Compensatory evolution of Pseudomonas aeruginosa’s slow growth phenotype suggests mechanisms of adaptation in cystic fibrosis

Long-term infection of the airways of cystic fibrosis patients with Pseudomonas aeruginosa is often accompanied by a reduction in bacterial growth rate. This reduction has been hypothesised to increase within-patient fitness and overall persistence of the pathogen. Here, we apply adaptive laboratory evolution to revert the slow growth phenotype of P. aeruginosa clinical strains back to a high growth rate. We identify several evolutionary trajectories and mechanisms leading to fast growth caused by transcriptional and mutational changes, which depend on the stage of adaptation of the strain. Return to high growth rate increases antibiotic susceptibility, which is only partially dependent on reversion of mutations or changes in the transcriptional profile of genes known to be linked to antibiotic resistance. We propose that similar mechanisms and evolutionary trajectories, in reverse direction, may be involved in pathogen adaptation and the establishment of chronic infections in the antibiotic-treated airways of cystic fibrosis patients.

The SKI complex is a broad-spectrum antiviral drug target

Starting from a yeast suppressor screening platform, we have identified the SKI complex as a potential broad-spectrum antiviral target. We found that the NS1 protein of influenza A virus (IAV) and the ORF4a protein of Middle East respiratory syndrome coronavirus (MERS-CoV), which both function to bind double-strand RNA and inhibit cellular interferon responses, cause a slow growth phenotype when expressed in yeast. Knockout of the components of the yeast SKI complex caused a loss of this slow growth phenotype, suggesting a functional link between the viral proteins and the SKI complex. The SKI complex is a helicase that unwinds double-strand RNA and sends it to the RNA exosome for degradation. We next investigated whether the highly conserved human SKI complex was important for replication of IAV and MERS-CoV. RNAi based experiments showed that both viruses were inhibited when the SKI complex was removed, suggesting the complex has a proviral role in replication. Through in silico modelling using the published crystal structure of the SKI complex, we looked for potential binding pockets for chemical compounds. We screened a selection of these compounds for antiviral activity and have found four different chemicals capable of inhibiting IAV infection. Our most studied of these also inhibits not only MERS-CoV, but also Ebolavirus Makona. Our data suggests the SKI complex may be a target for broad-spectrum antiviral therapy and we have multiple chemical structures from which to work to develop therapeutic approaches.

Identification of Genes Required for Glucan Exopolysaccharide Production in Lactobacillus johnsonii Suggests a Novel Biosynthesis Mechanism

ABSTRACT Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides—a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δeps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene (242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsonii. IMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

The crystal structure of the C-terminal domain of the Ebola virus nucleoprotein

Ebola virus (EBOV) and Marburg virus are members of the family Filoviridae. Both are highly pathogenic and cause hemorrhagic fever, lethal in 90% of infected people. There is fear that the viruses can be used as bioterrorism agents. There are no approved vaccines, and intense effort is underway to discover drugs targeting these viruses. The EBOV genome encodes seven proteins, two of which have no known structures: RNA polymerase (L) and nucleoprotein (NP). NP is essential for packaging viral genomic RNA into the nucleocapsid. Other viruses also contain nucleoproteins, but only the Ebola and Marburg NP proteins contain two distinct domains. The C-terminal domain (Ct; ~100 residues) has no homologues; it acts as a hub for protein-protein interactions important for the assembly of the nucleocapsid and for the interaction with the VP40 matrix protein, embedded in the viral membrane. We obtained three distinct crystal forms of the Ct domain of NP from EBOV, and solved the structures using anomalous scattering from Se, and Molecular Replacement. High-quality NMR data were also collected. The models were refined at 1.6-2.0 Å resolution to R factors ~20%. The protein has a novel fold, with topology distantly related to the β-grasp fold. In spite of its small size, the Ct domain shows high melting temperature of ~60°C. Our efforts focus on the identification of how the C-terminal domain of NP binds to its partners. As part of an effort towards anti-filovirus drug discovery, proteins NP, VP24, VP35 and VP40 are being targeted for small molecule inhibition using a yeast-based phenotypic assay. Each protein, when expressed in budding yeast, produces a slow-growth phenotype. Chemical suppressors of the slow-growth phenotype will be identified and used in viral growth assays to confirm their antiviral activity. The structure of NP will be used to complement small molecule screening methods.

The Ras/Protein Kinase A Pathway Acts in Parallel with the Mob2/Cbk1 Pathway To Effect Cell Cycle Progression and Proper Bud Site Selection

ABSTRACT In Saccharomyces cerevisiae, Ras proteins connect nutrient availability to cell growth through regulation of protein kinase A (PKA) activity. Ras proteins also have PKA-independent functions in mitosis and actin repolarization. We have found that mutations in MOB2 or CBK1 confer a slow-growth phenotype in a ras2Δ background. The slow-growth phenotype of mob2Δ ras2Δ cells results from a G1 delay that is accompanied by an increase in size, suggesting a G1/S role for Ras not previously described. In addition, mob2Δ strains have imprecise bud site selection, a defect exacerbated by deletion of RAS2. Mob2 and Cbk1 act to properly localize Ace2, a transcription factor that directs daughter cell-specific transcription of several genes. The growth and budding phenotypes of the double-deletion strains are Ace2 independent but are suppressed by overexpression of the PKA catalytic subunit, Tpk1. From these observations, we conclude that the PKA pathway and Mob2/Cbk1 act in parallel to determine bud site selection and promote cell cycle progression.