The bhuTUV and bhuO genes play vital roles in the ability of Brucella abortus to use heme as an iron source and are regulated in an iron-responsive manner by RirA and Irr

Brucella abortus is a Gram negative intracellular pathogen that causes the zoonotic disease brucellosis. Antibiotic treatment for brucellosis in humans is prolonged and sometimes followed by relapses. Currently, the United States employs prevention of the illness in humans through cattle vaccinations, eliminating the bacterium in its natural host. Unfortunately, these vaccine strains cause the disease in humans, and Brucella research ultimately aims to identify new vaccine targets as well as alternative treatment options. Brucella abortus resides in the phagosomal compartment of the host macrophage where essential nutrients such as iron are limited. Most bacteria need iron, and within the macrophage, heme is a likely source of iron due to the breakdown of red blood cells by the host macrophage. Heme transporters in Gram negative bacteria are highly conserved, and include components for outer membrane, periplasmic, and cytoplasmic membrane transport. BhuA has been previously characterized as the outer membrane heme transporter of Brucella abortus and here we report that BhuT, BhuU, and BhuV (BhuTUV) are the periplasmic and cytoplasmic heme transport components and that they are required in order for Brucella abortus to transport heme as an iron source. Utilization of heme as an iron source requires the breakdown of heme into ferrous iron, carbon monoxide, and biliverdin by a heme oxygenase. BhuO has been identified as a heme oxygenase in Brucella abortus , and although there seems to be more than one heme oxygenase in Brucella , this study shows that BhuO is needed for the use of heme as an iron source under iron starvation conditions in vitro. Further, both bhuTUV and bhuO are regulated in an iron-responsive manner. The iron responsive regulator Irr directly represses bhuO, which shares an operon with rirA . Then the rhizobial iron regulator RirA in turn represses the bhuTUV operon. Together, these regulators help to maintain iron homeostasis within the bacterial cell, protecting it from damaging hydroxyl radicals produced by Fenton chemistry.Ph.D

A high-content imaging assay for the quantification of the Burkholderia pseudomallei induced multinucleated giant cell (MNGC) phenotype in murine macrophages

BACKGROUND: Burkholderia pseudomallei (Bp), a Gram-negative, motile, facultative intracellular bacterium is the causative agent of melioidosis in humans and animals. The Bp genome encodes a repertoire of virulence factors, including the cluster 3 type III secretion system (T3SS-3), the cluster 1 type VI secretion system (T6SS-1), and the intracellular motility protein BimA, that enable the pathogen to invade both phagocytic and non-phagocytic cells. A unique hallmark of Bp infection both in vitro and in vivo is its ability to induce cell-to-cell fusion of macrophages to form multinucleated giant cells (MNGCs), which to date are semi-quantitatively reported following visual inspection. RESULTS: In this study we report the development of an automated high-content image acquisition and analysis assay to quantitate the Bp induced MNGC phenotype. Validation of the assay was performed using T6SS-1 (∆hcp1) and T3SS-3 (∆bsaZ) mutants of Bp that have been previously reported to exhibit defects in their ability to induce MNGCs. Finally, screening of a focused small molecule library identified several Histone Deacetylase (HDAC) inhibitors that inhibited Bp-induced MNGC formation of macrophages. CONCLUSIONS: We have successfully developed an automated HCI assay to quantitate MNGCs induced by Bp in macrophages. This assay was then used to characterize the phenotype of the Bp mutants for their ability to induce MNGC formation and identify small molecules that interfere with this process. Successful application of chemical genetics and functional reverse genetics siRNA approaches in the MNGC assay will help gain a better understanding of the molecular targets and cellular mechanisms responsible for the MNGC phenotype induced by Bp, by other bacteria such as Mycobacterium tuberculosis, or by exogenously added cytokines

The bhuTUV and bhuO genes play vital roles in the ability of Brucella abortus to use heme as an iron source and are regulated in an iron-responsive manner by RirA and Irr

Brucella abortus is a Gram negative intracellular pathogen that causes the zoonotic disease brucellosis. Antibiotic treatment for brucellosis in humans is prolonged and sometimes followed by relapses. Currently the United States employs prevention of the illness in humans through cattle vaccinations eliminating the bacterium in its natural host. Unfortunately these vaccine strains cause the disease in humans and Brucella research ultimately aims to identify new vaccine targets as well as alternative treatment options. Brucella abortus resides in the phagosomal compartment of the host macrophage where essential nutrients such as iron are limited. Most bacteria need iron and within the macrophage heme is a likely source of iron due to the breakdown of red blood cells by the host macrophage. Heme transporters in Gram negative bacteria are highly conserved and include components for outer membrane periplasmic and cytoplasmic membrane transport. BhuA has been previously characterized as the outer membrane heme transporter of Brucella abortus and here we report that BhuT BhuU and BhuV (BhuTUV) are the periplasmic and cytoplasmic heme transport components and that they are required in order for Brucella abortus to transport heme as an iron source. Utilization of heme as an iron source requires the breakdown of heme into ferrous iron carbon monoxide and biliverdin by a heme oxygenase. BhuO has been identified as a heme oxygenase in Brucella abortus and although there seems to be more than one heme oxygenase in Brucella this study shows that BhuO is needed for the use of heme as an iron source under iron starvation conditions in vitro. Further both bhuTUV and bhuO are regulated in an iron-responsive manner. The iron responsive regulator Irr directly represses bhuO which shares an operon with rirA . Then the rhizobial iron regulator RirA in turn represses the bhuTUV operon. Together these regulators help to maintain iron homeostasis within the bacterial cell protecting it from damaging hydroxyl radicals produced by Fenton chemistry

