M. Kamran Azim

Isolation and characterization of the prokaryotic proteasome homolog HslVU (ClpQY) from Thermotoga maritima and the crystal structure of HslV.

Heat-shock locus VU (HslVU) is an ATP-dependent proteolytic system and a prokaryotic homolog of the proteasome. It consists of HslV, the protease, and HslU, the ATPase and chaperone. We have cloned, sequenced and expressed both protein components from the hyperthermophile Thermotoga maritima. T. maritima HslU hydrolyzes a variety of nucleotides in a temperature-dependent manner, with the optimum lying between 75 and 80 degrees C. It is also nucleotide-unspecific for activation of HslV against amidolytic and caseinolytic activity. The Escherichia coli and T. maritima HslU proteins mutually stimulate HslV proteins from both sources, suggesting a conserved activation mechanism. The crystal structure of T. maritima HslV was determined and refined to 2.1-A resolution. The structure of the dodecameric enzyme is well conserved compared to those from E. coli and Haemophilus influenzae. A comparison of known HslV structures confirms the presence of a cation-binding site, although its exact role in the proteolytic mechanism of HslV remains unclear. Amongst factors responsible for the thermostability of T. maritima HslV, extensive ionic interactions/salt-bridge networks, which occur specifically in the T. maritima enzyme in comparison to its mesophilic counterparts, seem to play an important role.

Honey modulates oxidative burst of professional phagocytes.

The effects of natural honey and its major sugar constituents (i.e. D‐fructose, D‐glucose, maltose and sucrose) on phagocytic respiratory burst have been studied. Pre‐incubated whole blood and isolated leukocytes with honey samples and sugars were induced for phagocytosis and the level of reactive oxygen species (ROS) was monitored by using chemiluminescence assays. Honey samples were found to decrease the luminol‐enhanced chemiluminescence in opsonized zymosan‐stimulated whole blood and isolated leukocytes with statistically significant differences; indicating inhibition of ROS production including hydrogen peroxide, hydroxyl free radical and hypochlorous acid. Thus honey appears to modify the oxidative burst process by inhibiting phagocytic myeloperoxidase activity. Chemiluminescence assays further showed that among the major sugar constituents of honey, D‐fructose in high concentration exerted an inhibitory effect on exocytosis‐associated myeloperoxidase catalyzed ROS formation. These results pointed out an immuno‐modulatory potential of honey in the course of phagocytosis. Copyright

Characterization of the nematicidal activity of natural honey.

Antimicrobial activities of honey against bacteria and fungi are extensively reported in the scientific literature. However, its nematicidal potential has not been characterized so far. This study examined the effect of natural honey on model nematode Caenorhabditis elegans and analyzed the honey component(s) responsible for nematicidal activity. Characterization of honey-treated C. elegans was done using fluorescence and phase contrast microscopy. Egg-laying and egg-hatching defects of honey-treated C. elegans were studied. For identification of nematicidal component(s), bioactivity-directed fractionation of honey samples was carried out using dialysis, ultrafiltration, chromatographic, and spectroscopic techniques. Natural honeys of different floral sources showed nematicidal activity against different developmental stages of C. elegans. The nematicidal components of honey induced cell death in intestinal lumen and gonads of C. elegans as revealed by microscopy. The nematicidal action of honey was found to be due to reproductive anomaly as manifested by defects in egg-laying and -hatching by C. elegans. Honey with concentration as low as 0.03% exerted profound egg-laying defects, whereas 6% honey showed defects in egg hatching. The major sugar components of honey were not involved in observed nematicidal activity. The bioactive components responsible for anti-C. elegans activity were found in the 2-10 kDa fraction of honey, which was resolved into ∼25 peaks by reverse phase HPLC. LC-MS followed by further spectroscopic characterization revealed a glycoconjugate with the molecular mass of 5511 as the major nematicidal component of honey.

