Contribution of the Periplasmic Chaperone Skp to Efficient Presentation of the Autotransporter IcsA on the Surface of Shigella flexneri

IcsA is an outer membrane protein in the autotransporter family that is required for Shigella flexneri pathogenesis. Following its secretion through the Sec translocon, IcsA is incorporated into the outer membrane in a process that depends on YaeT, a component of an outer membrane β-barrel insertion machinery. We investigated the role of the periplasmic chaperone Skp in IcsA maturation. Skp is required for the presentation of the mature amino terminus (alpha-domain) of IcsA on the bacterial surface and contributes to cell-to-cell spread of S. flexneri in cell culture. A mutation in skp does not prevent the insertion of the β-barrel into the outer membrane, suggesting that the primary role of Skp is the folding of the IcsA alpha-domain. In addition, the requirement for skp can be partially bypassed by disrupting icsP, an ortholog of Escherichia coli ompT, which encodes the protease that processes IcsA between the mature amino terminus and the β-barrel outer membrane anchor. These findings are consistent with a model in which Skp plays a critical role in the chaperoning of the alpha-domain of IcsA during transit through the periplasm.Type V secretion apparatuses (also called autotransporters) consist of an extensive class of large, outer membrane proteins of gram-negative bacteria, typically virulence factors, found in all subdivisions of proteobacteria (28). Although originally designated as “autotransporters” because they were thought to mediate their own insertion into and translocation across the outer membrane, more recent evidence suggests that autotransporter secretion and insertion requires the aid of accessory factors (21, 29). Secretion involves the insertion of the carboxy-terminal β-barrel domain into the outer membrane and translocation of the mature passenger (alpha) domain across the outer membrane (Fig. ​(Fig.1).1). Whether these two events occur sequentially or simultaneously is unclear. Analysis of crystal structures indicates that the carboxy-terminal end of the passenger domain is present within the central pore of the β-barrel (4, 27). Several studies provide evidence that at least some autotransporters are partially folded in the periplasm (7, 20), and one of these studies provides strong evidence that the passenger domain may be partially or fully incorporated into the β-barrel prior to incorporation of the mature protein into the outer membrane (20).Open in a separate windowFIG. 1.Schematic of the autotransporter IcsA. (A) Linear diagram showing the signal peptide (SP), alpha-domain (IcsA_53-757), and carboxy-terminal β-barrel domain. (B) IcsA in the outer membrane. The carboxy-terminal β-barrel is inserted into the outer membrane, and the mature amino-terminal alpha-domain is exposed on the bacterial surface. N′, mature amino terminus; C, carboxyl terminus; OM, outer membrane; arrow, proposed site of cleavage between residues 757 and 758 by IcsP.Shigella flexneri is a gram-negative human pathogen which, upon passage through the lower digestive tract, gains entry into colonic epithelial cells. Once S. flexneri is intracellular, it spreads to adjacent cells by secreting IcsA, a surface-associated autotransporter that is required for the polymerization of host cell actin on the bacterial surface. Actin polymerization occurs at a single pole of the bacterium and is required for infection of adjacent cells and disease pathogenesis (5, 24, 33).IcsA is encoded on a large virulence plasmid. The full-length protein is approximately 120 kDa and has three assigned functional and structural domains (25): an atypical Sec secretion signal (IcsA_1-52), the alpha-domain (IcsA_53-757), which is exposed on the bacterial surface and contains sequences that are required for actin polymerization, and the beta-domain (IcsA_758-1102), which forms a β-barrel structure in the outer membrane (Fig. ​(Fig.1A)1A) (21, 25). In vivo, a fraction of IcsA molecules are proteolytically processed at the junction between the alpha- and beta-domains by the protease IcsP (SopA), a protein which is also encoded on the virulence plasmid (14, 34). IcsA_53-757 is found in the supernatant of liquid cultures, while mature full-length IcsA (IcsA_53-1102), IcsA_758-1102 (14, 34), and some IcsA_53-757 (this work) remain cell associated. IcsA, like other autotransporters, is secreted at the bacterial pole (22), the site at which actin tail assembly occurs. As it is for other β-barrel-containing outer membrane proteins, insertion of IcsA and other autotransporters into the outer membrane requires the outer membrane insertase YaeT (BamA, Omp85) (21).Skp, DegP, and SurA are periplasmic chaperones that, like YaeT, appear to function in the targeting and/or insertion of outer membrane proteins (35). Evidence based on synthetic phenotypes suggests that during outer membrane protein insertion Skp and DegP act in one pathway and that SurA acts in a distinct but parallel pathway (35).We investigated the role of the periplasmic chaperone Skp in the folding and secretion of IcsA in S. flexneri. We found that in the absence of skp, IcsA is inefficiently presented on the surface of S. flexneri, leading to a cellular spread defect. Surprisingly, the protein was still efficiently cleaved by the outer membrane protease IcsP, as wild-type levels of IcsA_53-757 were detected in the culture supernatants. We found that introduction of the icsP mutation into the skp strain background led to an increase in the levels of full-length IcsA presented on the bacterial cell surface of the skp mutant, and we present models that could explain our results.     

