致病性大肠杆菌UPEC CFT073全基因组分析及致病机制的新认识

尿道致病性大肠杆菌UPEC CFT073菌株(uropathogenic Escherichia coli CFT073)于2002年被完全测序并注释。但是,对其基因组的研究还很不完善,首先表现在基因组注释的系统性错误和滞后性。作者运用一系列生物信息学方法和工具,从编码蛋白质基因、编码RNA基因等角度对RefSeq数据库的基因组注释进行了系统的修正和增补,并在此基础上鉴别了一批新的候选致病因子基因。进一步的分析表明,得到的基因组注释对CFT073致病相关的一些重要调控关系和机制能够给出更准确、完整的描述。     

Chemoreceptors of Escherichia coli CFT073 Play Redundant Roles in Chemotaxis toward Urine

Community-acquired urinary tract infections (UTIs) are commonly caused by uropathogenic Escherichia coli (UPEC). We hypothesize that chemotaxis toward ligands present in urine could direct UPEC into and up the urinary tract. Wild-type E. coli CFT073 and chemoreceptor mutants with tsr, tar, or aer deletions were tested for chemotaxis toward human urine in the capillary tube assay. Wild-type CFT073 was attracted toward urine, and Tsr and Tar were the chemoreceptors mainly responsible for mediating this response. The individual components of urine including L-amino acids, D-amino acids and various organic compounds were also tested in the capillary assay with wild-type CFT073. Our results indicate that CFT073 is attracted toward some L- amino acids and possibly toward some D-amino acids but not other common compounds found in urine such as urea, creatinine and glucuronic acid. In the murine model of UTI, the loss of any two chemoreceptors did not affect the ability of the bacteria to compete with the wild-type strain. Our data suggest that the presence of any strong attractant and its associated chemoreceptor might be sufficient for colonization of the urinary tract and that amino acids are the main chemoattractants for E. coli strain CFT073 in this niche.     

DsdX is the second D-serine transporter in uropathogenic Escherichia coli clinical isolate CFT073

d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.     

Toxoplasma Co-opts Host Cells It Does Not Invade

Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.     

S. mansoni Bolsters Anti-Viral Immunity in the Murine Respiratory Tract

The human intestinal parasite Schistosoma mansoni causes a chronic disease, schistosomiasis or bilharzia. According to the current literature, the parasite induces vigorous immune responses that are controlled by Th2 helper cells at the expense of Th1 helper cells. The latter cell type is, however, indispensable for anti-viral immune responses. Remarkably, there is no reliable literature among 230 million patients worldwide describing defective anti-viral immune responses in the upper respiratory tract, for instance against influenza A virus or against respiratory syncitial virus (RSV). We therefore re-examined the immune response to a human isolate of S. mansoni and challenged mice in the chronic phase of schistosomiasis with influenza A virus, or with pneumonia virus of mice (PVM), a mouse virus to model RSV infections. We found that mice with chronic schistosomiasis had significant, systemic immune responses induced by Th1, Th2, and Th17 helper cells. High serum levels of TNF-α, IFN-γ, IL-5, IL-13, IL-2, IL-17, and GM-CSF were found after mating and oviposition. The lungs of diseased mice showed low-grade inflammation, with goblet cell hyperplasia and excessive mucus secretion, which was alleviated by treatment with an anti-TNF-α agent (Etanercept). Mice with chronic schistosomiasis were to a relative, but significant extent protected from a secondary viral respiratory challenge. The protection correlated with the onset of oviposition and TNF-α-mediated goblet cell hyperplasia and mucus secretion, suggesting that these mechanisms are involved in enhanced immune protection to respiratory viruses during chronic murine schistosomiasis. Indeed, also in a model of allergic airway inflammation mice were protected from a viral respiratory challenge with PVM.     

