Current Genetics最新文献

Aspergillus aculeatus produces cellulolytic enzymes in the presence of their substrates. We screened a library of 12,000 A. aculeatus T-DNA-inserted mutants to identify a regulatory factor involved in the expression of their enzyme genes in response to inducers. We found one mutant that reduced the expression of FIII-avicelase (chbI) in response to cellulose. T-DNA was inserted into a putative protein kinase gene similar to AN10082 in A. nidulans, serine-arginine protein kinase F, SrpkF. Fold increases in srpkF gene expression in response to various carbon sources were 2.3 (D-xylose), 44 (Avicel^®), 59 (Bacto^™ Tryptone), and 98 (no carbon) compared with D-glucose. Deletion of srpkF in A. aculeatus resulted in a significant reduction in cellulose-responsive expression of chbI, hydrocellulase (cel7b), and FIb-xylanase (xynIb) genes at an early induction phase. Further, the srpkF-overexpressing strain showed upregulation of the srpkF gene from four- to nine-fold higher than in the control strain. srpkF overexpression upregulated cbhI and cel7b in response to cellobiose and the FI-carboxymethyl cellulase gene (cmc1) and xynIb in response to D-xylose. However, the srpkF deletion did not affect the expression of xynIb in response to D-xylose due to the less expression of srpkF under the D-xylose condition. Our data demonstrate that SrpkF is primarily involved in cellulose-responsive expression, though it has a potential to stimulate gene expression in response to both cellobiose and D-xylose in A. aculeatus.

The development of nucleic-acid-based antimicrobials such as RNA-cleaving DNAzyme (RCD), a short catalytically active nucleic acid, is a promising alternative to the current antibiotics. The current rapid spread of antimicrobial resistance (AMR) in bacteria renders some antibiotics useless against bacterial infection, thus creating the need for alternative antimicrobials such as DNAzymes. This review summarizes recent advances in the use of RCD as a diagnostic and therapeutic agent against AMR. Firstly, the recent diagnostic application of RCD for the detection of bacterial cells and the associated resistant gene(s) is discussed. The next section summarises the therapeutic application of RCD in AMR bacterial infections which includes direct targeting of the resistant genes and indirect targeting of AMR-associated genes. Finally, this review extends the discussion to challenges of utilizing RCD in real-life applications, and the potential of combining both diagnostic and therapeutic applications of RCD into a single agent as a theranostic agent.

Cryptococcus neoformans, basidiomycetous pathogenic yeast, is basically an environmental fungus and, therefore, challenged by ever changing environments. In this study, we focused on how C. neoformans responds to stress caused by cadmium that is one of high-risk pollutants. By tracking phenotypes of the resistance or sensitivity to cadmium, we undertook forward and reverse genetic studies to identify genes involved in cadmium metabolism in C. neoformans. We found that the main route of Cd²⁺ influx is through Mn²⁺ ion transporter, Smf1, which is an ortholog of Nramp (natural resistance-associated macrophage protein 1) of mouse. We found that serotype A strains are generally more resistant to cadmium than serotype D strains and that cadmium resistance of H99, a representative of serotype A strains, was found to be due to a partial defect in SMF1. We found that calcium channel has a subsidiary role for cadmium uptake. We also showed that Pca1 (P-type-ATPase) functions as an extrusion pump for cadmium. We examined the effects of some metals on cadmium toxicity and suggested (i) that Ca²⁺ and Zn²⁺ could exert their protective function against Cd²⁺ via restoring cadmium-inhibited cellular processes and (ii) that Mg²⁺ and Mn²⁺ could have antagonistic roles in an unknown Smf1-independent Cd²⁺ uptake system. We proposed a model for Cd2⁺-response of C. neoformans, which will serve as a platform for understanding how this organism copes with the toxic metal.

The role of general transcription factor TFIIB in transcription extends well beyond its evolutionarily conserved function in initiation. Chromatin localization studies demonstrating binding of TFIIB to both the 5' and 3' ends of genes in a diverse set of eukaryotes strongly suggested a rather unexpected role of the factor in termination. TFIIB indeed plays a role in termination of transcription. TFIIB occupancy of the 3' end is possibly due to its interaction with the termination factors residing there. Interaction of the promoter-bound TFIIB with factors occupying the 3' end of a gene may be the basis of transcription-dependent gene looping. The proximity of the terminator-bound factors with the promoter in a gene loop has the potential to terminate promoter-initiated upstream anti-sense transcription thereby conferring promoter directionality. TFIIB, therefore, is emerging as a factor with pleiotropic roles in the transcription cycle. This could be the reason for preferential targeting of TFIIB by viruses. Further studies are needed to understand the critical role of TFIIB in viral pathogenesis in the context of its newly identified roles in termination, gene looping and promoter directionality.

