The fliL gene significantly affects the motility and sporulation abilities of Clostridioides difficile / 生物工程学报

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.

Contribution of flagella and motility to gut colonisation and pathogenicity of Salmonella Enteritidis in the chicken

ABSTRACT Salmonella Enteritidis causes fowl paratyphoid in poultry and is frequently associated to outbreaks of food-borne diseases in humans. The role of flagella and flagella-mediated motility into host-pathogen interplay is not fully understood and requires further investigation. In this study, one-day-old chickens were challenged orally with a wild-type strain Salmonella Enteritidis, a non-motile but fully flagellated (SE ΔmotB) or non-flagellated (SE ΔfliC) strain to evaluate their ability to colonise the intestine and spread systemically and also of eliciting gross and histopathological changes. SE ΔmotB and SE ΔfliC were recovered in significantly lower numbers from caecal contents in comparison with Salmonella Enteritidis at early stages of infection (3 and 5 dpi). The SE ΔmotB strain, which synthesises paralysed flagella, showed poorer intestinal colonisation ability than the non-flagellated SE ΔfliC. Histopathological analyses demonstrated that the flagellated strains induced more intense lymphoid reactivity in liver, ileum and caeca. Thus, in the present study the flagellar structure and motility seemed to play a role in the early stages of the intestinal colonisation by Salmonella Enteritidis in the chicken.

Morphology, distribution and abundance of antennal sensilla of the oyster mushroom fly, Coboldia fuscipes (Meigen) (Diptera: Scatopsidae)

ABSTRACT We investigated the distribution, morphology and abundance of antennae sensilla of Coboldia fuscipes (Meigen) using scanning electron microscopy. Antennae of C. fuscipes consisted of scape, pedicel, and flagellum with eight flagellomeres. Antennal scape and pedicel had only one type of sensillum, i.e., sensilla chaetica. Significant differences were found between the number and distribution of these sensilla. Four types of morphologically distinct sensilla on the flagellum were identified, including sensilla chaetica, sensilla trichoidea, sensilla coeloconica, and sensilla basiconica (three subtypes). Significant differences were found in the abundance and distribution of sensilla among the antennal flagella and diverse flagellomeres in both sexes. Sensilla trichoidea is the most abundant of sensilla discovered on the antennal flagellum. Sensilla chaetica is the largest and longest sensilla among all the types of sensilla found on the antennal surface of C. fuscipes. Sensilla coeloconica is widely distributed all over the flagellum surface except for the first of female. Some significant differences in the abundance and distribution were also observed among sensilla basiconica of flagellum. The probable biological function of each sensillum type was deduced based on the basis of their structure. These results serve as important basis for further studies on the host location mechanism and mating behavior of C. fuscipes.

Mechanisms and molecular typing of carbapenem-resistant Proteus mirabilis strains deficient in swarming motility / 中华微生物学和免疫学杂志

Objective To investigate the mechanism and epidemiological characteristics of carbap-enem-resistant Proteus mirabilis ( PM) strains deficient in swarming motility. Methods PM strains were isolated from Hangzhou General Hospital of CAPF ( Chinese People′s Armed Police Forces) during January 2013 to December 2014. Bacterial motility and flagella of the PM strains were observed through semi-solid agar culture and flagella staining. Pulsed-field gel electrophoresis ( PFGE) was performed for homology anal-ysis. Antimicrobial susceptibility test and phenotypic confirmatory test were also carried out. PCR analysis and DNA sequencing were performed to confirm the genotype of resistant genes. Plasmid electroporation and S1-PFGE in combination with Southern blot hybridization were used to determine the location of the carbap-enem-resistant genes. Genetic structure of the blaKPC-2 gene was obtained by PCR mapping. Results A total of 42 PM isolates deficient in swarming motility were screened out and the resistance rates to imipenem and meropenem were 57. 1% and 52. 4%, respectively. PCR analysis and DNA sequencing confirmed that 24 carbapenem-resistant PM isolates deficient in swarming motility carried blaKPC-2 gene and belonged to three clones as indicated by the results of PFGE. Southern blot hybridization indicated that the blaKPC-2 gene was located on plasmids varying in size (26 kb, 55 kb and 139 kb). In addition, some of the strains harbored several resistant genes, such as blaTEM-1 , blaCTX-M-65 and rmtB. The genetic structures of strains carrying blaKPC-2 gene were ISKpn8, blaKPC-2 and ISKpn6-like from upstream to downstream. Conclusion Compared with the PM strains with swarming motility, the carbapenem-resistance rate was significantly higher in these PM strains deficient in swarming motility. Carbapenemases KPC-2 played an important role in the carbapen-em-resistant PM strains deficient in swarming motility. There was a cloning spread trend for carbapenem-re-sistant PM strains in our hospital. Clinicians should pay more attention to the risk of spreading.