Pomegranate materials inhibit flagellin gene expression and flagellar-propelled motility of uropathogenic Escherichia coli strain CFT073

This report describes the inhibitory effect of pomegranate rind extract (PGRE) on the motility of uropathogenic Escherichia coli (UPEC), a common agent of uncomplicated urinary tract infections (UTIs). To this end, a fliC-lux reporter, as well as Western blot analysis and scanning electron microscopy, was used to demonstrate that when UPEC strain CFT073 is exposed to PGRE, expression of the flagellin gene, fliC, and flagellin production decrease. In agreement with these results, the swimming and swarming motilities of UPEC were observed to be hindered in the presence of PGRE. To evaluate the effect of other pomegranate materials (PMs), the hydrolysable tannins in pomegranate (PG; punicalagin) and pomegranate fruit powder (PGP) were also investigated. Of the materials tested, PGRE had the strongest inhibitory effect on fliC expression and motility. Moreover, a fractionation of PGRE showed fractions with a molecular weight between 1000 and 3000?kDa to be the strongest inhibitors of fliC expression. Because flagellum-mediated motility has been suggested to enable UPEC to disseminate to the upper urinary tract; we propose that PGRE might be therapeutically beneficial in the treatment and prevention of UTIs.     

NOS2 Is Critical to the Development of Emphysema in Sftpd Deficient Mice but Does Not Affect Surfactant Homeostasis

Rationale

Surfactant protein D (SP-D) has important immuno-modulatory properties. The absence of SP-D results in an inducible NO synthase (iNOS, coded by NOS2 gene) related chronic inflammation, development of emphysema-like pathophysiology and alterations of surfactant homeostasis.

Objective

In order to test the hypothesis that SP-D deficiency related abnormalities in pulmonary structure and function are a consequence of iNOS induced inflammation, we generated SP-D and iNOS double knockout mice (DiNOS).

Methods

Structural data obtained by design-based stereology to quantify the emphysema-like phenotype and disturbances of the intracellular surfactant were correlated to invasive pulmonary function tests and inflammatory markers including activation markers of alveolar macrophages and compared to SP-D (Sftpd^−/−) and iNOS single knockout mice (NOS2^−/−) as well as wild type (WT) littermates.

Measurements and Results

DiNOS mice had reduced inflammatory cells in BAL and BAL-derived alveolar macrophages showed an increased expression of markers of an alternative activation as well as reduced inflammation. As evidenced by increased alveolar numbers and surface area, emphysematous changes were attenuated in DiNOS while disturbances of the surfactant system remained virtually unchanged. Sftpd^−/− demonstrated alterations of intrinsic mechanical properties of lung parenchyma as shown by reduced stiffness and resistance at its static limits, which could be corrected by additional ablation of NOS2 gene in DiNOS.

Conclusion

iNOS related inflammation in the absence of SP-D is involved in the emphysematous remodeling leading to a loss of alveoli and associated alterations of elastic properties of lung parenchyma while disturbances of surfactant homeostasis are mediated by different mechanisms.     

大肠杆菌CFT073中Curli系统重要基因csgF的克隆、表达、纯化和结构分析

【目的】对大肠杆菌CFT073中Culri系统的重要蛋白CsgF进行高效表达,探索其纯化条件和三维结构,为研究Curli生物合成机制提供理论基础。【方法】以大肠杆菌CFT073基因组为模板扩增csgF基因,构建pET28a-csg F(nsp)-N-6His、pET28a-csg F(20-129)-N-6His、pET28a-csg F-C-6His和p ET28a-csg F(nsp)-C-6His等重组质粒,转化到大肠杆菌DH5α并在BL21(DE3)中诱导表达;通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳鉴定CsgF蛋白在大肠杆菌中的表达情况,用Ni-NTA His Bind Resin和凝胶排阻层析色谱纯化重组蛋白CsgF,SDS-PAGE和Western blotting方法鉴定分析;用Pull down实验研究CsgF与CsgG蛋白的相互作用,同源模建方法分析重组蛋白CsgF的三级结构。【结果】克隆了目的基因csg F,并筛选出稳定CsgF蛋白的条件:50 mmol/L Sodium acetate(pH 5.0)、150 mmol/L NaCl、5%Glyercol;CsgF与CsgG存在相互作用,CsgF三维结构模型显示为(β/α)。【结论】获得了高纯度稳定的CsgF重组蛋白及其三维结构,为进一步研究CsgF结构与功能奠定了基础。     

