Strengthening of enterococcal biofilms by Esp.

Multidrug-resistant (MDR) Enterococcus faecalis are major causes of hospital-acquired infections. Numerous clinical strains of E. faecalis harbor a large pathogenicity island that encodes enterococcal surface protein (Esp), which is suggested to promote biofilm production and virulence, but this remains controversial. To resolve this issue, we characterized the Esp N-terminal region, the portion implicated in biofilm production. Small angle X-ray scattering indicated that the N-terminal region had a globular head, which consisted of two DEv-Ig domains as visualized by X-ray crystallography, followed by an extended tail. The N-terminal region was not required for biofilm production but instead significantly strengthened biofilms against mechanical or degradative disruption, greatly increasing retention of Enterococcus within biofilms. Biofilm strengthening required low pH, which resulted in Esp unfolding, aggregating, and forming amyloid-like structures. The pH threshold for biofilm strengthening depended on protein stability. A truncated fragment of the first DEv-Ig domain, plausibly generated by a host protease, was the least stable and sufficient to strengthen biofilms at pH ≤ 5.0, while the entire N-terminal region and intact Esp on the enterococcal surface was more stable and required a pH ≤ 4.3. These results suggested a virulence role of Esp in strengthening enterococcal biofilms in acidic abiotic or host environments.

Increased Innate Immune Susceptibility in Hyperpigmented Bacteriophage-Resistant Mutants of Pseudomonas aeruginosa.

Bacteriophage (phage) therapy is an alternative to traditional antibiotic treatments that is particularly important for multidrug-resistant pathogens, such as Pseudomonas aeruginosa. Unfortunately, phage resistance commonly arises during treatment as bacteria evolve to survive phage predation. During in vitro phage treatment of a P. aeruginosa-type strain, we observed the emergence of phage-resistant mutants with brown pigmentation that was indicative of pyomelanin. As increased pyomelanin (due to hmgA gene mutation) was recently associated with enhanced resistance to hydrogen peroxide and persistence in experimental lung infection, we questioned if therapeutic phage applications could inadvertently select for hypervirulent populations. Pyomelanogenic phage-resistant mutants of P. aeruginosa PAO1 were selected for upon treatment with three distinct phages. Phage-resistant pyomelanogenic mutants did not possess increased survival of pyomelanogenic ΔhmgA in hydrogen peroxide. At the genomic level, large (~300 kb) deletions in the phage-resistant mutants resulted in the loss of ≥227 genes, many of which had roles in survival, virulence, and antibiotic resistance. Phage-resistant pyomelanogenic mutants were hypersusceptible to cationic peptides LL-37 and colistin and were more easily cleared in human whole blood, serum, and a murine infection model. Our findings suggest that hyperpigmented phage-resistant mutants that may arise during phage therapy are markedly less virulent than their predecessors due to large genomic deletions. Thus, their existence does not present a contraindication to using anti-pseudomonal phage therapy, especially considering that these mutants develop drug susceptibility to the familiar FDA-approved antibiotic, colistin.

Bacterial Cytological Profiling Identifies Rhodanine-Containing PAINS Analogs as Specific Inhibitors of Escherichia coli Thymidylate Kinase In Vivo.

In this study, we sought to determine whether an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against Escherichia coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays, as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the half-maximal inhibitory concentration in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. Although it is thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. Moreover, perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that bacterial cytological profiling can identify multiple pathways that are inhibited by an individual molecule. IMPORTANCE We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and nonspecific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false-positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

COVID-19 threatens faculty diversity: postdoctoral scholars call for action.

Despite substantial investment and effort by federal agencies and institutions to improve the diversity of the professoriate, progress is excruciatingly slow. One program that aims to enhance faculty diversity is the Institutional Research and Academic Career Development Award (IRACDA) funded by the National Institutes of Health/National Institute of General Medical Sciences. IRACDA supports the training of a diverse cohort of postdoctoral scholars who will seek academic research and teaching careers. The San Diego IRACDA program has trained 109 postdoctoral scholars since its inception in 2003; 59% are women and 63% are underrepresented (UR) Black/African-American, Latinx/Mexican-American, and Indigenous scientists. Sixty-four percent obtained tenure-track faculty positions, including a substantial 32% at research-intensive institutions. However, the COVID-19 pandemic crisis threatens to upend IRACDA efforts to improve faculty diversity, and academia is at risk of losing a generation of diverse, talented scholars. Here, a group of San Diego IRACDA postdoctoral scholars reflects on these issues and discusses recommendations to enhance the retention of UR scientists to avoid a "lost generation" of promising UR faculty scholars.

Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals.

The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcare's most potent antibiotics are becoming obsolete. Approximately two-thirds of the world's antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 104 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.

Review of the genus Fontidessus Miller & Spangler, 2008 (Coleoptera, Dytiscidae, Hydroporinae, Bidessini) with description of four new species.

The genus Fontidessus Miller & Spangler, 2008 (Coleoptera: Dytiscidae: Hydroporinae: Bidessini) is reviewed. The genus now includes seven species with three previously described, and four new species described here: F. microphthalmus Miller & Montano, sp. n.; F. bettae Miller & Montano, sp. n.; F. christineae Miller & Montano, sp. n., and F. aquarupe Miller & Montano, sp. n. Each species is diagnosed and described, including the previously known species, based on new specimens and new information. Habitus, male genitalia and other diagnostic features are illustrated for each species. A key to the seven species is provided. Fontidessus species are unique to hygropetric habitats in the Guiana Shield craton of northern South American.

ResumenEl género Fontidessus Miller & Spangler, 2008 (Coleoptera: Dytiscidae: Hydroporinae: Bidessini) es revisado. El género incluye siete especies, con tres descritas anteriormente, y cuatro nuevas especies descritas aquí: F. microphthalmus Miller & Montano, sp. n., F. bettae Miller & Montano, sp. n., F. christineae Miller & Montano, sp. n., y F. aquarupe Miller & Montano, sp. n. Las especies se diagnostican y se describen, incluyendo las especies previamente conocidas, basado en nuevos ejemplares e información. Habitus, genitales masculinos y otras características diagnósticas se ilustran para cada especie. Se presenta una clave para las siete especies. Fontidessus son exclusivos de hábitats hygropétricos en el Cratón de Guayana Escudo del norte de América del Sur.