Mohammad Riazul Islam

Evaluation of essential and variable residues of nukacin ISK‐1 by NNK scanning

Nukacin ISK‐1, a type‐A(II) lantibiotic, comprises 27 amino acids with a distinct linear N‐terminal and a globular C‐terminal region. To identify the positional importance or redundancy of individual residues responsible for nukacin ISK‐1 antimicrobial activity, we replaced the native codons of the parent peptide with NNK triplet oligonucleotides in order to generate a bank of nukacin ISK‐1 variants. The bioactivity of each peptide variant was evaluated by colony overlay assay, and hence we identified three Lys residues (Lys1, Lys2 and Lys3) that provided electrostatic interactions with the target membrane and were significantly variable. The ring structure of nukacin ISK‐1 was found to be crucially important as replacing the ring‐forming residues caused a complete loss of bioactivity. In addition to the ring‐forming residues, Gly5, His12, Asp13, Met16, Asn17 and Gln20 residues were found to be essential for antimicrobial activity; Val6, Ile7, Val10, Phe19, Phe21, Val22, Phe23 and Thr24 were relatively variable; and Ser4, Pro8, His15 and Ser27 were extensively variable relative to their positions. We obtained two variants, Asp13Glu and Val22Ile, which exhibited a twofold higher specific activity compared with the wild‐type and are the first reported type‐A(II) lantibiotic mutant peptides with increased potency.

Ring A of nukacin ISK-1: a lipid II-binding motif for type-A(II) lantibiotic.

Ring A of nukacin ISK-1, which is also present in different type-A(II) lantibiotics, resembles a lipid II-binding motif (TxS/TxD/EC, x denotes undefined residues) similar to that present in mersacidin (type-B lantibiotics), which suggests that nukacin ISK-1 binds to lipid II as a docking molecule. Results from our experiments on peptidoglycan precursor (UDP-MurNAc-pp) accumulation and peptide antagonism assays clearly indicated that nukacin ISK-1 inhibits cell-wall biosynthesis, accumulating lipid II precursor inside the cell, and the peptide activity can be repressed by lipid I and lipid II. Interaction analysis of nukacin ISK-1 and different ring A variants with lipid II revealed that nukacin ISK-1 and nukacin D13E (a more active variant) have a high affinity (K(D) = 0.17 and 0.19 μM, respectively) for lipid II, whereas nukacin D13A (a less active variant) showed a lower affinity, and nukacin C14S (a negative variant lacking the ring A structure) exhibited no interaction. Therefore, on the basis of the structural similarity and positional significance of the amino acids in this region, we concluded that nukacin ISK-1 binds lipid II via its ring A region and may lead to the inhibition of cell-wall biosynthesis.

Antimicrobial mechanism of lantibiotics.

Lantibiotics are ribosomally synthesized antimicrobial peptides that commonly target the cell wall precursor lipid II during their antimicrobial mechanism and exert their inhibitory activity by (i) inhibition of cell wall biosynthesis, and (ii) stable pore formation in the target membrane. Type-A(I) (i.e. nisin) and two-component (i.e. lacticin 3147) lantibiotics initially interact with lipid II to stabilize the complex, which then proceeds to inhibit cell wall biosynthesis and pore formation. Type-A(II) (i.e. nukacin ISK-1) and type-B (i.e. mersacidin) lantibiotics also use lipid II as a docking molecule, but can only inhibit cell wall biosynthesis without forming pores. In the present paper, we review the antimicrobial mechanism of different types of lantibiotics, their current progress and future prospect.

Mapping and identification of the region and secondary structure required for the maturation of the nukacin ISK-1 prepeptide.

