RIL-seq reveals extensive involvement of small RNAs in virulence and capsule regulation in hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create 'superbugs' that are challenging to treat. Understanding virulence regulation of hvKp is thus critical. Accumulating evidence suggest that posttranscriptional regulation by small RNAs (sRNAs) plays a role in bacterial virulence, but it has hardly been studied in K. pneumoniae. We applied RIL-seq to a prototypical clinical isolate of hvKp to unravel the Hfq-dependent RNA-RNA interaction (RRI) network. The RRI network is dominated by sRNAs, including predicted novel sRNAs, three of which we validated experimentally. We constructed a stringent subnetwork composed of RRIs that involve at least one hvKp virulence-associated gene and identified the capsule gene loci as a hub target where multiple sRNAs interact. We found that the sRNA OmrB suppressed both capsule production and hypermucoviscosity when overexpressed. Furthermore, OmrB base-pairs within kvrA coding region and partially suppresses translation of the capsule regulator KvrA. This agrees with current understanding of capsule as a major virulence and fitness factor. It emphasizes the intricate regulatory control of bacterial phenotypes by sRNAs, particularly of genes critical to bacterial physiology and virulence.

Carbapenem-resistant hypervirulent ST23 Klebsiella pneumoniae with a highly transmissible dual-carbapenemase plasmid in Chile.

BACKGROUND: The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid. RESULTS: Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM-1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st -5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems. CONCLUSIONS: We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.

Glutathione catabolism by Enterobacteriaceae species to hydrogen sulphide adversely affects the viability of host systems in the presence of 5'fluorodeoxyuridine.

Reduced glutathione (GSH) plays an essential role in relieving oxidative insult from the generation of free radicals via normal physiological processes. However, GSH can be exploited by bacteria as a signalling molecule for the regulation of virulence. We describe findings arising from a serendipitous observation that when GSH and Escherichia coli were incubated with 5'fluorodeoxyuridine (FUdR)-synchronised populations of Caenorhabditis elegans, the nematodes underwent rapid death. Death was mediated by the production of hydrogen sulphide mainly through the action of tnaA, a tryptophanase-encoding gene in E. coli. Other Enterobacteriaceae species possess similar cysteine desulfhydrases that can catabolise l-cysteine-containing compounds to hydrogen sulphide and mediate nematode killing when worms had been pre-treated with FUdR. When colonic epithelial cell lines were infected, hydrogen sulphide produced by these bacteria in the presence of GSH was also able to inhibit ATP synthesis in these cells particularly when cells had been treated with FUdR. Therefore, bacterial production of hydrogen sulphide could act in concert with a commonly used genotoxic cancer drug to exert host cell impairment. Hydrogen sulphide also increases bacterial adhesion to the intestinal cells. These findings could have implications for patients undergoing chemotherapy using FUdR analogues that could result in intestinal damage.

Membrane Potential-Dependent Uptake of Cationic Oligoimidazolium Mediates Bacterial DNA Damage and Death.

The treatment of bacterial infections is becoming increasingly challenging with the emergence of antimicrobial resistance. Thus, the development of antimicrobials with novel mechanisms of action is much needed. Previously, we designed several cationic main-chain imidazolium compounds and identified the polyimidazolium PIM1 as a potent antibacterial against a wide panel of multidrug-resistant nosocomial pathogens, and it had relatively low toxicity against mammalian epithelial cells. However, little is known about the mechanism of action of PIM1. Using an oligomeric version of PIM1 with precisely six repeating units (OIM1-6) to control for consistency, we showed that OIM1-6 relies on an intact membrane potential for entry into the bacterial cytoplasm, as resistant mutants to OIM1-6 have mutations in their electron transport chains. These mutants demonstrate reduced uptake of the compound, which can be circumvented through the addition of a sub-MIC dose of colistin. Once taken up intracellularly, OIM1-6 exerts double-stranded DNA breaks. Its potency and ability to kill represents a promising class of drugs that can be combined with membrane-penetrating drugs to potentiate activity and hedge against the rise of resistant mutants. In summary, we discovered that cationic antimicrobial OIM1-6 exhibits an antimicrobial property that is dissimilar to the conventional cationic antimicrobial compounds. Its killing mechanism does not involve membrane disruption but instead depends on the membrane potential for uptake into bacterial cells so that it can exert its antibacterial effect intracellularly.

