Motility Expression Research Articles

The Effects of Negative Pressure on the Gene Expression of Motility Related Proteins in Boar Spermatozoa during Liquid Storage at 17oC

Background: The present study was aimed to investigate the effects of negative pressure applied before storage on the gene expression of motility related proteins in boar spermatozoa.Methods: Boar semen samples were collected and pooled and diluted with Modena solution containing 0.4% (w/v) of bovine serum albumin. Negative pressure was applied for 2-5 min using a vacuum pump with a barometer. The pressure applied was 0 (Control), -0.02 MPa (P2), -0.04 MPa (P4) and -0.08 MPa (P8). The expression of AQN-1, AQN-3, AWN, PSP-I, PSP-II gene in boar spermatozoa was evaluated.Result: Application of -0.04 MPa improved the sperm motility compared with the other groups. In conclusion, our results confirmed thatnegative pressure preservation at 17oC had an effect on the expression of boar sperm adhesion protein gene. The relative expression of AWN0PSP-I and PSP-II genes in boar sperm were lower under -0.04 MPa and -0.08 MPa pressure, which was beneficial to protect spermatozoa motility.

Characterization of native Escherichia coli populations from bovine vagina of healthy heifers and cows with postpartum uterine disease.

Even though Escherichia coli are common bacteria of the bovine vaginal microbiota, they represent an important pathogen that causes diseases in the reproductive tract and subfertility. However, the actual endometrial virulence profile of E. coli is poorly understood. The present study aims to characterize the phylogenetic structure and virulence potential of native vaginal populations of E. coli from healthy heifers (H), and cows with postpartum uterine diseases (PUD), such as metritis/endometritis (MT) or repeat breeder cows (RB). To this end, the virulence repertoire of 97 E. coli isolates was genotypically and phenotypically assessed. Most of them were assigned to phylogenetic group A (74%), followed by B1 (17%) and D (9%); RB strains were significantly (p < 0.05) more represented by B1. Seven of the 15 evaluated virulence genes (VFG) were detected and the most prevalent were fimH (87%), agn43 (41%) and csgA (35%); while traT (27%), fyuA (11%), hlyA (5%) and kpsMT II (5%) were observed in a lower proportion. Particularly, fyuA was significantly higher (p < 0.05) in MT cows whereas csgA showed the same behavior in PUD animals (p < 0.05). When comparing H and PUD strains, these last ones were associated to positive expression of biofilm, fimbriae curli/cellulose and motility; yet RB strains did not show motility. Vaginal B1 E. coli populations, that possess VFG (fyuA and csgA) as well as the expression of motility, curli fimbriae/cellulose and biofilm, may represent risk factors for endometrial disorders; specifically, those that also, have kpsMT II may have a pathogenic potential for causing the RB syndrome. Future research focusing on the detection of these strains in the vaginal microbiota of cows with postpartum uterine diseases should be done since the control of their presence in vagina could reduce the risk that they access the uterus during the postpartum period.

Abstract 653: Ovarian cancer persister cells: 2D and 3D in-depth characterization and analysis

Abstract Ovarian cancer is one of the five cancer types with highest incidence of death in women. Due to a lack of diagnostic options, timely detection of ovarian cancer is difficult and 75% of patients are diagnosed at a late stage. The standard therapy for ovarian cancer is resection of the affected tissue, followed by chemotherapy. However, patients frequently suffer from relapse of the disease, as some tumor cells persist chemotherapy and grow into a new, more aggressive tumor. To offer therapeutic approaches that prevent relapse by eradicating persister cells we aimed to identify the molecular mechanisms and determinants that are characteristic for persister cells. As initial cellular model system OVCAR-3 cells were used to select ovarian cancer persister cells. Selection was performed analogously to serum levels of chemotherapy patients, i.e. OVCAR-3 cells were incubated twice in the presence of 13 µM Cisplatin for 4 hours. Cisplatin-selection identified clones were collected and in further analyses compared to randomly collected untreated control clones. In order to account the native tumor microenvironment and heterogeneity within the tumor and among patients we cultured precision-cut tissue slices from primary ovarian tumor tissue with 280 µm thickness. Tumor tissue slices were cultured either under control conditions or exposed to 13 µM Cisplatin. Slices were further processed and cultured in Matrigel producing tumor-derived organoids, i.e. tumoroids. In contrast to OVCAR-3 control clones, the Cisplatin-selected OVCAR-3 persister cells showed filopodia-like structures. A scratch assay demonstrated enhanced motility of OVCAR-3-persister cells. Interestingly, cell death analysis (Annexin V) of Cisplatin exposed control and persister clonal cell lines showed the presence of surviving cells in both, persister and control groups. To identify the molecular determinants specific for persister cells we performed RNA sequencing. Data showed that, in line with initial results, the motility associated genes Snail and Vimentin are up regulated in persister clones. We successfully generated tumoroids from patient-derived ovarian cancer tissue slices. Tumoroids were cultured for more than two months and displayed a high viability. After exposure to Cisplatin less tumoroids generated from tumor slices were found. OVCAR-3-derived persister clones show morphological alterations (filopodia-like structures), enhanced motility and expression of motility associated genes. We expect the identification of genetic determinants for persister cell characteristics from comparison of RNAseq results of OVCAR-3 persister clones and tumor-derived organoids. The identification of genetic determinants will direct the development of therapeutics approaches targeting persister cells in ovarian cancer to prevent recurrence of tumors. Citation Format: Kathrin Boepple, Meng Dong, Andrea Gaissler, Bernd Winkler, Markus Kleih, Frank Essmann, Walter E. Aulitzky. Ovarian cancer persister cells: 2D and 3D in-depth characterization and analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 653.

