Characterization of meningitis-causing bacteria, with focus on genomic and pangenomic study of multi-drug resistant Streptococcus pneumoniae from cerebrospinal fluid.

Streptococcus pneumoniae is a major cause of meningitis in under developed countries with low vaccination rates and high antibiotic resistance. This study aimed to analyze 83 suspected meningitis patients in Karachi for the detection of S. pneumoniae followed by its whole genome sequencing and Pan Genome analysis. Out of the 83 samples collected, 33 samples with altered physical (turbidity), cytological (white blood cell count) and biochemical (total protein and total glucose concentrations) parameters indicated potential meningitis cases, while these parameters were within normal healthy ranges in remaining 50 samples. Latex particle agglutination (LPA) was performed on the 33 samples, revealing 20 positive cases of bacterial meningitis. The PCR and culturing methods revealed 5 S. pneumoniae isolates. Antibiotic susceptibility tests showed that one S. pneumoniae strain was resistant to erythromycin, levofloxacin, and tetracycline. Whole-genome sequencing of this resistant strain was performed and S. pneumoniae was confirmed with MLST analysis, while it had > 2.3 Mb genome and a single repUS43 plasmid. In CARD analysis, the strain had tet(M), ermB, RlmA(II), patB, pmrA, and patA ARGs, which could provide resistance against tetracycline, macrolide, fluoroquinolone, and glycopeptide antibiotics. Phylogenetic analysis revealed that the isolate was closely related to strains from Hungary and the USA. Pan-genome analysis with 144 genome assemblies from NCBI database showed that 1101 non-redundant core genes were shared between all strains. This study gives valuable understanding into the prevalence and characterization of meningitis-causing bacteria in Karachi, Pakistan  with prime focus on multi-drug resistant S. pneumoniae.

Exploring the extrachromosomal plasmid rDNA of Naegleria fowleri AY27 genotype II: A human brain-eating amoeba via high-throughput sequencing.

Naegleria fowleri, also known as brain-earing amoeba, causes severe and rapidly fatal CNS infection in humans called primary amebic meningoencephalitis (PAM). The DNA from the N. fowleri clinical isolate was sequenced for circular extrachromosomal ribosomal DNA (CERE - rDNA). The CERE contains 18 S, 5.8 S, and 28 S ribosomal subunits separated by internal transcribed spacers, 5 open reading frames (ORFs), and mostly repeat elements comprising 7268 bp out of 15,786 bp (46%). A wide variety of variations and recombination events were observed. Finally, the ORFs that comprised only 4 hypothetical proteins were modeled and screened against Zinc drug-like compounds. Two compounds [ZINC77564275 (ethyl 2-(((4-isopropyl-4 H-1,2,4-triazol-3-yl) methyl) (methyl)amino) oxazole-4-carboxylate) and ZINC15022129 (5-(2-methoxyphenoxy)-[2,2'-bipyrimidine]-4,6(1 H,5 H)-dione)] were finalized as potential druggable compounds based on ADME toxicity analysis. We propose that the compounds showing the least toxicity would be potential drug candidates after laboratory experimental validation is performed.

Bridging drug discovery through hierarchical subtractive genomics against asd, trpG, and secY of pneumonia causing MDR Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is an opportunistic gram-positive, non-motile, and non-sporulating bacteria that induces pneumonia, a provocative lung infection affecting mainly the terminal bronchioles and the small air sacs known as alveoli. Recently, it has developed antibiotic resistance to the available consortium as per the WHO reports; thereby, novel remedial targets and resilient medications to forestall and cure this illness are desperately needed. Here, using pan-genomics, a total of 1,387 core proteins were identified. Subtractive proteome analyses further identified 12 proteins that are vital for bacteria. One membrane protein (secY) and two cytoplasmic proteins (asd and trpG) were chosen as possible therapeutic targets concerning minimum % host identity, essentiality, and other cutoff values, such as high resistance in the MDR S. aureus. The UniProt AA sequences of the selected targets were modelled and docked against 3 drug-like chemical libraries. The top-ranked compounds i.e., ZINC82049692, ZINC85492658 and 3a of Isosteviol derivative for Aspartate-semialdehyde dehydrogenase (asd); ZINC38222743, ZINC70455378, and 5 m Isosteviol derivative for Anthranilate synthase component II (trpG); and finally, ZINC72292296, ZINC85632684, and 7 m Isosteviol derivative for Protein translocase subunit secY (secY), were further subjected to molecular dynamics studies for thermodynamic stability and energy calculation. Our study proposes new therapeutic targets in S. aureus, some of which have previously been reported in other pathogenic microorganisms. Owing to further experimental validation, we anticipate that the adapted methodology and the predicted results in this work could make major contributions towards novel drug discovery and their targets in S. aureus caused pneumonia.

