Comparative experimental infection of Listeria monocytogenes and Listeria ivanovii in bovine trophoblasts.

Listeria monocytogenes is a Gram-positive, facultative intracellular and invasive bacterium that has tropism to the placenta, and causes fetal morbidity and mortality in several mammalian species. While infection with L. monocytogenes and L. ivanovii are known as important causes of abortion and reproductive failure in cattle, the pathogenesis of maternal-fetal listeriosis in this species is poorly known. This study used the bovine chorioallantoic membrane explant model to investigate the kinetics of L. monocytogenes, L. ivanovii, and L. innocua infections in bovine trophoblastic cells for up to 8 h post infection. L. monocytogenes and L. ivanovii were able to invade and multiply in trophoblastic cells without causing cell death or inducing expression of pro-inflammatory genes. Although L. innocua was unable to multiply in bovine trophoblastic cells, it induced transcription of the pro-inflammatory mediator CXCL6. This study demonstrated for the first time the susceptibility of bovine trophoblastic cells to L. monocytogenes and L. ivanovii infection

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

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

Proteomic Profile of <i>Brucella abortus</i>-Infected Bovine Chorioallantoic Membrane Explants

<div><p><i>Brucella abortus</i> is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with <i>B</i>. <i>abortus</i> (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with <i>B</i>. <i>abortus</i> were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation.</p></div

Functional classification, subcellular localization and the experimental group which were identified the proteins expressed in uninfected CAM explants or CAM explants infected with <i>Brucella abortus</i> 2308 at 0.5 and 4 h post inoculation.

<p>Functional classification, subcellular localization and the experimental group which were identified the proteins expressed in uninfected CAM explants or CAM explants infected with <i>Brucella abortus</i> 2308 at 0.5 and 4 h post inoculation.</p

Two-dimensional gels stained with Coomassie Blue with samples from bovine CAM explants uninfected or infected with <i>B</i>. <i>abortus</i> at 0.5 or 4 h post infection.

<p>A. Number of spots selected for identification by mass spectrometry after DIGE analysis in infected and uninfected CAM explants at different time intervals after <i>B</i>. <i>abortus</i> 2308 infection. Differentially expressed spots were selected (i.e. spots that were present in one experimental group—infected or uninfected controls—and absent in the other). B. After analysis of DIGE gels using DeCyder^™2-D Differential Analysis v7.0 software (GE Healthcare, UK) for determination of protein expression levels, new two-dimensional gels were prepared, stained with Coomassie Brilliant Blue G-250, and scanned for selecting spots of interest. Numbers refer to the spot identification used in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154209#pone.0154209.s002" target="_blank">S1 Table</a>.</p

Functional classification and Venn diagram of differentially expressed proteins identified by mass spectrometry (MALDI TOF/TOF).

<p>A. Distribution of functional classification of proteins selected by DIGE analysis and identified by mass spectrometry in extracts of bovine CAM explants uninfected or infected with <i>Brucella abortus</i> at 0.5 h and 4 h post inoculation. B. Venn diagram showing differentially expressed proteins in each experimental group. Abbreviations: 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1), 3-hydroxyisobutyrate dehydrogenase, mitochondrial precursor (HIBADH), aldose 1-epimerase (GALM); abhydrolase domain-containing protein 14B (ABHD14B), activator of 90 kDa heat shock protein ATPase homolog 1 (AHSA1), adenosylhomocysteinase (AHCY), aldose reductase (AKR1B1), alpha-actinin-4 (ACTN4), alpha-fetoprotein precursor (AFP), beta-actin (ACTB), beta-hexosaminidase subunit beta preproprotein (HEXB), biliverdin reductase A (BLVRA), calmodulin (CALM1), cathepsin D (CTSD), complement component 1 Q subcomponent-binding protein, mitochondrial precursor (C1QBP), creatine kinase B-type (CKB), cytokeratin 8 (KRT8), dynein light chain roadblock-type 1 (DYNLRB1), endoplasmin precursor (HSP90B1), F-actin-capping protein subunit beta (CAPZB), galectin-7-like (LGALS7), gelsolin isoform a (GSNA), gelsolin isoform b (GSNB) heat shock cognate 71 kDa protein (HSC70), heat shock protein beta-1 (HSPB1), hemoglobin subunit beta (HBB), high-mobility group box 1-like (HMGB1), inositol-3-phosphate synthase 1 (ISYNA1), keratin 14-like (KRT14), keratin, type II cytoskeletal 7 (KRT7), LDLR chaperone MESD (MESDC2), malate dehydrogenase, cytoplasmic (MDH1), NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 (NDUFS8), ornithine aminotransferase, mitochondrial precursor (OAT), placental prolactin (PRCII), proactivator polypeptide/prosaposin (PSAPL1/PSAP), protein disulfide-isomerase A3 precursor (PDIA3), prostaglandin reductase 2 (PTGR2), ras-related protein Rab-11A (RAB11A), ribosomal protein P1-like isoform 1 (RPLP1), secretory carrier-associated membrane protein 2 (SCAMP2), serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 5 (<b>S</b>ERPINB5), similar to BolA-like protein 2 isoform 1 (BOLA2), thioredoxin-dependent peroxide reductase, mitochondrial precursor (PRDX3), toll-interacting protein (TOLLIP), transgelin-2 (TAGLN), transitional endoplasmic reticulum ATPase (VCP), transthyretin precursor (TTR), tropomyosin 4 (TPM4).</p