Determining the Relative Contribution and Hierarchy of hha and qseBC in the Regulation of Flagellar Motility of Escherichia coli O157:H7

In recent studies, we demonstrated that a deletion of hha caused increased secretion of locus of enterocyte encoded adherence proteins and reduced motility of enterohemorrhagic Escherichia coli (EHEC) O157:H7. In addition to the importance of hha in positive regulation of motility, a two-component quorum sensing pathway encoded by the qseBC genes has been shown to activate bacterial motility in response to mammalian stress hormones epinephrine and norepinephrine as well as bacterially produced autoinducer-3. In this study, we compared regulatory contribution and hierarchy of hha, a member of the Hha/YmoA family of nucleoid-associated proteins, to that of qseBC in the expression of EHEC O157:H7 motility. Since norepinephrine affects motility of EHEC O157:H7 through a qseBC-encoded two-component quorum sensing signaling, we also determined whether the hha-mediated regulation of motility is affected by norepinephrine and whether this effect is qseBC dependent. We used single (Δhha or ΔqseC) and double (Δhha ΔqseC) deletion mutants to show that hha exerts a greater positive regulatory effect in comparison to qseBC on the expression of motility by EHEC O157:H7. We also show that Hha is hierarchically superior in transcriptional regulation of motility than QseBC because transcription of qseC was significantly reduced in the hha deletion mutant compared to that in the parental and the hha-complemented mutant strains. These results suggest that hha regulates motility of EHEC O157:H7 directly as well as indirectly by controlling the transcription of qseBC.

Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella.

The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC. SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant. Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently. A form of BarA lacking the two N-terminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA. Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters. Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB. The sirA gene did not regulate csrA but did activate csrB expression. Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter. We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB.

FliU and fliV: two flagellar genes essential for biosynthesis of Salmonella and Escherichia coli flagella

The possible functions of two recently described flagellar genes, fliU and fliV, have been examined. Introduction of gene fliC, encoding the bacterial flagellin protein, into a number of flagellin-deficient Salmonella and Escherichia coli strains failed to complement the mutations in these strains, and the FliC flagellin was accumulated in the bacterial cytoplasm. Complementation with fliU and fliV, which map downstream of fliC, restored motility to some of the mutants which became flagellated. After inactivation of either fliU or fliV, such complementation no longer occurred and the flagellin protein accumulated in the cytoplasm, which suggested that both genes are required for the secretion of flagellin and expression of motility. Expression of these genes from high copy number plasmids resulted in the synthesis of exceptionally long flagella and in detection of the FliV protein on polyacrylamide gels.

Atypical characteristics of the salmonid pathogen Aeromonas salmonicida

Incubation of Aeromonas salmonicida at supra-optimal temperatures, i.e. 30-37 degrees C, resulted in the expression of motility by polar flagella, and changes in sugar fermentation patterns, e.g. loss of acid production from mannitol, loss of the ability to degrade complex molecules (aesculin, DNA, elastin and gelatin), and an increase in antibiotic resistance (notably co-trimoxazole). Motility was enhanced in cultures grown in brain heart infusion broth supplemented with 18% (w/v) Ficoll.

Expression of motility in strains of the non-motile species Aeromonas media

Four isolates of the non-motile species Aeromonas media, icluding the type strain, were demonstrated by transmission electronk microcopy to possess polar flagella. Motility appeared to be restricted to old, regularly subcultured strains, and coincided with an increase in colony size and a concomitant reduction in the amount of brown diffusible pigment produced by colonies on tryptone soya agar.

Volume change associated with the G-F transformation of flagellin

The volume change of the solution, Δ V, associated with the polymerization at neutral pH of the protein, flagellin, to flagella, a typical G-F transformation, was measured with a Carlsberg-type dilatometer. A large positive volume change, greater than 150 ml. per mole of flagellin polymerized, was observed for this process. The effect of variations in protein concentration and temperature on Δ V was studied. Both the final value of Δ V observed after complete polymerization of flagellin had occurred, Δ V max, as well as the rate of the volume change on polymerizing flagellin, estimated as the reciprocal of the mid-plateau value ( tΔ V max 2) −1 , were found to be temperature-dependent. Between 22 and 28 °C the polymerization, rate increased with increasing temperature. However, over this temperature range ΔV max was unchanged and had a value of 157 ± 4 ml. per mole of flagellin polymerized. Above 28 °C the polymerization rate decreased with increasing temperature but ΔV max increased to a maximum value at 35 °C of 306 ml. per mole of flagellin polymerized. At temperatures higher than 35 °C, the rate for the volume change decreases markedly and polymerization, as measured by changes in specific viscosity, does not occur. The effect of increasing temperature on the rate and magnitude of the volume changes observed is explained by assuming that the protein can be reversibly transformed to a state which lacks the capacity to reconstitute flagella, and that at temperatures above 28 °C this transformation becomes rate limiting for the polymerization process. Such a reversible transformation of flagellin can explain some of the well-known effects of temperature on the expression of motility and the formation of flagella in vivo.