Exploring and targeting potential druggable antimicrobial resistance targets ArgS, SecY, and MurA in Staphylococcus sciuri with TCM inhibitors through a subtractive genomics strategy.

Staphylococcus sciuri (also currently Mammaliicoccus sciuri) are anaerobic facultative and non-motile bacteria that cause significant human pathogenesis such as endocarditis, wound infections, peritonitis, UTI, and septic shock. Methicillin-resistant S. sciuri (MRSS) strains also infects animals that include healthy broilers, cattle, dogs, and pigs. The emergence of MRSS strains thereby poses a serious health threat and thrives the scientific community towards novel treatment options. Herein, we investigated the druggable genome of S. sciuri by employing subtractive genomics that resulted in seven genes/proteins where only three of them were predicted as final targets. Further mining the literature showed that the ArgS (WP_058610923), SecY (WP_058611897), and MurA (WP_058612677) are involved in the multi-drug resistance phenomenon. After constructing and verifying the 3D protein homology models, a screening process was carried out using a library of Traditional Chinese Medicine compounds (consisting of 36,043 compounds). The molecular docking and simulation studies revealed the physicochemical stability parameters of the docked TCM inhibitors in the druggable cavities of each protein target by identifying their druggability potential and maximum hydrogen bonding interactions. The simulated receptor-ligand complexes showed the conformational changes and stability index of the secondary structure elements. The root mean square deviation (RMSD) graph showed fluctuations due to structural changes in the helix-coil-helix and beta-turn-beta changes at specific points where the pattern of the RMSD and root mean square fluctuation (RMSF) (< 1.0 Å) support any major domain shifts within the structural framework of the protein-ligand complex and placement of ligand was well complemented within the binding site. The ß-factor values demonstrated instability at few points while the radius of gyration for structural compactness as a time function for the 100-ns simulation of protein-ligand complexes showed favorable average values and denoted the stability of all complexes. It is assumed that such findings might facilitate researchers to robustly discover and develop effective therapeutics against S. sciuri alongside other enteric infections.

Genomic landscape of the emerging XDR Salmonella Typhi for mining druggable targets clpP, hisH, folP and gpmI and screening of novel TCM inhibitors, molecular docking and simulation analyses.

Typhoid fever is transmitted by ingestion of polluted water, contaminated food, and stool of typhoid-infected individuals, mostly in developing countries with poor hygienic environments. To find novel therapeutic targets and inhibitors, We employed a subtractive genomics strategy towards Salmonella Typhi and the complete genomes of eight strains were primarily subjected to the EDGAR tool to predict the core genome (n = 3207). Human non-homology (n = 2450) was followed by essential genes identification (n = 37). The STRING database predicted maximum protein-protein interactions, followed by cellular localization. The virulent/immunogenic ability of predicted genes were checked to differentiate drug and vaccine targets. Furthermore, the 3D models of the identified putative proteins encoded by the respective genes were constructed and subjected to druggability analyses where only "highly druggable" proteins were selected for molecular docking and simulation analyses. The putative targets ATP-dependent CLP protease proteolytic subunit, Imidazole glycerol phosphate synthase hisH, 7,8-dihydropteroate synthase folP and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase gpmI were screened against a drug-like library (n = 12,000) and top hits were selected based on H-bonds, RMSD and energy scores. Finally, the ADMET properties for novel inhibitors ZINC19340748, ZINC09319798, ZINC00494142, ZINC32918650 were optimized followed by binding free energy (MM/PBSA) calculation for ligand-receptor complexes. The findings of this work are expected to aid in expediting the identification of novel protein targets and inhibitors in combating typhoid Salmonellosis, in addition to the already existing therapies.

Mining therapeutic targets from the antibiotic-resistant Campylobacter coli and virtual screening of natural product inhibitors against its riboflavin synthase.

Campylobacter coli resides in the intestine of several commonly consumed animals, as well as water and soil. It leads to campylobacteriosis when humans eat raw/undercooked meat or come into contact with infected animals. A common manifestation of the infection is fever, nausea, headache, and diarrhea. Increasing antibiotic resistance is being observed in this pathogen. The increased incidence of C. coli infection, and post-infection complications like Guillain-Barré syndrome, make it an important pathogen. It is essential to find novel therapeutic targets and drugs against it, especially with the emergence of antibiotic-resistant strains. In the current study, genomes of 89 antibiotic-resistant strains of C. coli were downloaded from the PATRIC database. Potent drug targets (n = 36) were prioritized from the core genome (n = 1,337 genes) of this species. Riboflavin synthase was selected as a drug target and pharmacophore-based virtual screening was performed to predict its inhibitors from the NPASS (n = ~ 30,000 compounds) natural product library. The top three docked compounds (NPC115144, NPC307895, and NPC470462) were selected for dynamics simulation (for 50 ns) and ADMET profiling. These identified compounds appear safe for targeting this pathogen and can be further validated by experimental analysis before clinical trials.

