The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis

The ability of an isogenic set of mutants of Salmonella enterica serovar Typhimurium L354 (SL1344) with defined deletions in genes encoding components of tripartite efflux pumps, including acrB, acrD, acrF and tolC, to colonize chickens was determined in competition with L354. In addition, the ability of L354 and each mutant to adhere to, and invade, human embryonic intestine cells and mouse monocyte macrophages was determined in vitro. The tolC and acrB knockout mutants were hyper-susceptible to a range of antibiotics, dyes and detergents; the tolC mutant was also more susceptible to acid pH and bile and grew more slowly than L354. Complementation of either gene ablated the phenotype. The tolC mutant poorly adhered to both cell types in vitro and was unable to invade macrophages. The acrB mutant adhered, but did not invade macrophages. In vivo, both the acrB mutant and the tolC mutant colonized poorly and did not persist in the avian gut, whereas the acrD and acrF mutant colonized and persisted as well as L354. These data indicate that the AcrAB-TolC system is important for the colonization of chickens by S. Typhimurium and that this system has a role in mediating adherence and uptake into target host cells.     

Low expression of AcrB in the deoxycholate-sensitive strains of Salmonella enterica subspecies enterica serovar Pullorum

We investigated the mechanism responsible for bile susceptibility in three deoxycholate-sensitive (DCs) strains of Salmonella enterica subspecies enterica serovar Pullorum isolated in 1958 in Japan. Of the genes encoding the AcrAB-TolC efflux system, the expression of acrB mRNA was 10-fold lower in the DCs strains than in a deoxycholate-resistant (DCr) strain, whereas those of the acrA and tolC genes were two-fold lower. These results suggested that low expression of acrB was strongly correlated with bile susceptibility in the DCs strains. In addition, the increase in tolC expression levels was not detected in the DCr mutants derived from the DCs strains, suggesting that difference in the expression levels of tolC is not associated with bile susceptibility.     

Suppression of hypersensitivity of Escherichia coli acrB mutant to organic solvents by integrational activation of the acrEF operon with the IS1 or IS2 element

The AcrAB-TolC efflux pump plays an intrinsic role in resistance to hydrophobic solvents in Escherichia coli. E. coli OST5500 is hypersensitive to solvents due to inactivation of the acrB gene by insertion of IS30. Suppressor mutants showing high solvent resistance were isolated from OST5500. These mutants produced high levels of AcrE and AcrF proteins, which were not produced in OST5500, and in each mutant an insertion sequence (IS1 or IS2) was found integrated upstream of the acrEF operon, coding for the two proteins. The suppressor mutants lost solvent resistance on inactivation of the acrEF operon. The solvent hypersensitivity of OST5500 was suppressed by introduction of the acrEF operon with IS1 or IS2 integrated upstream but not by introduction of the operon lacking the integrated IS. It was concluded that IS integration activated acrEF, resulting in functional complementation of the acrB mutation. The acrB mutation was also complemented by a plasmid containing acrF or acrEF under the control of Plac. The wild-type tolC gene was found to be essential for complementation of the acrB mutation by acrEF. Thus, it is concluded that in these cells a combination of the proteins AcrA, AcrF, and TolC or the proteins AcrE, AcrF, and TolC is functional in solvent efflux instead of the AcrAB-TolC efflux pump.     

A mouse transgenic approach to induce ��-catenin signaling in a temporally controlled manner

