Evaluating the role of Burkholderia pseudomallei K96243 toxins BPSS0390, BPSS0395, and BPSS1584 in persistent infection.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with a mortality rate of up to 40% even with treatment. Despite the ability of certain antibiotics to control initial infection, relapse occurs in treated patients. The inability of antibiotics to clear this bacterial infection is in part due to persistence, an evasion mechanism against antibiotics and the effect of host defenses. Evaluation of antibiotic efficacy against B. pseudomallei revealed that up to 48% of in vitro grown populations can survive in a persister state. Toxin-antitoxin (TA) systems have been previously implicated in modulating bacterial persistence. We generated three isogenic TA mutants and found that loss of each toxin gene did not alter antibiotic persistence or macrophage survival. In response to macrophage-induced persistence, all three toxin mutants demonstrated increased intracellular susceptibility to levofloxacin which in part was due to the inability of the mutants to induce persistence after nitric oxide or nutrient starvation. In an inhalational model of murine melioidosis, both ΔBPSS0395 and ΔBPSS1584 strains were attenuated, and treatment with levofloxacin led to significant reduction in lung colonisation and reduced splenic colonisation by ΔBPSS0395. Based on our findings, these toxins deserve additional evaluation as putative therapeutic targets.

Increased Mortality in Mice following Immunoprophylaxis Therapy with High Dosage of Nicotinamide in Burkholderia Persistent Infections.

Bacterial persistence, known as noninherited antibacterial resistance, is a factor contributing to the establishment of long-lasting chronic bacterial infections. In this study, we examined the ability of nicotinamide (NA) to potentiate the activity of different classes of antibiotics against Burkholderia thailandensis persister cells. Here we demonstrate that addition of NA in in vitro models of B. thailandensis infection resulted in a significant depletion of the persister population in response to various classes of antibiotics. We applied microfluidic bioreactors with a continuous medium flow to study the effect of supplementation with an NA gradient on the recovery of B. thailandensis persister populations. A coculture of human neutrophils preactivated with 50 µM NA and B. thailandensis resulted in the most efficient reduction in the persister population. Applying single-cell RNA fluorescence in situ hybridization analysis and quantitative PCR, we found that NA inhibited gene expression of the stringent response regulator relA, implicated in the regulation of the persister metabolic state. We also demonstrate that a therapeutic dose of NA (250 mg/kg of body weight), previously applied as immunoprophylaxis against antibiotic-resistant bacterial species, produced adverse effects in an in vivo murine model of infection with the highly pathogenic bacterium Burkholderia pseudomallei, indicating that therapeutic dose and metabolite effects have to be carefully evaluated and tailored for every case of potential clinical application.

Differential expression of small RNAs from Burkholderia thailandensis in response to varying environmental and stress conditions.

BACKGROUND: 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. RESULTS: 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 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. In particular, the trans-encoded BTH_s1 and s39 exhibited differential expression profiles dependent on growth phase and cell stimuli, such as antibiotics and serum. Furthermore, knockdown of the highly-expressed BTH_s39 by antisense transcripts reduced B. thailandensis cell growth and attenuated host immune response upon infection, indicating that BTH_s39 functions in bacterial metabolism and adaptation to the host. In addition, expression of cis-encoded BTH_s13 and s19 found in the 5' untranslated regions of their cognate genes correlated with tight regulation of gene transcript levels. This sRNA-mediated downregulation of gene expression may be a conserved mechanism of post-transcriptional gene dosage control. CONCLUSIONS: These studies provide a broad analysis of differential Burkholderia sRNA expression profiles and illustrate the complexity of bacterial gene regulation in response to different environmental stress conditions.

Binding of nucleoid-associated protein fis to DNA is regulated by DNA breathing dynamics.

