Chaperone-tip adhesin complex is vital for synergistic activation of CFA/I fimbriae biogenesis.

Colonization factor CFA/I defines the major adhesive fimbriae of enterotoxigenic Escherichia coli and mediates bacterial attachment to host intestinal epithelial cells. The CFA/I fimbria consists of a tip-localized minor adhesive subunit, CfaE, and thousands of copies of the major subunit CfaB polymerized into an ordered helical rod. Biosynthesis of CFA/I fimbriae requires the assistance of the periplasmic chaperone CfaA and outer membrane usher CfaC. Although the CfaE subunit is proposed to initiate the assembly of CFA/I fimbriae, how it performs this function remains elusive. Here, we report the establishment of an in vitro assay for CFA/I fimbria assembly and show that stabilized CfaA-CfaB and CfaA-CfaE binary complexes together with CfaC are sufficient to drive fimbria formation. The presence of both CfaA-CfaE and CfaC accelerates fimbria formation, while the absence of either component leads to linearized CfaB polymers in vitro. We further report the crystal structure of the stabilized CfaA-CfaE complex, revealing features unique for biogenesis of Class 5 fimbriae.

Structural and Functional Insights into PpgL, a Metal-Independent ß-Propeller Gluconolactonase That Contributes to Pseudomonas aeruginosa Virulence.

Biofilm formation is a critical determinant in the pathopoiesis of Pseudomonas aeruginosa It could significantly increase bacterial resistance to drugs and host defense. Thus, inhibition of biofilm matrix production could be regarded as a promising attempt to prevent colonization of P. aeruginosa and the subsequent infection. PpgL, a periplasmic gluconolactonase, has been reported to be involved in P. aeruginosa quorum-sensing (QS) system regulation. However, the detailed function and catalysis mechanism remain elusive. Here, the crystal structure of PpgL is described in the current study, along with biochemical analysis, revealing that PpgL is a typical ß-propeller enzyme with unique metal-independent lactone hydrolysis activity. Consequently, comparative analysis of seven-bladed propeller lactone-catalyzing enzymes and mutagenesis studies identify the critical sites which contribute to the diverse catalytic and substrate recognition functions. In addition, the reduced biofilm formation and attenuated invasion phenotype resulting from deletion of ppgL confirm the importance of PpgL in P. aeruginosa pathogenesis. These results suggest that PpgL is a potential target for developing new agents against the diseases caused by P. aeruginosa.

Mechanistic insights into the allosteric regulation of Pseudomonas aeruginosa aspartate kinase.

In plants and microorganisms, aspartate kinase (AK) catalyzes an initial commitment step of the aspartate family amino acid biosynthesis. Owing to various structural organizations, AKs from different species show tremendous diversity and complex allosteric controls. We report the crystal structure of AK from Pseudomonas aeruginosa (PaAK), a typical α2ß2 hetero-tetrameric enzyme, in complex with inhibitory effectors. Distinctive features of PaAK are revealed by structural and biochemical analyses. Essentially, the open conformation of Lys-/Thr-bound PaAK structure clarifies the inhibitory mechanism of α2ß2-type AK. Moreover, the various inhibitory effectors of PaAK have been identified and a general amino acid effector motif of AK family is described.

[Spatial Distribution, Speciation, and Ecological Risk Assessment of Heavy Metals in Surface Sediments of Dongjiang Lake, Hunan Province].

