Project description:In prokaryotes and eukaryotes, cell-cell communication and recognition of self are critical to coordinate multicellular functions. While kin and kind discrimination are increasingly appreciated to shape naturally occurring microbe populations, the underlying mechanisms that govern these interbacterial interactions are insufficiently understood. Here we identify a mechanism of interbacterial signal transduction that is mediated by contact-dependent growth inhibition (CDI) system proteins. CDI systems have been characterized by their ability to deliver a polymorphic protein toxin into the cytoplasm of a neighboring bacterium, resulting in growth inhibition or death unless the recipient bacterium produces a corresponding immunity protein. Using the model organism Burkholderia thailandensis, we show that delivery of a catalytically active CDI system toxin to immune (self) bacteria results in gene expression and phenotypic changes within the recipient cells. Termed contact-dependent signaling (CDS), this response promotes biofilm formation and other community-associated behaviors. Examination of wild-type Burkholderia thailandensis and two mutant strains, each in triplicate (9 samples total). mutant BtEKA contains two amino acid substitutions (E3064A and K3066A) within the coding sequence of gene Bth_I2723. In mutant PS12-WT, the native promoter of gene Bth_I2723 has been replaced with the strong constitutive promoter of the E264 rpsL gene, PS12.
Project description:In prokaryotes and eukaryotes, cell-cell communication and recognition of self are critical to coordinate multicellular functions. While kin and kind discrimination are increasingly appreciated to shape naturally occurring microbe populations, the underlying mechanisms that govern these interbacterial interactions are insufficiently understood. Here we identify a mechanism of interbacterial signal transduction that is mediated by contact-dependent growth inhibition (CDI) system proteins. CDI systems have been characterized by their ability to deliver a polymorphic protein toxin into the cytoplasm of a neighboring bacterium, resulting in growth inhibition or death unless the recipient bacterium produces a corresponding immunity protein. Using the model organism Burkholderia thailandensis, we show that delivery of a catalytically active CDI system toxin to immune (self) bacteria results in gene expression and phenotypic changes within the recipient cells. Termed contact-dependent signaling (CDS), this response promotes biofilm formation and other community-associated behaviors.
Project description:Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with downstream target mRNAs to attenuate translation of mRNA into protein at the post-transcriptional level. In response to specific environmental changes, sRNAs can modulate the expression levels of target genes, thus enabling adaptation of cellular physiology. We profiled sRNA expression in the Gram-negative bacteria Burkholderia thailandensis cultured under 54 distinct growth conditions using a Burkholderia-specific microarray that contains probe sets to all intergenic regions (IGRs) greater than 90 bases. We identified 38 novel sRNAs and performed experimental validation on five sRNAs that play a role in adaptation of Burkholderia to cell stressors.
Project description:Innate immunity responds to pathogens by producing alarm signals and activating pathways that make host cells inhospitable for pathogen replication. The intracellular bacterium Burkholderia thailandensis invades the cytosol, hijacks host actin, and induces cell fusion to spread to adjacent cells, forming multinucleated giant cells (MNGCs) which promotes bacterial replication. We show that type I interferon (IFN) restricts macrophage MNGC formation during B. thailandensis infection. Guanylate-binding proteins (GBPs) expressed downstream of type I IFN were required to restrict MNGC formation through inhibition of Arp2/3-dependent actin motility during infection. GTPase activity and the CAAX prenylation domain were required for GBP2 recruitment to B. thailandensis, which restricted bacterial actin polymerization required for MNGC formation. Consistent with in vitro macrophages, Gbp2-/- Gbp5-/-, GbpChr3-KO mice were more susceptible to intranasal infection with B. thailandensis than wildtype mice. Our findings reveal that IFN and GBPs play a critical role in restricting cell-cell fusion during infection
Project description:Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, and are often fatal to humans and animals. Owing to the high fatality rate, potential for spread by aerosolization, and the lack of efficacious therapeutics, B. pseudomallei and B. mallei are considered biothreat agents of concern. In this study, we investigate the proteome of Burkholderia thailandensis, a closely related surrogate for the two more virulent Burkholderia species, during infection of host cells, and compare to that of B. thailandensis in culture. Studying the proteome of intracellular Burkholderia spp. is expected to reveal molecular mechanisms of intracellular survival and host immune evasion; but proteomic profiling of intracellular Burkholderia is challenging. Proteomic analyses of intracellular bacteria are typically hindered by the overwhelming host protein content recovered from infected cultures. To address this problem, we have applied bio-orthogonal noncanonical amino acid tagging (BONCAT) to B. thailandensis, enabling the enrichment of newly expressed bacterial proteins from virtually any growth condition, most importantly intracellular contexts. In this study, we show that B. thailandensis proteins were selectively labeled and efficiently enriched from infected host cells using BONCAT. We also demonstrate that this method can be used to label bacteria in situ by fluorescent tagging. Finally, we present a global proteomic profile of B. thailandensis as it infects host cells and a list of proteins that are differentially regulated in infection conditions as compared to bacterial monoculture. Among the identified proteins are quorum sensing regulated genes as well as homologs to previously identified virulence factors. This method provides a powerful tool to study the molecular processes during Burkholderia infection, a much-needed addition to the Burkholderia molecular toolbox.
