十字花科黑腐病菌pilY基因与涌动有关

细胞的运动在细菌的趋化、粘附、生物膜形成以及致病等过程中发挥关键作用。细菌的运动方式主要有三种:游动(Swimming motility)、涌动(Swarming motility)和颤动(Twitching motility)。游动和涌动由鞭毛介导,而颤动由Ⅳ型纤毛介导。全基因组测序结果显示,十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)存在一套完整的合成Ⅳ型纤毛的基因,而且能够产生Ⅳ型纤毛;但有证据表明Xcc不能进行颤动。在实验室之前的研究中发现,Ⅳ型纤毛基因pilH和pilG与Xcc的游动和涌动有关。在本研究中,通过缺失突变体和互补菌的构建,以及表型分析策略,对Ⅳ型纤毛基因pilY进行了生物学功能鉴定。结果发现,pilY缺失突变体的涌动能力比野生型菌株显著增强,而互补菌株的涌动能力与野生型的相似,证明pilY基因具有抑制Xcc涌动的功能。此外,本研究还发现pilY缺失不影响Xcc的生长繁殖、致病力以及致病因子的产生。

Molecular epidemiology of piliated pneumococcal isolates at a major tertiary hospital in the Klang Valley,Malaysia

Objective:To characterise a collection of pili-carrying and none pili-carrying pneumococcal isolates of clinical origin for serotypes,antibiotic resistance and genotype.Methods:In total,42 clinical isolates were collected between October 2017 and December 2019.Those isolates were analysed for antimicrobial susceptibility,serotype distribution,detection of pneumococcal virulence and pilus genes.Multilocus sequence typing was performed only for piliated isolates,followed by phylogenetic analysis.Results:The common isolation sites among the pneumococcal isolates were tracheal aspirate(28.6%),blood(26.2%),and sputum(23.8%).Fifty percent isolates were resistant to erythromycin,tetracycline(50.0%)and trimethoprim-sulfamethoxazole(43.0%).The most frequent were serotypes 19F(28.6%),6A/B(23.8%)and 19A(14.3%).Piliated isolates were detected in a small proportion(33.3%);64.3%were multidrug-resistant.ST320 was the prevalent sequence type among the piliated isolates and genetically related to the Pneumococcal Molecular Epidemiology Network clones Taiwan19F-14(CC271).In the phylogenetic analysis,some piliated isolates showed a close association having similar ST320,carrying serotype 19A and both pilus genes indicating their clonal spread.Conclusions:Pneumococcal lineages of piliated isolates have been globally disseminated and pili could have played a role in the spread of antibiotic resistant clones.

Adhesion of <i>Gallibacterium anatis</i>to Chicken Oropharyngeal Epithelial Cells and the Identification of Putative Fimbriae

Microbial infections are typically initiated by the colonization of tissues by a specific mechanism that promotes adherence to host cells or tissues. In this work, we characterized the ability of Gallibacterium anatis F149T to express fimbriae that may be involved in mucosal attachment. Using transmission electron microscopy, the fimbriae-like structures could be observed on the surface of negatively stained G. anatis F149T, and these structures were further visualized after being released by physical shaking. When the fimbriae-like structures were separated by SDS-PAGE, the proteins comprising them were isolated and sized at 13 and 25 kDa. G. anatis F149T was able to adhere to chicken oropharyngeal epithelial cells. Adhesion could be completely inhibited by pretreatment of the bacterial cells with trypsin, whereas 25% inhibition was attained after pretreatment with an antiserum against the 13 kDa protein. We demonstrated by immuno-gold electron microscopy that the antibodies from the antiserum were specifically associated with the fimbria-like structures on G. anatis. These results indicated that G. anatis F149T expresses fimbriae that contribute to its adhesion to chicken oropharyngeal epithelial cells and may be important for colonization of the upper respiratory tract.

Genomic characteristics of Dickeya fangzhongdai isolates from pear and the function of type Ⅳ pili in the chromosome