Although constitutive murine transgenic models have provided important insights into β-catenin signaling in tissue morphogenesis and tumorigenesis, these models are unable to express activated β-catenin in a temporally controlled manner. Therefore, to enable the induction (and subsequent de-induction) of β-catenin signaling during a predetermined time-period or developmental stage, we have generated and characterized a TETO-ΔN89β-catenin responder transgenic mouse. Crossed with the MTB transgenic effector mouse, which targets the expression of the reverse tetracycline transactivator (rtTA) to the mammary epithelium, we demonstrate that the stabilized (and activated) form of β-catenin (ΔN89β-catenin) is expressed only in the presence doxycycline-activated rtTA in the mammary epithelial compartment. Furthermore, we show that transgene-derived ΔN89β-catenin elicits significant mammary epithelial proliferation and precocious alveologenesis in the virgin doxycycline-treated MTB/TETO-ΔN89β-catenin bitransgenic. Remarkably, deinduction of TETO-ΔN89β-catenin transgene expression (through doxycycline withdrawal) results in the reversal of these morphological changes. Importantly, continued activation of the TETO-ΔN89β-catenin transgene results in palpable mammary tumors (within 7-9?months) in the doxycycline-treated virgin MTB/TETO-ΔN89β-catenin bigenic but not in the same bitransgenic without doxycycline administration. Collectively, these mammary epithelial responses to ΔN89β-catenin expression agree with previous reports using conventional transgenesis and therefore confirm that ΔN89β-catenin functions as expected in this doxycycline-responsive bigenic system. In sum, our mammary gland studies demonstrate "proof-of-principle" for using the TETO-ΔN89β-catenin transgenic responder to activate (and then de-activate) β-catenin signaling in any tissue of interest in a spatiotemporal specific fashion.     

Erwinia chrysanthemi tolC is involved in resistance to antimicrobial plant chemicals and is essential for phytopathogenesis

TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.     

Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from food-producing animals

Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.     

Mechanistic and spectroscopic studies of metallo-β-lactamase NDM-1

In an effort to biochemically characterize metallo-β-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)-NDM-1 fusion proteins with different N-termini (full-length, Δ6, Δ21, and Δ36). All MBP-NDM-1 fusion proteins were soluble; however, only one, MBP-NDM-1Δ36, exhibited high activity and bound 2 equiv of Zn(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1Δ36 variant, which exhibited a k(cat) of 16 s(-1) and a K(m) of 1.1 μM when using nitrocefin as a substrate, bound 2 equiv of Zn(II), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1Δ36 variant indicate the average metal binding site for Zn(II) in this variant consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn-Zn distance of 3.38 ?. This metal binding site is very similar to those of other metallo-β-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1Δ36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-β-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-β-lactamase CcrA.     

The effect of deleting residue C269 in the β12-β13 loop of protein phosphatase 2A (PP2A)catalytic subunit on the interaction between PP2A and metal ions, especially Mn(2+)

Protein phosphatase 2A (PP2A) is one of the most important Ser/Thr phosphatases in eukaryotic cells. The enzymatic core of PP2A (PP2A(D)) consists of a scaffold subunit (A subunit) and a catalytic subunit (C subunit). When residue Cys269 in the β12-β13 loop of the PP2A C subunit was deleted (ΔC269), the activity and the intrinsic fluorescence intensity of PP2A(D) decreased. Specify the effects of some metal ions on PP2A(D) were also changed. Mn(2+) in particular was an efficient activator of ΔC269 and altered the intrinsic fluorescence spectrum of ΔC269. Remarkably, after pre-treatment of ΔC269 with Mn(2+), the effects of other metal ions showed the same trends as they had on the WT. Molecular dynamics (MD) simulations showed that deletion of Cys269 decreased the polarity of the β12-β13 loop of PP2A Cα. We conclude that deletion of residue Cys269 alters the conformation and activity of PP2A(D) and influences the interaction between PP2A and various metal ions, notably Mn(2+).     

Charge requirements for proton gradient-driven translocation of anthrax toxin

Anthrax lethal toxin is used as a model system to study protein translocation. The toxin is composed of a translocase channel, called protective antigen (PA), and an enzyme, called lethal factor (LF). A proton gradient (ΔpH) can drive LF unfolding and translocation through PA channels; however, the mechanism of ΔpH-mediated force generation, substrate unfolding, and establishment of directionality are poorly understood. One recent hypothesis suggests that the ΔpH may act through changes in the protonation state of residues in the substrate. Here we report the charge requirements of LF's amino-terminal binding domain (LF(N)) using planar lipid bilayer electrophysiology. We found that acidic residues are required in LF(N) to utilize a proton gradient for translocation. Constructs lacking negative charges in the unstructured presequence of LF(N) translocate independently of the ΔpH driving force. Acidic residues markedly increase the rate of ΔpH-driven translocation, and the presequence is optimized in its natural acidic residue content for efficient ΔpH-driven unfolding and translocation. We discuss a ΔpH-driven charge state Brownian ratchet mechanism for translocation, where glutamic and aspartic acid residues in the substrate are the "molecular teeth" of the ratchet. Our Brownian ratchet model includes a mechanism for unfolding and a novel role for positive charges, which we propose chaperone negative charges through the PA channel during ΔpH translocation.     