Electrotransfection of Escherichia coli with λgt10 DNA

We have used electroporation to introduce lambda gt10 DNA into E. coli C600 (Electrotransfection). We obtained approximately 10(7) pfu (plaque forming units) per micrograms of lambda gt10 DNA. This frequency is 100-fold higher than the maximum reported for classical calcium chloride-induced transfection. We have also compared electrotransfection with in vitro packaging in cloning experiments using relatively small amounts (less than or equal to 10 ng) of DNA. Our results slow that electrotransfection can generate approximately 1000-fold more plaques than in vitro packaging at these concentrations.     

Characterization of Two Homologous Disulfide Bond Systems Involved in Virulence Factor Biogenesis in Uropathogenic Escherichia coli CFT073

Disulfide bond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic Escherichia coli (UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide and is in turn reoxidized by DsbB. DsbA has broad substrate specificity and reacts readily with reduced unfolded proteins entering the periplasm. The DsbLI system also comprises a functional redox pair; however, DsbL catalyzes the specific oxidative folding of the large periplasmic enzyme arylsulfate sulfotransferase (ASST). In this study, we characterized the DsbLI system of the prototypic UPEC strain CFT073 and examined the contributions of the DsbAB and DsbLI systems to the production of functional flagella as well as type 1 and P fimbriae. The DsbLI system was able to catalyze disulfide bond formation in several well-defined DsbA targets when provided in trans on a multicopy plasmid. In a mouse urinary tract infection model, the isogenic dsbAB deletion mutant of CFT073 was severely attenuated, while deletion of dsbLI or assT did not affect colonization.Disulfide bonds bridging cysteine pairs impart structural stability and protease resistance to secreted and membrane-associated proteins. Most organisms contain specific mechanisms for the formation of disulfide bonds in proteins, a process called oxidative protein folding. In bacteria, this folding process is catalyzed by the disulfide bond family of proteins (18, 22). The best-characterized bacterial disulfide bond machinery is the Escherichia coli K-12 oxidative system, which consists of two enzymes, the periplasmic DsbA and the inner-membrane DsbB (25, 35). DsbA is a monomeric protein comprising a thioredoxin (TRX) domain with an embedded helical insertion and a redox-active CPHC motif (34). This highly oxidizing protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide (2, 4, 5), and as a result, the two cysteines contained in the CPHC catalytic motif become reduced. DsbB reoxidizes this cysteine pair and restores the oxidizing activity of DsbA, enabling it to assist the folding of a new substrate protein (21).The DsbAB oxidative protein folding system plays a well-documented part in bacterial virulence. Several studies have demonstrated a direct role for both enzymes, particularly DsbA, in the biogenesis of virulence factors utilized by bacterial pathogens in various stages of the infection process (19). The protein forming the P-ring of E. coli flagella, FlgI, was one of the first DsbA substrates identified (10) and flagellum-mediated motility was subsequently demonstrated to require the presence of functional DsbA in several gram-negative pathogens, including Salmonella enterica (1), Proteus mirabilis (8), Erwinia carotovora subsp. atroseptica (9), Burkholderia cepacia (17), and Campylobacter jejuni (42). In Yersinia pestis, S. enterica, Shigella flexneri, and enteropathogenic E. coli, deletion of dsbA results in defective type III secretion, a major virulence mechanism employed by these enteric pathogens to manipulate the host during infection. The defect was shown in each case to involve the outer membrane secretin (YscC, SpiA, Spa32, and EscC, respectively), which requires a single intramolecular disulfide bond to adopt a functional conformation (23, 36, 37, 49). Fimbria-mediated adhesion is a crucial first step of the infection process as it allows host colonization by mucosal pathogens. DsbA is required for functional assembly of several types of fimbriae, including P fimbriae of uropathogenic E. coli (UPEC) (24), bundle-forming pili (Bfp) of enteropathogenic E. coli (55), mannose-resistant Proteus-like (MR/P) fimbriae of Proteus mirabilis (8), plasmid-encoded fimbriae (Pef) of