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Identification of a PadR-type regulator essential for intracellular pathogenesis of Burkholderia pseudomallei.

ABSTRACT: Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease endemic to the tropics. Melioidosis manifests in various ways ranging from acute skin lesions to pneumonia and, in rare cases, infection of the central nervous system. Bp is a facultative intracellular pathogen and it can infect various cell types. The Bp intracellular lifecycle has been partially elucidated and is highly complex. Herein, we have identified a transcriptional regulator, BP1026B_II1198, that is differentially expressed as Bp transits through host cells. A deletion mutant of BP1026B_II1198 was attenuated in RAW264.7 cell and BALB/c mouse infection. To further characterize the function of this transcriptional regulator, we endeavored to determine the regulon of BP1026B_II1198. RNA-seq analysis showed the global picture of genes regulated while ChIP-seq analysis identified two specific BP1026B_II1198 binding regions on chromosome II. We investigated the transposon mutants of these genes controlled by BP1026B_II1198 and confirmed that these genes contribute to pathogenesis in RAW264.7 murine macrophage cells. Taken together, the data presented here shed light on the regulon of BP1026B_II1198 and its role during intracellular infection and highlights an integral portion of the highly complex regulation network of Bp during host infection.

SUBMITTER: McMillan IA

PROVIDER: S-EPMC8128862 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Burkholderia pseudomallei (Bp) causes the tropical disease melioidosis that afflicts an estimated 165,000 people each year. Bp is a facultative intracellular pathogen that transits through distinct intracellular stages including attachment to host cells, invasion through the endocytic pathway, escape from the endosome, replication in the cytoplasm, generation of protrusions towards neighboring cells, and host cell fusion allowing Bp infection to spread without exiting the intracellular environment. We have identified a TetR-like transcriptional regulator, BP1026B_II1561, that is up-regulated during the late stages of infection as Bp protrudes toward neighboring cells. We have characterized BP1026B_II1561 and determined that it has a role in pathogenesis. A deletional mutant of BP1026B_II1561 is attenuated in RAW264.7 macrophage and BALB/c mouse models of infection. Using RNA-seq, we found that BP1026B_II1561 controls secondary metabolite biosynthesis, fatty acid degradation, and propanoate metabolism. In addition, we identified that BP1026B_II1561 directly controls expression of an outer membrane porin and genes in the shikimate biosynthetic pathway using ChIP-seq. Transposon mutants of genes within the BP1026B_II1561 regulon show defects during intracellular replication in RAW264.7 cells confirming the role of this transcriptional regulator and the pathways it controls in pathogenesis. BP1026B_II1561 also up-regulates the majority of the enzymes in shikimate and tryptophan biosynthetic pathways, suggesting their importance for Bp physiology. To investigate this, we tested fluorinated analogs of anthranilate and tryptophan, intermediates and products of the shikimate and tryptophan biosynthetic pathways, respectively, and showed inhibition of Bp growth at nanomolar concentrations. The expression of these pathways by BP1026b_II1561 and during intracellular infection combined with the inhibition of Bp growth by fluorotryptophan/anthranilate highlights these pathways as potential targets for therapeutic intervention against melioidosis. In the present study, we have identified BP1026B_II1561 as a critical transcriptional regulator for Bp pathogenesis and partially characterized its role during host cell infection.

Project description:Burkholderia pseudomallei is a soil-dwelling bacterium which has to survive not only under harsh environmental conditions, but also within various hosts where it can cause the infectious disease melioidosis. The ability, to quickly adapt to these different conditions, is based on its huge genome which encodes for complex regulatory networks. Among them are more than 60 genes belonging to the group of LysR-type transcriptional regulators (LTTRs). Here we analyzed a B. pseudomallei mutant harboring a transposon in the gene BPSL0117 annotated as a LTTR, which we named gvmR (globally acting virulence and metabolism regulator). The gvmR mutant displayed a growth defect in minimal medium and macrophages in comparison with the wild type. Moreover, inactivation of GvmR rendered B. pseudomallei avirulent in mice indicating a critical role of GvmR in infection. These defects of the mutant were rescued by ectopic expression of gvmR. To identify genes whose expression is modulated by GvmR, global transcriptome analysis of the B. pseudomallei wild type and gvmR mutant was performed using whole genome tiling microarrays. Transcript levels of 190 and 142 genes were found to be up- and downregulated in the gvmR mutant relative to the wild type. Among the most downregulated genes in the gvmR mutant were important virulence factor genes (T3SS3, T6SS1 and T6SS2), which might provide an explanation for the virulence defect of the gvmR mutant. In addition, expression of genes related to amino acid synthesis, glyoxylate shunt, iron-sulfur cluster assambly and syrbactin metabolism (secondary metabolite) was decreased in the mutant. Furthermore, inactivation of GvmR increased expression of genes involved in pyruvate metabolism, ATP synthesis, malleobactin and porin genes. Quantitative RT-PCR verified the differential expression of 27 selected genes. In summary, our data show that GvmR acts as an activating and repressing global regulator that is required to coordinate expression of a diverse set of metabolic and virulence genes for survival in the animal host and under nutrient limitation.

Project description:Burkholderia pseudomallei is a soil-dwelling bacterium able to survive not only under adverse environmental conditions, but also within various hosts which can lead to the disease melioidosis. The capability of B. pseudomallei to adapt to environmental changes is facilitated by the large number of regulatory proteins encoded by its genome. Among them are more than 60 uncharacterized LysR-type transcriptional regulators (LTTRs). Here we analyzed a B. pseudomallei mutant harboring a transposon in the gene BPSL0117 annotated as a LTTR, which we named gvmR (globally acting virulence and metabolism regulator). The gvmR mutant displayed a growth defect in minimal medium and macrophages in comparison with the wild type. Moreover, disruption of gvmR rendered B. pseudomallei avirulent in mice indicating a critical role of GvmR in infection. These defects of the mutant were rescued by ectopic expression of gvmR. To identify genes whose expression is modulated by GvmR, global transcriptome analysis of the B. pseudomallei wild type and gvmR mutant was performed using whole genome tiling microarrays. Transcript levels of 190 genes were upregulated and 141 genes were downregulated in the gvmR mutant relative to the wild type. Among the most downregulated genes in the gvmR mutant were important virulence factor genes (T3SS3, T6SS1, and T6SS2), which could explain the virulence defect of the gvmR mutant. In addition, expression of genes related to amino acid synthesis, glyoxylate shunt, iron-sulfur cluster assembly, and syrbactin metabolism (secondary metabolite) was decreased in the mutant. On the other hand, inactivation of GvmR increased expression of genes involved in pyruvate metabolism, ATP synthesis, malleobactin, and porin genes. Quantitative real-time PCR verified the differential expression of 27 selected genes. In summary, our data show that GvmR acts as an activating and repressing global regulator that is required to coordinate expression of a diverse set of metabolic and virulence genes essential for the survival in the animal host and under nutrient limitation.

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Identification of a PadR-type regulator essential for intracellular pathogenesis of Burkholderia pseudomallei.

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