Burkholderia pseudomallei produces 2-alkylquinolone derivatives important for host virulence and competition with bacteria that employ naphthoquinones for aerobic respiration

Melioidosis is caused by <i>Burkholderia pseudomallei</i>, an opportunistic Gram-negative pathogen that inhabits soil and water in tropical and subtropical regions. <i>B. pseudomallei</i> infections often occur following contact with contaminated water or soil or by inhalation of contaminated dust and water droplets. There is limited knowledge about how <i>B. pseudomallei</i> is able to survive in harsh environmental conditions and compete with the microbes that inhabit these niches. Previous research demonstrated that 3-methyl-2-alkylquinolones (MAQs), and their corresponding <i>N</i>-oxides (MAQNOs), are produced by <i>B. pseudomallei</i> and provide a competitive advantage when grown in the presence of Gram-positive bacteria. In this study, 39 Gram-negative environmental bacteria in the <i>Pseudomonadota</i> and <i>Bacteroidota</i> phyla were isolated and characterized. Intriguingly, <i>B. pseudomallei</i> inhibited 71% of bacteria in the phylum <i>Bacteroidota</i> in zone of inhibition and coculture competition assays, but no <i>Pseudomonadota</i> isolates were similarly inhibited. Transposon mutagenesis was utilized to identify <i>B. pseudomallei</i> genes required for the inhibition of <i>Sphingobacterium</i> sp. ST4, a representative member of the <i>Bacteroidota</i>. Three mutations mapped to <i>hmqA-G</i>, the locus encoding 2-alkylquinolone derivatives, and two mutations were identified in <i>scmR</i>, a gene encoding a quorum-sensing controlled LysR-type transcriptional regulator. <i>B. pseudomallei</i> strains with deletion mutations in <i>hmqD</i> and <i>scmR</i> were unable to produce 2-alkylquinolone derivatives or inhibit <i>Bacteroidota</i> isolates in competition assays. RAW264.7 murine macrophage cells were infected with <i>B. pseudomallei</i> 1026b and 1026b <i>ΔhmqD</i> and there was a 94-fold reduction in the number of intracellular 1026b <i>ΔhmqD</i> bacteria relative to 1026b. The 50% lethal dose (LD₅₀) of 1026b and 1026b <i>ΔhmqD</i> in BALB/c mice was determined to be 3 x 10⁵ colony forming units (CFU) and > 1 x 10⁶ CFU, respectively. Taken together, the results indicate that the products of the <i>B. pseudomallei hmqA-G</i> locus are important for intracellular replication in murine macrophages, virulence in a mouse model of melioidosis, and competition with bacteria that utilize naphthoquinones for aerobic respiration.