Salmonella enterica (6), type IV pili of Neisseria meningitidis (47), and toxin-coregulated pili (Tcp) of Vibrio cholerae (41). A number of studies have reported that dsbA and/or dsbB mutants are attenuated in infection models (9, 16, 41, 48, 52).The recent exponential increase in sequenced genomes has offered a first glimpse at the diversity of disulfide bond systems present in bacteria (13). In addition, it is now evident that several bacterial species encode multiple DsbA paralogues, often with demonstrated differences in substrate specificity. Neisseria meningitidis, for example, encodes three DsbA oxidoreductases: two inner membrane-associated lipoproteins (DsbA1 and DsbA2) and one periplasmic enzyme (DsbA3). While redundancy was observed in the oxidative folding of virulence-associated proteins by DsbA1 and DsbA2, DsbA3 alone was unable to restore important meningococcal virulence traits, such as type IV pilus-mediated adhesion to human endothelial cells (47). Recently, a second E. coli disulfide bond system (DsbLI) was identified in the genome-sequenced UPEC strain CFT073 and was demonstrated to be a functional paralogue of the prototypic DsbAB system (14). The oxidoreductase DsbL has the strongest oxidizing potential of all DsbA homologues characterized to date. Although the crystal structure of DsbL revealed a similar overall fold and domain architecture to DsbA, DsbL contains a longer helical insertion and deletions in the TRX domain that result in a truncated peptide binding groove. Moreover, DsbL shows different surface properties, including a distinct basic patch around the active site, which was suggested to allow stricter substrate specificity than the highly hydrophobic surface surrounding the active site of DsbA. Grimshaw and colleagues (14) demonstrated the specificity of the DsbLI system for the periplasmic enzyme arylsulfate sulfotransferase (ASST) encoded by assT, a gene found immediately upstream of dsbL and dsbI on the CFT073 chromosome. ASST belongs to a group of poorly characterized large bacterial ASSTs that are proposed to mediate detoxification of phenolic substances by catalyzing the transfer of sulfuryl groups from phenolic sulfates to phenol (26-28, 30). A reason for the specificity of DsbLI for ASST folding could be the presence of an allosteric disulfide bond, recently revealed by the enzyme''s crystal structure (33). This class of disulfide bond forms between C_α atoms of cysteines in unusually close proximity (3.8 Å in the case of ASST) and has higher steric strain energy than catalytic or structural disulfide bonds, thus explaining the requirement for the stronger DsbL oxidase for its formation (33). The activity of DsbL and DsbI was studied using plasmids introduced into E. coli K-12 strains with the native DsbAB system deleted. As yet, the role of the DsbLI system in UPEC virulence has not been investigated.E. coli CFT073 is a prototypic UPEC strain isolated from a female patient with acute pyelonephritis (38). UPEC strains are the causative agent of >80% of community-acquired urinary tract infections (UTIs) and >30% of nosocomial infections (7). The uropathogenic lifestyle of UPEC CFT073 is reflected in its genome, which contains several factors with an established role in urovirulence, including the well-studied type 1 and P fimbriae (50). Genomic comparison of CFT073—and other recently sequenced UPEC strains—with E. coli strains with distinct lifestyles (gut commensals, enteric pathogens, and avian pathogens) allows the discovery of genes unique to genomes of uropathogenic bacteria that are potentially novel urovirulence factors. One such UPEC-specific gene is assT, the gene located upstream of dsbL and dsbI in the chromosome of CFT073 (32).Here we characterize the DsbLI system in its native genetic background of UPEC CFT073 and compare and contrast the contribution of each of the two paralogous disulfide bond systems of CFT073 in the production of UPEC-associated virulence factors and in vivo uropathogenesis. Using isogenic dsbAB and dsbLI deletion mutants of CFT073, we demonstrate that the recently identified DsbLI oxidative protein folding machinery of UPEC CFT073 plays a secondary role in the production of urovirulence factors and does not appear to contribute to virulence in the mouse infection model used in this study. We also show that in the same infection model, an isogenic assT deletion mutant of CFT073 is not attenuated.     

