Predicting drug targets by homology modelling of Pseudomonas aeruginosa proteins of unknown function

The efficacy of antibiotics to treat bacterial infections declines rapidly due to antibiotic resistance. This problem has stimulated the development of novel antibiotics, but most attempts have failed. Consequently, the idea of mining uncharacterized genes of pathogens to identify potential targets for entirely new classes of antibiotics was proposed. Without knowing the biochemical function of a protein, it is difficult to validate its potential for drug targeting; therefore, the functional characterization of bacterial proteins of unknown function must be accelerated. Here, we present a paradigm for comprehensively predicting the biochemical functions of a large set of proteins encoded by hypothetical genes in human pathogens to identify candidate drug targets. A high-throughput approach based on homology modelling with ten templates per target protein was applied to the set of 2103 P. aeruginosa proteins encoded by hypothetical genes. The >21000 homology modelling results obtained and available biological and biochemical information about several thousand templates were scrutinized to predict the function of reliably modelled proteins of unknown function. This approach resulted in assigning one or often multiple putative functions to hundreds of enzymes, ligand-binding proteins and transporters. New biochemical functions were predicted for 41 proteins whose essential or virulence-related roles in P. aeruginosa were already experimentally demonstrated. Eleven of them were shortlisted as promising drug targets that participate in essential pathways (maintaining genome and cell wall integrity), virulence-related processes (adhesion, cell motility, host recognition) or antibiotic resistance, which are general drug targets. These proteins are conserved in other WHO priority pathogens but not in humans; therefore, they represent high-potential targets for preclinical studies. These and many more biochemical functions assigned to uncharacterized proteins of P. aeruginosa, made available as PaPUF database, may guide the design of experimental screening of inhibitors, which is a crucial step towards the validation of the highest-potential targets for the development of novel drugs against P. aeruginosa and other high-priority pathogens.

Predicting drug targets by homology modelling of Pseudomonas aeruginosa proteins of unknown function

Efficacies of antibiotics to treat bacterial infections rapidly decline due to antibiotic resistance. This stimulated the development of novel antibiotics, but most attempts failed. As a response, the idea of mining uncharacterised genes of pathogens to identify potential targets for entirely new classes of antibiotics raised. Without knowing the biochemical function of a protein it is difficult to validate its potential for drug targeting; therefore progress in the functional characterisation of bacterial proteins of an unknown function must be accelerated. Here we present a paradigm for comprehensively predicting biochemical functions of a large set of proteins encoded by hypothetical genes in human pathogens, to identify candidate drug targets. A high-throughput approach based on homology modelling with ten templates per target protein was applied on the set of 2103 P. aeruginosa proteins encoded by hypothetical genes. Obtained >21000 homology modelling results and available biological and biochemical information about several thousand templates was scrutinised to predict the function of reliably modelled proteins of unknown function. This approach resulted in assigning, one or often multiple, putative functions to hundreds of enzymes, ligand-binding proteins and transporters. New biochemical functions were predicted for 41 proteins whose essential or virulence-related roles in P. aeruginosa were already experimentally demonstrated. Eleven of them were shortlisted as promising drug targets which participate in essential pathways (maintaining genome and cell wall integrity), virulence-related processes (adhesion, cell motility, host recognition) or antibiotic resistance, which are general drug targets. These proteins are conserved among other WHO priority pathogens but not in humans, therefore they represent high-potential targets for pre-clinical studies. These and many more biochemical functions assigned to uncharacterised proteins of P. aeruginosa, available as PaPUF database may guide the design of experimental screening of inhibitors which is a crucial step toward validation of the most potential targets for the development of novel drugs against P. aeruginosa and other high-priority pathogens.

Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ

Biofilms are community structures of bacteria enmeshed in a self-produced matrix of exopolysaccharides. The biofilm matrix serves numerous roles, including resilience and persistence, making biofilms a subject of research interest among persistent clinical pathogens of global health importance. Our current understanding of the underlying biochemical pathways responsible for biosynthesis of these exopolysaccharides is largely limited to Gram-negative bacteria. Clostridia are a class of Gram-positive, anaerobic and spore-forming bacteria and include the important human pathogens Clostridium perfringens, Clostridium botulinum and Clostridioides difficile, among numerous others. Several species of Clostridia have been reported to produce a biofilm matrix that contains an acetylated glucan linked to a series of hypothetical genes. Here, we propose a model for the function of these hypothetical genes, which, using homology modelling, we show plausibly encode a synthase complex responsible for polymerization, modification and export of an O-acetylated cellulose exopolysaccharide. Specifically, the cellulose synthase is homologous to that of the known exopolysaccharide synthases in Gram-negative bacteria. The remaining proteins represent a mosaic of evolutionary lineages that differ from the described Gram-negative cellulose exopolysaccharide synthases, but their predicted functions satisfy all criteria required for a functional cellulose synthase operon. Accordingly, we named these hypothetical genes ccsZABHI, for the Clostridial cellulose synthase (Ccs), in keeping with naming conventions for exopolysaccharide synthase subunits and to distinguish it from the Gram-negative Bcs locus with which it shares only a single one-to-one ortholog. To test our model and assess the identity of the exopolysaccharide, we subcloned the putative glycoside hydrolase encoded by ccsZ and solved the X-ray crystal structure of both apo- and product-bound CcsZ, which belongs to glycoside hydrolase family 5 (GH-5). Although not homologous to the Gram-negative cellulose synthase, which instead encodes the structurally distinct BcsZ belonging to GH-8, we show CcsZ displays specificity for cellulosic materials. This specificity of the synthase-associated glycosyl hydrolase validates our proposal that these hypothetical genes are responsible for biosynthesis of a cellulose exopolysaccharide. The data we present here allowed us to propose a model for Clostridial cellulose synthesis and serves as an entry point to an understanding of cellulose biofilm formation among class Clostridia.

Identifying biofilm regulators as novel targets for antimicrobial drug design

<p>Antibiotic treatment regularly fails to cure patients suffering from infections caused by adaptively resistant microbial communities, referred to as biofilms. Even though at least two thirds of all clinical infections are associated with biofilms, there are no biofilm-specific therapies on the market or in clinical trials. <em>Pseudomonas aeruginosa</em> is a remarkably antibiotic resistant, nosocomial pathogen and biofilm-former that causes morbidity and mortality especially in cystic fibrosis and immunocompromised patients. This project aims to identify regulatory genes associated with drug resistance in <em>P. aeruginosa</em> biofilms to provide novel biofilm-specific targets for the design of potent drugs. A genome-wide screen of <em>P. aeruginosa</em> burn wound isolate UCBPP-PA14 identified 362 genes involved in biofilm formation, including dozens of regulatory and hypothetical genes. I will discuss regulatory as well as metabolic genes corresponding to the known resistome of antimicrobials.</p>

Novel mechanisms for phosphate acquisition in abundant rhizosphere-dwelling Bacteroidetes

Global food production is reliant on the application of finite phosphorus (P) fertilisers. Numerous negative consequences associated with intensive P fertilisation have resulted in a high demand to find alternative sustainable methods that will enhance crop P uptake. Bacteroidetes, primarily from the genus Flavobacterium, have recently been shown to be abundant members of the plant microbiome, but their general ecological role and potential to mobilise P in the rhizosphere remains very poorly characterised. Here, we sought to determine the P mobilisation potential of Flavobacterium strains isolated from the rhizosphere of oilseed rape (Brassica napus L.). We show that these Flavobacterium strains possess novel mechanisms for P mobilisation and subsequent acquisition. These include the constitutive and inducible expression of completely novel and phylogenetically distinct phosphatases, the phosphate starvation inducible expression of uncharacterised and hypothetical genes and gene clusters and, for the first time, the involvement of outer membrane SusCD transport complexes (usually associated with carbohydrate transport) in P acquisition. The genes encoding these unusual phosphate starvation inducible proteins were enriched in plant-associated Flavobacterium strains suggesting that this machinery represents niche-adaptive strategies for overcoming P scarcity in this genus. We propose that abundant rhizosphere-dwelling Flavobacterium spp. have evolved unique mechanisms for coping with Pi-stress which may provide novel solutions for future sustainable agricultural practices.

A Specific Sugar Moiety in the Lactococcus lactis Cell Wall Pellicle Is Required for Infection by CHPC971, a Member of the Rare 1706 Phage Species

ABSTRACT Lactococcus lactis is a Gram-positive bacterium widely used as a starter culture for the production of different dairy products, especially a large variety of cheeses. Infection of lactococcal starter cultures by bacteriophages is one of the major causes of fermentation failure and often leads to production halt. Lactococcal bacteriophages belonging to the c2, 936, and P335 species are the most commonly isolated in dairy plants and have been extensively investigated in the past three decades. Information regarding bacteriophages belonging to less commonly isolated species is, on the other hand, less extensive, although these phages can also contribute to starter culture infection. Here, we report the nucleotide sequence of the newly isolated L. lactis phage CHPC971, belonging to the rare 1706 species of lactococcal phages. We investigated the nature of the host receptor recognized by the phage and collected evidence that strongly suggests that it binds to a specific sugar moiety in the cell wall pellicle of its host. An in silico analysis of the genome of phage CHPC971 identified the hypothetical genes involved in receptor binding. IMPORTANCE Gathering information on how lactococcal bacteriophages recognize their host and proliferate in the dairy environment is of vital importance for the establishment of proper starter culture rotation plans and to avoid fermentation failure and consequent great economic losses for dairy industries. We provide strong evidence on the type of receptor recognized by a newly isolated 1706-type lactococcal bacteriophage, increasing knowledge of phage-host interactions relevant to dairying. This information can help to prevent phage infection events that, so far, are hard to predict and avoid.

Genetics

Genetics studies the problem of heredity, namely why offspring resemble their parents. The field emerged in 1900 with the rediscovery of the 1865 work of Gregor Mendel. William Bateson called the new field ‘genetics’ in 1905, and W. Johannsen used the term ‘gene’ in 1909. By analysing data about patterns of inheritance of characters, such as yellow and green peas, Mendelian geneticists infer the number and type of hypothetical genes. The major components of the theory of the gene, which proposed the model of genes as beads on a string, were in place by the 1920s. In the 1930s, the field of population genetics emerged from the synthesis of results from Mendelian genetics with Darwinian natural selection. Population geneticists study the distribution of genes in the gene pool of a population and changes caused by selection and other factors. The 1940s and 1950s saw the development of molecular genetics, which investigates problems about gene reproduction, mutation and function at the molecular level. Philosophical issues arise: the question about the evidence for the reality of hypothetical genes, and the status of Mendel’s laws, given that they are not universal generalizations. Debates have occurred about the nature of the relation between Mendelian and molecular genetics. Population genetics provides the perspective of the gene as the unit of selection in evolutionary theory. Molecular genetics and its accompanying technologies raise ethical issues about humans’ genetic information, such as the issue of privacy of information about one’s genome and the morality of changing a person’s genes. The nature–nurture debate involves the issue of genetic determinism, the extent to which genes control human traits and behaviour.

Broad-spectrum adaptive antibiotic resistance associated with Pseudomonas aeruginosa mucin-dependent surfing motility

Surfing motility is a novel form of surface adaptation exhibited by the nosocomial pathogen, Pseudomonas aeruginosa, in the presence of the glycoprotein mucin that is found in high abundance at mucosal surfaces especially the lungs of cystic fibrosis and bronchiectasis patients. Here we investigated the adaptive antibiotic resistance of P. aeruginosa under conditions in which surfing occurs compared to cells undergoing swimming. P. aeruginosa surfing cells were significantly more resistant to several classes of antibiotics including aminoglycosides, carbapenems, polymyxins, and fluroquinolones. This was confirmed by incorporation of antibiotics into growth medium, which revealed a concentration-dependent inhibition of surfing motility that occurred at concentrations much higher than those needed to inhibit swimming. To investigate the basis of resistance, RNA-Seq was performed and revealed that surfing influenced the expression of numerous genes. Included amongst genes dysregulated under surfing conditions were multiple genes from the Pseudomonas resistome, which are known to affect antibiotic resistance when mutated. Screening transposon mutants in these surfing-dysregulated resistome genes revealed that several of these mutants exhibited changes in susceptibility to one or more antibiotics under surfing conditions, consistent with a contribution to the observed adaptive resistance. In particular, several mutants in resistome genes, including armR, recG, atpB, clpS, nuoB, and certain hypothetical genes such as PA5130, PA3576 and PA4292, showed contributions to broad-spectrum resistance under surfing conditions and could be complemented by their respective cloned genes. Therefore, we propose that surfing adaption led to extensive multidrug adaptive resistance as a result of the collective dysregulation of diverse genes.