Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low

Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis.

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low

Antibiotic-resistance profiles of pathogenic and symbiotic <i>Burkholderia</i> species.

<p>Relative resistance to a panel of antibiotics for the four environmental and symbiotic strains emphasized in this study compared to the pathogenic <i>B. thailandensis</i> E264, the opportunistic pathogen <i>B. vietnamiensis</i> G4, and the plant pathogen <i>B. gladioli</i> BSR3. The average clearing of five replicate experiments at the highest antibiotic concentration are represented in the heat map. Full resistance (no clearing) is indicated with an asterisk.</p

Phylogenetic analysis of Type 4 secretion system clusters in <i>Burkholderia</i> species.

<p>Three clusters with a unique gene organization have the characteristic VirB and VirD4 proteins found in the <i>Agrobacterium tumefaciens</i> cluster. A fourth, truncated cluster (yellow) is also found in many <i>Burkholderia</i> strains, including <i>B. tuberum</i> STM678^T, but is unlikely to contribute to secretion.</p

Relationship between the <i>Burkholderia</i> Type 6 secretion systems and arrangement of gene clusters.

<p>Clusters were identified with BLASTP against the <i>B. pseudomallei</i> T6SS-2, and a concatenated neighbor-joining phylogenetic tree was generated in MEGA 5.1 using highly divergent protein sequences–VgrG, Hcp, ClpV, IcmF, and the lysozyme-like protein VC_A0109. The gene arrangements of the cluster were manually verified. Although <i>B. oklahomensis</i> EO147 was found to have five T6SS clusters (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083779#pone-0083779-g001" target="_blank">Fig. 1</a>), it was excluded from the analysis because the draft quality of the genome sequence did not allow for full reconstruction of the gene arrangement of the operon. The four symbiotic strains emphasized in this study are highlighted in bold. The <i>R. leguminosarum imp</i> region clusters with the T6SS-4 group and is indicated with an asterisk. The T6SSa and TG6SSb clusters are found only in the environmental and symbiotic <i>Burkholderia</i> species.</p

Distribution of virulence and symbiotic loci across <i>Burkholderia</i> species.

<p>Virulence-associated loci were identified using BLASTP against characteristic sequences (<i>B. pseudomallei</i> or <i>B. cenocepacia</i> ATPases for secretion systems, <i>B. pseudomallei</i> pilus genes for pilus-related clusters (red), and <i>B. tuberum nifH</i> and <i>nodA</i> sequences (green)). Non-canonical or truncated clusters are indicated in pink, and clusters highly associated with virulence for the Type 3 and Type 6 secretion systems are denoted with an asterisk.</p

Symbiotic <i>Burkholderia</i> species are not toxic to HeLa cells in culture.

<p>Symbiotic <i>Burkholderia</i> species (<i>B. tuberum</i> STM678^T, <i>B. silvatlantica</i> PVA5, <i>B. silvatlantica</i> SRMrh20^T, <i>B. unamae</i> MTI641^T) were compared to <i>B. thailandensis</i> E264 to determine if they were toxic to mammalian cells grown in culture. HeLa cells were grown until confluent and inoculated with an MOI of 50. After 8 and 24 h, samples of the supernatant of the inoculated and sham-inoculated cells were examined for LDH release using the Cyto-Tox 96 assay kit and spectrophotometric reading at 490 nm. At 8 h, only the positive control cells treated with detergent showed significant cell lysis. <i>B. thailandensis</i> E264 caused cell lysis about three times greater than that of the negative control (medium only). The effect of the symbiotic species was not statistically different from that of the negative control. At 24 h, <i>B. thailandensis</i> E264-induced morbidity had surpassed the detergent control, while the cytotoxicity caused by the symbiotic species increased only slightly from the earlier time point. Error bars indicate standard error.</p

Flagellar gene clusters in <i>Burkholderia</i>.

<p>All <i>Burkholderia</i> strains share a highly similar chemotaxis and flagellar gene cluster (<i>fla1</i>) on chromosome 1. Although the A group and the pathogenic B group share high homology in gene sequence and chromosomal arrangement, a phylogenetic analysis of five concatenated protein sequences (FliC, FlgM, FlgE, FlhB, FlgJ) shows a distinct clustering of the two lineages [A]. Additionally, the A group cluster is arranged entirely sequentially [B] whereas the pathogenic B cluster is split into four different regions throughout chromosome 1 (the <i>B. mallei</i> cluster is split into 5 regions)[C]. The <i>fla1</i> gene cluster is responsible for <i>Burkholderia</i> motility on soft agar, but not for intracellular motility or plaque formation in models of infection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083779#pone.0083779-French1" target="_blank">[32]</a>. A second flagellar gene cluster on chromosome 2 (<i>fla2</i>) is necessary for this intracellular motility. This second cluster is present only in the pathogenic strains, i.e. <i>B. pseudomallei, B. thailandensis</i>, and <i>B. oklahomensis</i> [D], and a similar cluster exists on chromosome 2 of the opportunistic pathogen <i>B. cenocepacia</i> [E].</p

Symbiotic <i>Burkholderia</i> species are not pathogenic <i>in vitro</i> to the nematode <i>C. elegans</i> using the slow-killing assay.

<p>The edge of bacterial lawns of test <i>Burkholderia</i> strains were seeded with age-synchronized <i>C. elegans</i> N2 juvenile worms on nematode growth agar (NGM) plates. An initial count of live worms was made, and again after 24, 48, and 72 h. The percent of survivors were enumerated based on a comparison of live worms present after 72 h versus the initial count. Four symbiotic species of <i>Burkholderia</i> (<i>B. tuberum</i> STM678^T, <i>B. silvatlantica</i> PVA5, <i>B. silvatlantica</i> SRMrh20^T, <i>B. unamae</i> MTI641^T) and one pathogenic species (<i>B. thailandensis</i> E264) were compared against the control, <i>E coli</i> OP50. Only the pathogenic species showed a significant and dramatic reduction in the number of live worms over the course of the experiments. All other test strains showed nearly 100% survival. Data from 48 h after the start of the experiment are shown. Error bars indicate standard error.</p

Symbiotic <i>Burkholderia</i> species are the preferred nutritional source of <i>C. elegans</i> compared to a pathogenic species.

<p><i>E. coli</i> and symbiotic <i>Burkholderia</i> species were individually compared to <i>B. thailandensis</i> E264 to examine the response of the nematode <i>C. elegans</i> to a nutritional preference. Live worms were age-synchronized and seeded into the center of a nematode growth agar (NGM) plate equidistant from a lawn of <i>B. thailandensis</i> E264 and the aforementioned non-pathogenic strains. Counts of live worms were taken initially after seeding, and at 24, 48, and 72 h intervals. In each competition assay, the <i>B. thailandensis</i> lawn was either avoided or contained mostly dead worms, whereas live worms thrived within the lawns of <i>E. coli</i> OP50 or symbiotic <i>Burkholderia</i> species. Error bars indicate standard error.</p