A hydrophilic polyimidazolium antibiotic targeting the membranes of Gram-negative bacteria.

OBJECTIVES: The rise of MDR Gram-negative bacteria (GNB), especially those resistant to last-resort drugs such as carbapenems and colistin, is a global health risk and calls for increased efforts to discover new antimicrobial compounds. We previously reported that polyimidazolium (PIM) compounds exhibited significant antimicrobial activity and minimal mammalian cytotoxicity. However, their mechanism of action is relatively unknown. We examined the efficacy and mechanism of action of a hydrophilic PIM (PIM5) against colistin- and meropenem-resistant clinical isolates. METHODS: MIC and time-kill testing was performed for drug-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates. N-phenyl-1-naphthylamine and propidium iodide dyes were employed to determine membrane permeabilization. Spontaneous resistant mutants and single deletion mutants were generated to understand potential resistance mechanisms to the drug. RESULTS: PIM5 had the same effectiveness against colistin- and meropenem-resistant strains as susceptible strains of GNB. PIM5 exhibited a rapid bactericidal effect independent of bacterial growth phase and was especially effective in water. The polymer disrupts both the outer and cytoplasmic membranes. PIM5 binds and intercalates into bacterial genomic DNA upon entry of cells. GNB do not develop high resistance to PIM5. However, the susceptibility and uptake of the polymer is moderately affected by mutations in the two-component histidine kinase sensor BaeS. PIM5 has negligible cytotoxicity on human cells at bacterial-killing concentrations, comparable to the commercial antibiotics polymyxin B and colistin. CONCLUSIONS: PIM5 is a potent broad-spectrum antibiotic targeting GNB resistant to last-resort antibiotics.

Bacterial-induced cell fusion is a danger signal triggering cGAS-STING pathway via micronuclei formation.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease in the tropics and subtropics with high morbidity and mortality. The facultative intracellular bacterium induces host cell fusion through its type VI secretion system 5 (T6SS5) as an important part of its pathogenesis in mammalian hosts. This allows it to spread intercellularly without encountering extracellular host defenses. We report that bacterial T6SS5-dependent cell fusion triggers type I IFN gene expression in the host and leads to activation of the cGAMP synthase-stimulator of IFN genes (cGAS-STING) pathway, independent of bacterial ligands. Aberrant and abortive mitotic events result in the formation of micronuclei colocalizing with cGAS, which is activated by double-stranded DNA. Surprisingly, cGAS-STING activation leads to type I IFN transcription but not its production. Instead, the activation of cGAS and STING results in autophagic cell death. We also observed type I IFN gene expression, micronuclei formation, and death of chemically induced cell fusions. Therefore, we propose that the cGAS-STING pathway senses unnatural cell fusion through micronuclei formation as a danger signal, and consequently limits aberrant cell division and potential cellular transformation through autophagic death induction.

Designer broad-spectrum polyimidazolium antibiotics.

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

Dominant Carbapenemase-Encoding Plasmids in Clinical Enterobacterales Isolates and Hypervirulent Klebsiella pneumoniae, Singapore.

Dissemination of carbapenemase-encoding plasmids by horizontal gene transfer in multidrug-resistant bacteria is the major driver of rising carbapenem-resistance, but the conjugative mechanics and evolution of clinically relevant plasmids are not yet clear. We performed whole-genome sequencing on 1,215 clinical Enterobacterales isolates collected in Singapore during 2010-2015. We identified 1,126 carbapenemase-encoding plasmids and discovered pKPC2 is becoming the dominant plasmid in Singapore, overtaking an earlier dominant plasmid, pNDM1. pKPC2 frequently conjugates with many Enterobacterales species, including hypervirulent Klebsiella pneumoniae, and maintains stability in vitro without selection pressure and minimal adaptive sequence changes. Furthermore, capsule and decreasing taxonomic relatedness between donor and recipient pairs are greater conjugation barriers for pNDM1 than pKPC2. The low fitness costs pKPC2 exerts in Enterobacterales species indicate previously undetected carriage selection in other ecological settings. The ease of conjugation and stability of pKPC2 in hypervirulent K. pneumoniae could fuel spread into the community.

