A 14.7 kDa Protein from <i>Francisella tularensis subsp. novicida</i> (Named FTN_1133), Involved in the Response to Oxidative Stress Induced by Organic Peroxides, Is Not Endowed with Thiol-Dependent Peroxidase Activity

<div><p><i>Francisella</i> genus comprises Gram-negative facultative intracellular bacteria that are among the most infectious human pathogens. A protein of 14.7 KDa named as FTN_1133 was previously described as a novel hydroperoxide resistance protein in <i>F. tularensis subsp. novicida</i>, implicated in organic peroxide detoxification and virulence. Here, we describe a structural and biochemical characterization of FTN_1133. Contrary to previous assumptions, multiple amino acid sequence alignment analyses revealed that FTN_1133 does not share significant similarity with proteins of the Ohr/OsmC family or any other Cys-based, thiol dependent peroxidase, including conserved motifs around reactive cysteine residues. Circular dichroism analyses were consistent with the <i>in silico</i> prediction of an all-α-helix secondary structure. The pK_a of its single cysteine residue, determined by a monobromobimane alkylation method, was shown to be 8.0±0.1, value that is elevated when compared with other Cys-based peroxidases, such as peroxiredoxins and Ohr/OsmC proteins. Attempts to determine a thiol peroxidase activity for FTN_1133 failed, using both dithiols (DTT, thioredoxin and lipoamide) and monothiols (glutathione or 2-mercaptoethanol) as reducing agents. Heterologous expression of <i>FTN_1133</i> gene in <i>ahpC</i> and <i>oxyR</i> mutants of <i>E. coli</i> showed no complementation. Furthermore, analysis of <i>FTN_1133</i> protein by non-reducing SDS-PAGE showed that an inter-molecular disulfide bond (not detected in Ohr proteins) can be generated under hydroperoxide treatment, but the observed rates were not comparable to those observed for other thiol-dependent peroxidases. All the biochemical and structural data taken together indicated that FTN_1133 displayed distinct characteristics from other thiol dependent peroxidases and, therefore, suggested that FTN_1133 is not directly involved in hydroperoxide detoxification.</p></div

Squematic electron flow of systems that were used to test a possible thiol-dependent peroxidase activity of recombinant FTN_1133.

<p><b>A.</b> Consumption of hydroperoxides determined by FOX assay, using DTT, β-mercaptoethanol or glutathione as electron donors. <b>B.</b> Oxidized DTT assay was used to monitor the ability of thioredoxin to support a putative FTN_1133 activity. In this case, thioredoxin would provide electrons to reduce FTN_1133 instead of DTT, which would be engaged to recycle the thioredoxin protein; <b>C.</b> For lipoamide-coupled assay, electrons originated from NADH are transferred to a Dihydrolipoamide dehydrogenase (Lpd), that through lipoamide would reduce FTN_1133; <b>D.</b> In the GR/Grx-coupled assay, electrons originated from NADPH would flow to FTN_1133, through GR/Grx system. β-ME, β-mercaptoethanol; Trx_E.c., Thioredoxin from <i>E. coli</i>; LpD_X.f., Dihydrolipoamide Dehydrogenase from <i>X. fastidiosa</i>; GR_S.c., Glutathione Reductase from baker's yeast <i>S. cerevisae</i>; GrxC_X.f., Glutaredoxin C, from <i>X. fastidiosa</i>.</p

Sequence alignment of FTN_1133.

<p>FTN_1133 sequence was run against non-redundant database of NCBI using PSI-Blast algorithm operating in a default mode. The alignment of sequences from PSI-Blast output was generated by using the Kalign algorithm and processed by Jalview. The location of the cysteine residue on <i>Francisella</i> sequences is indicated by red boxes. Dark and light blue shadings backgrounds illustrate regions with greater than 80% of similarity presented among all sequences. Protein sequence ID FTN_1133 from <i>F. tularensis novicida</i> U112 has 100% amino acid identity with FTE_0530 (<i>F. tularensis novicida</i> FTE), FTG_0533 (<i>F. tularensis novicida</i> FTG), FN3523_1168 (<i>F. tularensis novicida</i> 3523) and FNFX1_1179 (<i>F. tularensis novicida</i> Fx1). Protein sequence IDFTT_1152 from <i>F. tularensis tularensis SCHU4</i> has 100% identity with FTL_0803 (<i>F. tularensis holarctica</i> LVS), FTF_1152, (<i>F. tularensis tularensis</i> FSC198), FTH_0797 (<i>F. tularensis holarctica</i> OSU18), FTW_1191 (<i>F. tularensis tularensis</i> WY96_3418), FTA_0849 (<i>F. tularensis holarctica</i> FTNF002_00), FTM_0836 (<i>F. tularensis mediasiatica</i> FSC147), FTU_1185 (<i>F. tularensis tularensis</i> TIGB03), FTV_1101 (<i>F. tularensis tularensis</i> TI0902), FTS_0796 (<i>F. tularensis holarctica</i> FSC200) and FTHG_00746 (<i>F. tularensis holarctica</i> 257). Protein sequences IDOOM_1726, Fphi_1909 and F7308_1240 are from <i>F. noatunensis orientalis</i> str. Toba 04, <i>F. philomiragia philomiragia</i> ATCC 25017 and <i>Francisella sp.</i> TX077308, respectively. Protein sequence ID, gi|517110088 is from <i>Fangia hongkongensis</i> and protein sequences ID, gi|515947316 and gi|510837960, are from <i>Piscirickettsia salmonis</i>.</p

