Systematic inactivation and phenotypic characterization of two-component signal transduction systems of Enterococcus faecalis V583

The ability of enterococci to adapt and respond to different environmental stimuli, including the host environment, led us to investigate the role of two-component signal transduction in the regulation of Enterococcus faecalis physiology. Using a bioinformatic approach, we previously identified 17 two-component systems (TCS), consisting of a sensory histidine kinase and the cognate response regulator, as well as an additional orphan response regulator (L. E. Hancock and M. Perego, J. Bacteriol. 184:5819-5825, 2002). In an effort to identify the potential function of each TCS in the biology of E. faecalis clinical isolate strain V583, we constructed insertion mutations in each of the response regulators. We were able to inactivate 17 of 18 response regulators, the exception being an ortholog of YycF, previously shown to be essential for viability in a variety of gram-positive microorganisms. The biological effects of the remaining mutations were assessed by using a number of assays, including antibiotic resistance, biofilm formation, and environmental stress. We identified TCS related to antibiotic resistance and environmental stress and found one system which controls the initiation of biofilm development by E. faecalis.     

Bacterial histidine kinase as signal sensor and transducer

Adaptation to an environmental stress is essential for cell survival in all organisms, from E. coli to human. To respond to changes in their surroundings, bacteria utilize two-component systems (TCSs), also known as histidyl-aspartyl phosphorelay (HAP) systems that consist of a histidine kinase (HK) sensor and a cognate response regulator (RR). While mammals developed complex signaling systems involving serine/threonine/tyrosine kinases in stress response mechanisms, bacterial TCS/HAP systems represent a simple but elegant prototype of signal transduction machineries. HKs are known as a seductive target for anti-bacterial therapeutic development, because of their significance in pathological virulence in some bacteria such as Salmonella enterica. Recent molecular and structural studies have shed light on the molecular basis of the signaling mechanism of HK sensor kinases. This review will focus on recent advancements in structural investigation of signal sensing and transducing mechanisms by HKs, which is critical to our understanding of bacterial biology and pathology.     

Evolutionary history of the OmpR/IIIA family of signal transduction two component systems in Lactobacillaceae and Leuconostocaceae

Background  

Two component systems (TCS) are signal transduction pathways which typically consist of a sensor histidine kinase (HK) and a response regulator (RR). In this study, we have analyzed the evolution of TCS of the OmpR/IIIA family in Lactobacillaceae and Leuconostocaceae, two families belonging to the group of lactic acid bacteria (LAB). LAB colonize nutrient-rich environments such as foodstuffs, plant materials and the gastrointestinal tract of animals thus driving the study of this group of both basic and applied interest.     

Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases

Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (phosphatidylethanolamine) [corrected] and GPCho (phosphatidylcholine) [corrected] . Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The K(m) values for ethanolamine and ATP were found to be 18.4+/-0.9 and 219+/-29 microM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a K(m) 80 times lower than that of ethanolamine. The K(m) values for choline, ethanolamine and ATP were 31.4+/-2.6 microM, 2.56+/-0.31 mM and 20.6+/-1.96 microM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.     

Lipid metabolism in germinating seeds. Purification of ethanolamine kinase from soya bean.

Ethanolamine kinase has been purified to homogeneity from germinating soya bean (Glycine max L.) seeds. The purified enzyme had a molecular weight of 17--19 000 as estimated by gel filtration and sodium dodecyl suphate-polyacrylamide gel electrophoresis. It would not phosphorylate choline, had a Km for ethanolamine of 8 microM and utilised Mg-ATP. The kinase could be purified in a 37 000 molecular weight form (dimer) which would easily dissociate on storage. In contrast to ethanolamine kinase whose activity was unaffected by the presence of choline in the assay system, soya bean choline kinase, although not phosphorylating ethanolamine, was competitively inhibited by the latter. The purification of specific choline and ethanolamine kinases from germinating soya bean confirmed in vivo observations which had indicated separate enzymes.     

Ethanolamine Accumulation by Photoreceptor Cells of the Rabbit Retina

The rabbit retina accumulates ethanolamine by an overall process that has a high affinity for ethanolamine. This process is different from the choline uptake, since ethanolamine accumulation was unaffected by high choline concentrations. Autoradiography identified the major site of high-affinity uptake as the perinuclear region of the photoreceptor cells. Ethanolamine accumulated by the high-affinity uptake was not used for neurotransmission by photoreceptor cells but was used to synthesize phosphatidylethanolamine. However, only a small percentage of the accumulated ethanolamine was converted into phospholipid. The rate of phosphorylation may contribute to control of phospholipid synthesis, since choline kinase activity is much greater than ethanolamine kinase activity in the rabbit retina.     

Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver.

Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123-125]. Kinetic properties of the two activities were determined. Choline kinase had a Km for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a Km for ethanolamine of 7.7 mM and choline was a 'mixed' type of inhibitor with a Ki of 0.037 mM. Both enzymes activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93% inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negliglible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase.     

Occurrence of virulence factors among human intestinal enterococcal isolates

AIMS: The aim of this study was to investigate the frequency of enterococcal virulence factors among human intestinal Enterococcus faecalis strains and to find out whether the pattern differs from that seen in published reports on food and clinical isolates. METHODS AND RESULTS: The E. faecalis isolates were cultured from human faecal samples obtained from five ulcerative colitis patients in remission phase. The species identification was based on API120 strips and species-specific PCR primers. The isolates were further characterized using the pulsed-field gel electrophoresis. The presence of seven different known enterococcal virulence factors among the confirmed E. faecalis isolates were screened using PCR techniques and published primers. CONCLUSIONS: Among the 35 isolates representing nine different pulsotypes the most frequent virulence factors were cpd (33 isolates), agg (25 isolates), gelE (22 isolates) and esp (15 isolates). No complete sets of genes associated for the production of functional cytolysin were encountered indicating that intestinal enterococci may differ in this respect from clinical strains. SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results, the commensal enterococcal strains appear to differ from clinical isolates in their complement of presumed virulence factors.     

Development of a method for markerless genetic exchange in Enterococcus faecalis and its use in construction of a srtA mutant

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, genetic analysis of these organisms has thus far been limited in scope due to the lack of advanced genetic tools. To broaden the repertoire of genetic tools available for manipulation of E.faecalis, we investigated the use of phosphoribosyl transferases as elements of a counterselection strategy. We report here the development of a counterselectable markerless genetic exchange system based on the upp-encoded uracil phosphoribosyl transferase of E. faecalis. Whereas wild-type E. faecalis is sensitive to growth inhibition by the toxic base analog 5-fluorouracil (5-FU), a mutant bearing an in-frame deletion of upp is resistant to 5-FU. When a cloned version of upp was ectopically introduced into the deletion mutant, sensitivity to 5-FU growth inhibition was restored, thereby providing the basis for a two-step integration and excision strategy for the transfer of mutant alleles to the enterococcal chromosome by recombination. This method was validated by the construction of a DeltasrtA mutant of E. faecalis and by the exchange of the surface protein Asc10, encoded on the pheromone-responsive conjugative plasmid pCF10, with a previously isolated mutant allele. Analysis of the DeltasrtA mutant indicated that SrtA anchors Asc10 to the enterococcal cell wall, facilitating the pheromone-induced aggregation of E. faecalis cells required for high-frequency conjugative plasmid transfer in liquid matings. The system of markerless exchange reported here will facilitate detailed genetic analysis of these important pathogens.     

Intra-leukocyte expression of two-component systems in Ehrlichia chaffeensis and Anaplasma phagocytophilum and effects of the histidine kinase inhibitor closantel

The two-component system (TCS) composed of a pair of a sensor histidine kinase and a response regulator, allows bacteria to sense signals and respond to changes in their environment through specific gene activation or repression. The present study examined TCS in the obligatory intracellular bacteria Ehrlichia chaffeensis and Anaplasma phagocytophilum, that cause human monocytic ehrlichiosis (HME) and human granulocytic anaplasmosis (HGA) respectively. The genomes of E. chaffeensis and A. phagocytophilum were each predicted to encode three pairs of TCSs. All six genes encoding three histidine kinases and three response regulators were expressed in both E. chaffeensis and A. phagocytophilum cultured in human leukocytes. Pretreatment of host cell-free E. chaffeensis or A. phagocytophilum with closantel, an inhibitor of histidine kinases, completely blocked the infection of host cells. Treatment of infected cells 1 day post infection with closantel cleared infection in dose-dependent manner. All six genes in E. chaffeensis were cloned, recombinant proteins were expressed, and polyclonal antibodies were produced. Double immunofluorescence labelling and Western blot analysis revealed that all six proteins were expressed in cell culture. Autokinase activities of the three recombinant histidine kinases from E. chaffeensis were inhibited by closantel in vitro. A number of E. chaffeensis genes, including the six TCS genes, were downregulated within 5-60 min post closantel treatment. These results suggest that these TCSs play an essential role in infection and survival of E. chaffeensis and A. phagocytophilum in human leukocytes.     

