In this review article, we show that occurrence of fungicide resistance is one of the most important issues in modern agriculture. Fungicide resistance may be due to mutations of genes encoding fungicide targets (qualitative fungicide resistance) or to different mechanisms that are induced by sub-lethal fungicide stress. These mechanisms result in different and varying levels of resistance (quantitative fungicide resistance). We discuss whether or not extensive use of fungicides in agricultural environments is related to the occurrence of fungicide resistance in clinical environments. Furthermore, we provide recommendations of how development of fungicide resistant pathogen populations may be prevented or delayed.

Human heat shock protein of 90 kDa (hHsp90) is a homodimer that has an essential role in facilitating malignant transformation at the molecular level. Inhibiting hHsp90 function is a validated approach for treating different types of tumors. Inhibiting the dimerization of hHsp90 via its C-terminal domain (CTD) should provide a novel way to therapeutically interfere with hHsp90 function. Here, we predicted hot spot residues that cluster in the CTD dimerization interface by a structural decomposition of the effective energy of binding computed by the MM-GBSA approach and confirmed these predictions using in silico alanine scanning with DrugScorePPI. Mutation of these residues to alanine caused a significant decrease in the melting temperature according to differential scanning fluorimetry experiments, indicating a reduced stability of the mutant hHsp90 complexes. Size exclusion chromatography and multi-angle light scattering studies demonstrate that the reduced stability of the mutant hHsp90 correlates with a lower complex stoichiometry due to the disruption of the dimerization interface. These results suggest that the identified hot spot residues can be used as a pharmacophoric template for identifying and designing small-molecule inhibitors of hHsp90 dimerization.

Populations of the causal agent of wheat tan spot, Pyrenophora tritici-repentis, that are collected from fields frequently treated with reduced fungicide concentrations have reduced sensitivity to strobilurin fungicides and azole fungicides (C 14 -demethylase inhibitors). Energy-dependent efflux transporter activity can be induced under field conditions and after in vitro application of sublethal amounts of fungicides. Efflux transporters can mediate cross-resistance to a number of fungicides that belong to different chemical classes and have different modes of action. Resistant isolates can grow on substrata amended with fungicides and can infect plants treated with fungicides at levels above recommended field concentrations. We identified the hydroxyflavone derivative 2-(4-ethoxy-phenyl)-chromen-4-one as a potent inhibitor of energy-dependent fungicide efflux transporters in P. tritici-repentis. Application of this compound in combination with fungicides shifted fungicideresistant P. tritici-repentis isolates back to normal sensitivity levels and prevented infection of wheat leaves. These results highlight the role of energy-dependent efflux transporters in fungicide resistance and could enable a novel disease management strategy based on the inhibition of fungicide efflux to be developed.

A very large type I polypeptide begins to reel out from a ribosome; minutes later, the still unidentifiable polypeptide, largely lacking secondary structure, is now in some cases a thousand or more residues longer. Synthesis of the final hundred C-terminal residues commences. This includes the identity code, the secretion signal within the last 50 amino acids, designed to dock with a waiting ATP binding cassette (ABC) transporter. What happens next is the subject of this review, with the main, but not the only focus on hemolysin HlyA, an RTX protein toxin secreted by the type I system. Transport substrates range from small peptides to giant proteins produced by many pathogens. These molecules, without detectable cellular chaperones, overcome enormous barriers, crossing two membranes before final folding on the cell surface, involving a unique autocatalytic process.Unfolded HlyA is extruded posttranslationally, C-terminal first. The transenvelope "tunnel" is formed by HlyB (ABC transporter), HlyD (membrane fusion protein) straddling the inner membrane and periplasm and TolC (outer membrane). We present a new evaluation of the C-terminal secretion code, and the structure function of HlyD and HlyB at the heart of this nanomachine. Surprisingly, key details of the secretion mechanism are remarkably variable in the many type I secretion system subtypes. These include alternative folding processes, an apparently distinctive secretion code for each type I subfamily, and alternative forms of the ABC transporter; most remarkably, the ABC protein probably transports peptides or polypeptides by quite different mechanisms. Finally, we suggest a putative structure for the Hly-translocon, HlyB, the multijointed HlyD, and the TolC exit.

Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB.

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Type 1 secretion systems are one of the main machineries in Gram-negative bacteria involved in the secretion of a wide range of substrates from the cytoplasm across the inner and outer membrane in one step to the extracellular space. The range of substrates varies from small proteins up to large surface layer proteins of about 900 kDa. Most of the substrates have a non-cleavable C-terminal secretion signal and so-called GG repeats that are able to bind calcium ions. The translocator complex is composed of a trimeric outer membrane protein that provides a pore in the outer membrane. A multimeric membrane fusion protein spans the periplasm and forms a continuous channel connecting the outer membrane protein with a dimeric ATP-binding cassette transporter in the inner membrane. The ATP-binding cassette-transporter is thought to form a channel through the inner membrane and energizes the transport process. This review will provide a detailed view of the components of the translocator and will summarize structural as well as functional data.

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