No effective vaccine is available for any parasitic disease. The treatment to those is solely dependent on chemotherapy, which is always threatened due to development of drug resistance in bugs. This warrants identification of new drug targets. Here, we discuss Nucleoside diphosphate kinases (NDKs) of pathogens that alter host's intra and extracellular environment, as novel drug targets to simultaneously tackle multiple pathogens. NDKs having diverse functions, are highly conserved among prokaryotes and eukaryotes (the mammal NDKs are called NMEs [non-metastatic enzymes]). However, NDKs and NMEs have been separately analysed in the past for their structure and functions. The role of NDKs of pathogen in modulation of inflammation, phagocytosis, apoptosis, and ROS generation in host is known. Conversely, its combined contribution in host-pathogen interaction has not been studied yet. Through the sequence and domain analysis, we found that NDKs can be classified in two groups. One group comprised NMEs 1-4 and few NDKs of select essential protozoan parasites and the bacterium Mycobacterium tuberculosis. The other group included NME7 and the other NDKs of those parasites, posing challenges in the development of drugs specifically targeting pathogen NDKs, without affecting NME7. However, common drugs targeting group 2 NDKs of pathogens can be designed, as NME7 of group 2 is expressed only in ciliated host cells. This review thus analyses comparatively for the first time the structures and functions of human NMEs and pathogen NDKs and predicts the possibilities of NDKs as drug targets. In addition, pathogen NDKs have been now provided a nomenclature in alignment with the NMEs of humans.

Plant cell wall acts as a primary barrier for microbial pathogens during infection. A cell wall-degrading enzyme thus may be a crucial virulence factor, as it may aid the pathogen in successful host invasion. Nine genes coding for feruloyl esterases (Fae), likely involved in plant cell wall degradation, have been annotated in the genome of the cereal-blast fungus Magnaporthe oryzae. However, role of any Fae in pathogenicity of M. oryzae remains hitherto under explored. Here, we identified FAE1 gene (MGG_08737) that was significantly upregulated during host penetration and subsequent colonisation stages of infection. Accordingly, while deletion of FAE1 in M. oryzae did not affect the vegetative growth and asexual development, the fae1Δ mutant showed significantly reduced pathogenesis on rice plants, mainly due to impaired host invasion and colonisation. Very few (< 10%) fae1Δ appressoria that formed the primary invasive hyphae failed to elaborate from the first invaded cell to the neighbouring plant cells. Interestingly, exogenously added glucose, as a simple carbon source, or ferulic acid, a product of the Fae activity, significantly supported the invasive growth of the fae1Δ mutant. We show that the Fae1-based feruloyl esterase activity, by targeting the plant cell wall, plays an important role in accumulating ferulic acid and/or sugar molecules, as a likely energy source, to enable host invasion and colonisation by M. oryzae. Given its role in plant cell wall digestion and host colonisation, M. oryzae Fae1 could be a potential candidate for a novel antifungal strategy and a biotechnological application in biofuel production.

Magic spot synthetases are emerging targets to overcome persistence caused by stringent response. The 'stringent response' is a bacterial stress survival mechanism, which results in the accumulation of alarmones (also called Magic spots) leading to the formation of dormant persister cells. These 'sleeper cells' evade antibiotic treatment and could result in relapse of infection. This review broadly investigates the phenomenon of stringent response and persistence, and specifically discusses the distribution, classification, and nomenclature of proteins such as Rel/SpoT homologs (RSH), responsible for alarmone synthesis. The authors further explain the relevance of RSH as potential drug targets to break the dormancy of persister cells commonly seen in biofilms. One of the significant factors that initiate alarmone synthesis is nutrient deficiency. In a starved condition, ribosome-associated RSH detects deacylated tRNA and initiates alarmone synthesis. Accumulation of alarmones has a considerable effect on bacterial physiology, virulence, biofilm formation, and persister cell formation. Preventing alarmone synthesis by inhibiting RSH responsible for alarmone synthesis will prevent or reduce persister cells' formation. Magic spot synthetases are thus potential targets that could be explored to overcome persistence seen in biofilms.

The increase in multidrug-resistant pathogenic bacteria has become a problem worldwide. Currently there is a strong focus on the development of novel antimicrobials, including antimicrobial peptides (AMP) and antimicrobial antisense agents. While the majority of AMP have membrane activity and kill bacteria through membrane disruption, non-lytic AMP are non-membrane active, internalize and have intracellular targets. Antimicrobial antisense agents such as peptide nucleic acids (PNA) and phosphorodiamidate morpholino oligomers (PMO), show great promise as novel antibacterial agents, killing bacteria by inhibiting translation of essential target gene transcripts. However, naked PNA and PMO are unable to translocate across the cell envelope of bacteria, to reach their target in the cytosol, and are conjugated to bacteria penetrating peptides (BPP) for cytosolic delivery. Here, we discuss how non-lytic AMP and BPP-PMO/PNA conjugates translocate across the cytoplasmic membrane via receptor-mediated transport, such as the cytoplasmic membrane transporters SbmA, MdtM/YjiL, and/or YgdD, or via a less well described autonomous process.

Most organisms possess several cell cycle checkpoints to preserve genome stability in periods of stress. Upon starvation, the absence of chromosomal duplication in the bacterium Escherichia coli is ensured by holding off commencement of replication. During normal growth, accumulation of the initiator protein DnaA along with cell cycle changes in its activity, ensure that DNA replication starts only once per cell cycle. Upon nutrient starvation, the prevailing model is that an arrest in DnaA protein synthesis is responsible for the absence of initiation. Recent indications now suggest that DnaA degradation may also play a role. Here we comment on the implications of this potential new layer of regulation.