Design of a chimeric protein composed of FimH, FyuA and CNF-1 virulence factors from uropathogenic Escherichia coli and evaluation its biological activity and immunogenicity in vitro and in vivo.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most prevalent bacterial infections in humans. Antibiotic resistance among UPEC isolates is increasing, and designing an effective vaccine can prevent or reduce these infections. FimH adhesin, iron scavenger receptor FyuA, and cytotoxic necrotizing factor -1 (CNF-1) are among the most important virulence factors of UPEC strains. Thus, a novel multi-epitope protein composed of FimH, FyuA, and CNF-1 was designed to evaluate its biological activity and immunogenicity in vitro and in vivo, respectively. The final vaccine design had seven domains, including the N-terminal domain of FimH, four domains of FyuA, and two domains of CNF-1, as determined by immunoinformatics analysis. The results of tertiary structure prediction showed that the chimeric protein had a C-score of -0.25 and Z-score of -1.94. Molecular docking indicated that thirty six ligand residues of the chimeric protein interacted with 53 receptor residues of TLR-4 by hydrogen bonds and hydrophobic interactions. Analysis of protein expression by SDS-PAGE showed an approximately 44 kDa band with different concentrations of IPTG which were confirmed by Western blot. According to ELISA results, the level of IL-8 produced by stimulated Ht29 cells with the chimeric protein was significantly higher than the stimulated Ht29 cells with CNF-1 alone and un-stimulated Ht29 cells. Rabbits subcutaneously immunized with the chimeric protein admixed with Freund adjuvant induced higher level of serum IgG on day 14 after the first vaccination than control rabbits. Furthermore, the booster dose of the chimeric protein significantly enhanced the IgG levels as compared to day 14 and also controls. As, the chimeric protein has suitable B-cell epitopes and MHC-I and MHC-II binding epitopes to stimulate humoral and cellular immunity, it could be a promising vaccine candidate against UTIs caused by UPEC. Evaluating the multi-epitope protein in inducing humoral and cellular immune responses, as well as protection, is ongoing in the mice models.

Targeting effect of berberine on type I fimbriae of Salmonella Typhimurium and its effective inhibition of biofilm.

As a primary cause of food contamination and human diseases, Salmonella Typhimurium can easily form a biofilm that is difficult to remove from food surfaces, and often causes significant invisible threats to food safety. Although berberine has been widely used as an anti-infective drug in traditional medicine, some basic principles underlying its mechanism, especially the interaction between berberine and type I fimbriae genes, has not been verified yet. In this study, two strains of major fimbrial gene mutants (ΔfimA and ΔfimH) were constructed to demonstrate the possible action of berberine on type I fimbriae genes. The broth microdilution method was used to determine the antibacterial activity of berberine against selected strains (WT, ΔfimA, and ΔfimH). Cell agglutination experiments revealed that the number of S. Typhimurium type I fimbriae reduced after berberine treatment, which was consistent with transmission electron microscopy results. Quantitative real-time PCR experiments also confirmed that berberine reduced fimA gene expression, indicating a certain interaction between berberine and fimA gene. Furthermore, confocal laser scanning microscopy imaging of biofilm clearly revealed that berberine prevents biofilm formation by reducing the number of type I fimbriae. Overall, it is well speculated for us that berberine could be an excellent combating-biofilm drug in clinical microbiology and food preservation. KEY POINTS: • Reduce the number of fimbriae. • Berberine targeting fimA. • Effective biofilm inhibitor.

Antiadhesive natural products against uropathogenic E. coli: What can we learn from cranberry extract?

