Microarray-Based Identification of Differentially Expressed Genes in Intracellular <i>Brucella abortus</i> within RAW264.7 Cells

<div><p><i>Brucella spp</i>. is a species of facultative intracellular Gram-negative bacteria that induces abortion and causes sterility in domesticated mammals and chronic undulant fever in humans. Important determinants of <i>Brucella</i>’s virulence and potential for chronic infection include the ability to circumvent the host cell’s internal surveillance system and the capability to proliferate within dedicated and non-dedicated phagocytes. Hence, identifying genes necessary for intracellular survival may hold the key to understanding <i>Brucella</i> infection. In the present study, microarray analysis reveals that 7.82% (244/3334) of all <i>Brucella abortus</i> genes were up-regulated and 5.4% (180/3334) were down-regulated in RAW264.7 cells, compared to free-living cells in TSB. qRT-PCR verification further confirmed a >5-fold up-regulation for fourteen genes. Functional analysis classified <i>araC</i>, <i>ddp</i>, and <i>eryD</i> as to partake in information storage and processing, <i>alp</i>, <i>flgF</i> and <i>virB9</i> to be involved in cellular processes, <i>hpcd</i> and <i>aldh</i> to play a role in metabolism, <i>mfs</i> and <i>nikC</i> to be involved in both cellular processes and metabolism, and four hypothetical genes (<i>bruAb1_1814</i>, <i>bruAb1_0475</i>, <i>bruAb1_1926</i>, and <i>bruAb1_0292</i>) had unknown functions. Furthermore, we constructed a <i>B. abortus</i> 2308 mutant Δ<i>ddp</i> where the <i>ddp</i> gene is deleted in order to evaluate the role of <i>ddp</i> in intracellular survival. Infection assay indicated significantly higher adherence and invasion abilities of the Δ<i>ddp</i> mutant, however it does not survive well in RAW264.7 cells. <i>Brucella</i> may survive in hostile intracellular environment by modulating gene expression.</p></div

Heat map and hierarchical clustering.

<p>Hierarchical clustering was performed based on all differentially expressed gene data. The results of hierarchical clustering on conditions show distinguishable gene expression profiling between samples. “Red” indicates high relative expression, and “blue” indicates low relative expression. “S2308” refers to the groups of free-living bacteria in TSB, “C2308” refers to the groups of intracellular bacteria.</p

Significant pathway of differentially expressed genes.

<p>“P value” indicates the enrichment p-value of the Pathway ID determined by the Fisher's exact test. “Enrichment_Score” indicates the enrichment score value of the Pathway ID, which equals -log₁₀ (p-value). A: Significant pathways of up-regulated genes; B: Significant pathway of down-regulated genes.</p

Infection assay.

<p>RAW264.7 cells were infected with Δ<i>ddp</i> mutant or wild type strain 2308 at a MOI 100∶1. A: The CFUs of total bacteria adherence to RAW264.7 cells. B: The CFUs of total bacteria invasion of RAW264.7 cells. C: Bacterial survival in RAW264.7 cells at 1 h, 12 h, 24 h, 36 h and 48 h p.i. All data are expressed as mean ± standard deviation and subjected to Student’s t-test, **, p≤0.01.</p

Intracellular transcriptional level of <i>Brucella</i> genes obtained by qRT-PCR and analysis of gene functional characteristics.

a<p><i>B. abortus</i> ORFs listed are used locus tag of genes in <i>B. abortus</i> strain 9-941.</p>b<p>Subcellular locations were predicted by the PSORTb v.3.0 server. Available: <a href="http://www.psort.org/psortb/index.html" target="_blank">http://www.psort.org/psortb/index.html</a>. Accessed 10 December 2012.</p>c<p>Functional characterization of the proteins was predicted by the software COGnitor. Available: <a href="http://www.ncbi.nlm.nih.gov/COG/old/xognitor.html" target="_blank">http://www.ncbi.nlm.nih.gov/COG/old/xognitor.html</a>. Accessed 10 December 2012. Functional categories: (1) Information storage and processing: (J: Translation, ribosomal structure and biogenesis; K: Transcription; L: DNA replication, recombination and repair); (2) Cellular processes: (D: Cell division and chromosome partitioning; O: Posttranslational modification, protein turnover, chaperones; M: Cell envelope biogenesis, outer membrane; P: Inorganic ion transport and metabolism; T: Signal transduction mechanisms); (3) Metabolism: (C: Energy production and conversion; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism); (4) Poorly characterized: (R: General function prediction only; S: Function unknown).</p>d<p>-: No related COG.</p>e<p>Results are expressed as 2^−ΔΔCt. Figures = 1 indicate that the gene is expressed similarly in both conditions, figures >1 indicate that the gene is over expressed in intracellular <i>Brucella</i>, and figures <1 indicate that the gene is expressed less in intracellular <i>Brucella</i>.</p

Strains, plasmids, and primers used in this study.

