Shiga Toxin-Producing Enteroaggregative Escherichia coli Epidemic O104:H4 Strain in Mice.

An O104:H4 Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) strain caused a large outbreak of bloody diarrhea and the hemolytic uremic syndrome in 2011. We previously developed an ampicillin (Amp)-treated C57BL/6 mouse model to measure morbidity (weight loss) and mortality of mice orally infected with the prototype Stx-EAEC strain C227-11. Here, we hypothesized that mice fed C227-11 cured of the pAA plasmid or deleted for individual genes on that plasmid would display reduced virulence compared to animals given the wild-type (wt) strain. C227-11 cured of the pAA plasmid or deleted for the known pAA-encoded virulence genes aggR, aggA, sepA, or aar were fed to Amp-treated C57BL/6 mice at doses of 1010–1011CFU. Infected animals were then either monitored for morbidity and lethality for 28 days or euthanized to determine intestinal pathology and colonization levels at selected times. The pAA-cured, aggR, and aggA mutants of strain C227-11 all showed reduced colonization at various intestinal sites. However, the aggR mutant was the only mutant attenuated for virulence as it showed both reduced morbidity and mortality. The aar mutant showed increased expression of the aggregative adherence fimbriae (AAF) and caused greater systemic effects in infected mice when compared to the C227-11 wt strain. However, unexpectedly, both the aggA and aar mutants displayed increased weight loss compared to wt. The sepA mutant did not exhibit altered morbidity or mortality in the Amp-treated mouse model compared to wt. Our data suggest that the increased morbidity due to the aar mutant could possibly be via an effect on expression of an as yet unknown virulence-associated factor under AggR control.