A virulence activator of a surface attachment protein in Burkholderia pseudomallei acts as a global regulator of other membrane-associated virulence factors

Burkholderia pseudomallei (Bp), causing a highly fatal disease called melioidosis, is a facultative intracellular pathogen that attaches and invades a variety of cell types. We previously identified BP1026B_I0091 as a surface attachment protein (Sap1) and an essential virulence factor, contributing to Bp pathogenesis in vitro and in vivo. The expression of sap1 is regulated at different stages of Bp intracellular lifecycle by unidentified regulator(s). Here, we identified SapR (BP1026B_II1046) as a transcriptional regulator that activates sap1, using a high-throughput transposon mutagenesis screen in combination with Tn-Seq. Consistent with phenotypes of the Δsap1 mutant, the ΔsapR activator mutant exhibited a significant reduction in Bp attachment to the host cell, leading to subsequent decreased intracellular replication. RNA-Seq analysis further revealed that SapR regulates sap1. The regulation of sap1 by SapR was confirmed quantitatively by qRT-PCR, which also validated the RNA-Seq data. SapR globally regulates genes associated with the bacterial membrane in response to diverse environments, and some of the genes regulated by SapR are virulence factors that are required for Bp intracellular infection (e.g., type III and type VI secretion systems). This study has identified the complex SapR regulatory network and its importance as an activator of an essential Sap1 attachment factor

Gene content of four fosmid clones conferring natural competency and/or DNA catabolism.

<p>(A) The fosmid backbone is indicated in black and the red region depicts the extent of <i>Bp</i> 1026b genomic DNA inserted in each fosmid. Gene products with known predicted functions are indicated by gene names and those with unknown functions are labeled with gene identifications. (B) Synteny map comparing fosmid regions of <i>Bp</i> K96243 and 1026b. Red regions indicate the high level of similarity between fosmid regions in strain K96243 (non-competent) versus 1026b (competent) aligned using Artemis webACT. Identical regions are indicated in red, non-identical regions in white, and blue indicates a region that is inverted in the two strains.</p

Confirmation of the ability of four fosmid clones to individually confer natural competency or growth on DNA.

<p>(A) <i>gfp-</i>DNA uptake assay to assess natural competency. Individual fluorescent bacteria are visible in the representative images (green fluorescence and DIC overlay). The percentages of cells that fluoresce following <i>gfp-</i>DNA uptake are shown in (B). Numbers are from 3 representative images from duplicate experiments. Error bars represent the SEM. Asterisks indicate the fosmid clone gave rise to significant higher %GFP positive comparing to the corresponding empty vector control in three fields (**, <i>P</i><0.005 based on unpaired <i>t</i>-test). (C) Growth of various fosmid-containing strains in 1x M9 with 0.25% DNA done in triplicate. Error bars represent the SEM. Asterisks indicate the fosmid clone gave rise to significant higher growth in DNA comparing to the corresponding empty vector control (*, <i>P</i><0.05 based on unpaired <i>t</i>-test; **, <i>P</i><0.005 based on unpaired <i>t</i>-test).</p

DNA uptake and utilization.

<p>(A) Model of DNA uptake in Gram-negative bacteria (see text for detail). (B) Utilization of DNA as a sole carbon and energy source in selected <i>Burkholderia</i> species. <i>Bp</i> 1026b and <i>B</i>. <i>thiailandensis</i> E264 strains exhibited heavy growth; <i>Bp</i> K96243 showed intermediate growth, while <i>Bc</i> K56-2 and <i>B</i>. <i>mallei</i> ATCC23344 were unable to grow on DNA.</p

<i>Burkholderia pseudomallei</i> natural competency and DNA catabolism: Identification and characterization of relevant genes from a constructed fosmid library

