Early transcriptional responses of bovine chorioallantoic membrane explants to wild type, ΔvirB2 or ΔbtpB Brucella abortus infection

Abstract

The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; P<0.05) were functionally classified, and transcripts related to defense and inflammation were assessed by quantitative real time RT-PCR. Infection with wild type B. abortus resulted in slightly more genes with decreased than increased transcription levels. Conversely, infection of trophoblastic cells with the ΔvirB2 or the ΔbtpB mutant strains, that lack a functional T4SS or that has impaired inhibition of TLR signaling, respectively, induced more upregulated than downregulated genes. Wild type Brucella abortus impaired transcription of host genes related to immune response when compared to ΔvirB and ΔbtpB mutants. Our findings suggest that proinflammatory genes are negatively modulated in bovine trophoblastic cells at early stages of infection. The virB operon and btpB are directly or indirectly related to modulation of these host genes. These results shed light on the early interactions between B. abortus and placental tissue that ultimately culminate in inflammatory pathology and abortion.

Figure 1. Internalization of wild type, ΔvirB2 or ΔbtpB Brucella abortus by bovine trophoblastic cells.

Chorioallantoic membrane (CAM) explants were inoculated, incubated for 4 h, followed by 1 h incubation with gentamicin, and then lysed for intracellular CFU counting. Data represents the average log of CFU numbers from CAM explants, from three independent experiments performed in triplicates. Data underwent logarithmic transformation followed by analysis of variance (ANOVA) and the Tukey's multiple comparison test with significance level of P<0.05.

Figure 2. Gene transcription profiling of the host response to B. abortus strains at 4 hours after infection of bovine trophoblastic cells.

(A) A heat map of gene transcription changes in bovine trophoblastic cells infected with wild type, ΔvirB2 and ΔbtpB B. abortus– strains, compared to mock-infected controls. (B) Fold changes in gene transcription for genes that were significantly (P<0.05) up or downregulated during wild type, ΔvirB2 and ΔbtpB B. abortus infection compared to mock-infected controls. These data represent results from pools of total RNA obtained from triplicates of CAM explants obtained from four independent experiments. Increase or decrease in mRNA levels are indicated in red or green, respectively.

Figure 3. Venn diagram indicating the number of genes with significant changes in mRNA levels assessed by microarray analysis in bovine trophoblastic cells from CAM explants obtained from 4 placentas at the last trimester of pregnancy (n = 4) infected with wild type, ΔvirB2 and ΔbtpB B. abortus compared to mock-infected controls.

Changes in transcription higher than 2-fold and values of P<0.05 were considered significant. (A) Downregulated genes and (B) upregulated genes. Abbreviations: 4105740 BARC 9BOV cDNA clone 9BOV30_O11 5′ (CK974693), HNF1 homeobox B (HNF1B), Hypothetical protein LOC616908 (LOC616908), LB01613.CR_H15 GC_BGC-16 cDNA clone IMAGE:8082593 (DT810899), Homeobox A4 (HOXA4), N-myc downstream regulated 1 (NDRG1), Ovo-like 1 (Drosophila) (OVOL1), PREDICTED: wingless-type MMTV integration site family, member 8B (WNT8B), Hypothetical LOC516011 (MEI), Q9V5V8_DROME CG13214-PA, isoform A (Q9V5V8), BDP1 protein Fragment (BDP1), Chromosome 15 open reading frame 26 ortholog (C21H15ORF26), Calmodulin binding transcription activator 1 (CAMTA1), GRM1 protein Fragment (GRM1), hCG1653800-like (LOC526866), Na+/K+ transporting ATPase interacting 2 (NKAIN2), Ankyrin repeat domain 52 (ANKRD52), 001128BAMA005012HT BAMA cDNA (BAMA), Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), Heat shock 70 kDa protein 1-like (HSPA1L), Hypothetical LOC505551 (MYCBPAP), NDRG family member 4 (NDRG4), PG12B_HUMAN (Q9BX93) Group XIIB secretory phospholipase A2-like protein precursor [TC318659] (PG12B), WSC domain containing 1 (WSCD1), BP250013A20E1 Soares normalized bovine placenta cDNA (BF042192), Contactin 4, isoform c precursor (CNTN4), LysM, putative peptidoglycan-binding, domain containing 3 (LYSMD3), Transient receptor potential channel 2 (TRPC2).

