Iron acquisition in Bacillus cereus: the roles of IlsA and bacillibactin in exogenous ferritin iron mobilization

9siIn host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.openopenSegond D; Abi Khalil E; Buisson C; Daou N; Kallassy M; Lereclus D; Arosio P; Bou-Abdallah F; Nielsen Le Roux C.Segond, D; Abi Khalil, E; Buisson, C; Daou, N; Kallassy, M; Lereclus, D; Arosio, Paolo; Bou Abdallah, F; Nielsen Le Roux, C

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Iron Acquisition in Bacillus cereus: The Roles of IlsA and Bacillibactin in Exogenous Ferritin Iron Mobilization

In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis. Author Summary Iron homeostasis is important for all living organisms; too much iron confers cell toxicity, and too little iron results in reduced cell fitness. While crucial for many cellular processes in both man and pathogens, a battle for this essential nutrient erupts during infection between the host and the invading bacteria. Iron is principally stored in ferritin, a large molecule able to bind several thousand iron ions. Although host ferritins represent a mine of iron for pathogens, studies of the mechanisms involved in its acquisition by bacteria are scarce. In the human opportunistic pathogen Bacillus cereus, the surface protein IlsA is able to bind several host iron sources in vitro. In this study, we show that IlsA acts as a ferritin receptor and enhances iron release from the ferritin through direct interaction with each ferritin subunit. Moreover, we demonstrate that the siderophore bacillibactin, a small secreted iron chelator, is essential for ferritin iron acquisition and takes part in B. cereus virulence. We propose a new iron acquisition model that provides new insights into bacterial host adaptation

Iron acquisition from ferritin relies on bacillibactin production.

<p>Growth kinetics of <i>B. cereus</i> wild type (WT; black square), Δ<i>asb</i> petrobactin mutant (blue triangle), Δ<i>entA</i> bacillibactin mutant (red circle), complemented <i>ΔentAΩentA</i> strains (purple diamond) and double Δ<i>entA</i>Δ<i>asb</i> mutant (grey cross). The strains were grown at 37°C in LB medium (<b>A</b>) and in LB medium treated with 2,2′-dipyridyl without addition of iron sources (<b>B</b>) or supplemented with 0.3 µM ferritin only (<b>C</b>) or with 0.3 µM ferritin and 5 µM enterobactin (<b>D</b>). Bacterial growth was monitored during 16 hours by measuring the optical density (OD) at 600 nm every hour. Curves are averages of three independent experiments and error bars are SEM from mean values.</p

Strains and plasmids used in this work.

<p><i>Bc</i>, <i>B. cereus</i>; <i>Ec</i>, <i>E. coli</i>; Tet, tetracycline; Erm, erythromycin; Kan, kanamycin; Amp, ampicillin.</p

Effects of siderophore deficiency on <i>B. cereus</i> virulence in <i>G. mellonella</i> are dose- and time-dependent.

<p>Wild type and mutant strains were injected separately into the hemocoel. For each strain, twenty last-instar larvae were infected with 3×10⁴ (<b>A</b>), 1×10⁴ (<b>B</b>) or 3×10³ (<b>C</b>) of mid-log phase bacteria. The survival rate (% of alive/total number of infected larvae) was monitored for 72 hours after infection with the wild type (black square), Δ<i>asb</i> (blue diamond), Δ<i>entA</i> (red triangle), <i>ΔentAΩentA</i> (purple triangle), Δ<i>entA</i>Δ<i>asb</i> (grey circle) strains or PBS (green cross). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003935#s2" target="_blank">Results</a> are mean values of three to seven independent experiments and error bars indicate SEM from mean values. Based on these data, LD₅₀ were estimated and are reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003935#ppat-1003935-t002" target="_blank">Table 2</a>. (<b>D</b>) Illustrates white alive and dead black larvae.</p

Virulence of siderophore mutants in <i>G. mellonella.</i>

a<p>The 50% lethal doses, in number of colony forming units (cfu), were evaluated by Probit survival.</p><p>analysis (<i>p</i><0.05).</p

Best fit parameters for ITC measurements of IlsA binding to ferritins^a.

a<p>The reported thermodynamic quantities are apparent values and include the contributions to the overall equilibrium from ferritin and buffer species in different states of protonation.</p><p>Standard errors from replicate determinations are indicated.</p>b<p>Calculated from Δ<i>G</i>⁰ = −<i>RT</i>ln<i>K</i>.</p>c<p>Calculated from Δ<i>S</i>⁰ = (Δ<i>H</i>°−Δ<i>G</i>°)/<i>T</i>.</p><p>HuHF, recombinant human H-chain ferritin; HuLF, recombinant human L-chain ferritin; HuH/LF, recombinant heteropolymer ferritin of 21H-chains and 4L-chains; MoHF, recombinant mouse H-chain ferritin. All experiments were repeated two to four times.</p

Role of IlsA in iron mobilization from ferritin.

<p>Demineralization of recombinant holoHuHF (recombinant <u>Hu</u>man <u>H</u>-chain <u>F</u>erritin, 1 µM) containing 500 Fe/shell was followed by the absorption of the Fe(III)-DFO (<u>D</u>e<u>f</u>er<u>o</u>xamine B) complex at 425 nm in the presence (black line) or absence (dotted line) of IlsA (5 µM). The experiment was repeated in triplicate using different protein preparations. Curves are averages of three independent runs and error bars are SEM from mean values.</p

IlsA is essential for ferritin binding on <i>B. cereus</i> cell surface.

<p><i>B. cereus</i> wild type (WT; A–F), <i>ΔilsA</i> mutant (G–I) and the complemented <i>ΔilsAΩilsA</i> (J–L) strains were grown in LB+0,3 µM HoSF (<u>Ho</u>rse <u>S</u>pleen <u>F</u>erritin) medium (only the wild type; A–C) and in iron-depleted LB (Dip) +0,3 µM HoSF medium (D–L). Bacterial cells were washed before fluorescence microscopy analysis using HoSF Alexa Fluor 594 labelled polyclonal antibody (B,E,H,K) or DAPI to stain DNA. The merged images (C,F,I,L) show DAPI in blue and ferritin in red. Experiments were performed three times. In iron rich LB medium, IlsA is not produced <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003935#ppat.1003935-Daou1" target="_blank">[14]</a> and no ferritin is detected on the bacterial surface in these conditions (A–C). Ferritin aggregates only on the surface in iron-depleted medium supplemented with ferritin (D–F). Absence of llsA in the Δ<i>ilsA</i> mutant compromises ferritin binding on the bacterial surface (G–I) whereas <i>ilsA</i> complementation restores ferritin aggregation (J–L).</p