Naegleria fowleri from Pakistan Has Type-2 Genotype.

Background: Primary amoebic meningoencephalitis (PAM) is an acute and fulminant CNS infection caused by Naegleria fowleri. Recreational activities and ritual ablution with contaminated warm fresh water are the main reason of PAM. Pakistan ranked the second most affected country, where most of the PAM incidences were reported from Karachi, Pakistan. Methods: In May, 2019, a 28-yr-old suspected PAM patient came to the Imam Zain-Ul-Abdin Hospital, Karachi. Biochemical and cytological investigations of patient's CSF were carried out at Karachi Diagnostic Center and Molecular Biology Lab. Sequencing of Naegleria sp. specific (ITS) primer-based amplicons was performed from both patient's CSF and water samples followed by multiple sequence alignment and phylogenetic studies. Results: Biochemical and cytological investigations of patient's CSF showed 5 mg/dl glucose, 240 mg/dl total protein and 2260/mm3 TLC suggesting acute meningoencephalitis. PCR-based analyses of patient's CSF and his residential tap water samples using Naegleria sp. specific (ITS) and N. fowleri specific primers revealed the presence of N. fowleri DNA. Nucleotide sequences of ITS primer-based amplicons from both patient's CSF and water samples were submitted in GenBank under the accession numbers MT726981.1 and MT726226.1, respectively. According to phylogenetic analysis, N. fowleri isolate from Pakistan has shown the least node age of seven. Conclusion: Here, for the very first time in Pakistan, N. fowleri genotype has been identified as type-2. Phylogenetic analysis showed that N. fowleri isolate from Pakistan is among the latest descendants, i.e., evolved later in life.

Insights into genome evolution, pan-genome, and phylogenetic implication through mitochondrial genome sequence of Naegleria fowleri species.

In the current study, we have systematically analysed the mitochondrial DNA (mtDNA) sequence of Naegleria fowleri (N. fowleri) isolate AY27, isolated from Karachi, Pakistan. The N. fowleri isolate AY27 has a circular mtDNA (49,541 bp), which harbours 69 genes (46 protein-coding genes, 21 tRNAs and 2 rRNAs). The pan-genome analysis of N. fowleri species showed a Bpan value of 0.137048, which implies that the pan-genome is open. KEGG classified core, accessory and unique gene clusters for human disease, metabolism, environmental information processing, genetic information processing and organismal system. Similarly, COG characterization of protein showed that core and accessory genes are involved in metabolism, information storages and processing, and cellular processes and signaling. The Naegleria species (n = 6) formed a total of 47 gene clusters; 42 single-copy gene clusters and 5 orthologous gene clusters. It was noted that 100% genes of Naegleria species were present in the orthogroups. We identified 44 single nucleotide polymorphisms (SNP) in the N. fowleri isolate AY27 mtDNA using N. fowleri strain V511 as a reference. Whole mtDNA phylogenetic tree analysis showed that N. fowleri isolates AY27 is closely related to N. fowleri (Accession no. JX174181.1). The ANI (Average Nucleotide Identity) values presented a much clear grouping of the Naegleria species compared to the whole mtDNA based phylogenetic analysis. The current study gives a comprehensive understanding of mtDNA architecture as well as a comparison of Naegleria species (N. fowleri and N. gruberi species) at the mitochondrial genome sequence level.

Distinct genetic landscape and a low response to doxorubicin in a luminal-A breast cancer cell line of Pakistani origin.