Type 1 Fimbriae Contribute to Catheter-Associated Urinary Tract Infections Caused by Escherichia coli

Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI), which represent the most frequent nosocomial infections. Knowledge of genetic factors for catheter colonization is limited, since their role has not been assessed using physicochemical conditions prevailing in a catheterized human bladder. The current study aimed to combine data from a dynamic catheterized bladder model in vitro with in vivo expression analysis for understanding molecular factors relevant for CAUTI caused by Escherichia coli. By application of the in vitro model that mirrors the physicochemical environment during human infection, we found that an E. coli K-12 mutant defective in type 1 fimbriae, but not isogenic mutants lacking flagella or antigen 43, was outcompeted by the wild-type strain during prolonged catheter colonization. The importance of type 1 fimbriae for catheter colonization was verified using a fimA mutant of uropathogenic E. coli strain CFT073 with human and artificial urine. Orientation of the invertible element (IE) controlling type 1 fimbrial expression in bacterial populations harvested from the colonized catheterized bladder in vitro suggested that the vast majority of catheter-colonizing cells (up to 88%) express type 1 fimbriae. Analysis of IE orientation in E. coli populations harvested from patient catheters revealed that a median level of ∼73% of cells from nine samples have switched on type 1 fimbrial expression. This study supports the utility of the dynamic catheterized bladder model for analyzing catheter colonization factors and highlights a role for type 1 fimbriae during CAUTI.     

Enterococcus faecalis Subverts and Invades the Host Urothelium in Patients with Chronic Urinary Tract Infection

Bacterial urinary tract infections (UTI) are a major growing concern worldwide. Uropathogenic Escherichia coli has been shown to invade the urothelium during acute UTI in mice and humans, forming intracellular reservoirs that can evade antibiotics and the immune response, allowing recurrence at a later date. Other bacterial species, such as Staphylococcus saprophyticus, Klebsiella pneumonia and Salmonella enterica have also been shown to be invasive in acute UTI. However, the role of intracellular infection in chronic UTI causing more subtle lower urinary tract symptoms (LUTS), a particular problem in the elderly population, is poorly understood. Moreover, the species of bacteria involved remains largely unknown. A previous study of a large cohort of non-acute LUTS patients found that Enterococcus faecalis was frequently found in urine specimens. E. faecalis accounts for a significant proportion of chronic bladder infections worldwide, although the invasive lifestyle of this uropathogen has yet to be reported. Here, we wanted to explore this question in more detail. We harvested urothelial cells shed in response to inflammation and, using advanced imaging techniques, inspected them for signs of bacterial pathology and invasion. We found strong evidence of intracellular E. faecalis harboured within urothelial cells shed from the bladder of LUTS patients. Furthermore, using a culture model system, these patient-isolated strains of E. faecalis were able to invade a transitional carcinoma cell line. In contrast, we found no evidence of cellular invasion by E. coli in the patient cells or the culture model system. Our data show that E. faecalis is highly competent to invade in this context; therefore, these results have implications for both the diagnosis and treatment of chronic LUTS.     