Partial purification and properties of ethanolamine kinase from spinach leaf.

Ethanolamine kinase was purified 60-fold by fractionation with ammonium sulfate, freeze-thawing, and gel filtration from a 100,000g supernatant from spinach leaf. The 100,00g supernatant preparation was stable for weeks, but the partially purified preparation lost half of the ethanolamine kinase activity in 10–14 days at 0–4 °C or ?20 °C. A molecular weight of 110,000 was estimated by gel filtration on Sephadex G-200. The reaction required ethanolamine (K_m, 42 μm), MgATP (K_m, 63 μm), and free magnesium ions. The enzyme was inhibited by MgATP, with an apparent K_i of 6.7 mm. Ethanolamine kinase was inhibited by calcium (in the presence of magnesium) and o-phenanthroline. EDTA above 0.9 mm inhibited the formation of phosphorylethanolamine and EGTA stimulated at low concentrations (0.4-0.9 mm) and inhibited at 1.8 mm. Ethanolamine kinase was inhibited by monomethylethanolamine and dimethylethanolamine, but not by choline (5 mm). The ethanolamine kinase and choline kinase activities of the 100,000g supernatant preparation could be separated by gel electrophoresis     

Overexpression of a mammalian ethanolamine-specific kinase accelerates the CDP-ethanolamine pathway

Ethanolamine kinase (EKI) is the first committed step in phosphatidylethanolamine (PtdEtn) biosynthesis via the CDP-ethanolamine pathway. We identify a human cDNA encoding an ethanolamine-specific kinase EKI1 and the structure of the EKI1 gene located on chromosome 12. EKI1 overexpression in COS-7 cells results in a 170-fold increase in ethanolamine kinase-specific activity and accelerates the rate of [3H]ethanolamine incorporation into PtdEtn as a function of the ethanolamine concentration in the culture medium. Acceleration of the CDP-ethanolamine pathway does not result in elevated cellular PtdEtn levels, but rather the excess PtdEtn is degraded to glycerophosphoethanolamine. EKI1 has negligible choline kinase activity in vitro and does not influence phosphatidylcholine biosynthesis. Acceleration of the CDP-ethanolamine pathway also does not change the rate of PtdEtn formation via the decarboxylation of phosphatidylserine. The data demonstrate the existence of separate ethanolamine and choline kinases in mammals and show that ethanolamine kinase can be a rate-controlling step in PtdEtn biosynthesis.     

Articulospora sp. produces Art1, an inhibitor of bacterial histidine kinase

A two-component system (TCS) comprising a histidine kinase (HK) sensor and a response regulator (RR) plays important roles in regulating the virulence of many pathogenic bacteria. We used a new screening method to isolate novel inhibitor Art1 against bacterial sensory HK from an acetone extract of solid cultures of Articulospora sp., an aquatic hypomycete. Art1 inhibited the ATP-dependent autophosphorylation of recombinant glutathione S-transferase-fusion protein SasA, a cyanobacterial HK, with an IC50 value of 9.5 microg/ml.     

Antimicrobial resistance of Enterococcus sp. isolated from the intestinal tract of patients from a university hospital in Brazil

This study reports the results about antimicrobial resistance of Enterococcus spp. isolated from intestinal tract of patients from a university hospital in Brazil. The identification of strains at species level was performed by conventional biochemical tests, API 20 Strep (bioMérieux), and polymerase chain reaction assay. The species distribution was E. faecium (34%), followed by E. faecalis (33%), E. gallinarum (23.7%), E. casseliflavus (5.2%), E. avium (1%), and E. hirae (1%). Intrinsic resistance to vancomycin characterized by presence of vanC genes was found in E. gallinarum and E. casseliflavus. The high prevalence of VanC phenotype enterococci is very important because these species have been reported as causing a wide variety of infections. Vancomycin-resistant E. faecium or E. faecalis were not found and no one isolate of these species was a beta-lactamase producer. Thirteen clinical isolates of enterococci (13.4%) showed multiresistance patterns, which were defined by resistance to three classes of antibiotics plus resistance to at least one aminoglycoside (gentamicin and/or streptomycin). The resistance to several antimicrobials shown by enterococcal strains obtained in this study is of concern because of the decrease in the therapeutic options for treatment of infections caused by enterococci.