Cell envelope defects of different capsule-null mutants in K1 hypervirulent Klebsiella pneumoniae can affect bacterial pathogenesis.

Hypervirulent Klebsiella pneumoniae (hvKP) causes Klebsiella-induced liver abscess. Capsule is important for the pathogenesis of Klebsiella in systemic infection, but its role in gut colonisation is not well understood. By generating ΔwcaJ, Δwza and Δwzy capsule-null mutants in a prototypical K1 hypervirulent isolate, we show that inactivation of wza (capsule exportase) and wzy (capsule polymerase) confer cell envelope defects in addition to capsule loss, making them susceptible to bile salts and detergent stress. Bile salt resistance is restored when the initial glycosyltransferase wcaJ was inactivated together with wzy, indicating that build-up of capsule intermediates contribute to cell envelope defects. Mouse gut colonisation competition assays show that the capsule and its regulator RmpA were not required for hvKP to persist in the gut, although initial colonisation was decreased in the mutants. Both ΔrmpA and ΔwcaJ mutants gradually outcompeted the wild type in the gut, whereas Δwza and Δwzy mutants were less fit than wild type. Together, our results advise caution in using the right capsule-null mutant for determination of capsule's role in bacterial pathogenesis. With the use of ΔwcaJ mutant, we found that although the capsule is important for bacterial survival outside the gut environment, it imposes a fitness cost in the gut.

Acquisition of Plasmid with Carbapenem-Resistance Gene blaKPC2 in Hypervirulent Klebsiella pneumoniae, Singapore.

The convergence of carbapenem-resistance and hypervirulence genes in Klebsiella pneumoniae has led to the emergence of highly drug-resistant superbugs capable of causing invasive disease. We analyzed 556 carbapenem-resistant K. pneumoniae isolates from patients in Singapore hospitals during 2010-2015 and discovered 18 isolates from 7 patients also harbored hypervirulence features. All isolates contained a closely related plasmid (pKPC2) harboring blaKPC-2, a K. pneumoniae carbapenemase gene, and had a hypervirulent background of capsular serotypes K1, K2, and K20. In total, 5 of 7 first patient isolates were hypermucoviscous, and 6 were virulent in mice. The pKPC2 was highly transmissible and remarkably stable, maintained in bacteria within a patient with few changes for months in the absence of antimicrobial drug selection pressure. Intrapatient isolates were also able to acquire additional antimicrobial drug resistance genes when inside human bodies. Our results highlight the potential spread of carbapenem-resistant hypervirulent K. pneumoniae in Singapore.

TIP60 represses activation of endogenous retroviral elements.

TIP60 is a lysine acetyltransferase and is known to be a haplo-insufficient tumor suppressor. TIP60 downregulation is an early event in tumorigenesis which has been observed in several cancer types including breast and colorectal cancers. However, the mechanism by which it regulates tumor progression is not well understood. In this study, we identified the role of TIP60 in the silencing of endogenous retroviral elements (ERVs). TIP60-mediated silencing of ERVs is dependent on BRD4. TIP60 and BRD4 positively regulate the expression of enzymes, SUV39H1 and SETDB1 and thereby, the global H3K9 trimethylation (H3K9me3) level. In colorectal cancer, we found that the loss of TIP60 de-represses retrotransposon elements genome-wide, which in turn activate the cellular response to pathogens, mediated by STING, culminating in an induction of Interferon Regulatory Factor 7 (IRF7) and associated inflammatory response. In summary, this study has identified a unique mechanism of ERV regulation in cancer cells mediated by TIP60 and BRD4 through regulation of histone H3 K9 trimethylation, and a new tumor suppressive role of TIP60 in vivo.

