Building a Resilient Scientific Network for COVID-19 and Beyond.

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates that the global scientific community monitor, assess, and respond to the evolving coronavirus disease (COVID-19) pandemic. But the current reactive approach to emerging variants is ill-suited to address the quickly evolving and ever-changing pandemic. To tackle this challenge, investments in pathogen surveillance, systematic variant characterization, and data infrastructure and sharing across public and private sectors will be critical for planning proactive responses to emerging variants. Additionally, an emphasis on incorporating real-time variant identification in point-of-care diagnostics can help inform patient treatment. Active approaches to understand and identify "immunity gaps" can inform design of future vaccines, therapeutics, and diagnostics that will be more resistant to novel variants. Approaches where the scientific community actively plans for and anticipates changes to infectious diseases will result in a more resilient system, capable of adapting to evolving pathogens quickly and effectively.

Are COVID-19 Vaccine Boosters Needed? The Science behind Boosters.

Waning vaccine-induced immunity coupled with the emergence of SARS-CoV-2 variants has led to increases in breakthrough infections, prompting consideration for vaccine booster doses. Boosters have been reported to be safe and increase SARS-CoV-2-specific neutralizing antibody levels, but how these doses impact the trajectory of the global pandemic and herd immunity is unknown. Information on immunology, epidemiology, and equitable vaccine distribution should be considered when deciding the timing and eligibility for COVID-19 vaccine boosters.

Small-Molecule Acetylation by GCN5-Related N-Acetyltransferases in Bacteria.

Acetylation is a conserved modification used to regulate a variety of cellular pathways, such as gene expression, protein synthesis, detoxification, and virulence. Acetyltransferase enzymes transfer an acetyl moiety, usually from acetyl coenzyme A (AcCoA), onto a target substrate, thereby modulating activity or stability. Members of the GCN5- N -acetyltransferase (GNAT) protein superfamily are found in all domains of life and are characterized by a core structural domain architecture. These enzymes can modify primary amines of small molecules or of lysyl residues of proteins. From the initial discovery of antibiotic acetylation, GNATs have been shown to modify a myriad of small-molecule substrates, including tRNAs, polyamines, cell wall components, and other toxins. This review focuses on the literature on small-molecule substrates of GNATs in bacteria, including structural examples, to understand ligand binding and catalysis. Understanding the plethora and versatility of substrates helps frame the role of acetylation within the larger context of bacterial cellular physiology.

New AMP-forming acid:CoA ligases from Streptomyces lividans, some of which are posttranslationally regulated by reversible lysine acetylation.

In nature, organic acids are a commonly used source of carbon and energy. Many bacteria use AMP-forming acid:CoA ligases to convert organic acids into their corresponding acyl-CoA derivatives, which can then enter metabolism. The soil environment contains a broad diversity of organic acids, so it is not surprising that bacteria such as Streptomyces lividans can activate many of the available organic acids. Our group has shown that the activity of many acid:CoA ligases is posttranslationally controlled by acylation of an active-site lysine. In some cases, the modification is reversed by deacylases of different types. We identified eight new acid:CoA ligases in S. lividans TK24. Here, we report the range of organic acids that each of these enzymes can activate, and determined that two of the newly identified CoA ligases were under NAD+ -dependent sirtuin deacylase reversible lysine (de)acetylation control, four were not acetylated by two acetyltransferases used in this work, and two were acetylated but not deacetylated by sirtuin. This work provides insights into the broad organic-acid metabolic capabilities of S. lividans, and sheds light into the control of the activities of CoA ligases involved in the activation of organic acids in this bacterium.

Staphylococcus aureus modulates the activity of acetyl-Coenzyme A synthetase (Acs) by sirtuin-dependent reversible lysine acetylation.

Lysine acylation is a posttranslational modification used by cells of all domains of life to modulate cellular processes in response to metabolic stress. The paradigm for the role of lysine acylation in metabolism is the acetyl-coenzyme A synthetase (Acs) enzyme. In prokaryotic and eukaryotic cells alike, Acs activity is downregulated by acetylation and reactivated by deacetylation. Proteins belonging to the bacterial GCN5-related N-acetyltransferase (bGNAT) superfamily acetylate the epsilon amino group of an active site lysine, inactivating Acs. A deacetylase can remove the acetyl group, thereby restoring activity. Here we show the Acs from Staphylococcus aureus (SaAcs) activates acetate and weakly activates propionate, but does not activate >C3 organic acids or dicarboxylic acids (e.g. butyrate, malonate and succinate). SaAcs activity is regulated by AcuA (SaAcuA); a type-IV bGNAT. SaAcuA can acetylate or propionylate SaAcs reducing its activity by >90% and 95% respectively. SaAcuA also succinylated SaAcs, with this being the first documented case of a bacterial GNAT capable of succinylation. Inactive SaAcsAc was deacetylated (hence reactivated) by the NAD+ -dependent (class III) sirtuin protein deacetylase (hereafter SaCobB). In vivo and in vitro evidence show that SaAcuA and SaCobB modulate the level of SaAcs activity in S. aureus.

Insights into the Function of the N-Acetyltransferase SatA That Detoxifies Streptothricin in Bacillus subtilis and Bacillus anthracis.

Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that Bacillus subtilis and Bacillus anthracis encode the GCN5-related N-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of the B. anthracis Ames SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations into satA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCE This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation.

Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA+ restored streptothricin resistance to B. subtilissatA (BsSatA) strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-coenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd [dissociation constant] = 1 µM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilissatA+ in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil.IMPORTANCE Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in B. subtilis and B. anthracis The initial characterization of the enzyme provides insights into its mechanism of catalysis.

Genome-wide redistribution of H3K27me3 is linked to genotoxic stress and defective growth.

H3K9 methylation directs heterochromatin formation by recruiting multiple heterochromatin protein 1 (HP1)-containing complexes that deacetylate histones and methylate cytosine bases in DNA. In Neurospora crassa, a single H3K9 methyltransferase complex, called the DIM-5,-7,-9, CUL4, DDB1 Complex (DCDC), is required for normal growth and development. DCDC-deficient mutants are hypersensitive to the genotoxic agent methyl methanesulfonate (MMS), but the molecular basis of genotoxic stress is unclear. We found that both the MMS sensitivity and growth phenotypes of DCDC-deficient strains are suppressed by mutation of embryonic ectoderm development or Su-(var)3-9; E(z); Trithorax (set)-7, encoding components of the H3K27 methyltransferase Polycomb repressive complex-2 (PRC2). Trimethylated histone H3K27 (H3K27me3) undergoes genome-wide redistribution to constitutive heterochromatin in DCDC- or HP1-deficient mutants, and introduction of an H3K27 missense mutation is sufficient to rescue phenotypes of DCDC-deficient strains. Accumulation of H3K27me3 in heterochromatin does not compensate for silencing; rather, strains deficient for both DCDC and PRC2 exhibit synthetic sensitivity to the topoisomerase I inhibitor Camptothecin and accumulate γH2A at heterochromatin. Together, these data suggest that PRC2 modulates the response to genotoxic stress.