The prepeptide NukA of the lantibiotic nukacin ISK-1 consists of an N-terminal leader peptide followed by a propeptide moiety that undergoes post-translational modifications, that is, formation of unusual amino acids by NukM, cleavage of the leader peptide and transport by NukT to yield a mature peptide. To identify the region and conformation required for the maturation of prepeptide, we expressed a series of NukA mutants, mutants with the N-terminus-truncated leader peptide and site-directed mutants with conserved residues in the leader peptide of type A(II) lantibiotics, which were evaluated on the basis of the production of nukacin ISK-1. In addition, the secondary structure data of NukA mutants or fragments were obtained by circular dichroism spectra. The results indicated the importance of the alpha-helical leader peptide with hydrophobic and hydrophilic orientation consisting of the conserved residues in type A(II) lantibiotics. The expression data from various combinations of the chimeric prepeptides consisting of NukA and LctA (the prepeptide of lacticin 481, which shows high identity with NukA) further revealed that the amino acid difference at the C-terminus of the propeptide moiety between NukA and LctA, especially His at position 15 and Phe at position 19, was important for the maturation processes by the nukacin ISK-1 biosynthetic enzymes. Our findings suggest that the determinants in NukA were critically involved in the biosynthesis of nukacin ISK-1 and would thus be important for recognition by the nukacin ISK-1 biosynthetic enzymes.

Yield of Two Consecutive Sputum Specimens for the Effective Diagnosis of Pulmonary Tuberculosis

Background From long instances, it is debatable whether three sputum specimens are required for the diagnosis of pulmonary tuberculosis (TB) or TB can be diagnosed effectively using two consecutive sputum specimens. This study was set out to evaluate the significance of examining multiple sputum specimens in diagnosis of TB. Methods We retrospectively reviewed the acid-fast bacillus (AFB) smear and culture results of three consecutive days’ sputum specimens from 413 confirmed TB patients which were detected as part of a larger active case finding study in Dhaka Central Jail, the largest correctional facility in Bangladesh. Results AFB was detected from 81% (n = 334) patients, of which 89% (n = 297) were diagnosed from the first and additional 9% (n = 30) were from the second sputum specimen. M. tuberculosis growth was observed for 406 patients and 85% (n = 343) were obtained from the first sputum and additional 10% (n = 42) were from the second one. The third specimen didn’t show significant additional diagnostic value for the detection of AFB by microscopy or growth of the M. tuberculosis. Conclusions We concluded from our study results that examining two consecutive sputum specimens is sufficient enough for the effective diagnosis of TB. It can also decrease the laboratory workload and hence improve the quality of work in settings with high TB burden like Bangladesh.

An endophytic Basidiomycete, Grammothele lineata, isolated from Corchorus olitorius, produces paclitaxel that shows cytotoxicity

Grammothele lineata, an endophyte isolated in our laboratory from jute (Corchorus olitorius acc. 2015) was found to be a substantial paclitaxel producer. Taxol and its related compounds, produced by this endophyte were extracted by growing the fungus in simple nutrient media (potato dextrose broth, PDB). Taxol was identified and characterized by different analytical techniques (TLC, HPLC, FTIR, LC-ESI-MS/MS) following its extraction by ethyl acetate. In PDB media, this fungus was found to produce 382.2 μgL-1 of taxol which is about 7.6 x103 fold higher than the first reported endophytic fungi, Taxomyces andreanae. The extracted taxol exhibited cytotoxic activity in an in vitro culture of HeLa cancer cell line. The fungal extract also exhibited antifungal and antibacterial activities against different pathogenic strains. This is the first report of a jute endophytic fungus harboring the capacity to produce taxol and also the first reported taxol producing species that belongs to the Basidiomycota phylum, so far unknown to be a taxol producer. These findings suggest that the fungal endophyte, Grammothele lineata can be an excellent source of taxol and can also serve as a potential species for chemical and genetic engineering to enhance further the production of taxol.

