Potent modulation of the CepR quorum sensing receptor and virulence in a Burkholderia cepacia complex member using non-native lactone ligands.

The Burkholderia cepacia complex (Bcc) is a family of closely related bacterial pathogens that are the causative agent of deadly human infections. Virulence in Bcc species has been shown to be controlled by the CepI/CepR quorum sensing (QS) system, which is mediated by an N-acyl L-homoserine lactone (AHL) signal (C8-AHL) and its cognate LuxR-type receptor (CepR). Chemical strategies to block QS in Bcc members would represent an approach to intercept this bacterial communication process and further delineate its role in infection. In the current study, we sought to identify non-native AHLs capable of agonizing or antagonizing CepR, and thereby QS, in a Bcc member. We screened a library of AHL analogs in cell-based reporters for CepR, and identified numerous highly potent CepR agonists and antagonists. These compounds remain active in a Bcc member, B. multivorans, with one agonist 250-fold more potent than the native ligand C8-AHL, and can affect QS-controlled motility. Further, the CepR antagonists prolong C. elegans survival in an infection model. These AHL analogs are the first reported non-native molecules that both directly modulate CepR and impact QS-controlled phenotypes in a Bcc member, and represent valuable chemical tools to assess the role of QS in Bcc infections.

Malleilactone Is a Burkholderia pseudomallei Virulence Factor Regulated by Antibiotics and Quorum Sensing.

Burkholderia pseudomallei, the causative agent of melioidosis, encodes almost a dozen predicted polyketide (PK) biosynthetic gene clusters. Many of these are regulated by LuxR-I-type acyl-homoserine (AHL) quorum-sensing systems. One of the PK gene clusters, the mal gene cluster, is conserved in the close relative Burkholderia thailandensis The B. thailandensis mal genes code for the cytotoxin malleilactone and are regulated by a genetically linked LuxR-type transcription factor, MalR. Although AHLs typically interact with LuxR-type proteins to modulate gene transcription, the B. thailandensis MalR does not appear to be an AHL receptor. Here, we characterize the mal genes and MalR in B. pseudomallei We use chemical analyses to demonstrate that the B. pseudomallei mal genes code for malleilactone. Our results show that MalR and the mal genes contribute to the ability of B. pseudomallei to kill Caenorhabditis elegans In B. thailandensis, antibiotics like trimethoprim can activate MalR by driving transcription of the mal genes, and we demonstrate that some of the same antibiotics induce expression of B. pseudomallei malR We also demonstrate that B. pseudomallei MalR does not respond directly to AHLs. Our results suggest that MalR is indirectly repressed by AHLs, possibly through a repressor, ScmR. We further show that malleilactone is a B. pseudomallei virulence factor and provide the foundation for understanding how malleilactone contributes to the pathology of melioidosis infections.IMPORTANCE Many bacterially produced polyketides are cytotoxic to mammalian cells and are potentially important contributors to pathogenesis during infection. We are interested in the polyketide gene clusters present in Burkholderia pseudomallei, which causes the often-fatal human disease melioidosis. Using knowledge gained by studies in the close relative Burkholderia thailandensis, we show that one of the B. pseudomallei polyketide biosynthetic clusters produces a cytotoxic polyketide, malleilactone. Malleilactone contributes to B. pseudomallei virulence in a Caenorhabditis elegans infection model and is regulated by an orphan LuxR family quorum-sensing transcription factor, MalR. Our studies demonstrate that malleilactone biosynthesis or MalR could be new targets for developing therapeutics to treat melioidosis.

Inhibiting the BfrB:Bfd interaction in Pseudomonas aeruginosa causes irreversible iron accumulation in bacterioferritin and iron deficiency in the bacterial cytosol.

Iron is an essential nutrient for bacteria but the reactivity of Fe2+ and the insolubility of Fe3+ present significant challenges to bacterial cells. Iron storage proteins contribute to ameliorating these challenges by oxidizing Fe2+ using O2 and H2O2 as electron acceptors, and by compartmentalizing Fe3+. Two types of iron-storage proteins coexist in bacteria, the ferritins (Ftn) and the heme-containing bacterioferritins (Bfr), but the reasons for their coexistence are largely unknown. P. aeruginosa cells harbor two iron storage proteins (FtnA and BfrB), but nothing is known about their relative contributions to iron homeostasis. Prior studies in vitro have shown that iron mobilization from BfrB requires specific interactions with a ferredoxin (Bfd), but the relevance of the BfrB:Bfd interaction to iron homeostasis in P. aeruginosa is unknown. In this work we explore the repercussions of (i) deleting the bfrB gene, and (ii) perturbing the BfrB:Bfd interaction in P. aeruginosa cells by either deleting the bfd gene or by replacing the wild type bfrB gene with a L68A/E81A double mutant allele in the P. aeruginosa chromosome. The effects of the mutations were evaluated by following the accumulation of iron in BfrB, analyzing levels of free and total intracellular iron, and by characterizing the ensuing iron homeostasis dysregulation phenotypes. The results reveal that P. aeruginosa accumulates iron mainly in BfrB, and that the nutrient does not accumulate in FtnA to detectable levels, even after deletion of the bfrB gene. Perturbing the BfrB:Bfd interaction causes irreversible flow of iron into BfrB, which leads to the accumulation of unusable intracellular iron while severely depleting the levels of free intracellular iron, which drives the cells to an acute iron starvation response despite harboring "normal" levels of total intracellular iron. These results are discussed in the context of a dynamic equilibrium between free cytosolic Fe2+ and Fe3+ compartmentalized in BfrB, which functions as a buffer to oppose rapid changes of free cytosolic iron. Finally, we also show that P. aeruginosa cells utilize iron stored in BfrB for growth in iron-limiting conditions, and that the utilization of BfrB-iron requires a functional BfrB:Bfd interaction.