The minimal α‐crystallin domain of Mj Hsp16.5 is functional at non‐heat‐shock conditions

The small heat shock protein (sHSP) from Methanococcus jannaschii (Mj Hsp16.5) forms a monodisperse 24mer and each of its monomer contains two flexible N‐ and C‐terminals and a rigid α‐crystallin domain with an extruding β‐strand exchange loop. The minimal α‐crystallin domain with a β‐sandwich fold is conserved in sHSP family, while the presence of the β‐strand exchange loop is divergent. The function of the β‐strand exchange loop and the minimal α‐crystallin domain of Mj Hsp16.5 need further study. In the present study, we constructed two fragment‐deletion mutants of Mj Hsp16.5, one with both the N‐ and C‐terminals deleted (ΔNΔC) and the other with a further deletion of the β‐strand exchange loop (ΔNΔLΔC). ΔNΔC existed as a dimer in solution. In contrast, the minimal α‐crystallin domain ΔNΔLΔC became polydisperse in solution and exhibited more efficient chaperone‐like activities to prevent amorphous aggregation of insulin B chain and fibril formation of the amyloidogenic peptide dansyl‐SSTSAA‐W than the mutant ΔNΔC and the wild type did. The hydrophobic probe binding experiments indicated that ΔNΔLΔC exposed much more hydrophobic surface than ΔNΔC. Our study also demonstrated that Mj Hsp16.5 used different mechanisms for protecting different substrates. Though Mj Hsp16.5 formed stable complexes with substrates when preventing thermal aggregation, no complexes were detected when preventing aggregation under non‐heat‐shock conditions. Proteins 2014; 82:1156–1167. © 2013 Wiley Periodicals, Inc.     

Genetic Analysis of Carbon Isotope Discrimination and its Relation to Yield in a Wheat Doubled Haploid Population

Carbon isotope discrimination (Δ¹³C) is considered a useful indicator for indirect selection of grain yield (GY) in cereals. Therefore, it is important to evaluate the genetic variation in Δ¹³C and its relationship with GY. A doubled haploid (DH) population derived from a cross of two common wheat varieties, Hanxuan 10 (H10) and Lumai 14 (L14), was phenotyped for Δ¹³C in the flag leaf, GY and yield associated traits in two trials contrasted by water availability, specifically, rain‐fed and irrigated. Quantitative trait loci (QTLs) were identified by single locus and two locus QTL analyses. QTLs for Δ¹³C were located on chromosomes 1A, 2B, 3B, 5A, 7A and 7B, and QTLs for other traits on all chromosomes except 1A, 4D, 5A, 5B and 6D. The population selected for high Δ¹³C had an increased frequency of QTL for high Δ¹³C, GY and number of spikes per plant (NSP) when grown under rain‐fed conditions and only for high Δ¹³C and NSP when grown under irrigated conditions, which was consistent with agronomic performance of the corresponding trait values in the high Δ¹³C progeny; that is, significantly greater than that in the low Δ¹³C. Therefore, selection for Δ¹³C was beneficial in increasing grain yield in rain‐fed environments.     

Absence of PmrAB-mediated phosphoethanolamine modifications of Citrobacter rodentium lipopolysaccharide affects outer membrane integrity

The PmrAB two-component system of enterobacteria regulates a number of genes whose protein products modify lipopolysaccharide (LPS). The LPS is modified during transport to the bacterial outer membrane (OM). A subset of PmrAB-mediated LPS modifications consists of the addition of phosphoethanolamine (pEtN) to lipid A by PmrC and to the core by CptA. In Salmonella enterica, pEtN modifications have been associated with resistance to polymyxin B and to excess iron. To investigate putative functions of pEtN modifications in Citrobacter rodentium, ΔpmrAB, ΔpmrC, ΔcptA, and ΔpmrC ΔcptA deletion mutants were constructed. Compared to the wild type, most mutant strains were found to be more susceptible to antibiotics that must diffuse across the LPS layer of the OM. All mutant strains also showed increased influx rates of ethidium dye across their OM, suggesting that PmrAB-regulated pEtN modifications affect OM permeability. This was confirmed by increased partitioning of the fluorescent dye 1-N-phenylnaphthylamine (NPN) into the OM phospholipid layer of the mutant strains. In addition, substantial release of periplasmic β-lactamase was observed for the ΔpmrAB and ΔpmrC ΔcptA strains, indicating a loss of OM integrity. This study attributes a new role for PmrAB-mediated pEtN LPS modifications in the maintenance of C. rodentium OM integrity.     

