Edwardsiella tarda Attenuates Virulence upon Oxytetracycline Resistance.

The relationship between antibiotic resistance and bacterial virulence has not yet been fully explored. Here, we use Edwardsiella tarda as the research model to investigate the proteomic change upon oxytetracycline resistance (LTB4-ROTC). Compared to oxytetracycline-sensitive E. tarda (LTB4-S), LTB4-ROTC has 234 differentially expressed proteins, of which the abundance of 84 proteins is downregulated and 15 proteins are enriched to the Type III secretion system, Type VI secretion system, and flagellum pathways. Functional analysis confirms virulent phenotypes, including autoaggregation, biofilm formation, hemolysis, swimming, and swarming, are impaired in LTB4-ROTC. Furthermore, the in vivo bacterial challenge in both tilapia and zebrafish infection models suggests that the virulence of LTB4-ROTC is attenuated. Analysis of immune gene expression shows that LTB4-ROTC induces a stronger immune response in the spleen but a weaker response in the head kidney than that induced by LTB4-S, suggesting it's a potential vaccine candidate. Zebrafish and tilapia were challenged with a sublethal dose of LTB4-ROTC as a live vaccine followed by LTB4-S challenge. The relative percentage of survival of zebrafish is 60% and that of tilapia is 75% after vaccination. Thus, our study suggests that bacteria that acquire antibiotic resistance may attenuate virulence, which can be explored as a potential live vaccine to tackle bacterial infection in aquaculture.

α-Ketoglutarate downregulates thiosulphate metabolism to enhance antibiotic killing.

Potentiation of the effects of currently available antibiotics is urgently required to tackle the rising antibiotics resistance. The pyruvate (P) cycle has been shown to play a critical role in mediating aminoglycoside antibiotic killing, but the mechanism remains unexplored. In this study, we investigated the effects of intermediate metabolites of the P cycle regarding the potentiation of gentamicin. We found that α-ketoglutarate (α-KG) has the best synergy with gentamicin compared to the other metabolites. This synergistic killing effect was more effective with aminoglycosides than other types of antibiotics, and it was effective against various types of bacterial pathogens. Using fish and mouse infection models, we confirmed that the synergistic killing effect occurred in vivo. Furthermore, functional proteomics showed that α-KG downregulated thiosulphate metabolism. Upregulation of thiosulphate metabolism by exogenous thiosulphate counteracted the killing effect of gentamicin. The role of thiosulphate metabolism in antibiotic resistance was further confirmed using thiosulphate reductase knockout mutants. These mutants were more sensitive to gentamicin killing, and less tolerant to antibiotics compared to their parental strain. Thus, our study highlights a strategy for potentiating antibiotic killing by using a metabolite that reduces antibiotic resistance.

Triticum urartu MTP1: its ability to maintain Zn2+ and Co2+ homeostasis and metal selectivity determinants.

KEY MESSAGE: TuMTP1 maintains Zn2+ and Co2+ homeostasis by sequestering excess Zn2+ and Co2+ into vacuoles. The mutations NSEDD/VTVTT in the His-rich loop and I119F in TMD3 of TuMTP1 restrict metal selectivity. Mineral nutrients, such as zinc (Zn) and cobalt (Co), are essential or beneficial for plants but can be toxic at elevated levels. Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. However, the determinants of substrate selectivity have not been clarified due to the diversity of MTP1 substrates in various plants. In this study, Triticum urartu MTP1 was characterized. When expressed in yeast, TuMTP1 conferred tolerance to Zn2+ and Co2+ but not Fe2+, Cu2+, Ni2+ or Cd2+ in solid and liquid culture and localized on the vacuolar membrane. Furthermore, TuMTP1-expressing yeast accumulated more Zn2+ and Co2+ when treated. TuMTP1 expression in T. urartu roots was significantly increased under Zn2+ and Co2+ stresses. Determinants of substrate selectivity were then examined through site-directed mutagenesis. The exchange of NSEDD with VTVTT in the His-rich loop of TuMTP1 restricted its metal selectivity to Zn2+, whereas the I119F mutation confined specificity to Co2+. The mutations H74, D78, H268 and D272 (in the Zn2+-binding site) and Leu322 (in the C-terminal Leu-zipper) partially or completely abolished the transport function of TuMTP1. These results show that TuMTP1 might sequester excess cytosolic Zn2+ and Co2+ into yeast vacuoles to maintain Zn2+ and Co2+ homeostasis. The NSEDD/VTVTT and I119F mutations are crucially important for restricting the substrate specificity of TuMTP1, and the Zn2+-binding site and Leu322 are essential for its ion selectivity and transport function. These results can be employed to change metal selectivity for biofortification or phytoremediation applications.

Spectroscopic and molecular modeling studies on the interactions of fluoranthene with bovine hemoglobin.

This study aims to investigate the interaction between fluoranthene (FLA) and Bovine hemoglobin (BHb) by ultraviolet-visible (UV-vis) absorption, fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopy and molecular docking method. The results showed that the fluorescence intensity of BHb was declined with the increase of FLA concentration. The binding procedure was spontaneous mainly driven by hydrophobic force. The number of binding sites were 0.709 (298 K), and 1.41 (310 K). The binding constants were equal to 4.68 × 103 mol·L-1 at 298 K and 6.17 × 105 mol·L-1 at 310 K. The binding distance between FLA and the tryptophan residue of BHb was 4.50 nm. The results of UV-vis spectra, synchronous fluorescence and CD spectra revealed that FLA could change the conformation of BHb, which might affect the physiological functions of hemoglobin. Moreover, molecular modeling results showed that the fluorescence experimental results were in agreement with the results obtained by molecular docking.

[Causes and countermeasure of complications in operative management of intra - articular fracture of calcaneus].

OBJECTIVE: To discuss the probable causes of the post-surgery complications with the intra-artcular fracture of calcaneus, the proper steps for prevention and solution. METHODS: Seventy-one patients (76 injured feet) included 49 males and 22 females aged from 19 to 56 years old (mean 35.6 years). According to Sanders' classification, 23 cases (24 injuried feet) belonged to type II, 36 (38 injured feet) were type III, the remain 12 (14 injured feet) met the criteria of type IV. All the patients received the operation of open reduction, autogenous bone grafting and internal fixation with stainless steel plates. RESULTS: Thirteen injuried feet developed early complications. Two injuried feet got the superficial layer of the wound disrupted and infected, I had the deep layer of the wound disrupted and infected. Cutaneous necrosis at the pointed end of the wound occurred in 7 cases. Another 1 developed osteomyelitis. Two cases suffered from sural nerve damage. Two injuried feet developed late complications, both of them suffered from arthritis of talocalcaneal joint. All the patients were followed up at least 6 months (ranged from 6 to 42 months, mean 19 months). According to Kerr's post-surgery evaluation criteria, 34 injuried feet were excellent, 32 were fine, 9 were acceptable, only 1 was bad. CONCLUSION: If proper measures are taken, the post-surgery complications of intra-articular fracture of calcaneus will be reduced. This requires us to be strict in selecting operation indication, to make a good plan and preparation, to select a right time for operation, to improve surgical skills and pay more attention to peri-surgery nursing. If complications happen, according measures should be taken in order to get a better outcome.