Adaptive value of foot-and-mouth disease virus capsid substitutions with opposite effects on particle acid stability

Foot-and-mouth disease virus (FMDV) is a picornavirus that exhibits an extremely acid sensitive capsid. This acid lability is directly related to its mechanism of uncoating triggered by acidification inside cellular endosomes. Using a collection of FMDV mutants we have systematically analyzed the relationship between acid stability and the requirement for acidic endosomes using ammonium chloride (NH4Cl), an inhibitor of endosome acidification. A FMDV mutant carrying two substitutions with opposite effects on acid-stability (VP3 A116V that reduces acid stability, and VP1 N17D that increases acid stability) displayed a rapid shift towards acid lability that resulted in increased resistance to NH4Cl as well as to concanamicyn A, a different lysosomotropic agent. This resistance could be explained by a higher ability of the mutant populations to produce NH4Cl-resistant variants, as supported by their tendency to accumulate mutations related to NH4Cl-resistance that was higher than that of the WT populations. Competition experiments also indicated that the combination of both amino acid substitutions promoted an increase of viral fitness that likely contributed to NH4Cl resistance. This study provides novel evidences supporting that the combination of mutations in a viral capsid can result in compensatory effects that lead to fitness gain, and facilitate space to an inhibitor of acid-dependent uncoating. Thus, although drug-resistant variants usually exhibit a reduction in viral fitness, our results indicate that compensatory mutations that restore this reduction in fitness can promote emergence of resistance mutants.

Temperature-responded biological fitness of carbendazim-resistance Fusarium graminearum mutants conferring the F167Y, E198K and E198L substitutions

Understanding the effects of temperature on Fusarium graminearum infection can provide the theoretical guidance for chemical control of Fusarium head blight (FHB)．Here, we evaluated the effects of various temperatures on biological fitness development of wild-type sensitive strain 2021 and carbendazim-resistance mutants conferring β2-tubulin substitutions F167Y, E198K and E198L. The results showed that mycelial growth and conidiation of four strains increased with the increase of the temperature between 10°C and 25°C. Conidia of F167Y displayed strong adaptability to low temperature. The virulence of the four strains was largely similar at the same temperature, showing an upward trend between 10°C and 25°C. At 10°C, the hyphal growth of all strains was significantly inhibited, and metabolism slowed down and the accumulation of secondary metabolites decreased. Subsequently, the production of deoxynivalenol (DON) and its intermediate, pyruvate and aurofusarin decreased at low temperature, and the expression of DON biosynthesis-related genes Tri5, FgPK and AUR decreased accordingly. At the same temperature, the aurofusarin production of the strains F167Y and E198K was higher than that of strains 2021 and E198L. The contents of DON and pyruvic acid in carbendazim-resistance mutants were higher than that of the wild-type strain 2021. The sensitivity of four strains to different fungicides changed at various temperatures. The sensitivity to most fungicides increased with the temperature decreasing. The carbendazim-resistance mutants appeared positive cross-resistance with other benzimidazoles. But there was no cross-resistance to pyraclostrobin and azoles. These results would direct us to use fungicide preventing the infection of F. graminearum with changeable atmospheric temperature at wheat flower stage.

Morphological, molecular and resistance responses to soft-rot disease variability among plantlets of Phalaenopsis amabilis regenerated from irradiated protocorms

Putri HA, Purwito A, Sudarsono, Sukma D. 2021. Morphological, molecular and resistance responses to soft-rot disease variability among plantlets of Phalaenopsis amabilis regenerated from irradiated protocorms. Biodiversitas 22: 1077-1090. Phalaenopsis amabilis (L.) Blume is a prominent donor for the white petal and sepal trait in Phalaenopsis breeding. However, it has an undesirable character, such as susceptible to soft-rot disease. Therefore, developing soft-rot resistance mutants through gamma irradiation could be explored. This study aimed to evaluate the variability of plantlets regenerated from irradiated and non-irradiated protocorms using morphology, stomatal size and molecular markers and to test responses of the plantlets against soft-rot disease. The plantlets were regenerated from irradiated (5, 10, 15 or 20 Gy) and non-irradiated protocorms. The results showed that P. amabilis plantlet variants were successfully identified based on their leaf morphology and stomatal size variations. A few plantlets have low stomatal densities, large stomatal size, and high chloroplast numbers, which indicated they were polyploids. Leaf disc assay for soft-rot disease response grouped most of the plantlets into very susceptible or susceptible. Moreover, four soft-rot resistant plantlets regenerated from irradiated and non-irradiated protocorms were successfully identified. The resistant plantlets were identified after three consecutive periods of inoculations with pathogens causing soft-rot disease. The evaluation also confirmed nucleotide variation in the Pto gene isolated from different levels of plantlet variant resistance responses.