Physicochemical properties of DNA, such as shape, affect protein-DNA recognition. However, the properties of DNA that are most relevant for predicting the binding sites of particular transcription factors (TFs) or classes of TFs have yet to be fully understood. Here, using a model that accurately captures the melting behavior and breathing dynamics (spontaneous local openings of the double helix) of double-stranded DNA, we simulated the dynamics of known binding sites of the TF and nucleoid-associated protein Fis in Escherichia coli. Our study involves simulations of breathing dynamics, analysis of large published in vitro and genomic datasets, and targeted experimental tests of our predictions. Our simulation results and available in vitro binding data indicate a strong correlation between DNA breathing dynamics and Fis binding. Indeed, we can define an average DNA breathing profile that is characteristic of Fis binding sites. This profile is significantly enriched among the identified in vivo E. coli Fis binding sites. To test our understanding of how Fis binding is influenced by DNA breathing dynamics, we designed base-pair substitutions, mismatch, and methylation modifications of DNA regions that are known to interact (or not interact) with Fis. The goal in each case was to make the local DNA breathing dynamics either closer to or farther from the breathing profile characteristic of a strong Fis binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by gel-shift assay, changed in accordance with our expectations. We conclude that Fis binding is associated with DNA breathing dynamics, which in turn may be regulated by various nucleotide modifications.

Counting small RNA in pathogenic bacteria.

Here, we present a modification to single-molecule fluorescence in situ hybridization that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short (~200 nucleotide) nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. By neutralizing the fluorescence from unbound probes, we were able to significantly reduce the number of false positives, allowing for accurate quantification of sRNA levels. Exploiting an automated, mutli-color wide-field microscope and data analysis package, we analyzed the statistics of sRNA expression in thousands of individual bacteria. We found that only a small fraction of either Yersinia pseudotuberculosis or Yersinia pestis bacteria express the small RNAs YSR35 or YSP8, with the copy number typically between 0 and 10 transcripts. The numbers of these RNA are both increased (by a factor of 2.5× for YSR35 and 3.5× for YSP8) upon a temperature shift from 25 to 37 °C, suggesting they play a role in pathogenesis. The copy number distribution of sRNAs from bacteria-to-bacteria are well-fit with a bursting model of gene transcription. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in cellular regulatory networks.

c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response.

BACKGROUND: The pathogenic Yersinia species exhibit a primarily extracellular lifestyle through manipulation of host signaling pathways that regulate pro-inflammatory gene expression and cytokine release. To identify host genes that are targeted by Yersinia during the infection process, we performed an RNA interference (RNAi) screen based on recovery of host NF-κB-mediated gene activation in response to TNF-α stimulation upon Y. enterocolitica infection. RESULTS: We screened shRNAs against 782 genes in the human kinome and 26 heat shock genes, and identified 19 genes that exhibited ≥ 40% relative increase in NF-κB reporter gene activity. The identified genes function in multiple cellular processes including MAP and ERK signaling pathways, ion channel activity, and regulation of cell growth. Pre-treatment with small molecule inhibitors specific for the screen hits c-KIT and CKII recovered NF-κB gene activation and/or pro-inflammatory TNF-α cytokine release in multiple cell types, in response to either Y. enterocolitica or Y. pestis infection. CONCLUSIONS: We demonstrate that pathogenic Yersinia exploits c-KIT signaling in a T3SS-dependent manner to downregulate expression of transcription factors EGR1 and RelA/p65, and pro-inflammatory cytokines. This study is the first major functional genomics RNAi screen to elucidate virulence mechanisms of a pathogen that is primarily dependent on extracellular-directed immunomodulation of host signaling pathways for suppression of host immunity.

Discovery of DNA operators for TetR and MarR family transcription factors from Burkholderia xenovorans.

Determining transcription factor (TF) recognition motifs or operator sites is central to understanding gene regulation, yet few operators have been characterized. In this study, we used a protein-binding microarray (PBM) to discover the DNA recognition sites and putative regulons for three TetR and one MarR family TFs derived from Burkholderia xenovorans, which are common to the genus Burkholderia. We also describe the development and application of a more streamlined version of the PBM technology that significantly reduced the experimental time. Despite the genus containing many pathogenically important species, only a handful of TF operator sites have been experimentally characterized for Burkholderia to date. Our study provides a significant addition to this knowledge base and illustrates some general challenges of discovering operators on a large scale for prokaryotes.