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts from Cranberry fruits (Vaccinium macrocarpon) are traditionally used against urinary tract infections, mainly due to antiadhesive activity against uropathogenic E. coli (UPEC), but the exact mode of action and compounds, responsible for the activity, are unknown. AIM OF THE STUDY: i. To investigate if cranberry extract acts only by a single component or must be assessed as a multi-active-compound preparation; ii to screen isolated cranberry-related natural products under in vitro conditions to pinpoint natural products with antiadhesive effects against UPEC, followed by in silico calculations (QSAR) to predict potential antiadhesive compounds; iii. investigations by using urine samples from cranberry treated volunteers for evaluation on the bacterial transcriptome and the mannose-binding side of FimH, iv. to investigate if besides Tamm Horsfall Protein induction in the kidney, the extract acts also directly against UPEC. MATERIAL AND METHODS: Antiadhesive activity of 105 compounds was determined by flow cytometric adhesion assay (UPEC UTI89 on T24 bladder cells). Urine samples from 16 volunteers treated with cranberry extract (p.o., 7 days, 900 mg/day) were used for ex vivo testing concerning influence on the bacterial transcriptome (Illumina RNA-seq) and interaction with the mannose binding domain of type-1 fimbriae. RESULTS: i. The antiadhesive effect of cranberry extract cannot be attributed to a single compound or to a single fraction. ii. Unglycosylated flavones and flavonols with bulky substitution of the B ring contribute to the antiadhesive activity. 3'-8″-biflavones and flavolignans (not related to cranberry fruits) were identified as potent antiadhesive compounds against UPEC. iii. QSAR yielded a model with good statistical performance and sufficient internal and external predictive ability. iv. Urine samples from male cranberry-treated volunteers indicated significant interaction with the mannose binding domain of type-1 fimbriae, which correlated with the amount of Tamm-Horsfall Protein in the test samples. v Cranberry extract did not influence the UPEC transcriptome; gene expression of bacterial adhesins (P-, S-fimbrae, curli) was not influenced by the urine samples, while a slight, but non-significant upregulation of type 1 fimbriae was observed. CONCLUSIONS: B-ring substituted flavones and flavonols from cranberry contribute to the antiadhesive activity against UPEC by inhibition of the FimH-mediated interaction with the host cell bladder epithelium.

Expression of the Nontypeable Haemophilus influenzae Type IV Pilus Is Stimulated by Coculture with Host Respiratory Tract Epithelial Cells.

The type IV pilus (Tfp) of nontypeable Haemophilus influenzae (NTHI) mediates adherence, colonization, motility, and biofilm formation, and the major protein subunit, PilA, is a promising vaccine candidate. Thus, it is crucial to understand how Tfp expression is regulated within the microenvironments of the human nasopharynx, which NTHI colonizes asymptomatically, and the more distal regions of the respiratory tract where NTHI-induced diseases occur. Here, we examined the effects of coculture of NTHI with human airway epithelial cells and heme availability on Tfp expression at temperatures typical of the human nasopharynx (34°C) or warmer anatomical sites during infection (37°C). Tfp expression was estimated by pilA promoter activity, pilA gene expression, and relative abundances of PilA and pilin protein. The results revealed that at both temperatures, NTHI cocultured with airway epithelial cells demonstrated significantly greater expression of pilA, PilA/pilin protein, and likely, fully assembled Tfp than NTHI cultured on an abiotic surface. Because NTHI is a heme auxotroph, we hypothesized that availability of heme from host cells might be a signal for Tfp expression. Thereby, we cultured NTHI in iron-limited medium, and we observed that supplementation with heme significantly increased pilA promoter activity. Collectively, our data suggested that NTHI Tfp expression was stimulated by soluble factor(s) released by epithelial cells, which are present in all microenvironments of the respiratory tract. The expression of this target antigen under conditions that mimic the human airway strongly supports the rationale for the use of PilA as a vaccine immunogen to prevent NTHI-induced diseases of the respiratory tract.

The involvement of NF-κB/P38 pathways in Scutellaria baicalensis extracts attenuating of Escherichia coli K88-induced acute intestinal injury in weaned piglets.

The present study was carried out to evaluate the effect of dietary supplementation of Scutellaria baicalensis extracts (SBE) on intestinal health in terms of morphology, barrier integrity and immune responses in weaned piglets challenged with Escherichia coli K88. A total of seventy-two weaned piglets were assigned into two groups to receive a basal diet without including antibiotic additives or the basal diet supplemented 1000 mg SBE/kg diet for 14 d. On day 15, twelve healthy piglets from each group were selected to expose to oral administration of either 10 ml 1 × 109 colony-forming units of E. coli K88 or the vehicle control. After 48 h of E.coli K88 challenge, blood was sampled, and then all piglets were killed humanely for harvesting jejunal and ileal samples. Dietary supplementation of SBE significantly decreased diarrhoea frequency and improved feed conversion ratio (P < 0·05). SBE supplementation to E.coli K88-challenged piglets improved villous height and villous height/crypt depth (P < 0·05), recovered the protein expression of occludin and zonula occludens-2 in both the jejunum and ileum (P < 0·05), and mitigated the increases in plasma IL-1ß, TNF-α, IL-6, IgA and IgG (P < 0·05). Meanwhile, dietary SBE effectively inhibited the stimulation of NF-κB, P38 and TNF-α as well as IL-1ß in the small intestine of piglets challenged by E. coli K88 and prevented the activation of NF-κB/P38 signalling pathways (P < 0·05). Collectively, SBE supplementation can potently attenuate diarrhoea in weaning piglets and decrease inflammatory cytokine expressions through inhibiting the NF-κB and P38 signalling pathways.