Adding Insult to Injury: Mechanistic Basis for How AmpC Mutations Allow Pseudomonas aeruginosa To Accelerate Cephalosporin Hydrolysis and Evade Avibactam

ABSTRACT Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide and notorious for its broad-spectrum resistance to antibiotics. A key mechanism that provides extensive resistance to β-lactam antibiotics is the inducible expression of AmpC β-lactamase. Recently, a number of clinical isolates expressing mutated forms of AmpC have been found to be clinically resistant to the antipseudomonal β-lactam–β-lactamase inhibitor (BLI) combinations ceftolozane-tazobactam and ceftazidime-avibactam. Here, we compare the enzymatic activity of wild-type (WT) AmpC from PAO1 to those of four of these reported AmpC mutants, bearing mutations E247K (a change of E to K at position 247), G183D, T96I, and ΔG229–E247 (a deletion from position 229 to 247), to gain detailed insights into how these mutations allow the circumvention of these clinically vital antibiotic-inhibitor combinations. We found that these mutations exert a 2-fold effect on the catalytic cycle of AmpC. First, they reduce the stability of the enzyme, thereby increasing its flexibility. This appears to increase the rate of deacylation of the enzyme-bound β-lactam, resulting in greater catalytic efficiencies toward ceftolozane and ceftazidime. Second, these mutations reduce the affinity of avibactam for AmpC by increasing the apparent activation barrier of the enzyme acylation step. This does not influence the catalytic turnover of ceftolozane and ceftazidime significantly, as deacylation is the rate-limiting step for the breakdown of these antibiotic substrates. It is remarkable that these mutations enhance the catalytic efficiency of AmpC toward ceftolozane and ceftazidime while simultaneously reducing susceptibility to inhibition by avibactam. Knowledge gained from the molecular analysis of these and other AmpC resistance mutants will, we believe, aid in the design of β-lactams and BLIs with reduced susceptibility to mutational resistance.

Re-Understanding the Mechanisms of Action of the Anti-Mycobacterial Drug Bedaquiline

Bedaquiline (BDQ) inhibits ATP generation in Mycobacterium tuberculosis by interfering with the F-ATP synthase activity. Two mechanisms of action of BDQ are broadly accepted. A direct mechanism involves BDQ binding to the enzyme’s c-ring to block its rotation, thus inhibiting ATP synthesis in the enzyme’s catalytic α3β3-headpiece. An indirect mechanism involves BDQ uncoupling electron transport in the electron transport chain from ATP synthesis at the F-ATP synthase. In a recently uncovered second direct mechanism, BDQ binds to the enzyme’s ε-subunit to disrupt its ability to link c-ring rotation to ATP synthesis at the α3β3-headpiece. However, this mechanism is controversial as the drug’s binding affinity for the isolated ε-subunit protein is moderate and spontaneous resistance mutants in the ε-subunit cannot be isolated. Recently, the new, structurally distinct BDQ analogue TBAJ-876 was utilized as a chemical probe to revisit BDQ’s mechanisms of action. In this review, we first summarize discoveries on BDQ’s mechanisms of action and then describe the new insights derived from the studies of TBAJ-876. The TBAJ-876 investigations confirm the c-ring as a target, while also supporting a functional role for targeting the ε-subunit. Surprisingly, the new findings suggest that the uncoupler mechanism does not play a key role in BDQ’s anti-mycobacterial activity.

The influence of Rhus coriaria seeds Extracts on the Genetic Resistance of Aspergillus amstelodami

Introduction: This study aims to investigate the safe use of Rhus coriaria seed extracts (water and ethanol) genetically on fungi Aspergillus amstelodami because the extensive use of plant in many pharmaceutical and food fields. Materials and methods: In this study A1 (Wa1) strain of the fungus A.spergillus amstelodami was used in all genetic testing, Rhus coriaria seeds were obtained from the local markets and two types of extracts were prepared (water and ethanol extracts of Rhus coriaria seeds), two types of resistance mutants were isolated both spontaneous and induced by using mutation agent (nitrous acid). Results and Discussion: In this study 18 spontaneous resistance mutation were isolated in frequency 4.26×10-5 and 96 induced mutations were induced by nitrous acid in Frequency39.76×10-5at (MIC) 16 mg / ml of the ethanol extract, and 22 spontaneous mutations were isolate in frequency 4.59×10-5 and 91 nitrous acid induced mutation with an average recurrence 37.36×10-5at (MIC) 25 mg / ml of water extract. Conclusion: We conclude that presence of resistant ability in A. amstelodami toward Rhus coriaria water and ethanol seeds extracts. Therefore, we suggest further thorough studies to detect the activity to plant extract in order to be use in agricultural pest control.