Selection of Pseudomonas for industrial textile dyes decolourization

Pigment is the first contaminant to be recognised in bodies of water and wastewater. Besides the aesthetic problem, dyes obstruct light and reduce oxygen mass transfer. This paper describes the selection of Pseudomonas strains with the ability to remove colour from textile industrial dyes. Four Pseudomonas species were tested against 14 commercial industrial dyes. Pseudomonas cepacia exhibited no growth at all on plates containing dyes (1 g l^?1), whereas Pseudomonas aeruginosa, Pseudomonas oleovorans and Pseudomonas putida exhibited considerable growth. Decolourization in a liquid culture revealed that P. oleovorans is more viable for decolourizing textile dyes, as it achieved over 80% colour removal for two of the 14 dyes studied; it also proved to be more tolerant to high dye concentrations.     

Modulation of P2X7 purinergic receptor in macrophages by Leishmania amazonensis and its role in parasite elimination

The purinergic P2X₇ receptor is a membrane protein of leucocytes involved in the clearance of intracellular bacteria such as Chlamydia and Mycobacterium. In this work, we investigated the role and modulation of macrophage P2X₇R in intracellular infection with the protozoan parasite Leishmania amazonensis. Upon infection, isolated murine macrophages displayed enhanced expression of P2X₇R and were significantly more responsive to extracellular ATP (ATPe)-induced pore opening, as demonstrated by the increased uptake of Lucifer Yellow. This was extended to the in vivo situation, where cells from established cutaneous lesions were more sensitive to ATPe than cells from uninfected mice. ATP treatment of infected macrophages inhibited parasite growth, and this was prevented by pre-treatment with oxidized ATP, a selective antagonist of P2X₇R. Parasite killing was unlikely due to induction of nitric oxide production or cytolysis of infected macrophage, as those functions were unaltered with parasite-effective ATPe concentrations. A direct drug effect is also unlike, as ATPe enhanced axenic parasite growth. We found that leishmanial infection rendered wild-type but not P2X₇R-deficient macrophages more prone to ATP-induced apoptosis. These results show that macrophage infection with L. amazonensis leads to enhanced expression of functional P2X₇R, that upon ligation with ATPe helps in the elimination of the parasites by an as yet unclear mechanism possibly involving host cell apoptosis.     

Effects of Submerged Vegetation on Water Clarity Across Climates

A positive feedback between submerged vegetation and water clarity forms the backbone of the alternative state theory in shallow lakes. The water clearing effect of aquatic vegetation may be caused by different physical, chemical, and biological mechanisms and has been studied mainly in temperate lakes. Recent work suggests differences in biotic interactions between (sub)tropical and cooler lakes might result in a less pronounced clearing effect in the (sub)tropics. To assess whether the effect of submerged vegetation changes with climate, we sampled 83 lakes over a gradient ranging from the tundra to the tropics in South America. Judged from a comparison of water clarity inside and outside vegetation beds, the vegetation appeared to have a similar positive effect on the water clarity across all climatic regions studied. However, the local clearing effect of vegetation decreased steeply with the contribution of humic substances to the underwater light attenuation. Looking at turbidity on a whole-lake scale, results were more difficult to interpret. Although lakes with abundant vegetation (>30%) were generally clear, sparsely vegetated lakes differed widely in clarity. Overall, the effect of vegetation on water clarity in our lakes appears to be smaller than that found in various Northern hemisphere studies. This might be explained by differences in fish communities and their relation to vegetation. For instance, unlike in Northern hemisphere studies, we find no clear relation between vegetation coverage and fish abundance or their diet preference. High densities of omnivorous fish and coinciding low grazing pressures on phytoplankton in the (sub)tropics may, furthermore, weaken the effect of vegetation on water clarity.     