A Glycosylated Cationic Block Poly(ß-peptide) Reverses Intrinsic Antibiotic Resistance in All ESKAPE Gram-Negative Bacteria.

Carbapenem-resistant Gram-negative bacteria (GNB) are heading the list of pathogens for which antibiotics are the most critically needed. Many antibiotics are either unable to penetrate the outer-membrane or are excluded by efflux mechanisms. Here, we report a cationic block ß-peptide (PAS8-b-PDM12) that reverses intrinsic antibiotic resistance in GNB by two distinct mechanisms of action. PAS8-b-PDM12 does not only compromise the integrity of the bacterial outer-membrane, it also deactivates efflux pump systems by dissipating the transmembrane electrochemical potential. As a result, PAS8-b-PDM12 sensitizes carbapenem- and colistin-resistant GNB to multiple antibiotics in vitro and in vivo. The ß-peptide allows the perfect alternation of cationic versus hydrophobic side chains, representing a significant improvement over previous antimicrobial α-peptides sensitizing agents. Together, our results indicate that it is technically possible for a single adjuvant to reverse innate antibiotic resistance in all pathogenic GNB of the ESKAPE group, including those resistant to last resort antibiotics.

Protective Role of Kupffer Cells and Macrophages in Klebsiella pneumoniae-Induced Liver Abscess Disease.

Klebsiella pneumoniae-induced liver abscess (KLA) is emerging as a leading cause of pyogenic liver abscess worldwide. In recent years, the emergence of hypervirulent K. pneumoniae (hvKp) has been strongly associated with KLA. Unlike classical K. pneumoniae, which generally infects the immunocompromised population, hvKp can cause serious and invasive infections in young and healthy individuals. hvKp isolates are often associated with the K1/K2 capsular types and possess hypermucoviscous capsules. KLA is believed to be caused by K. pneumoniae colonizing the gastrointestinal tract of the host and translocating across the intestinal barrier via the hepatic portal vein into the liver to cause liver abscess. We optimized the isolation of the liver-resident macrophages called Kupffer cells in mice and examined their importance in controlling bacterial loads during hvKp infection in healthy mice. Our study reveals the high capability of Kupffer cells to kill hvKp in vitro despite the presence of the bacterial hypermucoviscous capsule, in contrast to other macrophages, which were unable to phagocytose the bacteria efficiently. Depletion of Kupffer cells and macrophages with liposome-encapsulated clodronate (liposomal clodronate) in both an intraperitoneal and an oral mouse infection model resulted in increased bacterial loads in the livers, spleens, and lungs and increased mortality of the infected mice. Thus, Kupffer cells and macrophages are critical for the control of hvKp infection.

The proteobacterial species Burkholderia pseudomallei produces ergothioneine, which enhances virulence in mammalian infection.

Bacteria use various endogenous antioxidants for protection against oxidative stress associated with environmental survival or host infection. Although glutathione (GSH) is the most abundant and widely used antioxidant in Proteobacteria, ergothioneine (EGT) is another microbial antioxidant, mainly produced by fungi and Actinobacteria. The Burkholderia genus is found in diverse environmental niches. We observed that gene homologs required for the synthesis of EGT are widely distributed throughout the genus. By generating gene-deletion mutants and monitoring production with isotope-labeled substrates, we show that pathogenic Burkholderia pseudomallei and environmental B. thailandensis are able to synthesize EGT de novo. Unlike most other bacterial EGT synthesis pathways described, Burkholderia spp. use cysteine rather than γ-glutamyl cysteine as the thiol donor. Analysis of recombinant EgtB indicated that it is a proficient sulfoxide synthase, despite divergence in the active site architecture from that of mycobacteria. The absence of GSH, but not EGT, increased bacterial susceptibility to oxidative stresses in vitro. However, deletion of EGT synthesis conferred a reduced fitness to B. pseudomallei, with a delay in organ colonization and time to death during mouse infection. Therefore, despite the lack of an apparent antioxidant role in vitro, EGT is important for optimal bacterial pathogenesis in the mammalian host.-Gamage, A. M., Liao, C., Cheah, I. K., Chen, Y., Lim, D. R. X., Ku, J. W. K., Chee, R. S. L., Gengenbacher, M., Seebeck, F. P., Halliwell, B., Gan, Y.-H. The proteobacterial species Burkholderia pseudomallei produces ergothioneine, which enhances virulence in mammalian infection.