Dickeya fangzhongdai, the causal agent of bleeding canker of pear, is a new member of the Dickeya genus and the only one that infects woody plants. Recent studies have reclassified several Dickeya isolates as D. fangzhongdai, which were isolated from various environments, including water, Phalaenopsis sp. and Aglaonema sp. To provide genomic characterization of D. fangzhongdai isolates from pear, the genomes of D. fangzhongdai strain JS5(=China General Microbiological Culture Collection Center, CGMCC 1.15464 ~T=DSM 101947 ~T), along with two other isolates, LN1 and QZH3, were sequenced and compared to those of other Dickeya spp. Homology greater than 99% was observed among three D. fangzhongdai strains. Plasmid, type IV secretion system(T4 SS) and type IV pili(TFPs) were found in genomes of D. fangzhongdai isolates. Comparative analysis of the type Ⅲ secretion systems(T3 SS), type Ⅲ secretion effectors(T3 SE), plant cell wall degradation enzymes(PCWDE) and membrane transport proteins of Dickeya spp. showed some differences which might reflect the variations of virulence, phylogenetic and phenotypic characteristics of Dickeya spp. In addition, deletion mutant of TFP in D. fangzhongdai JS5 showed no twitching motility and reduced virulence and biofilm formation. The fingdings of the distinctive plasmid, T4 SS and TFPs, as well as the differences of T3 SE, PCWDE and membrane transport proteins make D. fangzhongdai isolates unique. These results also suggested that acquisition of virulence genes by horizontal gene transfer might play some role in the genetic variation of D. fangzhongdai.

Detailed Histological Structure of Human Hair Follicle Bulge Region at Different Ages: A Visible Niche for Nesting Adult Stem Cells

In the bulge region of the hair follicle, a densely and concentrically packed cell mass is encircled by the arrector pili muscle (APM), which offers a specilized microenvironment (niche) for housing heterogeneous adult stem cells. However, the detailed histological architecture and the cellular composition of the bulge region warrants intensive study and may have implications for the regulation of hair follicle growth regulation. This study was designed to define the gene-expression pro-files of putative stem cells and lineage-specific precursors in the mid-portions of plucked hair follicles prepared according to the presence of detectable autofluorescence. The structure was also characterized by using a consecutive sectioning technique. The bulge region of the hair follicle with autofluorescence was precisely excised by employing a micro-dissection procedure. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to identify the gene expression profiles specific for epithelial, melanocyte and stromal stem cells in the bulge region of the hair follicle visualized by autofluorescence. The morphology and its age-dependent changes of bulge region of the hair follicles with autofluorescence segment were also examined in 9 scalp skin specimens collected from patients aged 30 weeks to 75 years, by serial sectioning and immuno-staining. Gene expression profile analysis revealed that there were cells with mRNA transcripts of DctHiTyraseLo-Tyrp1LoMC1RLoMITFLo/K15Hi/NPNTHi in the bulge region of the hair follicle with autofluorescence segments, which differed from the patterns in hair bulbs. Small cell-protrusions that sprouted from the outer root sheath (ORS) were clearly observed at the APM inserting level in serial sections of hair follicles by immunohistological staining, which were characteristically replete with K15+/K19+expressing cells. Likewise, the muscle bundles of APM positive for smooth muscle actin intimately encircled these cell-protrusions, and the occurrence frequency of the cell-protrusions was increased in fetal scalp skin compared with adult scalp skin. This study provided the evidence that the cell-protrusions occurring at the ORS relative to the APM insertion are more likely to be characteristic of the visible niches that are filled with abundant stem cells. The occurrence frequency of these cell-protrusions was significantly increased in fetal scalp skin samples (128%) as compared with the scalp skins of younger (49.4%) and older (25.4%) adults (P<0.01), but difference in the frequency between the two adult groups were not significant. These results indicated that these cell-protrusions function as a niche house for the myriad stem cells and/or precursors to meet the needs of the development of hair follicles in an embryo. The micro-dissection used in this study was simple and reliable in excising the bulge region of the hair follicle with autofluorescence segments dependent on their autofluorescence is of value for the study of stem cell culture.

Regulation of fim genes in uropathogenic Escherichia coli

Uropathogenic Escherichia coli(UPEC)is the leading cause of urinary tract infections in women,causing significant morbidity and mortality in this population.Adherence to host epithelial cells is a pivotal step in the pathogenesis of UPEC.One of the most important virulence factors involved in mediating this attachment is the type 1 pilus(type 1 fimbria)encoded by a set of fim genes arranged in an operon.The expression of type 1 pili is controlled by a phenomenon known as phase variation,which reversibly switches between the expression of type 1 pili(Phase-ON)and loss of expression(Phase-OFF).Phase-ON cells have the promoter for the fimA structural gene on an invertible DNA element called fimS,which lines up to allow transcription,whereas transcription of the structural gene is silenced in Phase-OFF cells.The orientation of the fimS invertible element is controlled by two site-specific recombinases,FimB and FimE.Environmental conditions cause transcriptional and post-transcriptional changes in UPEC cells that affect the level of regulatory proteins,which in turn play vital roles in modulating this phase switching ability.The role of fim gene regulation in UPEC pathogenesis will be discussed.