Methanol extract of Lonicera caerulea var. emphyllocalyx fruit has anti-motility and anti-biofilm activity against enteropathogenic Escherichia coli.

Foodborne diseases have become a worldwide problem that threatens public health and welfare. Enteropathogenic Escherichia coli (EPEC) is one of major pathogens of moderate to severe diarrhea. The increased prevalence of EPEC strains that produce extended spectrum ß-lactamase (ESBL) has deepened the problem. The fruit of Lonicera caerulea var. emphyllocalyx (LCE) has been used as a traditional food preservative and medicine in northern temperate zones such as Hokkaido Island, Japan. In this study, we investigated the antibacterial effect of LCE fruit extract (LCEE) against EPEC. The antibacterial activities of LCEE were examined by bacterial growth, time-kill curve, soft-agar motility, electron microscopy, and 96 well-microplate biofilm assays. We also investigated the bacterial mRNA expression of biofilm-associated genes (fliC, csgA, and fimA) by quantitative real-time PCR assays. LCEE was found to suppress the growth, time-kill curve, and spread of EPEC. It also reduced the biofilm formation in a dose-dependent manner. Morphological analysis using transmission and scanning electron microscopy revealed that LCEE diminished the function of flagella resulting in reduced motility and biofilm formation. The mRNA expression of all three biofilm associated genes was downregulated under LCEE treatment. Extracts of the fruit of LCE inhibit the motility and biofilm formation of EPEC as a result of the inhibition of flagella development and function. We propose LCEE as a therapeutic candidate for the effective therapy of EPEC-associated infectious diseases.

Phytochemicals As Uropathognic Escherichia Coli FimH Antagonist: In Vitro And In Silico Approach.

BACKGROUND: Urinary tract infection (UTI) is caused by uropathogenic Escherichia coli (UPEC). The UPEC initiate pathogenesis by expressing type 1 pili, which attach to membrane receptors on the uroepithelial cells. Inhibition of attachment can provide a valuable target for prophylaxis in symptom-free milieu. METHODS: The antibacterial efficacy of alcoholic, hydroalcoholic and aqueous extracts of four plants namely Achyranthes aspera, Andrographis paniculata, Artemissia vulgaris and Glycyrrhiza glabra was evaluated against seven isolated bacterial strains and procured E. coli (UTI89/UPEC) strain. Screening of isolated strains was based on morphological characteristics and biofilm forming ability followed by physiological and biochemical analysis. RESULTS: The hydroalcoholic extracts of G. glabra at 50 µg/ml showed an impending antioxidant (DPPH) effect of 95.65% compared to ascorbic acid. The MIC values of all the plant extracts against selected bacterial strains ranged between 125 to 1000 µg/ml. In silico molecular docking performed to make out the antiadhesive role of 115 documented phytochemicals from selected plants identified quercetin-3-glucoside, ethyl caffeate, liquiritoside, liquiritin and isoliquiritigenin as potential phytochemicals. Molecular dynamics simulation performed by PTRAJ module of Amber11 package to monitor the stability of hydrogen bond showed that quercetin-3-glucoside and ethyl caffeate are potential phytochemicals as antiadhesive forming H-bonds with the FimH protein ligand. CONCLUSIONS: Aforesaid phytochemicals demonstrate effective antibacterial activity through the anti-adhesion mechanism.

Trends in antimicrobial resistance in Neisseria gonorrhoeae and molecular characteristics of N. gonorrhoeae with decreased susceptibility to ceftriaxone in Shandong, China, 2007 to 2014.