3D models of human ERα and ERβ complexed with 5-androsten-3β,17β-diol

Recently, binding of 5-androsten-3β,17β-diol (Δ(5)-androstenediol) to human estrogen receptor-beta (ERβ) was found to repress microglia-mediated inflammation, which is associated with various neurodegenerative diseases, such as multiple sclerosis. In contrast, binding of estradiol to ERβ resulted in little or no repression of microglia-mediated inflammation. Binding of Δ(5)-androstenediol to ERβ, as well as to ERα, is unexpected because unlike estradiol, Δ(5)-androstenediol has a saturated A ring and a C19 methyl group. To begin to elucidate the interaction of Δ(5)-androstenediol with both ERs, we constructed 3D models of Δ(5)-androstenediol with human ERα and ERβ for comparison with the crystal structures of estradiol in ERα and ERβ. Conformational flexibility in human ERα and ERβ accommodates the C19 methyl on Δ(5)-androstenediol. This conformational flexibility may be relevant for binding of other Δ(5)-steroids with C19 methyl substituents, such as 25-hydroxycholesterol and 27-hydroxycholesterol, to ERs.     

Tricyclic nitrogen base 1,N6-ethenoadenine and its ribosides as substrates for purine-nucleoside phosphorylases: Spectroscopic and kinetic studies

The title compound is an excellent substrate for E. coli PNP, as well as for its D204N mutant. The main product of the synthetic reaction is N9-riboside, but some amount of N7-riboside is also present. Surprisingly, 1,N⁶-ethenoadenine is also ribosylated by both wild-type and mutated (N243D) forms of calf PNP, which catalyze the synthesis of a different riboside, tentatively identified as N6-β-D-ribosyl-1,N⁶-ethenoadenine. All ribosides are susceptible to phosphorolysis by the E. coli PNP (wild type). All the ribosides are fluorescent and can be utilized as analytical probes.     

A novel two-gene requirement for the octanoyltransfer reaction of Bacillus subtilis lipoic acid biosynthesis

The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM and ywfL, which we have renamed lplJ, lipM and lipL respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to Escherichia coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulphur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilisΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coliΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion article provide evidence that LipM and LipL catalyse sequential reactions in a novel pathway for lipoic acid biosynthesis.     

Molecular dynamics simulations to investigate the relationship between the structural stability and amyloidogenesis of the wild-type and N-terminal hexapeptide deletion ΔN6 β2-microglobulin

β2-Microglobulin (β2m) forms amyloid fibrils in patients undergoing long-term haemodialysis, leading to dialysis-related amyloidosis. Proteolysis of the N-terminal region of β2m results in a truncation of the six N-terminal residues (ΔN6 β2m) in ～30% of the β2m molecules extracted from ex vivo fibrils. The ΔN6 β2m has been shown to exhibit a higher tendency for self-association comparing to the wild-type (wt) β2m, particularly at neutral pH. In order to gain atomic insights into the early stages of amyloid formation of the wt and ΔN6 β2m, various molecular dynamics simulations were conducted to investigate the stability and dynamics of these two molecules at various temperatures and neutral pH in this study. Our results, in agreement with previous experimental results, indicate that the structural stability of the ΔN6 β2m is lower than that of the wt β2m. It can be attributed to fact that the removal of the N-terminal six residues results in the loss of the salt–bridge interaction between residues R3 and D59, leading to the increased solvent exposure of the K3 peptide. It further allows water molecules to destabilise the interior region of the K3 peptide, leading to the elongation between the B- and E-strands. It may further accelerate the conformational changes of the ΔN6 β2m, leading to the formation of amyloid fibrils more readily at neutral pH. Our results also suggest that the K3 peptide may be a potential initiation site of amyloid formation for the ΔN6 β2m due to its increased solvent exposure. We further suggest that fibril morphology of the ΔN6 β2m formed at neutral pH is similar to that of the wt β2m formed at low pH (1.5–3) since they adopt the similar conformation with the elongation between B- and E-strands for their partially unfolded amyloidogenic intermediates.     