Number of <i>Brucella abortus</i> internalized (Log CFU/mL) in bovine CAM explants.

<p>Chorioallantoic membrane (CAM) explants were inoculated with <i>Brucella abortus</i> 2308, centrifuged for 15 min at 1000 xg and maintained at 37°C in 5% CO₂ for 30 min to allow internalization of bacteria, followed by 1 h of incubation with gentamicin, and then lysed for intracellular CFU counting. Quantification of <i>B</i>. <i>abortus</i> internalized was performed using the drop count method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154209#pone.0154209.ref019" target="_blank">19</a>] and data represent mean and standard error of three explants for each fetuses (n = 3), after logarithmic transformation. Analysis of variance (ANOVA) and Tukey's multiple comparison test were performed and no statistically significant differences were observed (P > 0.05).</p

Comparative experimental infection of <i>Listeria monocytogenes</i> and <i>Listeria ivanovii</i> in bovine trophoblasts

<div><p><i>Listeria monocytogenes</i> is a Gram-positive, facultative intracellular and invasive bacterium that has tropism to the placenta, and causes fetal morbidity and mortality in several mammalian species. While infection with <i>L</i>. <i>monocytogenes</i> and <i>L</i>. <i>ivanovii</i> are known as important causes of abortion and reproductive failure in cattle, the pathogenesis of maternal-fetal listeriosis in this species is poorly known. This study used the bovine chorioallantoic membrane explant model to investigate the kinetics of <i>L</i>. <i>monocytogene</i>s, <i>L</i>. <i>ivanovii</i>, and <i>L</i>. <i>innocua</i> infections in bovine trophoblastic cells for up to 8 h post infection. <i>L</i>. <i>monocytogenes</i> and <i>L</i>. <i>ivanovii</i> were able to invade and multiply in trophoblastic cells without causing cell death or inducing expression of pro-inflammatory genes. Although <i>L</i>. <i>innocua</i> was unable to multiply in bovine trophoblastic cells, it induced transcription of the pro-inflammatory mediator CXCL6. This study demonstrated for the first time the susceptibility of bovine trophoblastic cells to <i>L</i>. <i>monocytogenes</i> and <i>L</i>. <i>ivanovii</i> infection.</p></div

Cell mortality rate of bovine trophoblasts infected with <i>Listeria</i> spp.

<p><b>(</b>A) Cell mortality was analyzed by measuring the LDH released in the supernatant of CAM explants infected with <i>L</i>. <i>monocytogenes</i> or non infected controls at 1 and 8 hpi. (B) Cell mortality was analyzed by measuring the LDH released in the supernatant of explants uninfected or infected with <i>L</i>. <i>monocytogenes</i>, <i>L</i>. <i>innocua</i>, or <i>L ivanovii</i> at 1 and 8 hpi. The value of 100% cell mortality was calculated as optical density measured for LDH of total trophoblastic cells from explants lysed with mix substrate Cytotox kit Non-Radioactive Cytotoxicity 96 Assay. Results are presented as median and range of the data of five independent experiments performed in triplicate (Mann Whitney, * p <0.05).</p