In this study, the trends of antimicrobial resistance in Neisseria gonorrhoeae were analysed in Shandong Province of China during 2007 to 2014. Furthermore, the ceftriaxone (CRO) genetic resistance determinants, including penA, mtrR, penB, ponA, and pilQ genes, were sequenced and the molecular mechanisms of decreased susceptibility or resistance to CRO in N. gonorrhoeae were elucidated. Overall, the increasing trends of resistance to penicillin (PEN), tetracycline (TET), and ciprofloxacin (CIP), and decreasing trends of susceptibility to CRO and azithromycin (AZM) were observed in Shandong Province between 2007 and 2014. The proportions of PPNG, TRNG, PP/TRNG, and MDR isolates increased sharply in this district. PenA mosaic structure, the substitution of A501V, and an insertion of aspartate in amino acid position 345 were identified in the N. gonorrhoeae isolates with decreased susceptibility or resistance to CRO. All the 28 N. gonorrhoeae isolates had substitutions at Gly-120 and Ala-121 of porin encoded by penB, substitution of L421P in PBP1, and a single nucleotide (A) deletion in the 13 bp inverted repeat located between the -10 and -35 sequences in the mtrR promoter. Additionally, 21 N.gonorrhoeae isolates had substitutions of A39T/G45D in MtrR, and three new substitutions of R44G, L47R, and/or H105F in MtrR were observed. Therefore, PenA mosaic structure in N. gonorrhoeae and the substitutions of Ala-501 in PBP2 may considerably increase CRO MICs. A close association between the genetic polymorphisms in mtrR, penB, and ponA and the development of decreased susceptibility to CRO might be confirmed.

Bioactive Immune Components of Anti-Diarrheagenic Enterotoxigenic Escherichia coli Hyperimmune Bovine Colostrum Products.

Diarrhea is a common illness among travelers to resource-limited countries, the most prevalent attributable agent being enterotoxigenic Escherichia coli (ETEC). At this time, there are no vaccines licensed specifically for the prevention of ETEC-induced traveler's diarrhea (TD), and this has propelled investigation of alternative preventive methods. Colostrum, the first milk expressed after birthing, is rich in immunoglobulins and innate immune components for protection of newborns against infectious agents. Hyperimmune bovine colostrum (HBC) produced by immunization of cows during gestation (and containing high levels of specific antibodies) is a practical and effective prophylactic tool against gastrointestinal illnesses. A commercial HBC product, Travelan, is available for prevention of ETEC-induced diarrhea. Despite its demonstrated clinical efficacy, the underlying immune components and antimicrobial activity that contribute to protection remain undefined. We investigated innate and adaptive immune components of several commercial HBC products formulated to reduce the risk of ETEC-induced diarrhea, including Travelan and IMM-124E, a newer product that has broader gastrointestinal health benefits. The immune components measured included total and ETEC-specific IgG, total IgA, cytokines, growth factors, and lactoferrin. HBC products contained high levels of IgG specific for multiple ETEC antigens, including O-polysaccharide 78 and colonization factor antigen I (CFA/I) present in the administered vaccines. Antimicrobial activity was measured in vitro using novel functional assays. HBC greatly reduced ETEC motility in soft agar and exhibited bactericidal activity in the presence of complement. We have identified immune components and antimicrobial activity potentially involved in the prevention of ETEC infection by HBC in vivo.

Prophylactic Efficacy of Hyperimmune Bovine Colostral Antiadhesin Antibodies Against Enterotoxigenic Escherichia coli Diarrhea: A Randomized, Double-Blind, Placebo-Controlled, Phase 1 Trial.

Background: Tip-localized adhesive proteins of bacterial fimbriae from diverse pathogens confer protection in animal models, but efficacy in humans has not been reported. Enterotoxigenic Escherichia coli (ETEC) commonly elaborate colonization factors comprising a minor tip adhesin and major stalk-forming subunit. We assessed the efficacy of antiadhesin bovine colostral IgG (bIgG) antibodies against ETEC challenge in volunteers. Methods: Adults were randomly assigned (1:1:1) to take oral hyperimmune bIgG raised against CFA/I minor pilin subunit (CfaE) tip adhesin or colonization factor I (CFA/I) fimbraie (positive control) or placebo. Two days before challenge, volunteers began a thrice-daily, 7-day course of investigational product administered in sodium bicarbonate 15 minutes after each meal. On day 3, subjects drank 1 × 109 colony-forming units of colonization factor I (CFA/I)-ETEC strain H10407 with buffer. The primary efficacy endpoint was diarrhea within 120 hours of challenge. Results: After enrollment and randomization, 31 volunteers received product, underwent ETEC challenge, and were included in the per protocol efficacy analysis. Nine of 11 placebos developed diarrhea, 7 experiencing moderate to severe disease. Protective efficacy of 63% (P = .03) and 88% (P = .002) was observed in the antiadhesin bIgG and positive control groups, respectively. Conclusions: Oral administration of anti-CFA/I minor pilin subunit (CfaE) antibodies conferred significant protection against ETEC, providing the first clinical evidence that fimbrial tip adhesins function as protective antigens.