To understand the heavy metal pollution status of Dongjiang Lake, the contents and species of heavy metals in the surface sediments were investigated during September 2021, and the heavy metal pollution level and potential ecological risk were evaluated. The results showed that Cd, Pb, As, Cu, Zn, Ni, and Cr contents were in the range of 0.40-34.1, 14.8-1688, 6.99-1155, 6.89-280, 26.2-1739, 6.29-55.4, and 23.3-44.8 mg·kg-1, respectively, with extremely uneven spatial distributions. The highest contents of Cd, Pb, As, Zn, Cu, and Ni were found in the site adjacent to Yaogangxian tungsten ore. The proportion of metal species with bioavailability was high, in which Cd in acid-soluble species was 46.7%-71.5% and Pb in reducible species was 46.8%-67.0%. The bioavailable species of Cu, Zn, Ni, and Cr were 35%-68%, 42%-72%, 26%-51%, and 6%-30%, respectively, although they primarily existed in residual species. According to the geo-accumulation index (Igeo), there was a moderate or extreme pollution status of Cd in all sites, moderate or extreme pollution status of Pb in 90% of sites, and moderate pollution status of As, Cu, and Zn in 30% of sites. The ecological risk factor (Eri) of Cd showed high potential ecological risk in all sites with significantly high potential ecological risk in 80% of sites. Moreover, As and Pb had significantly high potential ecological risk, and Cu had moderate potential ecological risk in S7, which was adjacent to Yaogangxian tungsten ore. There was a high total potential ecological risk in all sites and significantly high potential ecological risk in 50% of sites. Therefore, the surface sediments of Dongjiang Lake were under the combined pollution of Cd, Pb, As, Zn, and Cu with high bioavailability and high total potential ecological risk.

Nucleotide binding as an allosteric regulatory mechanism for Akkermansia muciniphila ß-N-acetylhexosaminidase Am2136.

ß-N-acetylhexosaminidases (EC3.2.1.52), which belong to the glycosyl hydrolase family GH20, are important enzymes for oligosaccharides modification. Numerous microbial ß-N-acetylhexosaminidases have been investigated for applications in biology, biomedicine and biotechnology. Akkermansia muciniphila is an anaerobic intestinal commensal bacterium which possesses specific ß-N-acetylhexosaminidases for gut mucosal layer colonization and mucin degradation. In this study, we assessed the in vitro mucin glycan cleavage activity of the A. muciniphila ß-N-acetylhexosaminidase Am2136 and demonstrated its ability that hydrolyzing the ß-linkages joining N-acetylglucosamine to a wide variety of aglycone residues, which indicated that Am2136 may be a generalist ß-N-acetylhexosaminidase. Structural and enzyme activity assay experiments allowed us to probe the essential function of the inter-domain interactions in ß23-ß33. Importantly, we revealed that the hydrolysis activity of Am2136 was enhanced by nucleotides. We further speculated that this activation mechanism might be associated with the conformational motions between domain III and IV. To our knowledge, this is the first report of nucleotide effector regulated ß-N-acetylhexosaminidase, to reveal its novel biological functions. These findings contribute to understanding the distinct properties within the GH20 family and lay a certain foundation to develop controllable glycan hydrolyzing catalysts.Abbreviations: OD600 - optical cell densities at 600 nm; LB - Luria-Bertani; IPTG - isopropyl ß-D-1-thiogalactopyranoside; PMSF - phenylmethanesulfonyl fluoride; rmsd - root mean square deviation; GlcNAc - N-acetyl-ß-D-glucosamine; GalNAc - N-acetyl-ß-D-galactosamine; Gal - galactose.

Structural and molecular dynamic studies of Pseudomonas aeruginosa OdaA reveal the regulation role of a C-terminal hinge element.

BACKGROUND: Crotonase superfamily members exhibit great catalytic diversity towards various acyl-CoA substrates. A common CoA moiety binding pattern is usually observed in this family, understanding the substrate-binding mechanism would facilitate the rational engineering of crotonases for improved properties. METHODS: We applied X-ray crystallography to investigate a putative enoyl-CoA hydratase/isomerase OdaA in Pseudomonas aeruginosa. Thermal shift assay (TSA) were performed to explore the binding of OdaA with CoA thioester substrates. Furthermore, we performed molecular dynamics (MD) simulations to elucidate the dynamics of its CoA-binding site. RESULTS: We solved the crystal structures of the apo and CoA-bound OdaA. Thermal shift assay (TSA) showed that CoA thioester substrates bind to OdaA with a different degree. MD simulations demonstrated that the C-terminal alpha helix underwent a structural transition and a hinge region would associate with this conformational change. CONCLUSIONS: TSA in combination with MD simulations elucidate that the dynamics of C-terminal alpha helix in CoA-binding, and a hinge region play an important role in conformational change. GENERAL SIGNIFICANCE: Those results help to extend our knowledge about the nature of crotonases and would be informative for future mechanistic studies and industry applications.