Mutations in Smooth Muscle Alpha-Actin (ACTA2) Cause Coronary Artery Disease, Stroke, and Moyamoya Disease, Along with Thoracic Aortic Disease

The vascular smooth muscle cell (SMC)-specific isoform of α-actin (ACTA2) is a major component of the contractile apparatus in SMCs located throughout the arterial system. Heterozygous ACTA2 mutations cause familial thoracic aortic aneurysms and dissections (TAAD), but only half of mutation carriers have aortic disease. Linkage analysis and association studies of individuals in 20 families with ACTA2 mutations indicate that mutation carriers can have a diversity of vascular diseases, including premature onset of coronary artery disease (CAD) and premature ischemic strokes (including Moyamoya disease [MMD]), as well as previously defined TAAD. Sequencing of DNA from patients with nonfamilial TAAD and from premature-onset CAD patients independently identified ACTA2 mutations in these patients and premature onset strokes in family members with ACTA2 mutations. Vascular pathology and analysis of explanted SMCs and myofibroblasts from patients harboring ACTA2 suggested that increased proliferation of SMCs contributed to occlusive diseases. These results indicate that heterozygous ACTA2 mutations predispose patients to a variety of diffuse and diverse vascular diseases, including TAAD, premature CAD, ischemic strokes, and MMD. These data demonstrate that diffuse vascular diseases resulting from either occluded or enlarged arteries can be caused by mutations in a single gene and have direct implications for clinical management and research on familial vascular diseases.     

Interaction of Mycobacterium tuberculosis CYP130 with Heterocyclic Arylamines

The Mycobacterium tuberculosis P450 enzymes are of interest for their pharmacological development potential, as evidenced by their susceptibility to inhibition by antifungal azole drugs that normally target sterol 14α-demethylase (CYP51). Although antifungal azoles show promise, direct screening of compounds against M. tuberculosis P450 enzymes may identify novel, more potent, and selective inhibitory scaffolds. Here we report that CYP130 from M. tuberculosis has a natural propensity to bind primary arylamines with particular chemical architectures. These compounds were identified via a high throughput screen of CYP130 with a library of synthetic organic molecules. As revealed by subsequent x-ray structure analysis, selected compounds bind in the active site by Fe-coordination and hydrogen bonding of the arylamine group to the carbonyl oxygen of Gly²⁴³. As evidenced by the binding of structural analogs, the primary arylamine group is indispensable, but synergism due to hydrophobic contacts between the rest of the molecule and protein amino acid residues is responsible for a binding affinity comparable with that of the antifungal azole drugs. The topology of the CYP130 active site favors angular coordination of the arylamine group over the orthogonal coordination of azoles. Upon substitution of Gly²⁴³ by an alanine, the binding mode of azoles and some arylamines reverted from type II to type I because of hydrophobic and steric interactions with the alanine side chain. We suggest a role for the conserved Ala(Gly)²⁴³-Gly²⁴⁴ motif in the I-helix in modulating both the binding affinity of the axial water ligand and the ligand selectivity of cytochrome P450 enzymes.CYP130 is one of the 20 Mycobacterium tuberculosis cytochrome P450 (P450, CYP)² enzymes and is one of three (CYP51, CYP121, and CYP130) that have been studied as individually expressed proteins at the structural level. Evidence has accumulated for the importance of M. tuberculosis P450 enzymes in virulence (CYP132) (1), host infection (CYP125) (2), and pathogen viability (CYP128, CYP121) (3, 4), although neither their exact biological functions nor any of the endogenous substrates upon which these enzymes operate have yet been established. However, it has recently been shown in vitro that CYP121 catalyzes a C–C coupling reaction between two tyrosine groups (5). CYP130 is absent from the genome of Mycobacterium bovis, suggesting that it might play specific role(s) in the infection of the human host and thus constitute a potential therapeutic target.The potential of M. tuberculosis P450 enzymes for pharmacologic development was initially suggested by their susceptibility to inhibition by antifungal azole drugs such as fluconazole, econazole, and clotrimazole. These drugs block sterol 14α-demethylase CYP51 in fungi (6), tightly bind to M. tuberculosis P450 proteins (7, 8), and display inhibitory potential against latent and multidrug-resistant forms of tuberculosis both in vitro and in tuberculosis-infected mice (9–14).The substantial differences between fungal CYP51 and the potential P450 targets in microbial pathogens, including M. tuberculosis, suggest that the direct screening of compounds against M. tuberculosis CYP enzymes could identify novel inhibitory scaffolds that are more potent and selective than antifungal drugs. Structurally characterized screening targets are advantageous, as the already defined purification and crystallization protocols can be applied to obtain co-crystal structures and to elucidate the binding modes of screening hits. This approach has been successfully applied to CYP51, resulting in identification of novel inhibitory scaffolds for CYP51 therapeutic targets (15, 16).Toward this goal, the property of P450 enzymes to shift the ferric heme iron Soret band on ligand binding (17) provides an experimental platform for high throughput screening of compound libraries to select chemotypes with high binding affinities for the target. Expulsion of the heme iron axial water ligand from the Fe-coordination sphere by the incoming substrate followed by transition of the ferric heme from the low-spin hexacoordinated to the high-spin pentacoordinated state characterize type I spectral shifts and are a prerequisite for P450 catalytic activity. Replacement of a weak axial ligand, the water molecule, with a stronger one possessing a nitrogen-containing aliphatic or aromatic group coordinating to the heme iron characterizes type II spectral shifts.To find new high affinity ligands of CYP130, a commercial library of 20,000 small organic molecules comprising a large selection of molecular scaffolds was screened against the enzyme. In contrast to the results with CYP51, no type I binding hits were identified. Screening produced about a dozen structurally diverse type II hits that were unexpectedly devoid of the usual aromatic nitrogen atoms readily accessible for axial coordination of the heme iron, suggesting an alternative coordination mode. High resolution x-ray structure analysis determined that two compounds coordinated to the heme iron via a primary arylamine group, providing the first structural evidence on P450-heterocyclic arylamine interactions.     