Antibiotic Prophylaxis Is Associated with Subsequent Resistant Infections in Children with an Initial Extended-Spectrum-Cephalosporin-Resistant Enterobacteriaceae Infection.

The objective of this study was to assess the association between previous antibiotic use, particularly long-term prophylaxis, and the occurrence of subsequent resistant infections in children with index infections due to extended-spectrum-cephalosporin-resistant Enterobacteriaceae We also investigated the concordance of the index and subsequent isolates. Extended-spectrum-cephalosporin-resistant Escherichia coli and Klebsiella spp. isolated from normally sterile sites of patients aged <22 years were collected along with associated clinical data from four freestanding pediatric centers. Subsequent isolates were categorized as concordant if the species, resistance determinants, and fumC-fimH (E. coli) or tonB (Klebsiella pneumoniae) type were identical to those of the index isolate. In total, 323 patients had 396 resistant isolates; 45 (14%) patients had ≥1 subsequent resistant infection, totaling 73 subsequent resistant isolates. The median time between the index and first subsequent infections was 123 (interquartile range, 43 to 225) days. In multivariable Cox proportional hazards analyses, patients were 2.07 times as likely to have a subsequent resistant infection (95% confidence interval, 1.11 to 3.87) if they received prophylaxis in the 30 days prior to the index infection. In 26 (58%) patients, all subsequent isolates were concordant with their index isolate, and 7 (16%) additional patients had at least 1 concordant subsequent isolate. In 12 of 17 (71%) patients with E. coli sequence type 131 (ST131)-associated type 40-30, all subsequent isolates were concordant. Subsequent extended-spectrum-cephalosporin-resistant infections are relatively frequent and are most commonly due to bacterial strains concordant with the index isolate. Further study is needed to assess the role prophylaxis plays in these resistant infections.

Cranberry extract inhibits in vitro adhesion of F4 and F18+Escherichia coli to pig intestinal epithelium and reduces in vivo excretion of pigs orally challenged with F18+ verotoxigenic E. coli.

F4+E. coli and F18+E. coli infections are an important threat for pig industry worldwide. Antibiotics are commonly used to treat infected piglets, but the emerging development of resistance against antibiotics raises major concerns. Hence, alternative therapies to prevent pigs from F4+E. coli and F18+E. coli infections need to be developed. Since cranberry previously showed anti-adhesive activity against uropathogenic E. coli, we aimed to investigate whether cranberry extract could also inhibit binding of F4+E. coli and F18+E. coli to pig intestinal epithelium. Using the in vitro villus adhesion assay, we found that low concentrations of cranberry extract (20µg or 100µg/ml) have strong inhibitory activity on F4+E. coli (75.3%, S.D.=9.31 or 95.8%, S.D.=2.56, respectively) and F18+E. coli adherence (100% inhibition). This effect was not due to antimicrobial activity. Moreover, cranberry extract (10mg or 100mg) could also abolish in vivo binding of F4 and F18 fimbriae to the pig intestinal epithelium in ligated loop experiments. Finally, two challenge experiments with F18+E. coli were performed to address the efficacy of in-feed or water supplemented cranberry extract. No effect could be observed in piglets that received cranberry extract only in feed (1g/kg or 10g/kg). However, supplementation of feed (10g/kg) and drinking water (1g/L) significantly decreased excretion and diarrhea. The decreased infection resulted in a decreased serum antibody response indicating reduced exposure to F18+E. coli.

Swimming and twitching motility are essential for attachment and virulence of Pantoea ananatis in onion seedlings.