Anti-Cancer Drug HMBA Acts as an Adjuvant during Intracellular Bacterial Infections by Inducing Type I IFN through STING.

The anti-proliferative agent hexamethylene bisacetamide (HMBA) belongs to a class of hybrid bipolar compounds developed more than 30 y ago for their ability to induce terminal differentiation of transformed cells. Recently, HMBA has also been shown to trigger HIV transcription from latently infected cells, via a CDK9/HMBA inducible protein-1 dependent process. However, the effect of HMBA on the immune response has not been explored. We observed that pretreatment of human peripheral blood mononuclear cells with HMBA led to a markedly increased production of IL-12 and IFN-γ, but not of TNF-α, IL-6, and IL-8 upon subsequent infection with Burkholderia pseudomallei and Salmonella enterica HMBA treatment was also associated with better intracellular bacterial control. HMBA significantly improved IL-12p70 production from CD14+ monocytes during infection partly via the induction of type I IFN in these cells, which primed an increased transcription of the p35 subunit of IL-12p70 during infection. HMBA also increased early type I IFN transcription in human monocytic and epithelial cell lines, but this was surprisingly independent of its previously reported effects on positive transcription elongation factor b and HMBA inducible protein-1. Instead, the effect of HMBA was downstream of a calcium influx, and required the pattern recognition receptor and adaptor STING but not cGAS. Our work therefore links the STING-IRF3 axis to enhanced IL-12 production and intracellular bacterial control in primary monocytes. This raises the possibility that HMBA or related small molecules may be explored as therapeutic adjuvants to improve disease outcomes during intracellular bacterial infections.

Complement-activated vitronectin enhances the invasion of nonphagocytic cells by bacterial pathogens Burkholderia and Klebsiella.

Burkholderia pseudomallei is a serum-resistant Gram-negative bacterium capable of causing disseminated infections with metastatic complications. However, its interaction with nonphagocytic cells is poorly understood. We observed that exposure of B. pseudomallei and the closely related yet avirulent B. thailandensis to human plasma increased epithelial cell invasion by >20 fold. Enhanced invasion was primarily driven by a plasma factor, which required a functional complement cascade, but surprisingly, was downstream of C3 mediated opsonisation. Receptor blocking studies with RGD-domain containing peptide and αV ß3 blocking antibody identified complement-activated vitronectin as the factor facilitating this invasion. Plasma treatment led to the recruitment of vitronectin onto the bacterial surface, and its conversion into the active conformation. Activation of vitronectin, as well as increased invasion, required the complement pathway and was not observed in C3 or C5 depleted serum. The integrin inhibitor cilengitide, currently in clinical trials as an anti-angiogenesis agent, suppresses plasma-mediated Burkholderia invasion by ~95%, along with a downstream reduction in intracellular bacterial replication. We extend these findings to serum-resistant Klebsiella pneumoniae as well. Thus, the potential use of commercially available integrin inhibitors as anti-infective agents during selective bacterial infections should be explored.

Burkholderia pseudomallei suppresses Caenorhabditis elegans immunity by specific degradation of a GATA transcription factor.

Burkholderia pseudomallei is a Gram-negative soil bacterium that infects both humans and animals. Although cell culture studies have revealed significant insights into factors contributing to virulence and host defense, the interactions between this pathogen and its intact host remain to be elucidated. To gain insights into the host defense responses to B. pseudomallei infection within an intact host, we analyzed the genome-wide transcriptome of infected Caenorhabditis elegans and identified ∼6% of the nematode genes that were significantly altered over a 12-h course of infection. An unexpected feature of the transcriptional response to B. pseudomallei was a progressive increase in the proportion of down-regulated genes, of which ELT-2 transcriptional targets were significantly enriched. ELT-2 is an intestinal GATA transcription factor with a conserved role in immune responses. We demonstrate that B. pseudomallei down-regulation of ELT-2 targets is associated with degradation of ELT-2 protein by the host ubiquitin-proteasome system. Degradation of ELT-2 requires the B. pseudomallei type III secretion system. Together, our studies using an intact host provide evidence for pathogen-mediated host immune suppression through the destruction of a host transcription factor.