Targeting the Bet-Hedging Strategy with an Inhibitor of Bacterial Efflux Capacity Enhances Antibiotic Efficiency and Ameliorates Bacterial Persistence In Vitro.

Persistence is a bet-hedging strategy in bacterial populations that increases antibiotic tolerance and leads to the establishment of latent infections. In this study, we demonstrated that a synthetic non-toxic taxane-based reversal agent (tRA), developed as an inhibitor of ABC transporter systems in mammalian cancer cells, enhanced antibiotic killing of persister populations from different pathogens, including Burkholderia, Pseudomonas, Francisella, and Yersinia. Acting as an inhibitor of bacterial efflux at 100 nM, tRA99020 enhanced antibiotic efficiency and suppressed the production of natural products of Burkholderia species polyketide synthase (PKS) function. We demonstrate that the metabolites produced by PKS in response to stress by different antibiotics act as inhibitors of mammalian histone deacetylase activity and stimulate cell death. Applying a single-molecule fluorescence in situ hybridization (smFISH) assay, we analyzed on a single-cell level the activation profiles of the persistence regulating pks gene in Burkholderia thailandensis treated with tRA99020 and antibiotics. We posit that a multi-pronged approach encompassing antibiotic therapies and inhibition of efflux systems and fatty acid catabolism will be required for efficient eradication of persistent bacterial populations.

Methods for Enrichment of Bacterial Persister Populations for Phenotypic Screens and Genomic Studies.

Transient phenotypic adaptations in bacteria that enable survival at bactericidal antibiotic concentrations give rise to bacterial persistence. Naturally, the abundance of persister cells is very low (about 1 in 105 cells) in actively growing bacterial populations. Therefore, in order to study bacterial persistence mechanisms for therapeutics development, persister cells need to be enriched from a larger culture. Here, we describe three enrichment methods for obtaining Burkholderia thailandensis persisters: (1) flow sorting for persisters from exponentially dividing cultures by fluorescent staining of bacterial cells with a translational membrane depolarization-specific DiBAC4(3) dye, (2) antibiotic lysis of nonpersisters, and (3) culture aging to induce persister survival. We also describe herein the lysis of persister cells obtained by all three methods for downstream bacterial RNA extraction and transcriptomics analysis.

Visualization and modeling of inhibition of IL-1ß and TNF-α mRNA transcription at the single-cell level.

IL-1ß and TNF-α are canonical immune response mediators that play key regulatory roles in a wide range of inflammatory responses to both chronic and acute conditions. Here we employ an automated microscopy platform for the analysis of messenger RNA (mRNA) expression of IL-1ß and TNF-α at the single-cell level. The amount of IL-1ß and TNF-α mRNA expressed in a human monocytic leukemia cell line (THP-1) is visualized and counted using single-molecule fluorescent in-situ hybridization (smFISH) following exposure of the cells to lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. We show that the small molecule inhibitors MG132 (a 26S proteasome inhibitor used to block NF-κB signaling) and U0126 (a MAPK Kinase inhibitor used to block CCAAT-enhancer-binding proteins C/EBP) successfully block IL-1ß and TNF-α mRNA expression. Based upon this single-cell mRNA expression data, we screened 36 different mathematical models of gene expression, and found two similar models that capture the effects by which the drugs U0126 and MG132 affect the rates at which the genes transition into highly activated states. When their parameters were informed by the action of each drug independently, both models were able to predict the effects of the combined drug treatment. From our data and models, we postulate that IL-1ß is activated by both NF-κB and C/EBP, while TNF-α is predominantly activated by NF-κB. Our combined single-cell experimental and modeling efforts show the interconnection between these two genes and demonstrates how the single-cell responses, including the distribution shapes, mean expression, and kinetics of gene expression, change with inhibition.

What Can Go Wrong When Applying Immune Modulation Therapies to Target Persistent Bacterial Infections.

