Commensal bacteria contribute to immune homeostasis in the gastrointestinal tract; however, the underlying mechanisms for this are not well understood. A single dose of exopolysaccharide (EPS) from the probiotic spore-forming bacterium Bacillus subtilis protects mice from acute colitis induced by the enteric pathogen Citrobacter rodentium. Adoptive transfer of macrophage-rich peritoneal cells from EPS-treated mice confers protection from disease to recipient mice. In vivo, EPS induces development of anti-inflammatory M2 macrophages in a TLR4-dependent manner, and these cells inhibit T cell activation both in vitro and in C. rodentium-infected mice. In vitro, the M2 macrophages inhibit both CD4+ and CD8+ T cells, and the inhibition of CD4+ T cells is dependent on TGF-β, whereas inhibition of CD8+ T cells is dependent on both TGF-β and PD-L1. We suggest that administration of B. subtilis EPS can be utilized to broadly inhibit T cell activation and thus control T cell-mediated immune responses in numerous inflammatory diseases.

Zymomonas mobilis produces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to the yeast Saccharomyces cerevisiae for industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle quickly and exhibit higher resistance to harmful chemicals than single cells. While spontaneous floc-forming Z. mobilis mutants have been described, little is known about the natural conditions that induce Z. mobilis floc formation or about the genetic factors involved. Here we found that wild-type Z. mobilis forms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential for both floc formation and survival in a minimal aerobic medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in a minimal aerobic medium, providing a physiological role for this enzyme, which has previously been found to be disadvantageous in a rich aerobic medium. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation. IMPORTANCE The bacterium Zymomonas mobilis is best known for its anaerobic fermentative lifestyle, in which it converts glucose into ethanol at a yield surpassing that of yeast. However, Z. mobilis also has an aerobic lifestyle, which has confounded researchers with its attributes of poor growth, accumulation of toxic acetic acid and acetaldehyde, and respiratory enzymes that are detrimental for aerobic growth. Here we show that a major Z. mobilis respiratory enzyme and the ability to form multicellular aggregates, called flocs, are important for survival, but only during aerobic growth in a medium containing a minimum set of nutrients required for growth. Supplements, such as vitamins or yeast extract, promote aerobic growth and, in some cases, inhibit floc formation. We propose that Z. mobilis likely requires aerobic respiration and floc formation in order to survive in natural environments that lack protective factors found in supplements such as yeast extract.

Sindbis virus (SINV) is an enveloped, single-stranded RNA virus, which is transmitted via mosquitos to a wide range of vertebrate hosts. SINV produced by vertebrate, baby hamster kidney (BHK) cells is more than an order of magnitude less infectious than SINV produced from mosquito (C6/36) cells. The cause of this difference is poorly understood. In this study, charge detection mass spectrometry was used to determine the masses of intact SINV particles isolated from BHK and C6/36 cells. The measured masses are substantially different: 52.88 MDa for BHK derived SINV and 50.69 MDa for C6/36 derived. Further analysis using several mass spectrometry-based methods and biophysical approaches indicates that BHK derived SINV has a substantially higher mass than C6/36 derived because in the lipid bilayer, there is a higher portion of lipids containing long chain fatty acids. The difference in lipid composition could influence the organization of the lipid bilayer. As a result, multiple stages of the viral lifecycle may be affected *

No abstract

The bacterium Zymomonas mobilis naturally produces ethanol at near theoretical maximum yields, making it of interest for industrial ethanol production. Zymomonas mobilis requires the vitamin pantothenate for growth. Here we characterized the genetic basis for the Z. mobilis pantothenate auxotrophy. We found that this auxotrophy is due to the absence of a single gene, panD, encoding aspartate-decarboxylase. Heterologous expression of Escherichia coli PanD in Z. mobilis or supplementation of the growth medium with the product of PanD activity, β-alanine, eliminated the need for exogenous pantothenate. We also determined that Z. mobilis IlvC, an enzyme better known for branched-chain amino acid synthesis, is required for pantothenate synthesis in Z. mobilis, as it compensates for the absence of PanE, another pantothenate synthesis pathway enzyme. In addition to contributing to an understanding of the nutritional requirements of Z. mobilis, our results have led to the design of a more cost-effective growth medium.

Zymomonas mobilis produces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to yeast for industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle 15 quickly and exhibit higher resistance to harmful chemicals. While spontaneous floc-forming Z. mobilis mutants have been described, little is known about the natural conditions that induce Z. mobilis floc formation and the genetic factors involved. Here we found that wild-type Z. mobilis forms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential 20 for both floc formation and survival in an aerobic minimal medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in an aerobic minimal medium, providing a physiological role for this enzyme which has previously been found to be disadvantageous in aerobic rich media. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation. 25

Enveloped viruses can cause devastating zoonotic diseases and are the most likely to cause global pandemics. We identified a new class of small-molecule sulfur-containing antiviral compounds (XM series) that broadly inhibit enveloped viruses. The antivirals’ mechanism of action was explored via various multidisciplinary approaches, concluding that the XM antivirals alter membrane lipid chemical compositions, increase membrane order deep within the hydrophobic region of the bilayer, and increase membrane phase transition temperatures. Such effects cause inhibition of membrane fusion and viral entry, while leaving the viral glycoproteins and genomes largely unaffected. Consequently, we tested whether these features would lead to effective whole inactivated enveloped virus vaccines. As a proof-of-principle, we generated an inactivated influenza virus (IIV) vaccine using compound XM-01 (XM-01-IIV). We compared this new vaccine to traditional paraformaldehyde-inactivated, to control compound-inactivated, and to live virus vaccines, using a mouse model of disease. Excitingly, compared to a traditional IIV vaccine, XM-01-IIV vaccination improved neutralizing antibody responses against both hemagglutinin and neuraminidase, and decreased mouse morbidity and mortality following influenza virus challenge. Therefore, this study uncovers a novel class of broadly acting antivirals that enhances influenza virus vaccine development and offers great potential for broadly generating future highly-potent inactivated enveloped virus vaccines.

The alphavirus Chikungunya virus is transmitted to humans via infected mosquitos. Most infected humans experience symptoms which can range from short-term fatigue and fever to debilitating arthritis that can last for months or years. Some patients relapse and experience symptoms months or years after the initial bout of disease. The capsid protein of Chikungunya virus forms a shell around the viral RNA genome; this structure is called the nucleocapsid core.The core protects the genome during virus transmission and with the correct environmental trigger, this proteinaceous shell dissociates and releases the viral genome to initiate infection. We hypothesized that targeting compounds to interfere with the nucleocapsid core's function would constrain virus spread either by inhibiting the release of viral genomes during entry or by reducing the number of infectious virus particles assembled. We implemented a high throughput, in vitro, FRET-based assay to monitor nucleic acid packaging by purified Chikungunya capsid protein as a proxy for nucleocapsid core assembly and disassembly. We screened 10,000 compounds and found 45 that substantially modulated the assembly of core-like particles. A subset of compounds was selected to study their effects in virus-infected vertebrate cells. Our results show that four compounds inhibit infectious virus production by at least 90% in a dose-dependent manner. The most promising inhibitor was tested and found to reduce the amount of nucleocapsid cores inside the cell during Chikungunya virus infection. These compounds could be the foundation for anti-viral therapeutics.Keywords (6 max): Chikungunya virus; antiviral compounds; nucleocapsid core; fluorescence quenching-based high throughput screen