Primers used in this study.

<p>MCS, multiple cloning site.</p

Bacterial strains used in this study.

<p>Cm, chloramphenicol; Gm, gentamicin; Ap, ampicillin; Tc, tetracycline; Km, kanamycin; Tp, Trimethoprim; Px, polymyxin; Sm, streptomycin; r  =  resistance; s  =  sensitive.</p

Plasmids used in this study.

<p>Cm, chloramphenicol; Gm, gentamicin; Ap, ampicillin; Tc, tetracycline; Km, kanamycin; Tp, Trimethoprim; Px, polymyxin; Sm, streptomycin; r  =  resistance; s  =  sensitive.</p

Characterization of BPSS1521 (<i>bprD</i>), a Regulator of <i>Burkholderia pseudomallei</i> Virulence Gene Expression in the Mouse Model

<div><p>The Gram-negative saprophytic bacterium <i>Burkholderia pseudomallei</i> is the causative agent of melioidosis, a severe infectious disease of both humans and animals. Severity of the disease is thought to be dependent on both the health status of the host, including diabetes mellitus and kidney disease, and bacterial-derived factors. To identify the bacterial factors important during an acute infection, gene expression profiles in the spleen, lung, and liver of BALB/c (Th2 prototype) and C57BL/6 mice (Th1 prototype) were determined using DNA microarrays. This analysis identified BPSS1521 (<i>bprD</i>), a predicted transcriptional regulator located in the type III secretion system (T3SS-3) operon, to be up regulated, specifically in C57BL/6 mice. BALB/c mice infected with a <i>bprD</i> mutant showed a shorter time to death and increased inflammation, as determined by histopathological analysis and enumeration of bacteria in the spleen. Elevated numbers of multinucleated giant cells (MNGCs), which is the hallmark of melioidosis, were detected in both the wild-type and the <i>bprD</i> mutants; a similar elevation occurs in melioidosis patients. One striking observation was the increased expression of BPSS1520 (<i>bprC</i>), located downstream of <i>bprD</i>, in the <i>bprD</i> mutant. BprC is a regulator of T6SS-1 that is required for the virulence of <i>B. pseudomallei</i> in murine infection models. Deletion of <i>bprD</i> led to the overexpression of <i>bprC</i> and a decreased time to death. <i>bprD</i> expression was elevated in C57BL/6 —as compared to BALB/c—mice, suggesting a role for BprD in the natural resistance of C57BL/6 mice to <i>B. pseudomallei</i>. Ultimately, this analysis using mice with different immune backgrounds may enhance our understanding of the outcomes of infection in a variety of models.</p></div

Fold changes in gene expression in the <i>B. pseudomallei</i> K96243 wild-type and <i>bprD</i>-mutant strains.

<p>The fold changes in expression in the <i>B. pseudomallei</i> K96243 wild-type (▪) and <i>bprD</i>-mutant (□) strains of the BPSS1520 (<i>bprC</i>) (A), BPSS1496 (<i>tssA</i>) (B), BPSS1497 (<i>tssB</i>) (C), and BPSS1498 (<i>hcp1</i>) (D) genes at mid-logarithmic phase in LB medium were measured by qRT-PCR. The bars indicate means ± standard error of two experiments; * significant difference.</p

Venn diagrams showing the number of genes differentially expressed in each organ.

<p>Genes expressed at higher levels in the spleen (pink); lung (green); and liver (yellow) of BALB/C (A) and C57BL/6 (B) mice compared to <i>in vitro</i>.</p

List of the <i>B. pseudomallei</i> genes that have >2SD fold change in expression (<i>in vivo/in vitro</i>).

<p>* Expression level change >2SD in the organs of mice and ND  =  no data.</p

Survival curves and numbers of bacteria in the spleen of BALB/c mice after infection with <i>B. pseudomallei</i>.

<p>(A) The virulence of the <i>B. pseudomallei</i> K96243 wild-type and <i>bprD</i> mutant strains was compared in BALB/c mice, which are highly susceptible to infection. X-axis, days after infection; Y-axis, % survival. BALB/c mice (groups of eight) were intraperitoneally infected with ∼10⁴ CFU of the <i>B. pseudomallei</i> K96243 wild-type (•), <i>bprD</i> mutant (∇) or <i>bprD</i> complemented (▪) strain, and their survival was monitored daily. The survival of mice infected with the wild-type strain was significantly different from those infected with the <i>bprD</i> mutant (<i>P</i> = 0.0015). (B) The numbers of <i>B. pseudomallei</i> K96243 wild-type, <i>bprD</i>-mutant, and <i>bprD</i>-complemented strains in the spleen of BALB/c mice. X-axis, days after infection; Y-axis, number of bacteria (CFU). A total of 1×10⁴ CFU of the wild-type (▪) and <i>bprD</i>-mutant (□) strains, and 0.5×10⁴ CFU of the <i>bprD</i>-complemented ( ) strain, were intraperitoneally injected into BALB/c mice. The number of bacteria in the spleen on days 2, 3, 7 and 13 was determined. The experiment was performed twice; average values of data are shown.</p

Schematic diagram of the <i>B. pseudomallei</i> K96243 <i>bprD</i> gene and agarose gel of RT-PCR products.

<p>Schematic diagram of the genomic organization of the <i>B. pseudomallei</i> K96243 region containing <i>bprD</i> (A), and RT-PCR analysis of expression of genes in this region (B). The arrows show the position and direction of genes. The positions of RT-PCR primers are indicated by black arrows. Lane 1, RT-PCR product from <i>B. pseudomallei</i> K96243 wild-type cDNA; lane 2, negative control for wild-type or DNase-treated wild-type RNA (to evaluate contamination of wild-type RNA with gDNA); lane 3, <i>B. pseudomallei</i> K96243 <i>bprD</i> mutant cDNA; lane 4, negative control for mutant or DNase-treated <i>bprD</i> mutant RNA (to evaluate contamination of mutant RNA with gDNA); lane 5, <i>B. pseudomallei</i> K96243 wild-type genomic DNA control; and lane 5, No DNA control.</p

Photomicrographs of hematoxylin-and-eosin-stained spleens from BALB/c mice infected with <i>B. pseudomallei</i>.

<p>The spleens were collected on day 3 from non-infected BALB/c mice (A; ×100) and mice infected with <i>B. pseudomallei</i> (B–F). The spleen of mice infected with the <i>B. pseudomallei</i> K96243 wild-type strain showed multifocal areas of inflammatory cell infiltration and neutrophil abscess formation (arrows in B; ×100). The neutrophils (arrow) and necrotic cells (arrow head) are shown at high magnification (C; ×400). Numerous multinucleated giant cells are observed (arrows in D; ×400). Mice infected with the <i>B. pseudomallei bprD</i> mutant showed multifocal to coalescent pyogranulomatous splenitis (E; ×100). Necrotic areas with neutrophils (arrow) are shown at high magnification (F; ×400).</p