FTN_1133 secondary structure.

<p><b>A.</b> Cartoon of predicted FTN_1133 secondary structure. Blue traces represent predicted loop regions and red traces represent predicted α-helix region of FTN_1133 sequence. <b>B.</b> Circular dichroism spectra of FTN_1133 were collected with 3 and 6 µM in the presence of NaCl (100 mM), sodium phosphate pH 7.4 (20 mM) and 10% glycerol using a 0.01 cm cell in a JASCO J-710 spectropolarimeter. <b>C.</b> Estimates of secondary structure elements of FTN_1133 from data obtained from item B., using SELCON3, CONTINLL, CDSSTR algorithms. NRSMD, denotes the <u>N</u>ormalized <u>R</u>oot-<u>M</u>ean-<u>S</u>quare <u>D</u>eviation.</p

Assay for FTN_1133 DTT-dependent peroxidase activity.

<p>Decomposition of different peroxides was monitored during 40<b>A.</b>, <b>B.</b> and <b>C.</b>, CuOOH, tBOOH and H₂O₂ decomposition, respectively, in the presence of FTN_1133 (10 µM), HEPES-HCl pH 7.4 (50 mM), DTT (0.5 mM), sodium azide (0.1 mM) and DTPA (0.1 mM). <b>D.</b>, <b>E.</b> and <b>F.</b>, CuOOH, tBOOH and H₂O₂ decomposition, respectively, in the presence of OsmC (10 µM), HEPES-HCl pH 7.4 (50 mM), DTT (0.5 mM), sodium azide (0.1 mM) and DTPA (0.1 mM). All reactions were started by addition of 200 µM of peroxide. Blue line, (blue triangle) enzyme+DTT+peroxide (catalyzed reaction); Green line, (green triangle) enzyme+peroxide without DTT; Black line, (black square) only peroxide and red line, (red circle) peroxide+DTT (uncatalyzed reaction). The figure is representative of at least two independent set of experiments, each one done in technical triplicates.</p

Inter-molecular disulfide bond formation in FTN_1133 upon hydroperoxide treatment. A.

<p>Non-reducing SDS-PAGE of freshly purified recombinant FTN_1133 (5 µM) that was incubated in the absence or presence of 0.06, 0.2, 0.5, 2, 10 and 100 mM of DTT during 10 minutes at 37°C. <b>B.</b> Representative non-reducing SDS-PAGE showing that the treatment of FTN_1133 with increasing amount of hydroperoxides. FTN_1133 inter-molecular disulfide bond formation was assessed by the appearance of a band corresponding to the dimer (∼34 kDa). Treatments, using pre-reduced FTN_1133 (5 µM), were performed for 10 minutes at 37°C with 0, 1, 5, 10, 20, 50 and 100 µM of CuOOH (a), tBOOH (b) or H₂O₂ (c). <b>C.</b> Time course of FTN_1133 oxidation towards hydroperoxide treatment. The assay was carried out with pre-reduced FTN_1133 (5 µM) treated with 100 µM CuOOH (b), tBOOH (c) or H₂O₂ (d), during 0, 0.5, 1, 2, 3, 4 and 22 hours at 30°C in a buffer containing 0.5 M of NaCl, 20 mM of sodium phosphate pH 7.4 and 1 mM of DTPA. (a) Represent the control reaction (no addition of hydroperoxide). Immediately after hydroperoxides treatments, all samples were alkylated with NEM (100 mM) for 30 minutes at room temperature to avoid oxidation artifacts due to protein denaturation by SDS. The figure is representative of three independent set of experiments.</p

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P &lt; 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)