Escherichia coli Peptidoglycan Structure and Mechanics as Predicted by Atomic-Scale Simulations

Bacteria face the challenging requirement to maintain their shape and avoid rupture due to the high internal turgor pressure, but simultaneously permit the import and export of nutrients, chemical signals, and virulence factors. The bacterial cell wall, a mesh-like structure composed of cross-linked strands of peptidoglycan, fulfills both needs by being semi-rigid, yet sufficiently porous to allow diffusion through it. How the mechanical properties of the cell wall are determined by the molecular features and the spatial arrangement of the relatively thin strands in the larger cellular-scale structure is not known. To examine this issue, we have developed and simulated atomic-scale models of Escherichia coli cell walls in a disordered circumferential arrangement. The cell-wall models are found to possess an anisotropic elasticity, as known experimentally, arising from the orthogonal orientation of the glycan strands and of the peptide cross-links. Other features such as thickness, pore size, and disorder are also found to generally agree with experiments, further supporting the disordered circumferential model of peptidoglycan. The validated constructs illustrate how mesoscopic structure and behavior emerge naturally from the underlying atomic-scale properties and, furthermore, demonstrate the ability of all-atom simulations to reproduce a range of macroscopic observables for extended polymer meshes.     

A unique arabinose 5-phosphate isomerase found within a genomic island associated with the uropathogenicity of Escherichia coli CFT073

Previous studies showed that deletion of genes c3405 to c3410 from PAI-metV, a genomic island from Escherichia coli CFT073, results in a strain that fails to compete with wild-type CFT073 after a transurethral cochallenge in mice and is deficient in the ability to independently colonize the mouse kidney. Our analysis of c3405 to c3410 suggests that these genes constitute an operon with a role in the internalization and utilization of an unknown carbohydrate. This operon is not found in E. coli K-12 but is present in a small number of pathogenic E. coli and Shigella boydii strains. One of the genes, c3406, encodes a protein with significant homology to the sugar isomerase domain of arabinose 5-phosphate isomerases but lacking the tandem cystathionine beta-synthase domains found in the other arabinose 5-phosphate isomerases of E. coli. We prepared recombinant c3406 protein, found it to possess arabinose 5-phosphate isomerase activity, and characterized this activity in detail. We also constructed a c3406 deletion mutant of E. coli CFT073 and demonstrated that this deletion mutant was still able to compete with wild-type CFT073 in a transurethral cochallenge in mice and could colonize the mouse kidney. These results demonstrate that the presence of c3406 is not essential for a pathogenic phenotype.     

Arabidopsis thaliana GYRB3 Does Not Encode a DNA Gyrase Subunit

Background

DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3.

Methodology/Principal Findings

We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer.

Conclusions/Significance

These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen.     

Lactobacillus gasseri SBT2055 Reduces Infection by and Colonization of Campylobacter jejuni

Campylobacter is a normal inhabitant of the chicken gut. Pathogenic infection with this organism in humans is accompanied by severe inflammation of the intestinal mucosal surface. The aim of this study was to evaluate the ability of Lactobacillus gasseri SBT2055 (LG2055) to inhibit the adhesion and invasion of Campylobacter jejuni in vitro and to suppress C. jejuni colonization of chicks in vivo. Pretreatment with LG2055 significantly reduced adhesion to and invasion of a human epithelial cell line, Intestine 407, by C. jejuni 81–176. Methanol (MeOH)-fixed LG2055 also reduced infection by C. jejuni 81–176. However, proteinase K (ProK)-treated LG2055 eliminated the inhibitory effects. Moreover, LG2055 co-aggregated with C. jejuni 81–176. ProK treatment prevented this co-aggregation, indicating that the co-aggregation phenotype mediated by the proteinaceous cell-surface components of LG2055 is important for reducing C. jejuni 81–176 adhesion and invasion. In an in vivo assay, oral doses of LG2055 were administered to chicks daily for 14 days after oral inoculation with C. jejuni 81–176. At 14 days post-inoculation, chicks treated with LG2055 had significantly reduced cecum colonization by C. jejuni. Reduction in the number of C. jejuni 81–176 cells adhering to and internalized by human epithelial cells demonstrated that LG2055 is an organism that effectively and competitively excludes C. jejuni 81–176. In addition, the results of the chick colonization assay suggest that treatment with LG2055 could be useful in suppressing C. jejuni colonization of the chicks at early growth stages.