Variations in intergenic spacer rpl20-rps12 of mango (Mangifera indica) chloroplast DNA: implications for cultivar identification and phylogenetic analysis

We characterized rpl20-rps12 and atp-rbcL intergenic spacers of chloroplast DNA from 19 mango cultivars to analyze suitability of these regions for assessing intraspecific variation. Analysis of the atp-rbcL region showed no variation, while rpl20-rps12 spacer sequences revealed both intravarietal and inter-subspecific polymorphisms. The intravarietal SNP was at position 360 of rpl20-rps12 spacer [SNP type C(G)]. The first inter-subspecific genotype detected was a C-A variation at position 755 in 16 out of 19 mango cultivars tested. The second inter-subspecific genotype was an insertion of C at position 714 of the rpl20-rps12 spacer, which was detected only in the ‘Langra’ cultivar. Phylogenetic tree construction based on the rpl20-rps12 region grouped ‘Shane-e-khuda,’ ‘Sonara,’ ‘Padri,’ ‘Neelum,’ ‘Gulab Khas,’ ‘Dusehri,’ ‘Chonsa,’ and ‘Cari Saloli’ cultivars together in one group; ‘Anwar Ratol,’ ‘Zafran,’ ‘Tota Pari,’ ‘Safada,’ ‘Kalapahar,’ and ‘Almas’ cultivars in a second group; and ‘Bangan Palli,’ ‘Lohatia,’ ‘Kesar,’ and ‘Bombay Alphonso’ cultivars in a third group. This study supports the applicability of the rpl20-rps12 intergenic region of cpDNA for mango cultivar identification and phylogenetics.

Characterisation of hydrazides and hydrazine derivatives as novel aspartic protease inhibitors

Virtual screening of an in-house virtual library of synthetic compounds using FlexX, followed by enzyme inhibition, identified hydrazide and hydrazine derivatives as novel aspartic protease inhibitors. These compounds inhibited human cathepsin D and Plasmodium falciparum plasmepsin-II with low micromolar concentrations (IC50 = 1-2.5 μM). Modelling studies with plasmepsin-II predicted binding of ligands at the centre of the extended substrate-binding cleft, where hydrazide/hydrazine parts of the inhibitors acted as the transition state mimic by forming electrostatic interactions with catalytic aspartates.

Antigiardial activity of glycoproteins and glycopeptides from Ziziphus honey

Natural honey contains an array of glycoproteins, proteoglycans and glycopeptides. Size-exclusion chromatography fractionated Ziziphus honey proteins into five peaks with molecular masses in the range from 10 to >200 kDa. The fractionated proteins exhibited in vitro activities against Giardia lamblia with IC50 values ≤ 25 μg/mL. Results indicated that honey proteins were more active as antiprotozoal agents than metronidazole. This study indicated the potential of honey proteins and peptides as novel antigiardial agents.

Structural Characterization of Metalloprotease Vibriolysin of Cholera Pathogen Vibrio cholerae

Vibriolysin is among several zinc metalloproteases produced by Vibrio cholerae. It is involved in the molecular pathogenicity of cholera. Here, we cloned and expressed full-length vibriolysin gene from V. cholerae. Electrophoretic and mass spectrometric data showed that the N-terminal pro-peptide was removed from pro-vibriolysin generating a 45-kDa segment containing the metalloprotease plus the C-terminal domains, and the 35 kDa metalloprotease. The 35 kDa metalloprotease segment of vibriolysin was purified to homogeneity using ion-exchange and gel filtration chromatography. Circular dichroism (CD) analysis of vibriolysin indicated α+β secondary structure, similar to other closely related metalloproteases of known structure. Positive dichroic absorption maxima in near-UV CD spectrum provided evidence for bound metal atom(s). Dynamic Light Scattering (DLS) measurements at different pHs were also performed to establish the aggregational properties of purified vibriolysin in solution. The results of DLS studies revealed that vibriolysin exists as a homomer with a hydrodynamic radius of 56.7 nm ± 2% under physiological conditions and remains catalytic when BSA was used as a protein substrate. While, extreme acidic (pH 3.5-5.4; R(H) = 65-239 nm) and alkaline (pH 8.5-9.4; R(H) = 57-72 nm) buffering conditions induced further aggregation of vibriolysin, without any trace of the monomeric state in solution.

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’.

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.

Prevalence of American foul brood disease of honeybee in north-west Pakistan

American foul brood (AFB) disease is a deleterious bacterial disease worldwide, caused by spore forming bacterium Peanibacillus larvae that affects honeybee larvae and causes a significant decrease in the honeybee population. Following symptomatical and bacteriological approaches combined with 16S rDNA sequencing, an assessment has been made to evaluate the presence of AFB disease in North-West Pakistan as no record for bee-associated bacterial disease from Pakistan is available. A total of 1276 samples from 1520 bee colonies (15 apiaries) were collected, of which 476 samples (37.30%) were found with symptoms of AFB. Biochemical and 16S rDNA analysis indicated that all these farms have Peanibacillus larvae infection. It is concluded that the prevalence of AFB bacterial disease to such an extent in these regions of Pakistan will devastate the apicultural industries in a large scale across the country.