Project description:Burkholderia thailandensis is a soil-dwelling bacterium that shares many metabolic pathways with the ecologically similar, but evolutionarily distant, Pseudomonas aeruginosa. Among the diverse nutrients it can utilize is choline, which can be converted into the osmoprotectant glycine betaine and further catabolized as a source of carbon and nitrogen, similar to P. aeruginosa. Orthologs of genes in the choline catabolic pathway in these two bacteria showed distinct differences in gene arrangement as well as an additional orthologous transcriptional regulator in B. thailandensis. In this study, we showed that multiple glutamine amidotransferase1 (GATase1)-containing AraC-family transcription regulators (GATRs) are involved in regulation of the B. thailandensis choline catabolic pathway (gbdR1, gbdR2, souR). Using genetic analyses and sequencing the transcriptome in the presence and absence of choline, we identified the likely regulons of gbdR1 (BTH_II1869) and gbdR2 (BTH_II0968). We also identified a functional ortholog for P. aeruginosa souR, a GATR that regulates the metabolism of sarcosine to glycine. GbdR1 is absolutely required for expression of the choline catabolic locus, similar to P. aeruginosa GbdR, while GbdR2 is important to increase expression of the catabolic locus. Additionally, the B. thailandensis SouR ortholog (BTH_II0994) is required for catabolism of choline and its metabolites as carbon sources, whereas in P. aeruginosa, SouR function can by bypassed by GbdR. The strategy employed by B. thailandensis represents a distinct regulatory solution to control choline catabolism and thus provides both an evolutionary counterpoint and an experimental system to compare the acquisition and regulation of this pathway during environmental growth and infection.
Project description:Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with downstream target mRNAs to attenuate translation of mRNA into protein at the post-transcriptional level. In response to specific environmental changes, sRNAs can modulate the expression levels of target genes, thus enabling adaptation of cellular physiology. We profiled sRNA expression in the Gram-negative bacteria Burkholderia thailandensis cultured under 54 distinct growth conditions using a Burkholderia-specific microarray that contains probe sets to all intergenic regions (IGRs) greater than 90 bases. We identified 38 novel sRNAs and performed experimental validation on five sRNAs that play a role in adaptation of Burkholderia to cell stressors. A custom Affymetrix microarray was designed using Burkholderia thailandensis E264 gene annotations from Pathema and included 8711 probe sets targeting 5557 protein-coding genes and 2908 IGRs longer than 90 bases on the plus strand. Experiments were performed in four classes of media: nutrient broth, M9-minimal media, Luria broth, and various host conditions such as brain-heart infusion, calf serum, or tryptic soy broth. There were 101 samples in four time course arrays measuring temperature increase, pH drop, salt stress, and phosphate starvation (21 distinct conditions). Each condition was grown at 37° C, except the initial temperature experiment at 25°C, and replicated five times. In order to survey a diverse range of environmental and stress conditions, another 10 replicated and 23 unreplicated treatments were also tested.
Project description:Examination of the 12-bis-THA stress response in Burkholderia thailandensis strain E555 to investigate its antimicrobial mechanism of action
Project description:Introduction. Burkholderia thailandensis is a clinically rare opportunistic pathogen in the genus Burkholderia, and the genomic features and virulence characteristics of B. thailandensis strains that cause human infection remain unclear.Gap Statement. B. thailandensis strains with different virulence induce different host innate immune responses in vitro.Aim. This work aimed to understand the sequence diversity, phylogenetic relationship, and virulence of B. thailandensis BPM causing human infection.Methodology. The comparative molecular and genomic analyses, and mouse infection studies were applied to analyse the virulence and genomic features of B. thailandensis BPM originating from China.Results. The whole genome sequence analysis showed that the genomes of BPM and other avirulent B. thailandensis strains were broadly similar, comprising two highly syntenic chromosomes with comparable numbers of coding regions (CDs), protein family distributions, and horizontally acquired genomic islands. By examining species-specific genomic regions, we obtained molecular explanations for previously known differences in virulence and discovered the potential specific virulence-associated genes of BPM, which likely work together to confer the virulence of BPM. Significantly reduced LD50 and survival rates during mouse infection experiments were found in BPM compared to the avirulent B. thailandensis E264 (BtE264).Conclusion. Taken together, the results of this study provide basic information on the genomic features and virulence characteristics of the virulent B. thailandensis strain BPM, which is helpful for understanding its evolution as it relates to pathogenesis and environmental adaptability.