Alveolar Macrophages Infected with Ames or Sterne Strain of <i>Bacillus anthracis</i> Elicit Differential Molecular Expression Patterns

<div><p>Alveolar macrophages (AMs) phagocytose <i>Bacillus anthracis</i> following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of <i>B. anthracis</i>, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of <i>B. anthracis</i>, which lacks the plasmid encoding capsule, is widely adapted as a vaccine strain. Although differences in the outcome of infection with the two strains may have originated from the presence or absence of an anti-phagocytic capsule, the disease pathogenesis following infection will be manifested via the host responses, which is not well understood. To gain understanding of the host responses at cellular level, a microarray analysis was performed using primary rhesus macaque AMs infected with either Ames or Sterne spores. Notably, 528 human orthologs were identified to be differentially expressed in AMs infected with either strain of the <i>B. anthracis.</i> Meta-analyses revealed genes differentially expressed in response to <i>B. anthracis</i> infection were also induced upon infections with multiple pathogens such as <i>Francisella Novicida</i> or <i>Staphylococcus aureus</i>. This suggests the existence of a common molecular signature in response to pathogen infections. Importantly, the microarray and protein expression data for certain cytokines, chemokines and host factors provide further insights on how cellular processes such as innate immune sensing pathways, anti-apoptosis versus apoptosis may be differentially modulated in response to the virulent or vaccine strain of <i>B. anthracis</i>. The reported differences may account for the marked difference in pathogenicity between these two strains.</p></div

Microarray analyses identified 528 human orthologs which were differentially expressed between Ames and Sterne infected AMs.

<p>AMs obtained from five rhesus macaque donors were infected with Ames or Sterne spores at an MOI of 10 for indicated time points. Total mRNAs were purified and hybridized to rhesus macaque cDNA microarrays. “Ames vs Sterne” bar depicts the ratio of gene expressions (in logarithmic scale) between Ames-infected AMs to Sterne-infected counterparts. “Ames” and “Sterne” bar depict fold change of a gene expression (in logarithmic scale) by normalizing Ames or Sterne treated AMs with the 0 h time point. The level of fold changes are colored coded, where red stands for high values (>1) and blue for low fold changes (<1). To highlight statistically significant changes for “Ames vs. Sterne” for the ease of visualization, we decreased the contrast by three folds for the portion of the heat map, where differential expression is not significant.</p

Gene ontology (GO) analysis.

<p>Statistically significant overrepresentation of selected functional classes and protein families based upon gene ontology (GO) analysis.</p

Overlap between the genes identified from current study and studies referenced.

<p>Explanation of the column headings in table are as follows: Pathogens: name of the pathogen used in the referenced microarray studies; # of overlapping genes: the number of genes that are overlapping between the current study and the referenced studies; Cells used in the study: the type of cells used in the referenced microarray studies; Selected Overlapping Genes: selected gene symbols that are overlapping between the two studies; P-value: the P-value of the overlap; PMID or GEO ID: the reference of the published microarray studies.</p

AMs infected with Ames or Sterne spores show differential pro-inflammatory cytokine/chemokine protein expression patterns.

<p>AMs were infected with either Ames or Sterne spores at an MOI of 10. Supernatants were collected at indicated time points and cytokine/chemokines levels were quantified. The experiment was performed at least three times and data for three rhesus macaque donors are shown. Scatter plots are presented as mean ± Standard Deviation. Experiments for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087201#pone-0087201-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087201#pone.0087201.s001" target="_blank">Figure S1</a> were performed concurrently and shared the same controls. P-value is calculated using a paired Student’s t-test. *represents p-value <0.05 and **represents p-value <0.01.</p

Time-dependent kinetic difference in the induction of COX-2 and PGE₂ expression (A) AMs were infected with either Ames or Sterne spores for 90 min, 4 h or 18 h.

<p>Cells were lysed and the mRNA was purified and quantified by real time PCR. Fold expression was calculated by normalizing to time 0. (B) AMs were infected with Ames or Sterne spores at an MOI of 10. The amount of PGE₂ was quantified by ELISA. Data shown in (A) and (B) are representative of n = 3 experiments. Scatter plots are presented as mean ± Standard deviation. P-value is calculated using a paired two tailed Student’s t-test. *represent p-value <0.05.</p