In the <i>irp2</i> knockout strain the production of yersiniabactin is blocked.

<p>Yersiniabactin production was tested using a GFP reporter assay with the knockout strain WA-CS <i>irp1</i>::Kan^r containing the pCJG3.3N plasmid as the reporter strain to detect yersiniabactin in supernatants. The number of bacteria versus the measured arbitrary amount of fluorescence from 50,000 counted bacteria is shown in panel A. A) Supernatants from the <i>irp2</i> knockout (E4Δ<i>irp2</i>) cultures (grey) induced a lower amount of GFP compared to supernatants from the wild-type (WT) cultures (white). This indicates that production of yersiniabactin was blocked in the <i>irp2</i> knockout. The experiments were performed in duplicate. B) The GFP reporter assay is dependent on yersiniabactin production, and production of yersiniabactin in the <i>irp2</i> knockout is blocked. Yersiniabactin production is only seen in the wild-type (WT) cells cultured in iron-depleted medium (NBD), while the <i>irp2</i> knockout (E4Δ<i>irp2</i>) strain cultured in NBD and iron-containing medium (NB), as well as the wild-type strain grown in NB, did not produce yersiniabactin. Three different experiments were performed, with each sample analyzed in duplicate. C) The expression of HMWP1 and HMWP2 is disrupted by the insertion of a kanamycin resistance gene into <i>irp2</i> using the Tagetron Knockout System. The <i>irp2</i> knockout (E4Δ<i>irp2</i>) strain was not able to produce HMWP1 and HMWP2 when cultured in NBD. M: marker; lane 1: wild-type strain E4; lane 2–5: <i>irp2</i> gene knockouts created using the wild-type strain E4; lane 6 wild-type EHOS strain 03-702; lane 7: wild-type EHOS strain 03-819 served as HPI-positive control because the HMWP2 of this strain was confirmed by Edman degradation.</p

Other iron chelators also reduce ROS production by PMNs.

<p>Along with yersiniabactin, aerobactin, deferoxamine, and deferiprone also reduce ROS production by PMNs. To determine the effect of other iron-binding molecules on ROS production by PMNs, PMNs were pre-incubated with aerobactin, deferoxamine, deferiprone, or yersiniabactin. The pre-treated PMNs were then activated using PMA, and the production of ROS was measured using luminol. Red line: the concentration-dependent decrease in ROS production following yersiniabactin treatment. Gray line: the decrease in ROS production following treatment with aerobactin. Yellow line: the concentration-dependent decrease in ROS production following deferiprone treatment. Green line: the concentration-dependent decrease in ROS production following deferoxamine treatment. The level of ROS produced by stimulated PMNs without additives was set as 100%. All experiments were replicated three times, and each sample was analyzed in duplicate.</p

Growth curves of the EHOS E4 isolate and its corresponding <i>irp2</i> knockout strain.

<p>The results are presented as the mean of three experiments performed in duplicate on different days. Cells were grown in: A) nutrient broth treated with 1% Chelex; B) nutrient broth treated with 1% Chelex and supplemented with 80% iron-saturated lactoferrin (∼6.2 µM, Fe-LF) or unsaturated lactoferrin (∼6.4 µM,0-LF); C) nutrient broth treated with 1% Chelex and supplemented with 17 µM holo-transferrin or 17 µM apo-transferrin; D) nutrient broth treated with 1% Chelex and supplemented with 10 µM or 30 µM hemin.</p

ROS production by PMNs is reduced by yersiniabactin.

<p>To determine the effect of yersiniabactin on the ROS production of PMNs, PMNs were pre-incubated with yersiniabactin. PMNs were then activated by PMA, and the production of ROS was measured using luminol. A) Absolute ROS production measured in arbitrary fluorescence units (A.U.) of PMNs after pre-incubation with yersiniabactin or iron-saturated yersiniabactin or the controls (PMNs that were only pre-incubated in RPMI/HSA medium and PMNs that were pre-incubated with yersiniabactin but not stimulated with PMA). B) The yersiniabactin-mediated inhibition of ROS production by PMNs is concentration dependent. Red line: the concentration-dependent decrease in production of ROS following treatment with yersiniabactin. Blue line: no decrease in ROS production following treatment with different concentrations of iron-saturated yersiniabactin. Black dotted line: negative control (PMNs incubated with yersiniabactin, but not stimulated with PMA); ***<i>p</i> value<0.0005, **<i>p</i> value<0.005, *<i>p</i> value<0.05. C) Treatment of PMNs with unsaturated lactoferrin or holo-transferrin partly inhibits the yersiniabactin-mediated inhibition of ROS production. Black bars: relative ROS production following treatment with yersiniabactin; gray bars: relative ROS production following treatment with yersiniabactin and unsaturated lactoferrin; white bars: relative ROS production following treatment with yersiniabactin and holo-transferrin. ^aConcentration of the iron chelator. D) Treatment of PMNs with saturated lactoferrin or apo-transferrin partly inhibits the yersiniabactin-mediated inhibition of ROS production. Black bars: relative ROS production following treatment with yersiniabactin; gray bars: relative ROS production following treatment with yersiniabactin and saturated lactoferrin; white bars: relative ROS production following treatment with yersiniabactin and apo-transferrin. All experiments were replicated three times, and each sample was analyzed in duplicate.</p

HBV sequences used in this study.