The condition-dependent transcriptional landscape of Burkholderia pseudomallei.

Burkholderia pseudomallei (Bp), the causative agent of the often-deadly infectious disease melioidosis, contains one of the largest prokaryotic genomes sequenced to date, at 7.2 Mb with two large circular chromosomes (1 and 2). To comprehensively delineate the Bp transcriptome, we integrated whole-genome tiling array expression data of Bp exposed to >80 diverse physical, chemical, and biological conditions. Our results provide direct experimental support for the strand-specific expression of 5,467 Sanger protein-coding genes, 1,041 operons, and 766 non-coding RNAs. A large proportion of these transcripts displayed condition-dependent expression, consistent with them playing functional roles. The two Bp chromosomes exhibited dramatically different transcriptional landscapes--Chr 1 genes were highly and constitutively expressed, while Chr 2 genes exhibited mosaic expression where distinct subsets were expressed in a strongly condition-dependent manner. We identified dozens of cis-regulatory motifs associated with specific condition-dependent expression programs, and used the condition compendium to elucidate key biological processes associated with two complex pathogen phenotypes--quorum sensing and in vivo infection. Our results demonstrate the utility of a Bp condition-compendium as a community resource for biological discovery. Moreover, the observation that significant portions of the Bp virulence machinery can be activated by specific in vitro cues provides insights into Bp's capacity as an "accidental pathogen", where genetic pathways used by the bacterium to survive in environmental niches may have also facilitated its ability to colonize human hosts.

Characterization and analysis of the Burkholderia pseudomallei BsaN virulence regulon.

BACKGROUND: Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis. A conserved type III secretion system (T3SS3) and type VI secretion system (T6SS1) are critical for intracellular survival and growth. The T3SS3 and T6SS1 genes are coordinately and hierarchically regulated by a TetR-type regulator, BspR. A central transcriptional regulator of the BspR regulatory cascade, BsaN, activates a subset of T3SS3 and T6SS1 loci. RESULTS: To elucidate the scope of the BsaN regulon, we used RNAseq analysis to compare the transcriptomes of wild-type B. pseudomallei KHW and a bsaN deletion mutant. The 60 genes positively-regulated by BsaN include those that we had previously identified in addition to a polyketide biosynthesis locus and genes involved in amino acid biosynthesis. BsaN was also found to repress the transcription of 51 genes including flagellar motility loci and those encoding components of the T3SS3 apparatus. Using a promoter-lacZ fusion assay in E. coli, we show that BsaN together with the chaperone BicA directly control the expression of the T3SS3 translocon, effector and associated regulatory genes that are organized into at least five operons (BPSS1516-BPSS1552). Using a mutagenesis approach, a consensus regulatory motif in the promoter regions of BsaN-regulated genes was shown to be essential for transcriptional activation. CONCLUSIONS: BsaN/BicA functions as a central regulator of key virulence clusters in B. pseudomallei within a more extensive network of genetic regulation. We propose that BsaN/BicA controls a gene expression program that facilitates the adaption and intracellular survival of the pathogen within eukaryotic hosts.

Type three secretion system-mediated escape of Burkholderia pseudomallei into the host cytosol is critical for the activation of NFκB.

BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three "injection type" type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NFκB in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NFκB activation is unknown. RESULTS: Known T3SS3 effectors are not responsible for NFκB activation. Furthermore, T3SS3-null mutants are able to activate NFκB almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NFκB activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection. CONCLUSIONS: T3SS3 does not directly activate NFκB but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NFκB activation.