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.

3-substituted coumarin derivatives over-activate the HslV protease: A potential drug target for antibacterial activity.

The bacterial HslVU complex consists of two different proteins, i.e., the HslV protease and the HslU ATPase. The functional HslVU enzyme complex forms only when the HslU c-terminal helix is inserted into the cavity located between two adjacent HslV monomers in order to allosterically activate the HslV protease. Based on its essential role in maintaining microbial proteostasis as well its absence from human beings, it is considered a promising therapeutic target for designing antibacterial agents. The goal of the present study was to find out potential drug candidates that could over-activate the HslV protease and produce aberrant proteolysis in pathogenic bacteria. Derivatives of 3-substituted coumarin have been identified as potential HslV protease activators based on their highest docking scores, ideal interaction patterns, and significant in-vitro HslV activation potential. Their ED50 values were in the sub-micromolar range, i.e., 0.4-0.48µM. The conformational stability of the contacts between the HslV dimer and the active compounds was further confirmed by molecular dynamics studies. Correspondingly, the ADMET characteristics of these lead molecules considerably demonstrated their significant non-toxic drug-like abilities. This research not only identified small non-peptidic HslV protease activators but also improved the understanding of the mode of action of 3-substituted coumarin derivatives as antibacterials.

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.

Identification and structural investigation of potential novel drug candidates against lethal human pathogen.

Neisseria meningtidis is responsible for causing meningococcal meningitis along with acute septicaemia in human beings. Functional genomics strategies proved cruciality of certain genes/proteins in Neisseria meningitidis pathogenesis. During the present studies, three important Neisseria meningitidis proteins i.e., Dead box RNA-Helicase, Polyribonucleotide nucleotidyl-transferase PNPase and Ribonuclease-III were targeted for homology modeling and protein-ligand docking studies not only to determine their three dimensional architectures but also to identify their potential novel inhibitors. The Biscoumarin, malonitrile and indole derivatives showed the best inhibitory mode against all of the three enzymes. Since, these enzymes are assembled in Gram-negative bacteria to form RNA degradosome assembly therefore their inhibition will definitely shut off the degradosome assembly and ultimately the decay of RNA, which is an essential life process. This is the first ever structural investigation of these drug targets along with identification of potential novel drug candidates. We believe that these small chemical compounds will be proved as better drugs and will provide an excellent barrier towards Neisseria meningitidis pathogenesis.

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.

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.

Anti-amoebic effects of synthetic acridine-9(10H)-one against brain-eating amoebae.

Pathogenic A. castellanii and N. fowleri are opportunistic free-living amoebae. Acanthamoeba spp. are the causative agents of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK), whereas Naegleria fowleri causes a very rare but severe brain infection called primary amebic meningoencephalitis (PAM). Acridinone is an important heterocyclic scaffold and both synthetic and naturally occurring derivatives have shown various valuable biological properties. In the present study, ten synthetic Acridinone derivatives (I-X) were synthesized and assessed against both amoebae for anti-amoebic and cysticidal activities in vitro. In addition, excystation, encystation, cytotoxicity, host cell pathogenicity was also performed in-vitro. Furthermore, molecular docking studies of these compounds with three cathepsin B paralogous enzymes of N. fowleri were performed in order to predict the possible docking mode with pathogen. Compound VII showed potent anti-amoebic activity against A. castellanii with IC50 53.46 µg/mL, while compound IX showed strong activity against N. fowleri in vitro with IC50 72.41 µg/mL. Compounds II and VII showed a significant inhibition of phenotypic alteration of A. castellanii, while compound VIII significantly inhibited N. fowleri cysts. Cytotoxicity assessment showed that these compounds caused minimum damage to human keratinocyte cells (HaCaT cells) at 100 µg/mL, while also effectively reduced the cytopathogenicity of Acanthamoeba to HaCaT cells. Moreover, Cathepsin B protease was investigated in-silico as a new molecular therapeutic target for these compounds. All compounds showed potential interactions with the catalytic residues. These results showed that acridine-9(10H)-one derivatives, in particular compounds II, VII, VIII and IX hold promise in the development of therapeutic agents against these free-living amoebae.

Small molecule activators of proteasome-related HslV peptidase.

