Global analysis of genes regulated by EvgA of the two-component regulatory system in Escherichia coli

The response regulator EvgA controls expression of multiple genes conferring antibiotic resistance in Escherichia coli (K. Nishino and A. Yamaguchi, J. Bacteriol. 184:2319-2323, 2002). To understand the whole picture of EvgA regulation, DNA macroarray analysis of the effect of EvgA overproduction was performed. EvgA activated genes related to acid resistance, osmotic adaptation, and drug resistance.     

Regulatory network of acid resistance genes in Escherichia coli

Overexpression of the response regulator EvgA confers an acid-resistant phenotype to exponentially growing Escherichia coli. This acid resistance is partially abolished by deletion of ydeP, yhiE or ydeO, genes induced by EvgA overexpression. Microarray analysis identified two classes of operons (genes). The first class contains seven operons induced by EvgA overexpression in the absence of ydeO, an AraC/XylS regulator gene. The second class contains 12 operons induced by YdeO overexpression. Operons in the second class were induced by EvgA overexpression only in the presence of ydeO. EvgA is likely to directly upregulate operons in the first class, and indirectly upregulate operons in the second class via YdeO. Analysis using the motif-finding program alignace identified an 18 bp inverted repeat motif in six upstream regions of all seven operons directly regulated by EvgA. Gel mobility shift assays showed the specific binding of EvgA to the six sequences. Introduction of mutations into the inverted repeats upstream of ydeP and b1500-ydeO resulted in reduction in EvgA-induced ydeP and ydeO expression and acid resistance. These results suggest that EvgA binds to the inverted repeats and upregulates the downstream genes. Overexpression of YdeP, YdeO and YhiE conferred acid resistance to exponentially growing cells, whereas GadX overexpression did not. Microarray analysis also identified several GadX-activated genes. Several genes induced by overexpression of YdeO and GadX overlapped; however, yhiE was induced only by YdeO. The acid resistance induced by YdeO overexpression was abolished by deletion of yhiE, gadC, slp-yhiF, hdeA or hdeD, genes induced by YdeO overexpression, suggesting that several genes orchestrate YdeO-induced acid resistance. We propose a model of the regulatory network of the acid resistance genes.     

Regulation of acid resistance by connectors of two-component signal transduction systems in Escherichia coli

Two-component signal transduction systems (TCSs), utilized extensively by bacteria and archaea, are involved in the rapid adaptation of the organisms to fluctuating environments. A typical TCS transduces the signal by a phosphorelay between the sensor histidine kinase and its cognate response regulator. Recently, small-sized proteins that link TCSs have been reported and are called "connectors." Their physiological roles, however, have remained elusive. SafA (sensor associating factor A) (formerly B1500), a small (65-amino-acid [65-aa]) membrane protein, is among such connectors and links Escherichia coli TCSs EvgS/EvgA and PhoQ/PhoP. Since the activation of the EvgS/EvgA system induces acid resistance, we examined whether the SafA-activated PhoQ/PhoP system is also involved in the acid resistance induced by EvgS/EvgA. Using a constitutively active evgS1 mutant for the activation of EvgS/EvgA, we found that SafA, PhoQ, and PhoP all contributed to the acid resistance phenotype. Moreover, EvgS/EvgA activation resulted in the accumulation of cellular RpoS in the exponential-phase cells in a SafA-, PhoQ-, and PhoP-dependent manner. This RpoS accumulation was caused by another connector, IraM, expression of which was induced by the activation of the PhoQ/PhoP system, thus preventing RpoS degradation by trapping response regulator RssB. Acid resistance assays demonstrated that IraM also participated in the EvgS/EvgA-induced acid resistance. Therefore, we propose a model of a signal transduction cascade proceeding from EvgS/EvgA to PhoQ/PhoP and then to RssB (connected by SafA and IraM) and discuss its contribution to the acid resistance phenotype.     

Specificity of the BvgAS and EvgAS phosphorelay is mediated by the C-terminal HPt domains of the sensor proteins

Despite the presence of highly conserved signalling modules, significant cross-communication between different two-component systems has only rarely been observed. Domain swapping and the characterization of liberated signalling modules enabled us to characterize in vitro the protein domains that mediate specificity and are responsible for the high fidelity in the phosphorelay of the unorthodox Bvg and Evg two-component systems. Under equimolar conditions, significant in vitro phosphorylation of purified BvgA and EvgA proteins was only obtained by their histidine kinases, BvgS and EvgS respectively. One hybrid histidine kinase consisting of the BvgS transmitter and HPt domains and of the EvgS receiver domain (BvgS-TO-EvgS-R) was able to phosphorylate BvgA but not EvgA. In contrast, the hybrid protein consisting of the BvgS transmitter and the EvgS receiver and HPt domains (BvgS-T-EvgS-RO) was unable to phosphorylate BvgA but efficiently phosphorylated EvgA. These results demonstrate that the C-terminal HPt domains of the sensor proteins endow the unorthodox two-component systems with a high specificity for the corresponding regulator protein. In the case of the response regulators, the receiver but not the output domains contribute to the specific interaction with the histidine kinases, because a hybrid protein consisting of the EvgA receiver and the BvgA output domain could only be phosphorylated by the EvgS protein.     

Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems

Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1. 24x10(6) M(-1).     