Antibiotics can treat the acute phase of a disease, but often do not completely clear the etiologic agent, allowing the pathogen to establish persistent infection that can revive the disease in a frustrating recurrence of infection. The mechanisms that control chronic bacterial infections are complex and involve pathogen adaptations that favor survival from both host immune responses and antibiotic bactericidal activity. Often, the causative agents of persistent infections are not drug-resistant species. Instead, bacterial persister cells temporarily enter a physiological state that is refractory to different classes of antibiotics. Supplemental therapies that potentiate antibiotic bactericidal efficiency and/or immune clearance of persistent pathogenic species may greatly improve the outcome of infectious disease. Here, we discuss the various outcomes in experimental studies in which a mega-dose of the energy-boosting vitamin B3 (nicotinamide) was applied in murine models of chronic infection to stimulate immune clearance of chronic infection or as an immune prophylactic treatment against the highly infectious pathogen, Burkholderia pseudomallei. It is our intent to raise awareness of the risks associated with immune modulation therapies. There is great variance in host immune responses to pathogenic bacteria. Each immune modulation approach needs to be tailored to a well-characterized host-pathogen interaction.

A Gene Cluster That Encodes Histone Deacetylase Inhibitors Contributes to Bacterial Persistence and Antibiotic Tolerance in Burkholderia thailandensis.

Persister cells are genetically identical variants in a bacterial population that have phenotypically modified their physiology to survive environmental stress. In bacterial pathogens, persisters are able to survive antibiotic treatment and reinfect patients in a frustrating cycle of chronic infection. To better define core persistence mechanisms for therapeutics development, we performed transcriptomics analyses of Burkholderia thailandensis populations enriched for persisters via three methods: flow sorting for low proton motive force, meropenem treatment, and culture aging. Although the three persister-enriched populations generally displayed divergent gene expression profiles that reflect the multimechanistic nature of stress adaptations, there were several common gene pathways activated in two or all three populations. These include polyketide and nonribosomal peptide synthesis, Clp proteases, mobile elements, enzymes involved in lipid metabolism, and ATP-binding cassette (ABC) transporter systems. In particular, identification of genes that encode polyketide synthases (PKSs) and fatty acid catabolism factors indicates that generation of secondary metabolites, natural products, and complex lipids could be part of the metabolic program that governs the persistence state. We also found that loss-of-function mutations in the PKS-encoding gene locus BTH_I2366, which plays a role in biosynthesis of histone deacetylase (HDAC) inhibitors, resulted in increased sensitivity to antibiotics targeting DNA replication. Furthermore, treatment of multiple bacterial pathogens with a fatty acid synthesis inhibitor, CP-640186, potentiated the efficacy of meropenem against the persister populations. Altogether, our results suggest that bacterial persisters may exhibit an outwardly dormant physiology but maintain active metabolic processes that are required to maintain persistence.IMPORTANCE The discovery of antibiotics such as penicillin and streptomycin marked a historic milestone in the 1940s and heralded a new era of antimicrobial therapy as the modern standard for medical treatment. Yet, even in those early days of discovery, it was noted that a small subset of cells (∼1 in 105) survived antibiotic treatment and continued to persist, leading to recurrence of chronic infection. These persisters are phenotypic variants that have modified their physiology to survive environmental stress. In this study, we have performed three transcriptomic screens to identify persistence genes that are common between three different stressor conditions. In particular, we identified genes that function in the synthesis of secondary metabolites, small molecules, and complex lipids, which are likely required to maintain the persistence state. Targeting universal persistence genes can lead to the development of clinically relevant antipersistence therapeutics for infectious disease management.

Soluble MD2 increases TLR4 levels on the epithelial cell surface.