BACKGROUND: Breast cancers exhibit genetic heterogeneity which causes differential responses to various chemotherapy agents. Given the unique demographic and genomic background in South Asia, genetic architecture in breast cancers is not fully explored. METHODS AND RESULTS: In this study, we determined the genetic landscape of our previously established luminal-A subtype breast cancer cell line (BC-PAK1), and compared it with a Caucasian origin MCF7 breast cancer cell line of the same molecular subtype. Deep whole-exome sequencing (100X) was performed from early passages of the primary cancer cells using the Illumina NextSeq500. Data analysis with in silico tools showed novel non-silent somatic mutations previously not described in breast cancers, including a frameshift insertion (p.Ala1591AlafsTer28) in CIC, and a frameshift deletion (p.Lys333LysfsTer21) in PABPC1. Five genes CDC27, PIK3CG, ARAP3, RAPGEF1, and EFNA3, related with cell cycle pathway (hsa04110), ErbB signaling pathway (hsa04012), Ras signaling pathway (hsa04014), and Rap1 signaling pathway (hsa04015) were found to have recurrent non-silent somatic mutations. Further, the major contribution of COSMIC signatures 3 (failure of DNA double-strand break repair by homologous recombination), and 12 (transcriptional strand-bias for T>C substitutions) was observed. Also, the somatic mutations landscape in BC-PAK1 was found to be different as compared to the MCF7 cell line. The unique genetic landscape of BC-PAK1 might be responsible for significantly reduced response to doxorubicin than the MCF7 cell line. CONCLUSION: This study presents a distinct genetic architecture in luminal-A breast cancer potentially responsible for differential response to chemotherapy. Further studies on large cohorts of breast cancer patients are suggested for implementation in personalized medicine.

In-silico three dimensional structure prediction of important Neisseria meningitidis proteins.

Pathogenic bacteria Neisseria meningitidis cause serious infection i.e. meningitis (infection of the brain) worldwide. Among five pathogenic serogroups, serogroup B causes life threatening illness as there is no effective vaccine available due to its poor immunogenicity. A total of 73 genes in N. meningitidis genome have identified that were proved to be essential for meningococcal disease and were considered as crucial drug targets. We targeted five of those proteins, which are known to involve in amino acid biosynthesis, for homology-based three dimensional structure determinations by MODELLER (v9.19) and evaluated the models by PROSA and PROCHECK programs. Detailed structural analyses of NMB0358, NMB0943, NMB1446, NMB1577 and NMB1814 proteins were carried out during the present research. Based on a high degree of sequence conservation between target and template protein sequences, excellent models were built. The overall three dimensional architectures as well as topologies of all the proteins were quite similar with that of the templates. Active site residues of all the homology models were quite conserved with respect to their respective templates indicating similar catalytic mechanisms in these orthologues. Here, we are reporting, for the first time, detailed three dimensional folds of N. meningitidis pathogenic factors involved in a crucial cellular metabolic pathway. Moreover, the three dimensional structural information of these important drug targets would be utilized in computer-aided drug designing in future.

Structural investigation of human dectin-1 receptor; A novel gateway in drug discovery.

Stimulation of C-type lectin domain of human dectin-1 receptor by fungal ß-glucans causes conformational changes in its cytoplasmic domain which initiates various cellular responses mediated by downstream signaling components. We aimed to build the three-dimensional structures of the cytoplasmic domain as well as C-type lectin domain of human Dectin-1along with their potential ligands through homology modeling.The overall three-dimensional fold of cytoplasmic domain was found to consist of mixed ß-sheet whereas,in case of C-type lectin domain antiparallel ß-sheets flanked by α-helices were observed. Protein-protein docking strategy was utilized to monitorkey interactions between cytoplasmic domainof dectin-1 receptor and PKCδ, as a prime regulator of Dectin-1 signaling. The interface was observed to have both hydrophilic and hydrophobic amino acid residues maintaining crucial contacts between the two proteins. The given three dimensional structural information can be implicated in structure-based drug designing to discover potential immunomodulators that can interfere with the immune responses and phagocytosis during inflammatory and infectious conditions.

Characterization of gut bacterial flora of Apis mellifera from north-west Pakistan.

Gut microbiota has been recognized to play a beneficial role in honey bees (Apis mellifera). Present study was designed to characterize the gut bacterial flora of honey bees in north-west Pakistan. Total 150 aerobic and facultative anaerobic bacteria from guts of 45 worker bees were characterized using biochemical assays and 16S rDNA sequencing followed by bioinformatics analysis. The gut isolates were classified into three bacterial phyla of Firmicutes (60%), Proteobacteria (26%) and Actinobacteria (14%). Most of the isolates belonged to genera and families of Staphylococcus, Bacillus, Enterococcus, Ochrobactrum, Sphingomonas, Ralstonia, Enterobacteriaceae, Corynebacterium and Micrococcineae. Many of these bacteria were tolerant to acidic environments and fermented sugars, hence considered beneficial gut inhabitants and involved the maintenance of a healthy microbiota. However, several opportunistic commensals that proliferate in the hive environment including members Staphylococcus haemolyticus group and Sphingomonas paucimobilis were also identified. This is the first report on bee gut microbiota from north-west Pakistan geographically situated at the crossroads of Indian subcontinent and central Asia.