Importance of sperm morphology during sperm transport and fertilization in mammals / 亚洲男科学杂志(英文版)

After natural or artificial insemination, the spermatozoon starts a journey from the site of deposition to the place of fertilization. However, only a small subset of the spermatozoa deposited achieves their goal: to reach and fertilize the egg. Factors involved in controlling sperm transport and fertilization include the female reproductive tract environment, cell-cell interactions, gene expression, and phenotypic sperm traits. Some of the significant determinants of fertilization are known (i.e., motility or DNA status), but many sperm traits are still indecipherable. One example is the influence of sperm dimensions and shape upon transport within the female genital tract towards the oocyte. Biophysical associations between sperm size and motility may influence the progression of spermatozoa through the female reproductive tract, but uncertainties remain concerning how sperm morphology influences the fertilization process, and whether only the sperm dimensions per se are involved. Moreover, such explanations do not allow the possibility that the female tract is capable of distinguishing fertile spermatozoa on the basis of their morphology, as seems to be the case with biochemical, molecular, and genetic properties. This review focuses on the influence of sperm size and shape in evolution and their putative role in sperm transport and selection within the uterus and the ability to fertilize the oocyte.

Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP)

Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP) assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

Los productos alternativos de LYT1 de Trypanosoma cruzi tienen un patrón de localización diferencial / The alternative products of Trypanosoma cruzi LYT1 have different localization patterns

LYT1 is a molecule with lytic activity under acidic conditions that, as genetically demonstrated, participates in the infection and stage transition of T. cruzi. The differing functions of this protein result from alternative trans-splicing, resulting in proteins that contain either a secretion and nuclear sequence (LYT1s) or the nuclear sequence alone (LYT1n). To determine the localization of different LYT1 products, transgenic parasites expressing LYT1s or LYT1n fused to the enhanced green fluorescence sequence were analyzed. LYT1s-EGFP localized to the flagellum, vacuoles, membrane and regions of the nucleus and kinetoplast; LYT1n-EGFP localized to the nucleus and kinetoplast, and occasionally in vacuoles. These results show that even though different LYT1 products localize to the same sites, they are also found in different intracellular organelles and microenvironments, which could influence their multifunctional behavior.

LYT1 es una molécula con actividad lítica en condiciones ácidas, que según se demostró genéticamente, participa en el proceso de infección y transición de estadio de T. cruzi. Su diferente funcionalidad es resultado de la producción de dos proteínas, obtenidas por trans-empalme alternativo, que contienen una secuencia de secreción y una nuclear (LYT1s) o únicamente la secuencia nuclear (LYT1n). Para evaluar la localización de los diferentes productos de LYT1, se analizaron parásitos transgénicos que expresan la secuencia de LYT1s o LYT1n fusionada con la secuencia de la verde fluorescente. LYT1s-EGFP se localiza en flagelo, vacuolas, membrana y región del núcleo y cinetoplasto; mientras que, LYT1n-EGFP se localiza en la región del núcleo y cinetoplasto, y ocasionalmente en vesículas. Estos resultados muestran que aún cuando los distintos productos de LYT1 comparten algunos sitios de localización, también se encuentran en distintos organelos y microambientes intracelulares que podrían influir en su comportamiento multifuncional.

Prokaryotic expression of Leptospira interrogans FliH/Ⅰ/Y/N genes and determination of the protein locations in leptospiral envelope / 中华微生物学和免疫学杂志

Objective To clone fliH, fliⅠ, fliY and fliN genes that encoding flagellum-associated proteins of L. interrogans for construction of their prokaryotic expression systems, and to determine the loca- tions of Flirt, FliⅠ, FIiY and FIiN. Methods The fliH, fliⅠ, fliY andfliN genes were amplified by PCR and sequenced after T-A cloning. Prokaryotie expression systems of the target genes were constructed subsequently. Expression of target recombinant proteins were demonstrated by SDS-PAGE and BioRad Gel Image Analyzer, and Ni-NTA affinity chromatography was performed to extract the target expression prod- ucts. Rabbits were subcutaneously immunized with the four recombinant proteins respectively to obtain anti- sera. ELISA was performed to measure the titers of antisera and Western blot assay was used to determine the immunobinding abilities among the antisera and their antigens. Immunoelectron microscopy was selected to locate the position of FliH, FliⅠ, FliY and FIiN. Results Segments of fliH, fliⅠ, fliY andfliN genes with 924, 1365, 1065 and 318 bp in size were successfully obtained by PCR. Similarities of nucleotide and puta- tive amino acid sequences from the four genes were 100% compared with the reported sequences. The con- structed prokaryotic systems efficiently expressed rFliH, rFliⅠ, rFliY and rFliN with the outputs of approxi- mate 20% of the total bacterial proteins. The rabbits immunized by rFliH, rFliⅠ, rFliY or rFliN could pro- duce antibody. The antisera had the titers above 1:100 000, and could recognize the corresponding recombi- nant proteins and membrane proteins of L interrogans to display positive Western hybridization bands. Flirt, FliⅠ, FliY and FliN were found to distribute on the external surface of inner envelope, the internal surface of outer envelope or the interspaces between the two layers of L. interrogans envelope. Conclusion The pro- karyotic expression systems was successfully constructed in this study, which could efficiently express flagel-lum-associated proteins FliH, FliⅠ, FliY and FliN of L. interrogans. The antisera with high titers to recognize their protein antigens were also obtained. Flagellum-associated proteins Flirt, FliⅠ, FIiY and FIiN are the inner envelope proteins and/or outer envelope proteins of L. interrogans.