Browsing by Subject "Motility"

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Stressful environments : motility and catecholamine response in Vibrio cholerae
(2014) Halang, Petra; Fritz-Steuber, Julia
The human pathogen Vibrio cholerae is able to inhabit a variety of environments. These include especially aquatic ecosystems, but the human intestine as well. V. cholerae is thus tolerant to a wide range of salinity and pH. Motility is achieved by a sodium driven polar flagellum. The affinity for Na+ to run the flagellum is determined by the stator complex PomAB, which is embedded in the cell membrane within the flagellar motor. A critical aminoacid residue for the binding of Na+ is aspartate 23 within the transmembrane helix of PomB. A mutation of this aminoacid residue leads to an immotile phenotype of V. cholerae. It was thus of interest to investigate if other polar or acidic aminoacid residues within PomB are important for the passage of Na+. Two potential candidates are serine at position 26 and aspartate at position 42 of PomB, both aminoacid residues are conserved within sodium driven flagellar stator complexes. To characterize the pathway of Na+ through the PomAB channel, the influence of chloride salts (Na+ and K+) and the pH on the motility of V. cholerae was studied. Motility decreased at elevated pH but increased if a chaotropic chloride salt was added, which excludes a direct Na+ and H+ competition in the process of binding to the conserved PomB D23 residue. Cells expressing the PomB S26A/T or D42N variants lost motility at low Na+ concentrations but regained motility in the presence of 170mM chloride. The swimming speeds of individual cells were also analyzed and revealed that S26 located within the membrane helix of PomB is required to promote very fast swimming of V. cholerae. Loss of hypermotility was observed with the S26T variant of PomB which was partially restored by lowering the pH of the external medium. Modification of PomA and PomB by N,N’-dicyclohexylcarbodiimide indicates the presence of protonated carboxyl groups in the hydrophobic regions of the two proteins. Na+ did not protect PomA and PomB from this modification. It could be demonstrated that the motility of V. cholerae is influenced by the pH and osmolality of the medium and thus, the aminoacid residues – S26 and D42 together with D23 – of PomB have a function in the passage of Na+ into the cell. The H+ rather than the Na+ concentration determines the efficiency of the motor, indicating the presence of a catalytical important hydrogen bond network in the motor channel. It is proposed that D23, S26 and D42 of PomB are part of an ion-conducting pathway formed by the PomAB stator complex. As mentioned above, V. cholerae is a pathogen which settles the human intestine. As other pathogens are able to respond specifically to the stress associated mammalian hormones epinephrine and norepinephrine it was of an interest to investigate the influence of these hormones on growth and motility of V. cholerae. The response to epinephrine and norepinephrine is mediated by the QseC sensor protein. The genome of V. cholerae comprises a gene which is homolog to qseC from E. coli. Growth and swarming of V. cholerae was enhanced in the presence of 0.1mM epinephrine or norepinephrine. qRT-PCR experiments revealed increased expression of the genes encoding the putative sensor kinase qseC and pomB, a component of the flagellar motor complex under the influence of catecholates. HPLC measurements of bacterial supernatant revealed that norepinephrine is completely degraded or metabolized after 48 h in the presence of V. cholerae, concomitant with the appearance of another, unidentified compound. On the other hand, V. cholerae seemed to stabilize epinephrine. After 48 h, 0.46% of the epinephrine added at the beginning of the growth experiment was retained. Again, a yet unidentified compound was detected. The experiments conducted in this work strongly indicate the presence of a catecholate receptor in V. cholerae.
Vergleichende Transkriptomanalyse und funktionelle Untersuchungen von enterohämorrhagischen Escherichia coli nach Kultivierung in Pflanzenmedium
(2020) Bufe, Thorsten; Schmidt, Herbert
Enterohemorrhagic Escherichia coli (EHEC) are human pathogens which are able to cause severe gastrointestinal diseases in humans. The gastrointestinal tract of cattle is considered as the main reservoir for EHEC and contaminated raw meat represents the primary source of infection. Yet there have been increasing reports over the last few decades of EHEC infections that were linked to the consumption of raw vegetables. Today it is generally accepted that EHEC bacteria are able to use plants as their secondary hosts, thus favouring the transmission to humans. To improve the understanding of this pathogen-plant interaction fundamental knowledge about the pathogens’ molecular adaptions towards plant material is urgently required. In the cope of this study the adaption of different EHEC strains towards components of the plant was examined. Therefore O157:H7 strain Sakai, O104:H4 strain C227-11phicu and O157:H strain 3072/96 were chosen as surrogates. In growth experiments performed with an artificial lettuce medium it could be shown that components of the lettuce were sufficient for the proliferation of the three strains. RNA-sequencing was performed to study the differential gene expression of the three strains after the growth in lettuce medium compared to the growth in M9 minimal medium. In order to compare genes according to standardized gene denotations, the differential gene expression analysis was performed on the basis of a shared genome including the genomes of the three pathogenic strains as well as the genome of Escherichia coli strain K-12 substrain MG1655. Analogous to the successful growth in presence of components of the plant an upregulation of genes involved in carbohydrate and peptide metabolism throughout all three strains was observed. Especially genes involved in the catabolism of lactose (lacZ), ribose (rbsAC) and xylose (xylF) were found to be uniformly upregulated. The greatest differences among the strains accounted for the regulation of motility and chemotaxis genes. O104:H4 strain C227-11phicu showed a strong upregulation of all three classes of the flagellar hierarchy (class I, II and III) in presence of plant derived compounds. These included genes involved in the establishment of the basal body hook structure (fli, flg), the synthesis of the flagellar filament (fliC), and the chemotaxis-system (che, tap, tar). In contrast, O157:H7 strain Sakai only featured upregulation of class I and class II genes. According to the transcriptional data both of these strains also showed increased swimming and swarming behaviour on motility plates in presence of lettuce extract. Solely O157:H- strain 3072/96, which is non-motile due to a deletion in the flhC gene, showed an upregulation of virulence factors encoded on the LEE pathogenicity island, including genes involved in the establishment of the T3SS (esc) and T3SS secreted effectors (esp). Interestingly, it was shown for O157:H- strain 3072/96 to have a powerful capacity to form biofilms in M9 minimal medium. Furthermore it was proven that the complementation of an intact flhC gene restored motility in O157:H- strain 3072/96. In this regard it could be shown that the deletion in flhC was not the mere reason for the augmented biofilm formation capacity. In addition to the biofilm formation, the strains’ potential to adhere to HT-29 cells was examined. Here a significantly increased adherence potential for O157:H- strain 3072/96 with respect to the motile strains could be observed, the lowest adherence potential was determined for O157:H7 strain Sakai. The results presented in this study clearly indicate that the different EHEC strains are capable to adapt towards the nutrient availability provided by their plantal host. It can be assumed that flagella and the chemotaxis system play a fundamental role in the finding and exploitation of the plant. Furthermore curli structures might play a crucial role in the initial adherence and the subsequent establishment of a biofilm on plant tissues. Presumably, besides the typical plant associated outbreak strain O157:H7 strain Sakai, there are further strains capable of utilizing their genetic repertoire in order to adapt towards the atypical environmental conditions within this niche. The findings of this study suggest that the strains, besides sharing multiple coinciding mechanisms, are able to adapt in a strain specific manner and use different strategies in coping with plants as their secondary hosts.