Enhanced production of nukacin D13E in Lactococcus lactis NZ9000 by the additional expression of immunity genes

Nukacin D13E (D13E) is a variant of type-A(II) lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1. D13E exhibited a twofold higher specific antimicrobial activity than nukacin ISK-1 against a number of Gram-positive bacteria. We previously reported the heterologous production of D13E in Lactococcus lactis NZ9000 under the control of nisin-controlled gene expression system. In this study, we demonstrated enhanced production of D13E by the additional expression of immunity genes, nukFEG. The nukacin ISK-1 immunity, conferred by the ABC transporter complex, NukFEG, and the lantibiotic-binding protein, NukH, was not overwhelmed by D13E. The additional NukFEG resulted in a fourfold increase in the immunity level of the strain and a 5.2-fold increase in D13E production. The additional NukFEGH-expressing strain with the highest D13E immunity showed reduced level of production. Further improvement in D13E production was achieved by using pH-controlled batch fermentation.

Bactericidal activity of nukacin ISK-1: an alternative mode of action

We previously reported bacteriostatic action of nukacin ISK-1 against Bacillus subtilis JCM 1465T. Here, we found its bactericidal activity against Micrococcus luteus DSM 1790 and Staphylococcus simulans 22, showing decrease in cell viability, cell lysis, and dissipation of the membrane potential. Moreover, leakage of small molecules such as K+, suggested the formation of small-sized or specific K+-conducting-pores by nukacin ISK-1.

LiaRS reporter assay: A simple tool to identify lipid II binding moieties in lantibiotic nukacin ISK-1

Binding to lipid II is an important step in the mode of action of most lantibiotics targeting the bacterial cell wall. We applied the Bacillus subtilis two-component system, LiaRS, that is known to respond to antibiotics interfering with lipid II cycle, in order to evaluate lipid II binding activity of known bacteriocins and also to identify lipid II binding moieties in lantibiotic nukacin ISK-1. Using this method, we confirmed that the methyllanthionine ring in nukacin ISK-1 is crucial for lipid II binding as previously indicated. In this study, we further identified that the three N-terminal lysine residues (K1, K2, and K3) and the glycine (G5) residue in nukacin ISK-1 are also important in lipid II binding.

Diversity of Plant Endophytic Volatile Organic Compound (VOC) and Their Potential Applications

Plant endophytes ranging from bacteria to fungi produce a diverse class of volatile organic compounds (VOCs) that are important for the development of symbiotic relation under highly competitive environment with the host. Not only that, they also play an important role in intraand inter-kingdom signalling. Chemically, this gas-phase mixture may contain acids, alcohols, aldehydes, aromatics, esters, heterocycles, ketones, terpenes, thiols, and so forth. Several evidences suggested their potential use for sustainable crop production and industrial applications. Many VOCs have been reported with significant effects for antibiosis and growth promotion. They provide for an alternative to chemicals F. T. Chowdhury · M. R. Islam · M. R. Islam · H. Khan (*) Department of Biochemistry and Molecular Biology, Faculty of Biological Sciences, University of Dhaka, Dhaka, Bangladesh e-mail: farhanatasnim@du.ac.bd; mriazulislam@du.ac.bd; rakibul_du@du.ac.bd; haseena@du.ac. bd # Springer Nature Switzerland AG 2018 S. Jha (ed.), Endophytes and Secondary Metabolites, Reference Series in Phytochemistry, https://doi.org/10.1007/978-3-319-76900-4_10-1 1 used to protect plants from pathogens and thus allow for better crop welfare. They also possess food and flavor properties which can be exploited in depth for food industries. Recent studies revealed that endophytes also produce diverse volatile hydrocarbons with fuel properties. They emit mixtures of volatile biofuel molecules comprising of alkanes, alkenes, acids, benzene derivatives, esters, etc. A vast diversity of endophytes are associated with plants for their ecology and fitness, and a systematic exploration of their VOCs will likely uncover novel use for their future utilization. In this chapter we highlight the nature and known or proposed functions of endophytic bacterial and fungal VOCs with a focus on the ones which have potential applications.