The accessory protein MD2 has been implicated in LPS-mediated activation of the innate immune system by functioning as a co-receptor with TLR4 for LPS binding at the cell surface. Epithelial cells that play a role in primary immune response, such as in the lung or gut, often express TLR4, but are dependent on circulating soluble MD2 (sMD2) to bind TLR4 to assemble the functional receptor. In this study, we show that sMD2 incubation with HEK293 epithelial cells transfected with TLR4 increases the cell surface levels of TLR4 in the absence of LPS. Dose response studies reveal that a threshold sMD2 concentration (approximately 450 nM) stimulates maximal TLR4 levels on the cell surface, whereas higher concentrations of sMD2 (approximately 1800 nM) reduce these enhanced TLR4 levels. We show evidence that MD2 multimer formation is increased at these higher concentrations of sMD2 and that addition of LPS to sMD2-stimulated cells masks the enhanced TLR4 cell surface levels, most likely due to the LPS-induced downregulation of TLR4 by endocytosis following receptor stimulation. All together, these results support a model in which sMD2 binds to TLR4 and increases TLR4 levels at the cell surface by preventing TLR4 turnover through the endocytic pathway. Thus, sMD2 may prime epithelial cells for enhanced immunoresponsive function prior to LPS exposure.

A role for cell adhesion in beryllium-mediated lung disease.

Chronic beryllium disease (CBD) is a debilitating lung disorder in which exposure to the lightweight metal beryllium (Be) causes the accumulation of beryllium-specific CD4+ T cells in the lung and formation of noncaseating pulmonary granulomas. Treatment for CBD patients who exhibit progressive pulmonary decline is limited to systemic corticosteroids, which suppress the severe host inflammatory response. Studies in the past several years have begun to highlight cell-cell adhesion interactions in the development of Be hypersensitivity and CBD. In particular, the high binding affinity between intercellular adhesion molecule 1 (I-CAM1) on lung epithelial cells and the beta(2) integrin LFA-1 on migrating lymphocytes and macrophages regulates the concerted rolling of immune cells to sites of inflammation in the lung. In this review, we discuss the evidence that implicates cell adhesion processes in onset of Be disease and the potential of cell adhesion as an intervention point for development of novel therapies.

Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation.

The heterogeneity of mRNA and protein expression at the single-cell level can reveal fundamental information about cellular response to external stimuli, including the sensitivity, timing, and regulatory interactions of genes. Here we describe a fully automated system to digitally count the intron, mRNA, and protein content of up to five genes of interest simultaneously in single-cells. Full system automation of 3D microscope scans and custom image analysis routines allows hundreds of individual cells to be automatically segmented and the mRNA-protein content to be digitally counted. Single-molecule intron and mRNA content is measured by single-molecule fluorescence in-situ hybridization (smFISH), while protein content is quantified though the use of antibody probes. To mimic immune response to bacterial infection, human monocytic leukemia cells (THP-1) were stimulated with lipopolysaccharide (LPS), and the expression of two inflammatory genes, IL1ß (interleukin 1ß) and TNF-α (tumor necrosis factor α), were simultaneously quantified by monitoring the intron, mRNA, and protein levels over time. The simultaneous labeling of cellular content allowed for a series of correlations at the single-cell level to be explored, both in the progressive maturation of a single gene (intron-mRNA-protein) and comparative analysis between the two immune response genes. In the absence of LPS stimulation, mRNA expression of IL1ß and TNF-α were uncorrelated. Following LPS stimulation, mRNA expression of the two genes became more correlated, consistent with a model in which IL1ß and TNF-α upregulation occurs in parallel through independent mechanistic pathways. This smFISH methodology can be applied to different complex biological systems to provide valuable insight into highly dynamic gene mechanisms that determine cell plasticity and heterogeneity of cellular response.

The bioinorganic chemistry and associated immunology of chronic beryllium disease.

Chronic beryllium disease (CBD) is a debilitating, incurable, and often fatal disease that is caused by the inhalation of beryllium particulates. The growing use of beryllium in the modern world, in products ranging from computers to dental prosthetics (390 tons of beryllium in the US in the year 2000) necessitates a molecular based understanding of the disease in order to prevent and cure CBD. We have investigated the molecular basis of CBD at Los Alamos National Laboratory during the past six years, employing a multidisciplinary approach of bioinorganic chemistry and immunology. The results of this work, including speciation, inhalation and dissolution, and immunology will be discussed.

Upregulation of I-CAM1 in response to beryllium exposure in small airway epithelial cells.