<p>Strains, plasmids, and primers used in this study.</p

<i>Riemerella anatipestifer</i> M949_1360 Gene Functions on the Lipopolysaccharide Biosynthesis and Bacterial Virulence - Fig 1

<p><b>Characterization of <i>R</i>. <i>anatipestifer</i> mutant strain RAΔ604 and complementation strain cRAΔ604</b> (A) Southern blot analysis. Lane 1: PEP<i>4351</i> (positive control); Lane 2: <i>R</i>. <i>anatipestifer</i> CH3 (negative control); Lane 3: mutant strain RAΔ604. (B) Schematic chart of Tn<i>4351</i> insertion in RAΔ604 chromosome at 810 bp of the M949_1360 gene. The flanking genes M949_1359, M949_1361, and M949_1362 were annotated as hypothetical protein, glycosyl transferase group 1 and lipopolysaccharide-modifying protein respectively. (C) qPCR analysis. Changes in mRNA levels are expressed as fold expression and calculated using the comparative CT (2^-ΔΔCT) method. The expression of M949_1360 was inactivated in RAΔ604. However, no significant difference was shown in expression levels of M949_1359 and M949_1361 between <i>R</i>. <i>anatipestifer</i> CH3 and RAΔ604 (ns, <i>p</i> > 0.05). Error bars represent standard deviations from three replicates (***, <i>p</i> < 0.01). (D) Identification of the complementation strain cRAΔ604 by PCR. Takara DL2000 marker; lanes 1–3: <i>R</i>. <i>anatipestifer</i> 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RAΔ604 (lane 2), and the complementation strain cRAΔ604 (lane 3), showing a 744-bp fragment of 16S rRNA; lanes 4–6: 963-bp fragment of M949_1360 gene was amplified from the WT strain CH3 (lane 4), and the complementation strain cRAΔ604 (lane 6), no 963-bp fragment of M949_1360 gene was amplified from the mutant strain RAΔ604 (lane 5); lanes 7–11: a 459-bp fragment of <i>R</i>. <i>anatipestifer</i> dnaB gene, but not a <i>E</i>. <i>coli</i> phoA gene was amplified from the WT strain CH3 (lane 7), the mutant strain RAΔ604 (lane 8) and the complementation strain cRAΔ604 (lane 9); lane 10: a 720-bp fragment of <i>E</i>. <i>coli</i> phoA gene was amplified from <i>E</i>. <i>coli</i> S17-1 strain; lane 11: distilled water, as a negative control.</p

Bacterial adherence and invasion assays.

<p>(A) Adherence assay. (B) Invasion assay. Each point represents the mean ± standard deviation. Both adherence and invasion capacities of the mutant strain RAΔ604 were decreased, as compared with those of <i>R</i>. <i>anatipestifer</i> WT strain CH3 (***, <i>p</i> < 0.01; **, <i>p</i> < 0.05). The complementation strain cRAΔ604 partially recovered adherence and invasion capacities (ns, <i>p</i> > 0.05).</p

LPS structure analysis.

<p>(A) Silver staining of purified LPS. Each lane contained 0.5 μg of purified LPS. Lane M: Prestained Protein Ladder (Thermo Fisher Scientific, Inc.); Lane 1: <i>R</i>. <i>anatipestifer</i> CH3 LPS; Lane 2: The mutant strain RAΔ604 LPS; Lane 3: The complementation strain cRAΔ604 LPS. (B) Western blot analysis. Each lane contained 10⁸−10⁹ CFU of bacteria in 10 μl volume. After SDS-PAGE and transferred to a NC membrane, the NC membrane was incubated with anti-CH3 LPS MAb 1C1 (1:2000 dilution). Lane M: Prestained Protein Ladder (Thermo Fisher Scientific, Inc.); Lane 1: <i>R</i>. <i>anatipestifer</i> WT strain CH3; Lane 2: The mutant strain RAΔ604 LPS; Lane 3: The complementation strain cRAΔ604.</p