Dipnath Baidyaroy, Gerald Brosch, Joong-hoon Ahn, Stefan Graessle, Sigrun Wegener, Nyerhovwo J. Tonukari, Oscar Caballero, Peter Loidl, Jonathan D. Walton, A Gene Related to Yeast HOS2 Histone Deacetylase Affects Extracellular Depolymerase Expression and Virulence in a Plant Pathogenic Fungus, The Plant Cell, Vol. 13, No. 7 (Jul., 2001), pp. 1609-1624

A gene, HDC1, related to the Saccharomyces cerevisiae histone deacetylase (HDAC) gene HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin. Engineered mutants of HDC1 had smaller and less septate conidia and exhibited an approximately 50% reduction in total HDAC activity. Mutants were strongly reduced in virulence as a result of reduced penetration efficiency. Growth of hdc1 mutants in vitro was normal on glucose, slightly decreased on sucrose, and reduced by 30 to 73% on other simple and complex carbohydrates. Extracellular depolymerase activities and expression of the corresponding genes were downregulated in hdc1 mutant strains. Except for altered conidial morphology, the phenotypes of hdc1 mutants were similar to those of C. carbonum strains mutated in ccSNF1 encoding a protein kinase necessary for expression of glucose-repressed genes. These results show that HDC1 has multiple functions in a filamentous fungus and is required for full virulence of C. carbonum on maize.

Dipnath Baidyaroy, Gerald Brosch, Joong-hoon Ahn, Stefan Graessle, Sigrun Wegener, Nyerhovwo J. Tonukari, Oscar Caballero, Peter Loidl, Jonathan D. Walton, A Gene Related to Yeast HOS2 Histone Deacetylase Affects Extracellular Depolymerase Expression and Virulence in a Plant Pathogenic Fungus, The Plant Cell, Vol. 13, No. 7 (Jul., 2001), pp. 1609-1624

A gene, HDC1, related to the Saccharomyces cerevisiae histone deacetylase (HDAC) gene HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin. Engineered mutants of HDC1 had smaller and less septate conidia and exhibited an approximately 50% reduction in total HDAC activity. Mutants were strongly reduced in virulence as a result of reduced penetration efficiency. Growth of hdc1 mutants in vitro was normal on glucose, slightly decreased on sucrose, and reduced by 30 to 73% on other simple and complex carbohydrates. Extracellular depolymerase activities and expression of the corresponding genes were downregulated in hdc1 mutant strains. Except for altered conidial morphology, the phenotypes of hdc1 mutants were similar to those of C. carbonum strains mutated in ccSNF1 encoding a protein kinase necessary for expression of glucose-repressed genes. These results show that HDC1 has multiple functions in a filamentous fungus and is required for full virulence of C. carbonum on maize.

Joong-Hoon Ahn, Jonathan D. Walton, Chromosomal Organization of TOX2, a Complex Locus Controlling Host-Selective Toxin Biosynthesis in Cochliobolus carbonum, The Plant Cell, Vol. 8, No. 5 (May, 1996), pp. 887-897

Rosmarinic acid (RA) is a phytochemical that exhibits a variety of biological activities. The biological synthesis pathway of RA has been well-established in plants. In this study, the shikimate pathway is engineered to enhance the availability of precursor substrates and the biosynthetic pathway is reconstructed to produce six RA derivatives: p-coumaroyl phenyllactic acid (PPLA), p-coumaroyl 4-hydroxyphenyllactic acid (P4PLA), caffeoyl phenyllactic acid (CPLA), p-coumaroyl salvinic acid (PSA), rosmarinic acid (RA), and isorinic acid (IA). To identify the optimal method for maximizing the production of the target compounds while minimizing the formation of unwanted byproducts, single-cell, stepwise, and coculture approaches are employed. Using the single-cell method, 145.1 mg L^-1 of PPLA and 361.6 mg L^-1 of P4PLA are synthesized. The stepwise method yields 28.0 mg L^-1 of CPLA, 23.4 mg L^-1 of PSA (which contains synthesized IA), and 10.9 mg L^-1 of RA. Finally, the coculture method results in the synthesis of 40.2 mg L^-1 of IA (which contains synthesized PSA).