Characterization of EvgAS-YdeO-GadE branched regulatory circuit governing glutamate-dependent acid resistance in Escherichia coli

Escherichia coli prefers growth in neutral pH environments but can withstand extremely acidic conditions (pH 2) for long periods. Of the four E. coli systems that contribute to acid resistance, one, the glutamate-dependent system, is remarkable in its efficacy and regulatory complexity. The resistance mechanism involves the intracellular consumption of protons by the glutamate decarboxylase isozymes GadA and GadB. The antiporter GadC then exports the product, gamma-aminobutyric acid, in exchange for fresh glutamate. A microarray study using overexpressed regulators uncovered evgAS and ydeO as potential regulators of gadE, now known to encode the essential activator of the gadA and gadBC genes. Examination of evgA and ydeO under normal expression conditions revealed that their products do activate gadE expression but only under specific conditions. They were important during exponential growth in acidified minimal medium containing glucose but were unnecessary for gadE expression in stationary-phase cells grown in complex medium. The response regulator EvgA activates gadE directly and indirectly via induction of the AraC-like regulator ydeO. Evidence obtained using gadE-lacZ operon fusions also revealed that GadE was autoinduced. Electrophoretic mobility shift assays indicated that EvgA, YdeO, and GadE bind to different regions upstream of gadE, indicating they all act directly at the gadE promoter. Since GadE controls the expression of numerous genes besides gadA and gadBC, the relevance of these regulatory circuits extends beyond acid resistance.     

A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA

The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter, MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26). In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance. In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations. These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA. We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed.     

Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA

SdiA is an Escherichia coli protein that regulates cell division in a cell density-dependent, or quorum-sensing, manner. We report that SdiA also controls multidrug resistance by positively regulating the multidrug resistance pump AcrAB. Overproduction of SdiA confers multidrug resistance and increased levels of AcrAB. Conversely, sdiA null mutants are hypersensitive to drugs and have decreased levels of AcrB protein. Our findings provide a link between quorum sensing and multidrug efflux. Combined with previously published reports, our data support a model in which a role of drug efflux pumps is to mediate cell-cell communication in response to cell density. Xenobiotics expelled by pumps may resemble the communication molecules that they normally efflux.     

Multidrug resistance in fungi

Antibiotic and synthetic chemotherapeutic resistance in pathogenic yeast becomes one of the biggest challenges for the modern chemotherapy. An increasing number of pathogenic yeast and filamentous fungi resistant to the action of the majority of currently used drugs is isolated in clinics nowadays. Among variety of the resistance mechanisms, the most dangerous grows to be the multidrug resistance. The most important mechanism of the multidrug resistance is the overexpression of membrane proteins participating in the active efflux of drugs out of the cells subjected to chemotherapy. Representatives of two classes of multidrug efflux transporters, ABC and MFS, have been identified in fungi. One of the most important strategies for overcome the phenomenon of multidrug resistance in pathogenic fungi, is the use of chemical compounds co-administrated with chemotherapeutics which are able to restore drug susceptibility in multidrug resistant cells. Mode of action of these chemical compounds may be very diverse, from the substrate competition, through the influence on the membrane fluidity, to the multidrug transporters activity modulation. This paper presents a review of the current knowledge on proteins contributing to fungal multidrug resistance and strategies for overcoming multidrug resistance by pharmacological intervention.     

AKT as locus of cancer multidrug resistance and fragility

Complexity and robustness of cancer hypoxic microenvironment are supported by the robust signaling networks of autocrine and paracrine elements creating powerful interactome for multidrug resistance. These elements generate a positive feedback loops responsible for the extreme robustness and multidrug resistance in solid cancer, leukemia, myeloma, and lymphoma. Phosphorylated AKT is a cancer multidrug resistance locus. Targeting that locus by oxidant/antioxidant balance modulation, positive feedback loops are converted into negative feedback loops, leading to disappearance of multidrug resistance. This is a new principle for targeting cancer multidrug resistance by the locus chemotherapy inducing a phenomenon of loops conversion. J. Cell. Physiol. 228: 671–674, 2013. © 2012 Wiley Periodicals, Inc.     

Biliary excretion of tauroursodeoxycholate-3-sulfate in the rat

Biliary organic anion excretion is mediated by an ATP-dependent primary active transporter, multidrug resistance protein 2. On the other hand, a multiplicity of canalicular organic anion transport has been suggested. Ursodeoxycholic acid, the 7beta-epimer of chenodeoxycholic acid, is clinically used for various hepatobiliary diseases. In our previous study, the contribution of multidrug resistance protein 2 for biliary excretion of taurine-conjugated bile acid sulfates depended on the numbers of hydroxyl residue. Therefore, to further examine the effect of hydrophobicity on the substrate specificity of multidrug resistance protein 2, we examined the effect of bile acid conjugates and organic anions on biliary excretion of tauroursodeoxycholate-3-sulfate, taurine and sulfonate-conjugated ursodeoxycholic acid, in rats. Biliary tauroursodeoxycholate-3-sulfate excretions was markedly delayed in Eisai hyperbilirubinemic rats. Taurolithocholate-3-sulfate inhibited but ursodeoxycholate-3,7-disulfate did not affect biliary tauroursodeoxycholate-3-sulfate excretion. Biliary tauroursodeoxycholate-3-sulfate excretion was inhibited by sulfobromophthalein, but was not inhibited by dibromosulfophthalein and cefpiramide. These findings indicate that tauroursodeoxycholate-3-sulfate is very specific for multidrug resistance protein 2.     

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.