Aminopeptidase secreted by Chromobacterium sp. Panama inhibits dengue virus infection by degrading the E protein.

Dengue virus (DENV) is the most prevalent and burdensome arbovirus transmitted by Aedes mosquitoes, against which there is only a limited licensed vaccine and no approved drug treatment. A Chromobacterium species, C. sp. Panama, isolated from the midgut of A. aegypti is able to inhibit DENV replication within the mosquito and in vitro. Here we show that C. sp. Panama mediates its anti-DENV activity through secreted factors that are proteinous in nature. The inhibitory effect occurs prior to virus attachment to cells, and is attributed to a factor that destabilizes the virion by promoting the degradation of the viral envelope protein. Bioassay-guided fractionation, coupled with mass spectrometry, allowed for the identification of a C. sp. Panama-secreted neutral protease and an aminopeptidase that are co-expressed and appear to act synergistically to degrade the viral envelope (E) protein and thus prevent viral attachment and subsequent infection of cells. This is the first study characterizing the anti-DENV activity of a common soil and mosquito-associated bacterium, thereby contributing towards understanding how such bacteria may limit disease transmission, and providing new tools for dengue prevention and therapeutics

Proteins recovered from active anti-DENV fraction of <i>C</i>. <i>sp</i>. Panama.

<p>Proteins recovered from active anti-DENV fraction of <i>C</i>. <i>sp</i>. Panama.</p

Protein extracts of culture supernatants of <i>C</i>. <i>sp</i>. Panama inhibit DENV entry by mediating degradation of the E protein.

<p>(<b>A</b>) DENV titers following exposure to a protein extract of the <i>C</i>. <i>sp</i>. Panama culture supernatant before or after viral attachment to BHK-21 cells; significance determined using unpaired t-tests. Raw underlying data available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006443#pntd.0006443.s002" target="_blank">S2 Table</a>. (<b>B</b>) Representative negative stained transmission EM images of control DENV particles (arrow head) and those treated with <i>C</i>. <i>sp</i>. Panama culture supernatant protein extract (empty arrow head); low magnification: 93,000X, high magnification: 245,000X. (<b>C</b>) Size distribution of the viral particles as measured from the transmission EM images collected in B; mean and standard deviation indicated; significance determined using unpaired t-test. (<b>D</b>) Western blot (anti-E) of DENV proteins when exposed to the <i>C</i>. <i>sp</i>. Panama culture supernatant protein extract. (<b>E</b>) Fluorescence microscopy showing DENV attachment to BHK-21 cells in the presence of the protein extract of the <i>C</i>. <i>sp</i>. Panama. Cells were fixed with 4% PFA, stained for DNA (DAPI-blue), and F-actin (Phalloidin-green). Membrane-bound DENV (arrow head) were immunostained with 4G2 anti-E antibody (Red). ns, not significant; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001.</p

Anti-DENV activity of <i>Chromobacterium sp</i>. Panama is mediated by secreted proteinous factors.

<p>DENV virus titers: (<b>A</b>) in the presence or absence of culture supernatant of <i>C</i>. <i>cp</i>. Panama following infection in BHK-21 and C6/36 cells, as determined by plaque or focus forming assays, respectively; (<b>B</b>) in BHK-21 cells upon exposure to different solvent extracts of culture supernatants of <i>C</i>. <i>sp</i>. Panama; (<b>C</b>) in BHK-21 cells in the presence or absence of culture supernatant of <i>C</i>. <i>cp</i>. Panama incubated for 1 hour at the indicated temperatures; and (<b>D</b>) upon exposure to protein extract of the culture supernatant of <i>C</i>. <i>sp</i>. Panama prepared by 70% ammonium sulfate precipitation (Pellet), the desalted supernatant of that extraction (Sup.) or control buffer (0.1M Tris-HCl). Significance determined using unpaired t-tests; (ns, not significant; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001). Raw underlying data available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006443#pntd.0006443.s002" target="_blank">S2 Table</a>.</p

Aminopeptidase secreted by <i>C</i>. <i>sp</i>. Panama induces DENV E protein degradation, thereby inhibiting viral entry.

<p>(<b>A</b>) Protein sequence alignment of CSPP0261 and orthologs in <i>Pseudomonas aeruginosa</i> (Paer, UniProt: P14756) and <i>Vibrio proteolyticus</i> (Vpro, UniProt: Q00971). Conserved active and metal binding sites highlighted in blue (in both orthologs) and red (in <i>P</i>. <i>aeruginosa</i> ortholog). (<b>B</b>) Protein sequence alignment of CSPP0262 and ortholog in <i>Vibrio proteolyticus</i>. Conserved active and metal binding sites highlighted in blue (Vpro, UniProt: Q01693). (<b>C</b>) Western blot (anti-E-DENV) of DENV2 proteins when exposed to the culture supernatant of <i>C</i>. <i>sp</i>. Panama with or without supplementation with 10 μM bestatin or 100 μM phosphoramidon (P-ramidon). (<b>D</b>) DENV titers in BHK-21 cells following incubation of the virus with the <i>C</i>. <i>sp</i>. Panama culture supernatant with or without supplementation with 10 μM bestatin or 100 μM phosphoramidon (P-ramidon); significance determined using unpaired t-tests (ns, not significant; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001). Raw underlying data available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006443#pntd.0006443.s002" target="_blank">S2 Table</a>.</p

The Roles, Challenges, and Merits of the p Value.

Since the 18th century, the p value has been an important part of hypothesis-based scientific investigation. As statistical and data science engines accelerate, questions emerge: to what extent are scientific discoveries based on p values reliable and reproducible? Should one adjust the significance level or find alternatives for the p value? Inspired by these questions and everlasting attempts to address them, here, we provide a systematic examination of the p value from its roles and merits to its misuses and misinterpretations. For the latter, we summarize modest recommendations to handle them. In parallel, we present the Bayesian alternatives for seeking evidence and discuss the pooling of p values from multiple studies and datasets. Overall, we argue that the p value and hypothesis testing form a useful probabilistic decision-making mechanism, facilitating causal inference, feature selection, and predictive modeling, but that the interpretation of the p value must be contextual, considering the scientific question, experimental design, and statistical principles