Fate of polyphenols in pili(Canarium ovatum Engl.) pomace after in vitro simulated digestion

Objective: To evaluate the stability and bioavailability of polyphenols in pili(Canarium ovatum Engl.) pomace during simulated in vitro digestion.Methods: Freeze-dried pili pomace was subjected to in vitro digestion simulating conditions in the stomach, small intestine and colon. Total polyphenols, anthocyanins, fl avonoids and condensed tannins, and its antioxidant activity – 1,1-diphenyl-2-picrylhydrazyl, 2,20-azinobis-3-ethylbenzothiozoline-6-sulfonic acid, and ferric reducing antioxidant power were measured using standard spectrophotometric methods.Results: In vitro digestion of pili pomace resulted in reduction of phenolic compounds.Condensed tannins and anthocyanins were released in the gastric and intestinal stages,while total polyphenols and flavonoids after fermentation simulating colonic conditions.Antioxidant values of the bioavailable fractions showed that more than 90% of activity was lost during simulated digestion.Conclusions: Findings indicate that pili pomace is a promising functional ingredient for food and dietary supplements which can furnish potentially bioavailable phenolic antioxidants to the body.

PCR Detection of Pilus-associated Genes and Serotype Identification of Pathogenic Escherichia coli Isolated from Chickens in the Jidong Area

[ Objective] This study aimed to explore the presence of type I pill （fimC gene） and P pill （papC gene） and identify the serotype of pathogenic E. coli isolated from chickens in the Jidong Area. [ Method] Type I pill （fimC gene） and P pill （papC gene） were detected by PCR. The serotype was identified by con- ventional agglutination test. [ Result ] The results showed that 100% of chicken-derived E. coil strains expressed type I pill （fimC gene） ; 39.1% （9/23） of chick- en-derived E. coli strains expressed P pill （papC gene）. In addition, 23 isolates of chicken-derived E. coil were assigned to 14 O serotypes, including O78, O93, O 45, O101, O38, O88, O24, O1, O163, O53, O15, O87, O34 and O29, among which O78 was the dominant serotype that accounted for 42.8% （6/14） of the total strain number. [ Conclusion] Chicken-derived E. coli strains in the Jidong Area belonged to 14 serotypes, and 078 was the dominant serotype; 83.3% of 078 serotvDe E. coli strains expressed both tvDe I Dill and P Dill.

Study on the mechanism of Porphyromonas gingivalis pili mediated ather osclerosis

Chronic periodontitis is the most common infectious periodontal disease caused by plaque biofilm.Pili is an important bacterial component of Porphyromonas gingivalis.Epidemiological studies have shown that chronic periodontitis can increase the risk of cardiovascular disease,and atherosclerosis is the pathological basis of cardiovascular disease.This article will summarize the mechanism of Porphyromonas gingivalis pili protein mediated chronic periodontitis and atherosclerosis,in order to expand new ideas of disease prevention and treatment.

Are type IV pili involved in <i>Vibrio anguillarum</i>virulence towards sea bass (<i>Dicentrarchus labrax</i>L.) larvae?

Vibrio anguillarum, an important bacterial fish pathogen, expresses a variety of virulence factors contributing to its ability to cause vibriosis in fish. Many virulence factors of this pathogen remain however unknown. For example, a type IV pilus system was previously reported to be potentially involved in the virulence of this bacterium but no experimental evidence was reported yet. In this study, complete genome sequencing of V. anguillarum strain VIB15, shown to be highly virulent towards sea bass (Dicen- trarchus labrax L.) larvae, revealed the presence of a PilA pilin. A V. anguillarum VIB15 pilA mutant was constructed and the pathogenicity of this mutant was assessed in a gnotobiotic sea bass system developed for virulence screening. Our results suggest that the V. anguillarum pilA gene is not crucial for virulence towards sea bass larvae. Possibly, another type IV pilus system identified in V. anguillarum, showing homology to the mannose-sensitive hemagglutinin pilin of Vibrio cholerae, may complement the pilA mutation. Alternatively, the type IV pilus system has a role in infection of juvenile or adult fish, rather than in the larval phase. As such, further research is required to unravel the potential role of type IV pili in V. anguillarum virulence.

迟缓爱德华菌菌毛蛋白原核表达及多抗血清制备

为研究迟缓爱德华菌菌毛蛋白PILI的功能和免疫原性,本研究利用PCR方法从迟缓爱德华菌基因组中克隆出pili基因,并将其构建到pET-32a原核表达载体上,重组载体转化BL21大肠杆菌诱导重组蛋白表达,通过SDSPAGE和Western blot方法验证重组蛋白表达。用亲和层析方法纯化蛋白,纯化的蛋白免疫小鼠,所得的多抗血清能够特异性识别迟缓爱德华菌PILI蛋白,为研究PILI蛋白奠定基础。