Variability in isotope discrimination factors in coral reef fishes: implications for diet and food web reconstruction

Interpretation of stable isotope ratios of carbon and nitrogen (δ(13)C and δ(15)N) is generally based on the assumption that with each trophic level there is a constant enrichment in the heavier isotope, leading to diet-tissue discrimination factors of 3.4‰ for (15)N (ΔN) and ～0.5‰ for (13)C (ΔC). Diet-tissue discrimination factors determined from paired tissue and gut samples taken from 152 individuals from 26 fish species at Ningaloo Reef, Western Australia demonstrate a large amount of variability around constant values. While caution is necessary in using gut contents to represent diet due to the potential for high temporal variability, there were significant effects of trophic position and season that may also lead to variability in ΔN under natural conditions. Nitrogen enrichment increased significantly at higher trophic levels (higher tissue δ(15)N), with significantly higher ΔN in carnivorous species. Changes in diet led to significant changes in ΔN, but not tissue δ(15)N, between seasons for several species: Acanthurus triostegus, Chromis viridis, Parupeneus signatus and Pomacentrus moluccensis. These results confirm that the use of meta-analysis averages for ΔN is likely to be inappropriate for accurately determining diets and trophic relationships using tissue stable isotope ratios. Where feasible, discrimination factors should be directly quantified for each species and trophic link in question, acknowledging the potential for significant variation away from meta-analysis averages and, perhaps, controlled laboratory diets and conditions.     

Fusion protein of Δ27LFn and EFn has the potential as a novel anthrax toxin inhibitor

PA-binding domain of LF (LFn) or PA-binding domain of EF (EFn) is the anthrax protective antigen (PA) binding domain of anthrax lethal factor (LF) or edema factor (EF). Here we show the development of a novel anthrax toxin inhibitor, fusion protein of N-terminal 27 amino acids deletion of LFn (Δ27LFn) and EFn. In a cell model of intoxication, fusion protein of Δ27LFn and EFn (Δ27LFn-EFn) was a 62-fold more potent toxin inhibitor than LFn or EFn, and this increased activity corresponded to a 39-fold higher PA-binding affinity by Biacore analysis. More importantly, Δ27LFn-EFn could protect the highly susceptible Fischer 344 rats from anthrax lethal toxin challenge. This work suggested that Δ27LFn-EFn has the potential as a candidate therapeutic agent against anthrax.

Structured summary

MINT-7014735, MINT-7014747, MINT-7014761: PA63 (uniprotkb:P13423) and LF (uniprotkb:P15917) bind (MI:0407) by surface plasmon resonance (MI:0107)     

A microfluidic system for investigation of extravascular transport and cellular uptake of drugs in tumors

Three-dimensional (3D) tumor models have been established in various microfluidic systems for drug delivery and resistance studies in vitro. However, one of the main drawbacks of these models is non-uniform distribution of cells, leaving regions with very low cell density within the 3D structures. As a result, molecular diffusion in the cell compartments is faster than that observed in solid tumors. To solve this problem, we developed a new technique for preparation of 3D tumor models in vitro. It was based on a microfluidic device containing three parallel channels separated by narrowly spaced posts. Tumor cells were loaded into the central channel at high density. To test the system, B16.F10 melanoma cells were perfusion-cultured overnight and the resulting 3D structure was characterized in terms of viability, density, and morphology of cells as well as transport properties of small fluorescent molecules. Immediately upon loading of tumor cells, the cell density was comparable to those observed in B16.F10 tumor tissues in vivo; and the viability of tumor cells was maintained through the overnight culture. The tumor model displayed low extracellular space and high resistance to diffusion of small molecules. For membrane-permeant molecules (e.g., Hoechst 33342), the rate of interstitial penetration was extremely slow, compared to membrane-impermeant molecules (e.g., sodium fluorescein). This versatile tumor model could be applied to in vitro studies of transport and cellular uptake of drugs and genes.