<div><p><i>Burkholderia</i> spp. are genetically and physiologically diverse. Some strains are naturally transformable and capable of DNA catabolism. <i>Burkholderia pseudomallei</i> (<i>Bp</i>) strains 1026b and K96243 and <i>B</i>. <i>thailandensis</i> strain E264 are able to utilize DNA as a sole carbon source for growth, while only strains 1026b and E264 are naturally transformable. In this study, we constructed low-copy broad-host-range fosmid library, containing <i>Bp</i> strain 1026b chromosomal DNA fragments, and employed a novel positive selection approach to identify genes responsible for DNA uptake and DNA catabolism. The library was transferred to non-competent <i>Bp</i> K96243 and <i>B</i>. <i>cenocepacia</i> (<i>Bc</i>) K56-2, harboring chromosomally-inserted <i>FRT</i>-flanked <i>sacB</i> and <i>pheS</i> counter-selection markers. The library was incubated with DNA encoding Flp recombinase, followed by counter-selection on sucrose and chlorinated phenylalanine, to select for clones that took up <i>flp</i>-DNA, transiently expressed Flp, and excised the <i>sacB-pheS</i> cassette. Putative clones that survived the counter-selection were subsequently incubated with <i>gfp</i>-DNA and bacteria were visualized via fluorescent microscopy to confirm natural competency. Fosmid sequencing identified several 1026b genes implicated in DNA uptake, which were validated using chromosomal mutants. One of the naturally competent clones selected in <i>Bc</i> K56-2 enabled <i>Bc</i>, <i>Bp</i> and <i>B</i>. <i>mallei</i> to utilize DNA as a sole carbon source, and all fosmids were used to successfully create mutations in non-naturally-competent <i>B</i>. <i>mallei</i> and <i>Bp</i> strains.</p></div

Characterization of selected genes from fosmids.

<p>Characterization of selected genes from fosmids.</p

Library production and counter-selection scheme.

<p>(A) Plasmid map of an in-lab created pBBR-FOSGAT and strategy for creating the fosmid library. <i>Bp</i> 1026b chromosomal DNA (1) was sheared (2), and approximately 50-Kbp size fragments were ligated to pBBR-FOSGAT fosmid vector DNA (3 and 4; fosmid vector indicated in red). The ligated DNA was then packaged into λ-phage particles (5) and the particles are used to infect <i>E</i>. <i>coli</i> host cells (6). (B) Counter-selection scheme to screen for fosmid clones enabling DNA uptake or DNA catabolism. (1) <i>pheS</i> and <i>sacB</i> counter-selectable markers were inserted into <i>Bc</i> K56-2 and <i>Bp</i> K96243 chromosomes through minTn7 integration. (2) The <i>E</i>. <i>coli</i> library containing fosmid pool from (A) was conjugated with <i>Bc</i> K56-2 and <i>Bp</i> K96243 containing <i>pheS</i> and <i>sacB</i> counter-selectable markers. (3) Colonies from these two <i>Burkholderia</i> strains were individually pooled and incubated with <i>flp</i>-DNA, allowing transiently expressed Flp to excise the chromosomally integrated counter-selection markers and counter-selected on media containing cPhe and sucrose. (4) Alternatively, the <i>Bc</i> K56-2 pool was selected on minimal medium containing purified salmon sperm DNA as a sole carbon source.</p

Allelic-replacement in <i>B</i>. <i>mallei</i> strain ATCC23344 and <i>B</i>. <i>pseudomallei</i> K96243 containing fosmids by natural transformation.

<p>(A) λ-Red plasmid pKaKa2 and individual fosmid clones were co-transformed into <i>B</i>. <i>mallei</i> ATCC23344 and <i>B</i>. <i>pseudomallei</i> K96243. (B) PCR products were generated containing the tellurite resistance cassette (<i>kilA-telA-telB</i>) flanked with 45-bp homologies to the <i>vacJ</i> gene. (C) Various numbers of distinct colonies were obtained. PCR was performed for the <i>kilA</i>-<i>vacJ</i> junction (D) or the <i>telA</i> gene (E) and all were positive. Two gels were shown here as representative results; “-” denotes negative control using wildtype <i>B</i>. <i>mallei</i> chromosomal DNA as template for <i>kilA</i>-<i>vacJ</i> junction PCR, and “+” denotes positive control using pwFRT-<i>tel</i> as template for <i>telA</i> PCR.</p

The Burkholderia pseudomallei intracellular 'TRANSITome'

Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a 'TRANSITomic' approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei 'TRANSITome' reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei 'TRANSITome' provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions