Nucleoside uptake in Vibrio cholerae and its role in the transition fitness from host to environment.

As it became evident recently, extracellular DNA could be a versatile nutrient source of the facultative pathogen Vibrio cholerae along the different stages of its life cycle. By the use of two extracellular nucleases and periplasmic phosphatases, V. cholerae degrades extracellular DNA to nucleosides. In this study, we investigated the nucleoside uptake via identification and characterization of VCA0179, VC1953 and VC2352 representing the three nucleoside transport systems in V. cholerae. Based on our results VC2352 seems to be the dominant nucleoside transporter. Nevertheless, all three transporters are functional and can contribute to the utilization of nucleosides as a sole source of carbon or nitrogen. We found that the transcriptional activity of these three distal genes is equally promoted or antagonized by CRP or CytR respectively. Finally, mutants impaired for nucleoside uptake exhibit decreased transition fitness from the host into low carbon environments along the life cycle of V. cholerae.

Multifaceted roles of extracellular DNA in bacterial physiology.

In textbooks, DNA is generally defined as the universal storage material for genetic information in all branches of life. Beyond this important intracellular role, DNA can also be present outside of living cells and is an abundant biopolymer in aquatic and terrestrial ecosystems. The origin of extracellular DNA in such ecological niches is diverse: it can be actively secreted or released by prokaryotic and eukaryotic cells by means of autolysis, apoptosis, necrosis, bacterial secretion systems or found in association with extracellular bacterial membrane vesicles. Especially for bacteria, extracellular DNA represents a significant and convenient element that can be enzymatically modulated and utilized for multiple purposes. Herein, we discuss briefly the main origins of extracellular DNA and the most relevant roles for the bacterial physiology, such as biofilm formation, nutrient source, antimicrobial means and horizontal gene transfer.

AAA+ proteases and their role in distinct stages along the Vibrio cholerae lifecycle.

The facultative human pathogen Vibrio cholerae has to adapt to different environmental conditions along its lifecycle by means of transcriptional, translational and post-translational regulation. This study provides a first comprehensive analysis regarding the contribution of the cytoplasmic AAA+ proteases Lon, ClpP and HslV to distinct features of V. cholerae behaviour, including biofilm formation, motility, cholera toxin expression and colonization fitness in the mouse model. While absence of HslV did not yield to any altered phenotype compared to wildtype, absence of Lon or ClpP resulted in significantly reduced colonization in vivo. In addition, a Δlon deletion mutant showed altered biofilm formation and increased motility, which could be correlated with higher expression of V. cholerae flagella gene class IV. Concordantly, we could show by immunoblot analysis, that Lon is the main protease responsible for proteolytic control of FliA, which is required for class IV flagella gene transcription, but also downregulates virulence gene expression. FliA becomes highly sensitive to proteolytic degradation in absence of its anti-sigma factor FlgM, a scenario reported to occur during mucosal penetration due to FlgM secretion through the broken flagellum. Our results confirm that the high stability of FliA in the absence of Lon results in less cholera toxin and toxin corgulated pilus production under virulence gene inducing conditions and in the presence of a damaged flagellum. Thus, the data presented herein provide a molecular explanation on how V. cholerae can achieve full expression of virulence genes during early stages of colonization, despite FliA getting liberated from the anti-sigma factor FlgM.

Identification of genes induced in Vibrio cholerae in a dynamic biofilm system.

The facultative human pathogen Vibrio cholerae, the causative agent of the severe secretory diarrheal disease cholera, persists in its aquatic reservoirs in biofilms during interepidemic periods. Biofilm is a likely form in which clinically relevant V. cholerae is taken up by humans, providing an infective dose. Thus, a better understanding of biofilm formation of V. cholerae is relevant for the ecology and epidemiology of cholera as well as a target to control the disease. Most previous studies have investigated static biofilms of V. cholerae and elucidated structural prerequisites like flagella, pili and a biofilm matrix including extracellular DNA, numerous matrix proteins and exopolysaccharide, as well as the involvement of regulatory pathways like two-component systems, quorum sensing and c-di-GMP signaling. However, aquatic environments are more likely to reflect an open, dynamic system. Hence, we used a biofilm system with constant medium flow and a temporal controlled reporter-system of transcription to identify genes induced during dynamic biofilm formation. We identified genes known or predicted to be involved in c-di-GMP signaling, motility and chemotaxis, metabolism, and transport. Subsequent phenotypic characterization of mutants with independent mutations in candidate dynamic biofilm-induced genes revealed novel insights into the physiology of static and dynamic biofilm conditions. The results of this study also reinforce the hypotheses that distinct differences in regulatory mechanisms governing biofilm development are present under dynamic conditions compared to static conditions.

[Morphology of low-velocity impact stains produced from single drops of blood]. / Zur Morphologie langsam beschleunigter Blutauftropfspuren.

Systematic variation of blood droplet volume, the distance fallen and the surface (paper, wood, plastics, tiles) led to the conclusion that the size and the shape of the stains ("fingers", satellites) allowed to deduce the distance fallen but only if the actual surface structure was known. We found that detailed photography at the crime scene was necessary, yet experiments have to be performed due to the extreme influence of the actual surface texture on all characteristics (size, spines, peripheral spatter) of the blood stains.

Characterization of Vibrio cholerae's Extracellular Nuclease Xds.

The Gram-negative bacterium Vibrio cholerae encodes two nucleases, Dns and Xds, which play a major role during the human pathogen's lifecycle. Dns and Xds control three-dimensional biofilm formation and bacterial detachment from biofilms via degradation of extracellular DNA and thus contribute to the environmental, inter-epidemic persistence of the pathogen. During intestinal colonization the enzymes help evade the innate immune response, and therefore promote survival by mediating escape from neutrophil extracellular traps. Xds has the additional function of degrading extracellular DNA down to nucleotides, which are an important nutrient source for V. cholerae. Thus, Xds is a key enzyme for survival fitness during distinct stages of the V. cholerae lifecycle and could be a potential therapeutic target. This study provides detailed information about the enzymatic properties of Xds using purified protein in combination with a real time nuclease activity assay. The data define an optimal buffer composition for Xds activity as 50 mM Tris/HCl pH 7, 100 mM NaCl, 10 mM MgCl2, and 20 mM CaCl2. Moreover, maximal activity was observed using substrate DNA with low GC content and ambient temperatures of 20-25°C. In silico analysis and homology modeling predicted an exonuclease domain in the C-terminal part of the protein. Biochemical analyses with truncated variants and point mutants of Xds confirm that the C-terminal region is sufficient for nuclease activity. We also find that residues D787 and H837 within the predicted exonuclease domain are key to formation of the catalytic center.

Regulated Proteolysis in Vibrio cholerae Allowing Rapid Adaptation to Stress Conditions.

The lifecycle of the causative agent of the severe secretory diarrheal disease cholera, Vibrio cholerae, is characterized by the transition between two dissimilar habitats, i.e., as a natural inhabitant of aquatic ecosystems and as a pathogen in the human gastrointestinal tract. Vibrio cholerae faces diverse stressors along its lifecycle, which require effective adaptation mechanisms to facilitate the survival fitness. Not surprisingly, the pathogen's transcriptome undergoes global changes during the different stages of the lifecycle. Moreover, recent evidence indicates that several of the transcription factors (i.e., ToxR, TcpP, and ToxT) and alternative sigma factors (i.e., FliA, RpoS, and RpoE) involved in transcriptional regulations along the lifecycle are controlled by regulated proteolysis. This post-translational control ensures a fast strategy by the pathogen to control cellular checkpoints and thereby rapidly respond to changing conditions. In this review, we discuss selected targets for regulated proteolysis activated by various stressors, which represent a key feature for fast adaptation of V. cholerae.

A Broad Spectrum Protein Glycosylation System Influences Type II Protein Secretion and Associated Phenotypes in Vibrio cholerae.

Protein secretion plays a crucial role for bacterial pathogens, exemplified by facultative human-pathogen Vibrio cholerae, which secretes various proteinaceous effectors at different stages of its lifecycle. Accordingly, the identification of factors impacting on protein secretion is important to understand the bacterial pathophysiology. PglLVc, a predicted oligosaccharyltransferase of V. cholerae, has been recently shown to exhibit O-glycosylation activity with relaxed glycan specificity in an engineered Escherichia coli system. By engineering V. cholerae strains to express a defined, undecaprenyl diphosphate-linked glycoform precursor, we confirmed functional O-linked protein glycosylation activity of PglLVc in V. cholerae. We demonstrate that PglLVc is required for the glycosylation of multiple V. cholerae proteins, including periplasmic chaperones such as DegP, that are required for efficient type II-dependent secretion. Moreover, defined deletion mutants and complementation strains provided first insights into the physiological role of O-linked protein glycosylation in V. cholerae. RbmD, a protein with structural similarities to PglLVc and other established oligosaccharyltransferases (OTases), was also included in this phenotypical characterization. Remarkably, presence or absence of PglLVc and RbmD impacts the secretion of proteins via the type II secretion system (T2SS). This is highlighted by altered cholera toxin (CT) secretion, chitin utilization and biofilm formation observed in ΔpglL Vc and ΔrbmD single or double mutants. This work thus establishes a unique connection between broad spectrum O-linked protein glycosylation and the efficacy of type II-dependent protein secretion critical to the pathogen's lifecycle.