Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus.

Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11) of the ergosterol biosynthesis pathway. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not trigger comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3ß,6α-diol, the "toxic diol" responsible for the fungistatic activity against yeasts, is not required for the fungicidal effects in A. fumigatus. While ERG3-lacking yeasts are azole resistant, ERG3-lacking A. fumigatus becomes more susceptible. Mutants lacking mitochondrial complex III functionality, which are much less effectively killed, but strongly inhibited in growth by azoles, convert eburicol more efficiently into the supposedly "toxic diol". We propose that the mode of action of azoles against A. fumigatus relies on accumulation of eburicol which exerts fungicidal effects by triggering cell wall carbohydrate patch formation.

The hidden burden of melioidosis in Nepal: a paradigm for the urgent need to implement a simple laboratory algorithm to detect Burkholderia pseudomallei in low-resource endemic areas.

Melioidosis, an emerging infectious disease caused by the Gram-negative bacillus Burkholderia pseudomallei, is massively underdiagnosed in many low- and middle-income countries. The disease is clinically extremely variable, has a high case fatality rate, and is assumed to be highly endemic in South Asian countries, including Nepal. The reasons for underdiagnosis include the lack of awareness among clinicians and laboratory staff and limited microbiological capacities. Because costly laboratory equipment and consumables are likely to remain a significant challenge in many melioidosis-endemic countries in the near future, it will be necessary to make optimum use of available tools and promote their stringent implementation. Therefore, we suggest that health facilities in resource-poor countries, such as Nepal, introduce a simple and low-cost diagnostic laboratory algorithm for the identification of B. pseudomallei cultures. This screening algorithm should be applied specifically to samples from patients with fever of unknown origin and risk factors for melioidosis, such as diabetes. In addition, there could also be a role of low-cost, novel, promising serological point-of-care tests, which are currently under research and development.

Only One of Three Bcs1 Homologs in Aspergillus fumigatus Confers Respiratory Growth.

The mitochondrial translocase Bcs1 is required for the correct assembly of complex III of the mitochondrial respiratory chain. Because of its importance, Bcs1 was recently proposed as a target for antifungal agents. The function of this AAA (ATPase Associated with diverse cellular Activities) protein has been extensively characterized in Saccharomyces cerevisiae. This yeast as well as previously studied mammals each encode only one homolog. In contrast, the pathogenic mold Aspergillus fumigatus encodes three putative Bcs1 homologs, none of which have been characterized to date. To study the role of these three homologs in A. fumigatus, conditional and deletion mutants of the respective genes AFUA_3G13000 (bcs1A), AFUA_4G01260 (bcs1B), and AFUA_2G14760 (bcs1C) were generated. A deletion or downregulation of bcs1A resulted in drastically reduced growth and sporulation rates and in a significantly altered susceptibility to azole antifungals. In contrast, mutants lacking Bcs1B or Bcs1C did not show any phenotypes differing from the wild type. Salicylhydroxamic acid-an inhibitor of the alternative oxidase that allows the respiratory chain to bypass complex III in some species-caused a complete growth arrest of the bcs1A deletion mutant. In a Galleria mellonella infection model, the deletion of bcs1A resulted in significantly decreased virulence. Only Bcs1A was able to partially complement a deletion of BCS1 in S. cerevisiae. The subcellular localization of Bcs1B and Bcs1C outside of mitochondria suggests that these Bcs1 homologs exert cellular functions different from that of Bcs1. Our data demonstrate that Bcs1A is the sole Bcs1 ortholog in A. fumigatus.

A head-to-head comparison of three MALDI-TOF mass spectrometry systems with 16S rRNA gene sequencing.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized diagnostics in culture-based microbiology. Commonly used MALDI-TOF MS systems in clinical microbiology laboratories are MALDI Biotyper (Bruker Daltonics) and Vitek MS (bioMérieux), but recently the new EXS2600 (Zybio) has been launched. This study aimed to evaluate the performance of the three devices by comparing the results to 16S rRNA gene sequencing. A set of 356 previously collected difficult-to-identify bacteria was tested in parallel with the three systems. Only the direct smear method and simple formic acid extraction were applied. Valid results were achieved for 98.6%, 94.4%, and 93.3% of all isolates by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Of all valid results, agreement with sequencing data was achieved in 98.9%, 98.5%, and 99.7% by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Considering only the isolates with valid measurements at the single-species level, misidentification rates were 0%, 2.6%, and 1.1% for MALDI Biotyper, EXS2600, and Vitek MS, respectively. Apart from minor performance differences, our data demonstrate that the three systems provide comparable results and are suitable for use in medical diagnostic laboratories.

Azole-induced cell wall carbohydrate patches kill Aspergillus fumigatus.

Azole antifungals inhibit the fungal ergosterol biosynthesis pathway, resulting in either growth inhibition or killing of the pathogen, depending on the species. Here we report that azoles have an initial growth-inhibitory (fungistatic) activity against the pathogen Aspergillus fumigatus that can be separated from the succeeding fungicidal effects. At a later stage, the cell wall salvage system is induced. This correlates with successive cell integrity loss and death of hyphal compartments. Time-lapse fluorescence microscopy reveals excessive synthesis of cell wall carbohydrates at defined spots along the hyphae, leading to formation of membrane invaginations and eventually rupture of the plasma membrane. Inhibition of ß-1,3-glucan synthesis reduces the formation of cell wall carbohydrate patches and delays cell integrity failure and fungal death. We propose that azole antifungals exert their fungicidal activity by triggering synthesis of cell wall carbohydrate patches that penetrate the plasma membrane, thereby killing the fungus. The elucidated mechanism may be potentially exploited as a novel approach for azole susceptibility testing.