Figure 4. Functional classification of genes with significant changes in transcription levels in bovine trophoblastic cells at 4 hours after infection with wild type, ΔvirB2, and ΔbtpB B. abortus.

(A) Downregulated genes, and (B) upregulated genes in comparison to mock-infected controls.

Figure 5. Changes in transcript abundance of defense and inflammation genes during infection of bovine trophoblastic cells with wild type, ΔvirB2 and ΔbtpB B. abortus, compared to mock-infected controls.

(A) Heat map of transcription changes during infections with wild type, ΔvirB2 and ΔbtpB B. abortus. (B–D) Fold changes in gene transcription of genes classified as defense and inflammation that were significantly (P<0.05) up or downregulated during wild type (B), ΔvirB2 (C), and ΔbtpB (D) B. abortus, compared to mock-infected controls. Fold changes >2 with P<0.05 were considered significant. Abbreviations: apolipoprotein L, 3 (APOL3), butyrophilin-like 9 (BTNL9), cathelicidin 4 (CATHL4), CCAAT/enhancer binding protein (C/EBP), epsilon (CEBPE), CD200 molecule (CD200), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-C motif) receptor-like 2 (CCRL2), chemokine (C-X-C motif) ligand 12 (CXCL12), chemokine (C-X-C motif) receptor 5 (CXCR5), chemokine binding protein 2 (CCBP2), complement component 3a receptor 1 (C3AR1), complement factor H (CFH), complement factor I (CFI), family with sequence similarity 19 (chemokine (C-C motif)-like), member A4 (FAM19A4), Fc fragment of IgG binding protein (FCGBP), fms-related tyrosine kinase 3-like (FLT3), heat shock 70 kDa protein 1-like (HSPA1L), HLX H2.0-like homeobox (HLX), IFN-alpha C (IFNAC), integrin, alpha M (complement component 3 receptor 3 subunit) (ITGAM), interferon alpha G (IFNAG), interferon-alphaomega-like (LOC787343), interleukin 1 family, member 6 (epsilon)-like (IL1F6), interleukin (IL15), interleukin 2 receptor, alpha (IL2RA), mitogen-activated protein kinase 14 (MAPK14),MPV17 mitochondrial membrane protein-like, nuclear gene encoding mitochondrial protein (MPV17L), N-myc downstream regulated 1 (NDRG1), pellino homolog 2 (PELI2), peptidoglycan recognition protein 1 (PGLYRP1), PIGR polymeric immunoglobulin receptor (PIGR), platelet/endothelial cell adhesion molecule (PECAM1), radical S-adenosyl methionine domain containing 2 (RSAD2), rCG28728-like (GBP4), serine peptidase inhibitor, Kazal type 5 (SPINK5), toll-like receptor 6 (TLR6), TRAF2 and NCK interacting kinase (TNIK), transient receptor potential cation channel subfamily V member 1-like, transcript, variant 2 (TRPV1), transmembrane 4 L six family member 19 (TM4SF19), tumor necrosis factor (ligand) superfamily, member 10-like (TNFSF10), tumor necrosis factor receptor superfamily, member 13C (TNFRSF13C), tumor necrosis factor receptor superfamily, member 9 (TNFRSF9).

Figure 6. Validation of the microarray results by real-time qRT-PCR.

Bars represent the average of fold change values from three independent experiments. Chorioallantoic membranes were infected with wild type B. abortus for evaluation of HSPA1L and PELI2 expression; with the ΔvirB2 Brucella abortus strain for evaluation of IL15, TNFRSF9, IL1F6, and TM4SF19; and with ΔbtpB Brucella abortus strain for evaluation of PECAM1. Abbreviations: interleukin 15 (IL15), heat shock 70 kDa protein 1-like (HSPA1L), tumor necrosis factor receptor superfamily, member 9 (TNFRSF9), apolipoprotein L, 3 (APOL3), pellino homolog 2 (PELI2), platelet/endothelial cell adhesion molecule (PECAM1), interleukin 1 family, member 6 (epsilon)-like (IL1F6), transmembrane 4 L six family member 19 (TM4SF19).