<b>Background/Objectives:</b> Previously, we reported that 3-<i>O</i>-alkyl difluoroquercetins (di-F-Q) potentiates the antimicrobial activity of aztreonam (ATM) against metallo-β-lactamase (MBL)-producing <i>P. aeruginosa</i> through simultaneous inhibition of MBLs and efflux pumps. However, the ATM-potentiating activity of the 3-<i>O</i>-alkyl di-F-Q was observed only at high and potentially toxic concentrations (32 mg/L). <b>Methods:</b> As both MBLs and efflux pumps reside in the periplasm of Gram-negative bacteria, their inhibitors should accumulate in the periplasmic space. However, the outer membrane porins, the major entry pathway in Gram-negative bacteria, allow the passive diffusion of hydrophilic polar molecules across the outer membrane. Thus, we reasoned that the introduction of a polar substituent at 7-OH position of 3-<i>O</i>-alkyl di-F-Q would enhance its periplasmic concentration to result in potentiation of ATM at lower concentrations. <b>Results:</b> The title compound <b>5</b> exhibited inhibitory activity against NDM-1 as well as the efflux pump of <i>P. aeruginosa</i>, which resulted in synergistical potentiation of ATM. A combination of ATM (8 mg/L) and <b>5</b> (8 mg/L) inhibited 80% of the ATM-resistant CPPA, while ATM alone did not show any inhibition. In addition, only 4 mg/L of <b>5</b> was needed to reduce the MIC₉₀ of ATM four-fold in ATM-resistant CPPA (n = 15). The time-kill data further supported the effectiveness of the combined treatment of ATM with <b>5</b>, and the combination of ATM (1xMIC) with 8 mg/L of <b>5</b> showed bactericidal effects in every bacterial strain tested (PA-002, <i>bla</i>_IMP, PA-003, <i>bla</i>_VIM, PA-014, <i>bla</i>_GES, and PA-017, <i>bla</i>_NDM) by reducing the bacterial loads by 5.1 log₁₀~8.9 log₁₀. <b>Conclusions:</b> The title compound <b>5</b> exhibited inhibitory activity against NDM-1 as well as the efflux pump of <i>P. aeruginosa</i>, and the combined inhibitory activity resulted in synergistical potentiation of ATM. It should be noted that most CPPA isolates tested were sensitized to 8 mg/L of ATM upon combination with 4~8 mg/L of <b>5</b>.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-β-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum β-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of β-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-β-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8–9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and β-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

The Gfo/Idh/MocA family enzyme DgpA was known to catalyze the regiospecific oxidation of puerarin to 3"-oxo-puerarin in the presence of 3-oxo-glucose. Here, we discovered that D3dgpA, <i>dgpA</i> cloned from the human gut bacterium <i>Dorea</i> sp. MRG-IFC3, catalyzed the regiospecific oxidation of various <i>C</i>-/<i>O</i>-glycosides, including puerarin, in the presence of methyl β-D-3-oxo-glucopyranoside. While <i>C</i>-glycosides were converted to 3"- and 2"-oxo-products by D3dgpA, <i>O</i>-glycosides resulted in the formation of aglycones and hexose enediolone from the 3"-oxo-products. From DFT calculations, it was found that isomerization of 3"-oxo-puerarin to 2"-oxo-puerarin required a small activation energy of 9.86 kcal/mol, and the <i>O</i>-glycosidic bond cleavage of 3"-oxo-products was also thermodynamically favored with a small activation energy of 3.49 kcal/mol. In addition, the reaction mechanism of D3dgpA was discussed in comparison to those of Gfo/Idh/MocA and GMC family enzymes. The robust reactivity of D3dgpA was proposed as a new general route for derivatization of glycosides.