Pantoea ananatis is a widespread phytopathogen with a broad host range. Despite its ability to infect economically important crops, such as maize, rice and onion, relatively little is known about how this bacterium infects and colonizes host tissue or spreads within and between hosts. To study the role of motility in pathogenicity, we analysed both swimming and twitching motility in P. ananatis LMG 20103. Genetic recombineering was used to construct four mutants affected in motility. Two flagellar mutants were disrupted in the flgK and motA genes, required for flagellar assembly and flagellar rotation, respectively. Similarly, two twitching motility mutants were generated, impaired in the structure (pilA) and functioning (pilT) of the type IV pili. The role of swimming and twitching motility during the infection cycle of P. ananatis in onion seedlings was determined by comparing the mutant- and wild-type strains using several in vitro and in planta assays. From the results obtained, it was evident that flagella aid P. ananatis in locating and attaching to onion leaf surfaces, as well as in pathogenicity, whereas twitching motility is instrumental in the spread of the bacteria on the surface once attachment has occurred. Both swimming and twitching motility contribute towards the ability of P. ananatis to cause disease in onions.

Preparation and preclinical evaluation of a freeze-dried formulation of a novel combined multivalent whole-cell/B-subunit oral vaccine against enterotoxigenic Escherichia coli diarrhea.

A promising liquid killed multivalent whole-cell plus enterotoxin B-subunit oral vaccine against enterotoxigenic Escherichia coli (ETEC), the primary cause of diarrhea among children in low-income countries and travelers to these areas, has recently been developed and tested in preclinical and phase-I and phase-II clinical studies. The vaccine contains killed E. coli bacteria over-expressing the main ETEC colonization factors (CFs) CFA/I, CS3, C5 and C6, and a recombinant enterotoxin B subunit protein (LCTBA) given together with a recently developed enterotoxin-derived adjuvant, dmLT. A dry-powder vaccine formulation should be advantageous especially for use in low-income countries. Here we describe a method to produce a dry-powder formulation by freeze-drying of the vaccine using inulin as stabilizer. Although not completely preventing aggregation of bacteria during freeze-drying, the stabilizer provided both improved overall bacterial morphology and almost complete recovery of the CF and B subunit antigens. Most importantly, oral-intragastric immunization of mice with the freeze-dried vaccine together with dmLT adjuvant elicited strong intestinal mucosal and serum antibody responses against all vaccine antigens, which were comparable to those achieved with the liquid vaccine. Our results indicate the feasibility to use freeze-drying with inulin as stabilizer for preparing a dry-powder formulation of the novel ETEC vaccine with retained oral-mucosal immunogenicity compared to the liquid formulation.

Transcriptional Repression of the VC2105 Protein by Vibrio FadR Suggests that It Is a New Auxiliary Member of the fad Regulon.

UNLABELLED: Recently, our group along with others reported that the Vibrio FadR regulatory protein is unusual in that, unlike the prototypical fadR product of Escherichia coli, which has only one ligand-binding site, Vibrio FadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of the vc2105 gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of the vc2105 gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates the vc2105 promoter from the FadR regulator. The expression level of the Vibrio cholerae vc2105 gene was elevated 2- to 3-fold in a fadR null mutant strain, validating that FadR is a repressor for the vc2105 gene. The ß-galactosidase activity of a vc2105-lacZ transcriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike the fadD gene, a member of the Vibrio fad regulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression of ctxAB (cholera toxin A/B) and tcpA (toxin coregulated pilus A). Given that the transcriptional regulation of vc2105 fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of the Vibrio fad regulon. IMPORTANCE: The Vibrio FadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterized in vitro and in vivo. An auxiliary fad gene (vc2105) from Vibrio was proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Further in vitro and in vivo experiments revealed that the Vibrio FadR is a repressor for the vc2105 gene. Unlike fadD, a member of the Vibrio fad regulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxAB and tcpA). Therefore, since transcriptional regulation of vc2105 fits the criteria for fad genes, it seems likely that vc2105 acts as a new auxiliary member of the Vibrio fad regulon.

Thermally activated charge transport in microbial protein nanowires.

The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors.

Imipenem Treatment Induces Expression of Important Genes and Phenotypes in a Resistant Acinetobacter baumannii Isolate.

Acinetobacter baumannii has emerged as a notorious multidrug-resistant pathogen, and development of novel control measures is of the utmost importance. Understanding the factors that play a role in drug resistance may contribute to the identification of novel therapeutic targets. Pili are essential for A. baumannii adherence to and biofilm formation on abiotic surfaces as well as virulence. In the present study, we found that biofilm formation was significantly induced in an imipenem-resistant (Imp(r)) strain treated with a subinhibitory concentration of antibiotic compared to that in an untreated control and an imipenem-susceptible (Imp(s)) isolate. Using microarray and quantitative PCR analyses, we observed that several genes responsible for the synthesis of type IV pili were significantly upregulated in the Imp(r) but not in the Imp(s) isolate. Notably, this finding is corroborated by an increase in the motility of the Imp(r) strain. Our results suggest that the ability to overproduce colonization factors in response to imipenem treatment confers biological advantage to A. baumannii and may contribute to clinical success.