<p>^† Details of the GenBank Accession Numbers for all HBV sequences are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132533#pone.0132533.s004" target="_blank">S1 Table</a>.</p><p>^‡ Five HBV/C sequences initially used in phylogenetic tree construction representing HBV/C7, C9, C10, C15, and C16 were not used in nucleotide divergence analysis because only single complete genome isolates were available for each of these subgenotypes.</p><p>^§ Core immune epitope analysis used sequences that cover the C gene region. Compared to the sequences used in the surface immune epitope analysis, only 16 HBV/C Asia sequences were used again in the core immune epitope analysis, while all S gene sequences from HBV/C Papua Pacific and the 87 HBV/C Indonesia of this study did not qualify for the core immune epitope analysis.</p><p>HBV sequences used in this study.</p

Phylogenetic tree of HBV/C isolates from different countries in East and Southeast Asia, and Papua-Pacific.

<p>A Bayesian phylogenetic tree analysis based on complete genome sequences showed that isolates from various subgenotypes (C1-C16) are clearly grouped into two major clusters, consistent with their geographical origins. Seven HBV/C subgenotypes (C1, C2, C5, C7, C8, C9, and C10) from East and Southeast Asia, and one (C14) from Papua (<i>light highlight</i>) were well-separated from those six subgenotypes (C6, C11, C12, C13, C15, and C16) from Papua, and from one subgenotype (C3) from Pacific, the more east region of the Papua (<i>dark highlight</i>). Although the root of subgenotype C3 phylogenetically is distanced from the subgenotypes of Papua, the isolate geographic origin, the immune epitope characteristics of surface and core proteins, and the HBsAg subtype gradient distribution showed these HBV/C3 isolates to be close to Papua subgenotypes. Therefore, the Papua and the Pacific subgenotypes are classified together into Papua-Pacific type. The diversification of the Asian type from the Papua-Pacific type started from Papua of Indonesia to the east. The other subgenotype, HBV/C4, was distanced from other subgenotypes. In this analysis, one strain (GQ358157) from Papua reported as C6 in our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132533#pone.0132533.ref023" target="_blank">23</a>] grouped into C12. We redefine this strain as a member of HBV/C12.</p

Distribution of HBsAg subtypes and HBV genotypes/subgenotypes of 271 HBV/C isolates according to their country/geographical origins in East/Southeast Asia and Papua-Pacific.

<p>^{# including 37 published complete genome sequences and 87 newly generated in this study; N. Caledonia: New Caledonia; PNG: Papua New Guinea.}</p><p>Distribution of HBsAg subtypes and HBV genotypes/subgenotypes of 271 HBV/C isolates according to their country/geographical origins in East/Southeast Asia and Papua-Pacific.</p

HBcAg amino acid motifs in B and T-cell epitopes of Asia and Papua-Pacific HBV/C isolates.

<p>For the sake of clarity, this figure was not drawn to scale. Among 15 amino acid positions examined within HBcAg immune epitopes of 143 isolates, we identified I/V at position c59 as the essential variation that classified HBV/C subgenotypes into two major clusters, the Asian and the Papua-Pacific (p-value <0.001; data not shown). HBV/C4 and C14 showed similar variation in most amino acids examined, with C4 and C14 having cI59 and cV59, respectively.</p

Distribution of HBV/C subtypes in the East and Southeast Asia and the Papua-Pacific.

<p>This study identified a west-to-east gradient in the distribution of HBsAg subtypes with <i>adrq+</i> (<i>red</i>) prominent in East-Southeast Asia and <i>adrq-</i> (<i>pink</i>) in the Pacific region (Vanuatu, Fiji, Tonga, and Kiribati). Interestingly, together with <i>adrq+</i>, <i>adrq-</i>indeterminate sA159/sA177 and a new pattern of <i>adrq-</i>indeterminate sV159/sV177 identified in this study were found in Papua and PNG, respectively, suggesting that the molecular admixture of HBV/C, particularly for subtype evolution, occurred in Papua and PNG with both <i>adrq-</i>indeterminate forms (<i>yellow</i>) as the transitional patterns.</p

Mean percentage nucleotide divergence of the complete genome between HBV/C subgenotypes.

<p>The total number of HBV/C isolates examined for each subgenotype is shown in bracket. Other existing subgenotypes (C7, C9, C10, C15, and C16) were not included in the genetic distance calculation since only single isolate was available for each subgenotype. Intrasubgenotype divergences are shown in bold.</p