The HslVU is the proteasome-related two component system composed of HslV peptidase and HslU chaperone. It is involved in the degradation of an array of intracellular proteins. The presence of HslVU homologs in pathogenic microbes and its absence in human makes it an antimicrobial drug target. The functional HslVU complex forms when HslV dodecamer is flanked at both ends by HslU hexamers. In the HslVU complex, eight residues at the carboxy termini of HslU subunits intercalate into a clefts between two adjacent HslV subunits causing a conformational change in the active site of HslV which in turn results in the allosteric activation of HslV peptidase. Here, we report small molecules capable of activating HslV peptidase in the absence of its natural activator HslU ATPase. For this purpose, virtual screening of an in-house library of synthetic and natural compounds was performed to find out ligands mimicking the interaction of HslU carboxy terminus with HslV dodecamer. The benzimidazole, quinazoline and chromone derivatives were suggested by ligand docking to bind at the HslU carboxy termini intercalation pockets in the HslV dodecamer. This was confirmed by HslV activation and isothermal titration calorimetry assays with these compounds that gave ED(50) in sub-micromolar range (0.6-1.5µM). The results showed for the first time that small, extracellular non-peptidic molecules can allosterically activate the peptide hydrolytic activity of HslV which in turn would initiate intracellular proteolysis.

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.

Self-Redirection of Metabolic Flux Toward Squalene and Ethanol Pathways by Engineered Yeast.

We have previously reported that squalene overproducing yeast self-downregulate the expression of the ethanol pathway (non-essential pathway) to divert the metabolic flux to the squalene pathway. In this study, the effect of co-production of squalene and ethanol on other non-essential pathways (fusel alcohol pathway, FA) of Saccharomyces cerevisiae was evaluated. However, before that, 13 constitutive promoters, like IRA1p, PET9p, RHO1p, CMD1p, ATP16p, USA3p, RER2p, COQ1p, RIM1p, GRS1p, MAK5p, and BRN1p, were engineered using transcription factor bindings sites from strong promoters HHF2p (-300 to -669 bp) and TEF1p (-300 to -579 bp), and employed to co-overexpress squalene and ethanol pathways in S. cerevisiae. The FSE strain overexpressing the key genes of the squalene pathway accumulated 56.20 mg/L squalene, a 16.43-fold higher than wild type strain (WS). The biogenesis of lipid droplets was stimulated by overexpressing DGA1 and produced 106 mg/L squalene in the FSE strain. AFT1p and CTR1p repressible promoters were also characterized and employed to downregulate the expression of ERG1, which also enhanced the production of squalene in FSE strain up to 42.85- (148.67 mg/L) and 73.49-fold (255.11 mg/L) respectively. The FSE strain was further engineered by overexpressing the key genes of the ethanol pathway and produced 40.2 mg/mL ethanol in the FSE1 strain, 3.23-fold higher than the WS strain. The FSE1 strain also self-downregulated the expression of the FA pathway up to 73.9%, perhaps by downregulating the expression of GCN4 by 2.24-fold. We demonstrate the successful tuning of the strength of yeast promoters and highest coproduction of squalene and ethanol in yeast, and present GCN4 as a novel metabolic regulator that can be manipulated to divert the metabolic flux from the non-essential pathway to engineered pathways.

Structural bioinformatics of Neisseria meningitidis LD-carboxypeptidase: implications for substrate binding and specificity.

Neisseria meningitidis, a gram negative bacterium, is the leading cause of bacterial meningitis and severe sepsis. Neisseria meningitidis genome contains 2,160 predicted coding regions including 1,000 hypothetical genes. Re-annotation of N. meningitidis hypothetical proteins identified nine putative peptidases. Among them, the NMB1620 protein was annotated as LD-carboxypeptidase involved in peptidoglycan recycling. Structural bioinformatics studies of NMB1620 protein using homology modeling and ligand docking were carried out. Structural comparison of substrate binding site of LD-carboxypeptidase was performed based on binding of tetrapeptide substrate 'L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine'. Inspection of different subsite-forming residues showed changeability in the S1 subsite across different bacterial species. This variability was predicted to provide a structural basis to S1-subsite for accommodating different amino acid residues at P1 position of the tetrapeptide substrate 'L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine'.