SARS-CoV-2 Infection in Captive Hippos (Hippopotamus amphibius), Belgium.

Two adult female hippos in Zoo Antwerp who were naturally infected with SARS-CoV-2 showed nasal discharge for a few days. Virus was detected by immunocytochemistry and PCR in nasal swab samples and by PCR in faeces and pool water. Serology was also positive. No treatment was necessary

A Translocated Bacterial Protein Protects Vascular Endothelial Cells from Apoptosis

The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane–associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium

The Streptococcus pneumoniae cia Regulon: CiaR Target Sites and Transcription Profile Analysis

The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S. pneumoniae genome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimal cia regulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and the htrA-spo0J region. In addition, the transcription profile of cia loss-of-function mutants and those with an apparent activated cia system representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed the cia-dependent expression of seven putative target loci and revealed three additional cia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, no cia-dependent expression could be detected. Furthermore, the competence regulon, including the comCDE operon required for induction of competence, was completely repressed by the cia system

Comparison of the Anti-Apoptotic Activities of BepA Homologs

<div><p>(A) Anti-apoptotic activities of wild-type and isogenic Δ<i>virB4</i> mutant strains of <i>Bh</i> in comparison with <i>Bq</i> and <i>Bt</i>.</p><p>(B) Domain structure of <i>Bh</i> BepA, its paralogs <i>Bh</i> BepB and <i>Bh</i> BepC, and the orthologs <i>Bt</i> BepA and <i>Bq</i> BepA1/<i>Bq</i> BepA2. These homologs contain conserved FIC and BID domains in their N-terminal and C-terminal regions, respectively, except for <i>Bq,</i> where the orthologous locus is split between these domains into two separate open reading frames by an internal stop codon.</p><p>(C) Anti-apoptotic activity of BepA homologs. HUVECs were infected with the indicated <i>Bh</i> strains for 24 h, followed by apoptotic induction with actinomycin D for 12 h. Caspase-3/-7 activities were then determined with a specific fluorogenic peptide substrate. Mean and SD are illustrated for one representative out of three independent experiments.</p></div

A Pharmacologically Increased cAMP Level in ECs Mimics the Anti-Apoptotic Effect of <i>Bh</i> BepA

<p>HUVECs were infected for 24 h with the indicated <i>Bh</i> strains or left uninfected (control) in the absence or presence of either (A) forskolin (1 μM) and IBMX (10 μM) or (B) dibutyryl cAMP (1 mM). If indicated, apoptosis was then induced with actinomycin D. Caspase-3/-7 activities were determined 9 h later. All strains were tested in triplicates a minimum of three times.</p

Anti-Apoptotic BepA Homologs Mediate an Increase in Intracellular cAMP and an Upregulation of cAMP Response Genes

<div><p>HUVECs were infected with the indicated <i>Bh</i> strains. The means and SD of one out of three independent replica experiments performed in triplicate samples are presented.</p><p>(A) IL-8 was determined in culture supernatants after infection for 54 h with MOI = 300.</p><p>(B) Expression of the cAMP-responsive genes <i>pde4B</i> and <i>crem</i> was determined by quantitative real-time PCR after infection for 54 h with MOI = 300.</p><p>(C) Intracellular cAMP levels were determined after infection for 30 h with MOI = 150.</p><p>In (B) and (C), samples marked with an asterisk (<i>p</i> < 0.05) differ statistically significantly from Δ<i>bepA–G</i> using an unpaired Student's <i>t</i>-test.</p></div

Delineation and Subcellular Localization of the Region of <i>Bh</i> BepA Required for Inhibition of Apoptosis

<div><p>(A) Schematic presentation of N-terminal GFP fusions to parts of <i>Bh</i> BepA.</p><p>(B) Determination of apoptosis following ectopic expression of the constructs illustrated in (A). GFP-BepA fusion proteins were ectopically expressed in HUVECs for 24 h, followed by 12 h incubation in the presence or absence of actinomycin D as indicated. The loss of membrane asymmetry in transfected cells (GFP-positive) was then quantified by flow cytometric analysis of APC-Annexin V– and PI-stained cells, allowing us to quantify the rate of apoptotic cells (Annexin V–positive and PI-negative). The means and SD of three independent replica experiments are shown. The <i>p</i>-values were determined by using an unpaired Student's <i>t</i>-test.</p><p>(C) The GFP–<i>Bh</i> BepA fusion proteins illustrated in (A) were ectopically expressed for 24 h in HEK293T cells. Cells were immunochemically stained to label the cell surface with Texas Red–conjugated WGA. Confocal pictures were taken for GFP (green channel) and WGA (red channel) in the <i>xy</i>-plane (upper image, overlay both channels, bar = 10 μm), and also in the <i>xz</i>-plane at the dashed stroke line (lower images, single channels and overlay channels).</p><p>(D) Fractionation of GFP–<i>Bh</i> BepA fusion proteins into membrane and cytosolic fractions by ultracentrifugation of post-nuclear extracts harvested from transfected HEK293T cells.</p></div

<i>Bh</i> BepA Is a Genuine VirB/VirD4 T4S Substrate That Is Translocated into ECs

<div><p>(A) The bars indicate the parts of <i>Bh</i> BepA or <i>Bh</i> BepD that were fused to Cya. These reporter fusions were used to monitor translocation via the VirB/VirD4 system. All constructs contain an N-terminal FLAG epitope for immunological detection of the encoded fusion protein.</p><p>(B) Quantification of the amount of intracellular cAMP in HUVECs infected for 20 h with the indicated bacterial strains (MOI = 300). Isogenic strains with a functional (wild-type) or non-functional (Δ<i>virB4</i>) VirB/VirD4 T4S system were used to express the different Cya reporter constructs. Mean and SD are shown for one representative out of three independent replica experiments.</p><p>(C) Steady-state FLAG-Cya fusion protein levels of the indicated <i>Bh</i> strains grown on IPTG-containing medium.</p></div