Characterization of the murine macrophage response to infection with virulent and avirulent Burkholderia species.

BACKGROUND: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease - from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. RESULTS: The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL-10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. CONCLUSIONS: These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions.

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.

Alveolar macrophages infected with Ames or Sterne strain of Bacillus anthracis elicit differential molecular expression patterns.

Alveolar macrophages (AMs) phagocytose Bacillus anthracis following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of B. anthracis, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of B. anthracis, 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 B. anthracis. Meta-analyses revealed genes differentially expressed in response to B. anthracis infection were also induced upon infections with multiple pathogens such as Francisella Novicida or Staphylococcus aureus. 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 B. anthracis. The reported differences may account for the marked difference in pathogenicity between these two strains.

The bhuQ gene encodes a heme oxygenase that contributes to the ability of Brucella abortus 2308 to use heme as an iron source and is regulated by Irr.

The Brucella BhuQ protein is a homolog of the Bradyrhizobium japonicum heme oxygenases HmuD and HmuQ. To determine if this protein plays a role in the ability of Brucella abortus 2308 to use heme as an iron source, an isogenic bhuQ mutant was constructed and its phenotype evaluated. Although the Brucella abortus bhuQ mutant DCO1 did not exhibit a defect in its capacity to use heme as an iron source or evidence of increased heme toxicity in vitro, this mutant produced increased levels of siderophore in response to iron deprivation compared to 2308. Introduction of a bhuQ mutation into the B. abortus dhbC mutant BHB2 (which cannot produce siderophores) resulted in a severe growth defect in the dhbC bhuQ double mutant JFO1 during cultivation under iron-restricted conditions, which could be rescued by the addition of FeCl(3), but not heme, to the growth medium. The bhuQ gene is cotranscribed with the gene encoding the iron-responsive regulator RirA, and both of these genes are repressed by the other major iron-responsive regulator in the alphaproteobacteria, Irr. The results of these studies suggest that B. abortus 2308 has at least one other heme oxygenase that works in concert with BhuQ to allow this strain to efficiently use heme as an iron source. The genetic organization of the rirA-bhuQ operon also provides the basis for the proposition that BhuQ may perform a previously unrecognized function by allowing the transcriptional regulator RirA to recognize heme as an iron source.

Angle-resolved Mueller matrix study of light scattering by B-cells at three wavelengths of 442, 633, and 850 nm.

Angle-resolved signals of polarized light scattered by biological cells provide rich information on cell morphology. Quantitative study of these signals can lead to new methods to develop and improve high-throughput instrumentation for cell probing such as scattering-based flow cytometry. We employ a goniometer system with a photoelastic modulation scheme to determine selected Mueller matrix elements of B-cell hydrosol samples. The angular dependence of S(11), S(12), and S(34) is determined from the scattered light signals between 10 and 160 deg at the three wavelengths 442, 633, and 850 nm. A finite-difference, time-domain (FDTD) method and coated-sphere model are used to investigate the effect of nuclear refractive index on the angle-resolved Mueller elements at different wavelengths using the 3-D structures of selected B cells reconstructed from confocal images. With these results, we demonstrate the value of the light-scattering method in obtaining the cell morphology information.

Chromosomal analysis and identification based on optical tweezers and Raman spectroscopy: reply.

We appreciate the authors' comments in their reply: "On the identification of chromosomes with Raman spectroscopy: a critical comment" [Opt. Express 15, 5997 (2007)]. Their main concern with our paper is asking if the collected spectra have shown the identification or differentiation between three human chromosomes. We think this comment is flawed because the authors misunderstood the main points of the original paper and interpreted the presented spectra data (Fig. 3 and Table 1) incorrectly.

Chromosomal analysis and identification based on optical tweezers and Raman spectroscopy.

The ability to identify specific chromosomes with certainty has been established by the development of several cytogenetic techniques based on staining. Here, we report the use of a new optical technique, laser tweezers and Raman spectroscopy (LTRS), to capture and manipulate chromosomes in order to obtain their spectral patterns for molecular analysis without the need for staining. The purpose of this study was to obtain Raman spectroscopy patterns for chromosomes number 1, 2, and 3 and to test if the Raman spectroscopy pattern could be used to distinguish these three chromosomes. In our experiment, optical tweezers were used to capture the individual chromosomes and the Raman spectral patterns were collected for the trapped chromosomes. Then, the captured chromosome was manipulated with the optical tweezers and moved to another chamber through a micro - channel, in which the chromosomes were G- banded for positive identification as chromosome number 1, 2, or 3. Generalized discriminate analysis (GDA) was used to compare the Raman signatures. This analysis revealed that chromosomes 1, 2, and 3 could be distinguished and identified based on their Raman spectra. Development of this approach will lead to more rapid automatic methods for chromosome analysis and identification without the use of prior staining. Moreover, the Raman spectral patterns may lend themselves to more detailed analysis of chromosomal structure than is currently available with standard staining protocols. Such analysis may some day be useful for rapid, automated screening and diagnosis for certain cancers.