Structural analysis of the sulfatase AmAS from Akkermansia muciniphila.

Akkermansia muciniphila, an anaerobic Gram-negative bacterium, is a major intestinal commensal bacterium that can modulate the host immune response. It colonizes the mucosal layer and produces nutrients for the gut mucosa and other commensal bacteria. It is believed that mucin desulfation is the rate-limiting step in the mucin-degradation process, and bacterial sulfatases that carry out mucin desulfation have been well studied. However, little is known about the structural characteristics of A. muciniphila sulfatases. Here, the crystal structure of the premature form of the A. muciniphila sulfatase AmAS was determined. Structural analysis combined with docking experiments defined the critical active-site residues that are responsible for catalysis. The loop regions I-V were proposed to be essential for substrate binding. Structure-based sequence alignment and structural superposition allow further elucidation of how different subclasses of formylglycine-dependent sulfatases (FGly sulfatases) adopt the same catalytic mechanism but exhibit diverse substrate specificities. These results advance the understanding of the substrate-recognition mechanisms of A. muciniphila FGly-type sulfatases. Structural variations around the active sites account for the different substrate-binding properties. These results will enhance the understanding of the roles of bacterial sulfatases in the metabolism of glycans and host-microbe interactions in the human gut environment.

Structural and biochemical analysis of 1-Cys peroxiredoxin ScPrx1 from Saccharomyces cerevisiae mitochondria.

BACKGROUND: ScPrx1 is a yeast mitochondrial 1-Cys peroxiredoxins (Prx), a type of Prx enzyme which require thiol-containing reducing agents to resolve its peroxidatic cysteine. ScPrx1 plays important role in protection against oxidative stress. Mitochondrial thioredoxin ScTrx3 and glutathione have been reported to be the physiological electron donor for ScPrx1. However, the mechanism underlying their actions, especially the substrate recognition of ScPrx1 requires additional elucidation. METHODS: The structure of ScPrx1 was obtained through crystallization experiments. The oligomeric state of ScPrx1 was monitored by Blue-Native PAGE. Mutations were generated by the QuikChange PCR-based method. The ScPrx1 activity assay was carried out by measuring the change of 340 nm absorption of the NADPH oxidation. RESULTS: ScPrx1 exist as a homodimer in solution. The structure adopts a typical Prx-fold core which is preceded by an N-terminal ß-hairpin and has a C-terminal extension. Mutations (Glu94Ala, Arg198Ala and Trp126) close to the active site could enhance the catalytic efficiency of ScPrx1 while His83Ala and mutations on α4-ß6 region exhibited reduced activity. The biochemical data also show that the deletion or mutations on ScPrx1 C-terminal have 2-4.56 fold increased activity. CONCLUSION: We inferred that conformational changes of ScPrx1 C-terminal segment were important for its reaction, and the α4-ß6 loop regions around the ScPrx1 active sites were important for the catalytic function of ScPrx1. Collectively, these structural features provides a basis for understanding the diverse reductant species usage in different 1-Cys Prxs.

Structural characterization of a Δ3, Δ2-enoyl-CoA isomerase from Pseudomonas aeruginosa: implications for its involvement in unsaturated fatty acid metabolism.

Gene PA4980 from Pseudomonas aeruginosa encodes a putative enoyl-coenzyme A hydratase/isomerase that is associated with the function of the biofilm dispersion-inducing signal molecule cis-2-decenoic acid. To elucidate the role of PA4980 in cis-2-decenoic acid biosynthesis, we reported the crystal structure of its protein product at 2.39 Å. The structural analysis and substrate binding prediction suggest that it acts as a monofunctional enoyl-coenzyme A isomerase, implicating an alternative pathway of the cis-2-decenoic acid synthesis.

Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

DspI, a putative enoyl-coenzyme A (CoA) hydratase/isomerase, was proposed to be involved in the synthesis of cis-2-decenoic acid (CDA), a quorum sensing (QS) signal molecule in the pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study provided a structural basis for the dehydration reaction mechanism of DspI during CDA synthesis. Structural analysis reveals that Glu126, Glu146, Cys127, Cys131 and Cys154 are important for its enzymatic function. Moreover, we show that the deletion of dspI results in a remarkable decreased in the pyoverdine production, flagella-dependent swarming motility, and biofilm dispersion as well as attenuated virulence in P. aeruginosa PA14. This study thus unravels the mechanism of DspI in diffusible signal factor (DSF) CDA biosynthesis, providing vital information for developing inhibitors that interfere with DSF associated pathogenicity in P. aeruginosa.

Author Correction: Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Structure-Function Relationship of Aminopeptidase P from Pseudomonas aeruginosa.

PepP is a virulence-associated gene in Pseudomonas aeruginosa, making it an attractive target for anti-P. aeruginosa drug development. The encoded protein, aminopeptidases P (Pa-PepP), is a type of X-prolyl peptidase that possesses diverse biological functions. The crystal structure verified its canonical pita-bread fold and functional tetrameric assembly, and the functional studies measured the influences of different metal ions on the activity. A trimetal manganese cluster was observed at the active site, elucidating the mechanism of inhibition by metal ions. Additionally, a loop extending from the active site appeared to be important for specific large-substrate binding. Based on the structural comparison and bacterial invasion assays, we showed that this non-conserved surface loop was critical for P. aeruginosa virulence. Taken together, these findings can extend our understanding of the catalytic mechanism and virulence-related functions of Pa-PepP and provide a solid foundation for the design of specific inhibitors against pathogenic-bacterial infections.

Economic burden and its associated factors of hospitalized patients infected with A (H7N9) virus: a retrospective study in Eastern China, 2013-2014.

BACKGROUND: H7N9 continues to cause human infections and remains a pandemic concern. Understanding the economic impacts of this novel disease is important for making decisions on health resource allocation, including infectious disease prevention and control investment. However, there are limited data on such impacts. METHODS: Hospitalized laboratory-confirmed H7N9 patients or their families in Jiangsu Province of China were interviewed. Patients' direct medical costs of hospitalization were derived from their hospital bills. A generalized linear model was employed to estimate the mean direct medical costs of patients with different characteristics. RESULTS: The mean direct cost of hospitalization for H7N9 was estimated to be ¥ 71 060 (95 % CI, 48 180-104 820), i.e., US$ 10 996 (95 % CI, 7 455-16 220), and was ¥12 060 (US$ 1 861), ¥136 120 (US$ 21 001) and ¥218 610 (US$ 33 728) for those who had mild or severe symptoms or who died, respectively. The principal components of the total fees differed among patients with different disease severity, although medication fees were always the largest contributors. Disease severity, proportion of reimbursement and family member monthly average income were identified as the key factors that contributed to a patient's direct medical cost of hospitalization. CONCLUSIONS: The direct medical costs of hospitalized patients with H7N9 are significant, and far surpass the annual per capita income of Jiangsu Province, China. The influencing factors identified should be taken into account when developing related health insurance policies and making health resource allocation. TRIAL REGISTRATION: Not applicable. This is a survey study with no health care intervention implemented on human participants.

The influence of fluoride on the expression of inhibitors of Wnt/ß-catenin signaling pathway in rat skin fibroblast Cells.

The effective therapy of fluoride-induced bone diseases requires an understanding of the mechanism of the disorders. Changes in the inhibitors of the Wnt/ß-catenin pathway, Dickkopf-1 (Dkk-1) and Sclerostin (SOST),were studied in supernatants harvested from rat skin fibroblasts cultured with varied doses of fluoride. The contents of SOST and Dkk-1 in fibroblast supernatants were assessed at four exposure time-points and investigated by using the method of ELISA. Compared to the relevant controls(0 mg F(−)/L), a significant decrease of the concentrations of SOST and Dkk-1 was observed as the fluoride concentration increased. Compared to the relevant time controls (24 h), a significant decrease of the concentrations of SOST and Dkk-1 was observed with the extension of time. Our results suggest that the Wnt/ß-catenin pathway inhibitors Dkk-1 and SOST play an important role in skeletal fluorosis. They can be used as important indications for diagnosing bone metabolism changes caused by fluoride exposure and therapeutic targets in diseases resulting from fluoride exposure.