In Vivo Consumption of Cranberry Exerts ex Vivo Antiadhesive Activity against FimH-Dominated Uropathogenic Escherichia coli: A Combined in Vivo, ex Vivo, and in Vitro Study of an Extract from Vaccinium macrocarpon.

For investigation of the molecular interaction of cranberry extract with adhesins of uropathogenic Escherichia coli (UPEC), urine from four volunteers consuming standardized cranberry extract (proanthocyanidin content = 1.24%) was analyzed within ex vivo experiments, indicating time-dependent significant inhibition of 40-50% of bacterial adhesion of UPEC strain NU14 to human T24 bladder cells. Under in vitro conditions a dose-dependent increase in bacterial adhesion was observed with proanthocyanidin-enriched cranberry Vaccinium macrocarpon extract (proanthocyanidin content = 21%). Confocal laser scanning microscopy and scanning electron microscopy proved that V.m. extract led to the formation of bacterial clusters on the outer plasma membrane of the host cells without subsequent internalization. This agglomerating activity was not observed when a PAC-depleted extract (V.m. extract(≠PAC)) was used, which showed significant inhibition of bacterial adhesion in cases where type 1 fimbriae dominated and mannose-sensitive UPEC strain NU14 was used. V.m. extract(≠PAC) had no inhibitory activity against P- and F1C-fimbriae dominated strain 2980. Quantitative gene expression analysis indicated that PAC-containing as well as PAC-depleted cranberry extracts increased the fimH expression in NU14 as part of a feedback mechanism after blocking FimH. For strain 2980 the PAC-containing extract led to up-regulation of P- and F1C-fimbriae, whereas the PAC-depleted extract had no influence on gene expression. V.m. and V.m. extract(≠PAC) did not influence biofilm and curli formation in UPEC strains NU14 and 2980. These data lead to the conclusion that also proanthocyanidin-free cranberry extracts exert antiadhesive activity by interaction with mannose-sensitive type 1 fimbriae of UPEC.

Presence of calcium-binding motifs in PilY1 homologs correlates with Ca-mediated twitching motility and evolutionary history across diverse bacteria.

Twitching motility, involving type IV pili, is essential for host colonization and virulence of many pathogenic bacteria. Studies of PilY1, a tip-associated type IV pili protein, indicate that PilY1 functions as a switch between pilus extension and retraction, resulting in twitching motility. Recent work detected a calcium-binding motif in PilY1 of some animal bacterial pathogens and demonstrated that binding of calcium to PilY1 with this motif regulates twitching. Though studies of PilY1 in non-animal pathogens are limited, our group demonstrated that twitching motility in the plant pathogen Xylella fastidiosa, which contains three PilY1 homologs, is increased by calcium supplementation. A study was conducted to investigate the phylogenetic relationship between multiple PilY1 homologs, the presence of calcium-binding motifs therein, and calcium-mediated twitching motility across diverse bacteria. Strains analyzed contained one to three PilY1 homologs, but phylogenetic analyses indicated that PilY1 homologs containing the calcium-binding motif Dx[DN]xDGxxD are phylogenetically divergent from other PilY1 homologs. Plant-associated bacteria included in these analyses were then examined for a calcium-mediated twitching response. Results indicate that bacteria must have at least one PilY1 homolog containing the Dx[DN]xDGxxD motif to display a calcium-mediated increase in twitching motility, which likely reflects an adaption to environmental calcium concentrations.

Expression and functional characterization of the Agrobacterium VirB2 amino acid substitution variants in T-pilus biogenesis, virulence, and transient transformation efficiency.

Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease by transferring transferred DNA (T-DNA) into the plant genome. The translocation process is mediated by the type IV secretion system (T4SS) consisting of the VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. Here, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2-) and tumorigenesis on tomato stems (Vir+ or Vir-), the mutants could be classified into three groups: ExB2-/Vir-, ExB2-/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2-/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation. Interestingly, although the five T-pilus-/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency.