Computational prediction of essential genes in an unculturable endosymbiotic bacterium, Wolbachia of Brugia malayi

New England Biolabs, 240 County Road, Ipswich, MA 01938-2723, USA.
BMC Microbiology (Impact Factor: 2.98). 11/2009; 9:243. DOI: 10.1186/1471-2180-9-243
Source: PubMed

ABSTRACT Wolbachia (wBm) is an obligate endosymbiotic bacterium of Brugia malayi, a parasitic filarial nematode of humans and one of the causative agents of lymphatic filariasis. There is a pressing need for new drugs against filarial parasites, such as B. malayi. As wBm is required for B. malayi development and fertility, targeting wBm is a promising approach. However, the lifecycle of neither B. malayi nor wBm can be maintained in vitro. To facilitate selection of potential drug targets we computationally ranked the wBm genome based on confidence that a particular gene is essential for the survival of the bacterium.
wBm protein sequences were aligned using BLAST to the Database of Essential Genes (DEG) version 5.2, a collection of 5,260 experimentally identified essential genes in 15 bacterial strains. A confidence score, the Multiple Hit Score (MHS), was developed to predict each wBm gene's essentiality based on the top alignments to essential genes in each bacterial strain. This method was validated using a jackknife methodology to test the ability to recover known essential genes in a control genome. A second estimation of essentiality, the Gene Conservation Score (GCS), was calculated on the basis of phyletic conservation of genes across Wolbachia's parent order Rickettsiales. Clusters of orthologous genes were predicted within the 27 currently available complete genomes. Druggability of wBm proteins was predicted by alignment to a database of protein targets of known compounds.
Ranking wBm genes by either MHS or GCS predicts and prioritizes potentially essential genes. Comparison of the MHS to GCS produces quadrants representing four types of predictions: those with high confidence of essentiality by both methods (245 genes), those highly conserved across Rickettsiales (299 genes), those similar to distant essential genes (8 genes), and those with low confidence of essentiality (253 genes). These data facilitate selection of wBm genes for entry into drug design pipelines.

Download full-text


Available from: Sanjay Kumar, Jul 15, 2015
  • Source
    • "The comparison with DBD resulted in the identification of those FDA approved drugs or drug like compounds for which the experimental evidence of binding with proteins similar to the S. aureus is available. Hence, these steps helped in advancing the identification of potential drug or lead like molecules for future medicinal chemistry based approaches (Holman et al., 2009). Out of 51 prioritized targets, all belonged to the unique metabolic pathways of MRSA including two component system, β lactamase resistance, secondary bile acid biosynthesis, nitrogen metabolism, methane metabolism, peptidoglycan biosynthesis, phosphotransferase system, benzoate degradation, Dalanine metabolism, limonene, bacterial secretion system and pinene degradation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Multiple Drug Resistant (MDR) bacteria are no more inhibited by the front line antibiotics due to extreme resistance. Methicillin Resistant Staphylococcus aureus (MRSA) is one of the MDR pathogens notorious for its widespread infection around the world. The high resistance acquired by MRSA needs a serious concern and efforts should be carried out for the discovery of better therapeutics. With this aim, we designed a comparison of the metabolic pathways of the pathogen, MRSA strain 252 (MRSA252) with the human host (i.e., Homo sapiens) by using well-established in silico methods. We identified several metabolic pathways unique to MRSA (i.e., absent in the human host). Furthermore, a subtractive genomics analysis approach was applied for retrieval of proteins only from the unique metabolic pathways. Subsequently, proteins of unique MRSA pathways were compared with the host proteins. As a result, we have shortlisted few unique and essential proteins that could act as drug targets against MRSA. We further assessed the druggability potential of the shortlisted targets by comparing them with the DrugBank Database (DBD). The identified drug targets could be useful for an effective drug discovery phase. We also searched the sequences of unique as well as essential enzymes from MRSA in Protein Data Bank (PDB). We shortlisted at least 12 enzymes for which there was no corresponding deposition in PDB, reflecting that their crystal structures are yet to be solved! We selected Glutamate synthase out of those 12 enzymes owing to its participation in significant metabolic pathways of the pathogen e.g., Alanine, Aspartate, Glutamate and Nitrogen metabolism and its evident suitability as drug target among other MDR bacteria e.g., Mycobacteria. Due to the unavailability of any crystal structure of Glutamate synthase in PDB, we generated the 3D structure by homology modeling. The modeled structure was validated by multiple analysis tools. The active site of Glutamate synthase was identified by not only superimposing the template structure (PDB ID: 1E0A) over each other but also by the Parallel-ProBiS algorithm. The identified active site was further validated by cross-docking the co-crystallized ligand (2-oxoglutaric acid; AKG) of PDB ID: 1LLW. It was concluded that the comparative metabolic in silico analysis together with structure-based methods provides an effective approach for the identification of novel antibiotic targets against MRSA. Copyright © 2014 Elsevier B.V. All rights reserved.
    Gene 11/2014; 556(2). DOI:10.1016/j.gene.2014.11.056 · 2.08 Impact Factor
  • Source
    • "In order to target focused library screening to drugs with predicted activity against Wolbachia, a bioinformatic analysis of predicted essential genes was undertaken. An essentiality score for each predicted gene of wBm was determined by two separate approaches (Holman et al. 2009). The first method compared each gene to entries in DEG (the Database of Essential Genes), a collection of *5000 experimentally identified essential genes from 15 different bacterial species, to predict essential genes that are mostly conserved across the bacterial domain. "
    [Show abstract] [Hide abstract]
    ABSTRACT: SUMMARY Anti-Wolbachia therapy delivers safe macrofilaricidal activity with superior therapeutic outcomes compared to all standard anti-filarial treatments, with the added benefit of substantial improvements in clinical pathology. These outcomes can be achieved, in principle, with existing registered drugs, e.g. doxycycline, that are affordable, available to endemic communities and have well known, albeit population-limiting, safety profiles. The key barriers to using doxycycline as an mass drug administration (MDA) strategy for widespread community-based control are the logistics of a relatively lengthy course of treatment (4-6 weeks) and contraindications in children under eight years and pregnancy. Therefore, the primary goal of the anti-Wolbachia (A·WOL) consortium is to find drugs and regimens that reduce the period of treatment from weeks to days (7 days or less), and to find drugs which would be safe in excluded target populations (pregnancy and children). A secondary goal is to refine regimens of existing antibiotics suitable for a more restricted use, prior to the availability of a regimen that is compatible with MDA usage. For example, for use in the event of the emergence of drug-resistance, in individuals with high loiasis co-infection and at risk of severe adverse events (SAE) to ivermectin, or in post-MDA 'endgame scenarios', where test and treat strategies become more cost effective and deliverable.
    Parasitology 07/2013; 141(1):1-9. DOI:10.1017/S0031182013001108 · 2.35 Impact Factor
  • Source
    • "We also calculated that the core set of orthologs shared between all of the completed Wolbachia genomes, including wOo, totals 575 genes (Supplemental Table S6). A comparison between this core set and those of Holman et al. (2009) (based on computational rankings of the essentiality of genes in the wBm genome by two different methods) displayed good concordance, as only three genes with high essentiality scores were absent from a Wolbachia genome (Supplemental Table S7). Notably, all of the genes encoding the type-IV secretion system are also intact in wOo (Supplemental Table S8) and conserved across all other Wolbachia genomes (Pichon et al. 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.
    Genome Research 08/2012; 22(12). DOI:10.1101/gr.138420.112 · 13.85 Impact Factor
Show more