Chronic Beryllium Disease (CBD) is a delayed-type hypersensitivity immune reaction that leads to granuloma formation in the lungs and potentially severe loss of pulmonary function. Although the molecular mechanisms that mediate beryllium (Be)-stimulated granuloma formation are not well understood, cell adhesion molecules are likely to play a key role in the migration of immune cells to sites of inflammation. In this study, we examined the role of the cell adhesion molecule I-CAM1 in Be-stimulated small airway epithelial cells (SAECs). These epithelial cells line the airway and represent the first point of contact for inhaled foreign substances. We find that Be exposure specifically induced I-CAM1 expression on the cell surface of SAEC and release of soluble I-CAM1 into the extracellular medium. Furthermore, anti-I-CAM1 antibodies inhibited Be-stimulated adhesion of SAEC to the macrophage cell-line THP1, indicating that the Be-induced adhesive properties of SAEC are at least partly due to I-CAM1 expression. These studies support a model in which I-CAM1 cell adhesion functions may play a role in directing immune cells to the lung and activating a Be-specific immune response in Be hypersensitivity disease.

Targeting toll-like receptor signaling pathways for design of novel immune therapeutics.

The Toll-like receptor (TLR) family plays a fundamental role in host innate immunity by mounting a rapid and potent inflammatory response to pathogen infection. TLRs recognize distinct microbial components and activate intracellular signaling pathways that induce expression of host inflammatory genes. Extensive research in the past decade to understand TLR-mediated mechanisms of innate immunity has enabled pharmaceutical companies to begin to develop novel therapeutics for the purpose of controlling inflammatory disease. Initially, extracellular TLR agonists were designed to compete with natural microbial ligands for binding to TLRs. More recently, basic research to identify new targets for drug development has begun to explore modulation of TLR intracellular signaling pathways, in addition to TLR ligand binding. In this review, we will discuss recent strategies, including the use of decoy peptides and mimetics, plant polyphenols, and chemically modified antisense oligonucleotides, that inhibit different molecular events in TLR signaling pathways to modulate the inflammatory response. The molecular mechanisms of these inhibitors range from interference with protein-protein interactions between signaling proteins, to inhibition of transcription factor activity, to perturbation of the plasma membrane, and are derived from host, pathogen, and plant sources and by rational design. Taken together, these studies represent promising avenues for the development of novel tailored immune therapeutics that can relieve the great toll inflicted by inflammatory disease on human health and quality of life.

PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis, we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host-targeted therapies against infectious disease caused by intracellular pathogens.

Chemokine regulation in response to beryllium exposure in human peripheral blood mononuclear and dendritic cells.

Exposure to beryllium (Be) induces a delayed-type hypersensitivity immune reaction in the lungs of susceptible individuals, which leads to the onset of Be sensitivity and Chronic Beryllium Disease (CBD). Although some mechanistic aspects of CBD have begun to be characterized, very little is known about the molecular mechanisms by which Be activates the host immune response. To gain insight into the cellular response to Be exposure, we have performed global microarray analysis using a mixture of peripheral blood mononuclear and dendritic cells (PBMC/DCs) from a non-CBD source to identify genes that are specifically upregulated in response to BeSO(4) stimulation, compared to a control metal salt, Al(2)(SO(4))(3). We identified a number of upregulated immunomodulatory genes, including several chemokines in the MIP-1 and GRO families. Using PBMC/DCs from three different donors, we demonstrate that BeSO(4) stimulation generally exhibits an increased rate of both chemokine mRNA transcription and release compared to Al(2)(SO(4))(3) exposure, although variations among the individual donors do exist. We show that MIP-1 alpha and MIP-1 beta neutralizing antibodies can partially inhibit the ability of BeSO(4) to stimulate cell migration of PBMC/DCs in vitro. Finally, incubation of PBMC/DCs with BeSO(4) altered the binding of the transcription factor RUNX to the MIP-1 alpha promoter consensus sequence, indicating that Be can regulate chemokine gene activation. Taken together, these results suggest a model in which Be stimulation of PBMC/DCs can modulate the expression and release of different chemokines, leading to the migration of lymphocytes to the lung and the formation of a localized environment for development of Be disease in susceptible individuals.