Targeting the Replication Initiator of the Second Vibrio Chromosome: Towards Generation of Vibrionaceae-Specific Antimicrobial Agents

The Vibrionaceae is comprised of numerous aquatic species and includes several human pathogens, such as Vibrio cholerae, the cause of cholera. All organisms in this family have two chromosomes, and replication of the smaller one depends on rctB, a gene that is restricted to the Vibrionaceae. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. Here, we carried out a high throughput cell-based screen to find small molecule inhibitors of RctB. We identified a compound that blocked growth of an E. coli strain bearing an rctB-dependent plasmid but did not influence growth of E. coli lacking this plasmid. This compound, designated vibrepin, had potent cidal activity against V. cholerae and inhibited the growth of all vibrio species tested. Vibrepin blocked RctB oriCII unwinding, apparently by promoting formation of large non-functional RctB complexes. Although vibrepin also appears to have targets other than RctB, our findings suggest that RctB is an attractive target for generation of novel antibiotics that only block growth of vibrios. Vibrio-specific agents, unlike antibiotics currently used in clinical practice, will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms

Evaluation of individual and ensemble probabilistic forecasts of COVID-19 mortality in the United States

Short-term probabilistic forecasts of the trajectory of the COVID-19 pandemic in the United States have served as a visible and important communication channel between the scientific modeling community and both the general public and decision-makers. Forecasting models provide specific, quantitative, and evaluable predictions that inform short-term decisions such as healthcare staffing needs, school closures, and allocation of medical supplies. Starting in April 2020, the US COVID-19 Forecast Hub (https://covid19forecasthub.org/) collected, disseminated, and synthesized tens of millions of specific predictions from more than 90 different academic, industry, and independent research groups. A multimodel ensemble forecast that combined predictions from dozens of groups every week provided the most consistently accurate probabilistic forecasts of incident deaths due to COVID-19 at the state and national level from April 2020 through October 2021. The performance of 27 individual models that submitted complete forecasts of COVID-19 deaths consistently throughout this year showed high variability in forecast skill across time, geospatial units, and forecast horizons. Two-thirds of the models evaluated showed better accuracy than a naïve baseline model. Forecast accuracy degraded as models made predictions further into the future, with probabilistic error at a 20-wk horizon three to five times larger than when predicting at a 1-wk horizon. This project underscores the role that collaboration and active coordination between governmental public-health agencies, academic modeling teams, and industry partners can play in developing modern modeling capabilities to support local, state, and federal response to outbreaks

The United States COVID-19 Forecast Hub dataset

Academic researchers, government agencies, industry groups, and individuals have produced forecasts at an unprecedented scale during the COVID-19 pandemic. To leverage these forecasts, the United States Centers for Disease Control and Prevention (CDC) partnered with an academic research lab at the University of Massachusetts Amherst to create the US COVID-19 Forecast Hub. Launched in April 2020, the Forecast Hub is a dataset with point and probabilistic forecasts of incident cases, incident hospitalizations, incident deaths, and cumulative deaths due to COVID-19 at county, state, and national, levels in the United States. Included forecasts represent a variety of modeling approaches, data sources, and assumptions regarding the spread of COVID-19. The goal of this dataset is to establish a standardized and comparable set of short-term forecasts from modeling teams. These data can be used to develop ensemble models, communicate forecasts to the public, create visualizations, compare models, and inform policies regarding COVID-19 mitigation. These open-source data are available via download from GitHub, through an online API, and through R packages

Neuraminidase Expressed by Streptococcus pneumoniae Desialylates the Lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae: a Paradigm for Interbacterial Competition among Pathogens of the Human Respiratory Tract

Both Neisseria meningitidis and Haemophilus influenzae are capable of mimicking host structures by decorating their lipopolysaccharides with sialic acid. We show that a neuraminidase expressed by Streptococcus pneumoniae (NanA) is able to desialylate the cell surfaces of both these species, which reside in and possibly compete for the same host niche

Plasmids used in this study.

<p>Plasmids used in this study.</p

Influence of vibrepin on <i>V. cholera</i>e.

<p>A) The optical density or number of colony forming units in cultures of the <i>V. cholerae</i> N16961 with the indicated concentrations of vibrepin. Representative results from at least 3 independent experiments are shown. B–E) Vibrepin induces aggregation of RctB <i>in vivo</i>. Representative fields of phase contrast (left) and GFP signal (right) of <i>V. cholerae</i> cells expressing RctB-GFP (B and C) or untagged GFP (D and E) are shown. (B and D) no treatment; (C and E) 1 µg of vibrepin was added 1 hr prior to the imaging. Bar = 2 µm.</p

Influence of vibrepin on an <i>E. coli</i> strain bearing an RctB-dependent plasmid in the absence of kanamycin.

<p>A–C) DH5α (A), DH5α/pYB289 (B), or DH5α/pYB376 (C) were incubated in LB media in test tubes with either vibrepin (16 µg/ml, thick black lines) or DMSO (gray lines). OD_{600 nm} and colony forming units (CFU) were determined at the indicated times. Representative growth curves from 3 or more independent experiments are presented. D) The relative amount of the <i>oriCII</i>-based plasmid pYB289 in DH5α after 4 hr of treatment with vibrepin (16 µg/ml) or DMSO. The amount of pYB289 relative to chromosomal DNA was determined before (t = 0) and 4 hrs after treatment (t = 4) using Southern hybridization. The relative amount of pYB289 at t = 0 was set as 1; the mean and standard deviations after 4 hr were calculated from 3 independent experiments.</p

Compound C2 does not inhibit growth of <i>V. cholerae</i> but C2 and vibrepin inhibit <i>B. subtilis</i> and <i>S. aureus</i> growth.

<p>A) Structure of compound C2. B–F) Growth curves of the indicated strains with addition of the indicated concentrations (µg/ml) of vibrepin (blue line), C2 (red line) or DMSO control (gray line). Representative growth curves (average of triplicate wells) from 3 or more independent experiments are presented.</p

Identification of a small molecule that inhibits RctB-dependent replication.

<p>A) Schematic of the RctB-dependent <i>oriCII</i>-bearing plasmid, pYB289, used in the high throughput screen for small molecule inhibitors of RctB. B) Structure of vibrepin, one of the compounds identified in the screen. C–G) growth curves of <i>E. coli</i> DH5α harboring no plasmid, pYB289 (encoding wt RctB), pYB340 (encoding RctB[L365I]), pYB344 (encoding RctB [P516Q]) and pWSK129 (a non-<i>oriCII</i>-based plasmid). Thick black lines represent growth in the presence of vibrepin (16 µg/ml) and gray lines represent growth in the presence of DMSO. Representative growth curves (average of triplicate wells from a plate reader) from 3 or more independent experiments are presented. The higher initial optical density of the cultures containing vibrepin is due to the incomplete solubility of this compound in LB media at 16 µg/ml. This is apparent in H) which shows the optical density generated by vibrepin (thick line), or DMSO (thin gray line) without the addition of cells.</p