Analysis of TNFAIP3, a feedback inhibitor of nuclear factor-κB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility

Introduction

Genome-wide association studies of rheumatoid arthritis (RA) have identified an association of the disease with a 6q23 region devoid of genes. TNFAIP3, an RA candidate gene, flanks this region, and polymorphisms in both the TNFAIP3 gene and the intergenic region are associated with systemic lupus erythematosus. We hypothesized that there is a similar association with RA, including polymorphisms in TNFAIP3 and the intergenic region.

Methods

To test this hypothesis, we selected tag-single nucleotide polymorphisms (SNPs) in both loci. They were analyzed in 1,651 patients with RA and 1,619 control individuals of Spanish ancestry.

Results

Weak evidence of association was found both in the 6q23 intergenic region and in the TNFAIP3 locus. The rs582757 SNP and a common haplotype in the TNFAIP3 locus exhibited association with RA. In the intergenic region, two SNPs were associated, namely rs609438 and rs13207033. The latter was only associated in patients with anti-citrullinated peptide antibodies. Overall, statistical association was best explained by the interdependent contribution of SNPs from the two loci TNFAIP3 and the 6q23 intergenic region.

Conclusions

Our data are consistent with the hypothesis that several RA genetic factors exist in the 6q23 region, including polymorphisms in the TNFAIP3 gene, like that previously described for systemic lupus erythematosus.     

Trace Element Composition of Selected Fertilizers Used in Chile: Phosphorus Fertilizers as a Source of Long-Term Soil Contamination

Anthropogenic activities like agriculture have resulted in increased concentrations of some trace elements of toxicological and environmental concern in soils. Application of fertilizers has been one of the major inputs of these contaminants to agricultural soils in developing countries. Twenty-two fertilizers, including straight nitrogen (N), phosphorus (P), potassium (K), and NK fertilizers and micronutrient sources, were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) for arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn). As expected, the trace element content of fertilizers was highly variable and related to the origin of the material. Phosphorus fertilizers, especially triple superphosphate, presented the highest As, Cd, Cu, Cr, Ni, V, and Zn concentrations. In some of these fertilizers, the Cr, V, and Zn contents reached values greater than 3475 mg kg^?1 of P, and the Cd content (up to 288 mg kg^?1 of P) was several times higher than the regulatory limits of different countries. Some micronutrient sources presented the highest concentrations of Mn and Pb. In the cases of N, K, and NK fertilizers, the trace element concentration was very low, sometimes below the detection limits. In some agricultural systems the input of trace elements such as As and Cd to the soil through P fertilizers application may be higher than the outputs through plant uptake and leaching; therefore the long-term use of these fertilizers may cause the trace element concentration to increase in the plow layer of agricultural soils.