Mining the nanotube-forming Bacillus amyloliquefaciens MR14M3 genome for determining anti- Candida auris and anti- Candida albicans potential by pathogenicity and comparative genomics analysis

Abstract

There is a global health concern associated with the emergence of the multidrug-resistant (MDR) fungus Candida auris, which has significant mortality rates. Finding innovative and distinctive anti-Candida compounds is essential for treating infections caused by MDR C. auris. A bacterial strain with anti-Candida activity was isolated and identified using 16 S rRNA gene sequencing. The whole genome was sequenced to identify biosynthesis-related gene clusters. The pathogenicity and cytotoxicity of the isolate were analyzed in Candida and HFF-1 cell lines, respectively. This study set out to show that whole-genome sequencing, cytotoxicity testing, and pathogenicity analysis combined with genome mining and comparative genomics can successfully identify biosynthesis-related gene clusters in native bacterial isolates that encode antifungal natural compounds active against Candida albicans and C. auris. The native isolate MR14M3 has the ability to inhibit C. auris (zone of inhibition 25 mm) and C. albicans (zone of inhibition 25 mm). The 16 S rRNA gene sequence of MR14M3 aligned with Bacillus amyloliquefaciens with similarity (100%). Bacillus amyloliquefaciens MR14M3 establishes bridges of intercellular nanotubes (L 258.56 ± 35.83 nm; W 25.32 ± 6.09 nm) connecting neighboring cells. Candida cell size was reduced significantly, and crushed phenotypes were observed upon treatment with the defused metabolites of B. amyloliquefaciens MR14M3. Furthermore, the pathogenicity of B. amyloliquefaciens MR14M3 on Candida cells was observed through cell membrane disruption and lysed yeast cells. The whole-genome alignment of the MR14M3 genome (3981,643 bp) using 100 genes confirmed its affiliation with Bacillus amyloliquefaciens. Genome mining analysis revealed that MR14M3-coded secondary metabolites are involved in the biosynthesis of polyketides (PKs) and nonribosomal peptide synthases (NRPSs), including 11 biosynthesis-related gene clusters with one hundred percent similarity. Highly conserved biosynthesis-related gene clusters with anti-C. albicans and anti-C. auris potentials and cytotoxic-free activity of B. amyloliquefaciens MR14M3 proposes the utilization of Bacillus amyloliquefaciens MR14M3 as a biofactory for an anti-Candida auris and anti-C. albicans compound synthesizer.

Keywords: Antifungal activity; Bacillus; Biofactory; Biosynthesis-related gene clusters; Candida albicans; Candida auris; Comparative genomics; Cytotoxicity; Genome mining.

Graphical abstract

Fig. 1

Scanning electron micrographs of representative Candida albicans and Candida auris cells. A: Budding yeast forms (blastospores) of Candida albicans CAL1; B: Electron-microscopic structure of Candida albicans CAL1 used for the study. C: Anti-Candida albicans activity of Bacillus amyloliquefaciens MR14M3; D and E: Electron-microscopic structure of well-defined, oval-shaped yeast morphology of Candida auris. Ring of double bud scars (arrow) located at one pole. F: Anti-Candida auris activity of Bacillus amyloliquefaciens MR14M3.

Fig. 2

Electron-microscopic structure of Bacillus amyloliquefaciens MR14M3 (length = 3428.3078 ± 1204.6725 nm; width = 568.9325 ± 39.6329 nm). A, C and E: Electron-microscopic structure of Bacillus amyloliquefaciens MR14M3; B and H: Cells of MR14M3. The yellow square indicates an intercellular nanotube connecting Bacillus amyloliquefaciens MR14M3. D: Single cell of Bacillus amyloliquefaciens MR14M3. The yellow arrow indicates the nanotubes (length = 258.5637 ± 35.8356 nm; width = 25.3264 ± 6.0908 nm) that bridge neighboring cells of Bacillus amyloliquefaciens MR14M3. E: Scanning electron micrographs and high magnification image of the nanotube phenotype of Bacillus amyloliquefaciens MR14M3.

Fig. 3

Electron-microscopic structure of Candida albicans and Bacillus amyloliquefaciens MR14M3 after antifungal susceptibility of C. albicans to MR14M3. A, B, C and D: Electron-microscopic structure of C. albicans from the edge of the zone of inhibition [T1]. C: Candida albicans cell membranes disrupted by Bacillus amyloliquefaciens MR14M3; length 3.6771 ± 0.2183 µm; width = 2.0323 ± 0.2168 µm; E, F, and G: Electron-microscopic structure of C. albicans from the zone of inhibition [T2]; length 3.3522 ± 0.3032 µm; width = 1.9165 ± 0.1911 µm. H and I: Electron-microscopic structure of C. albicans from the normal fungal growth region [T3]; length 3.754 ± 0.2617 µm; width = 3.2497 ± 0.1535 µm. J: Bacillus amyloliquefaciens MR14M3 from bacterial disc [T4]. K: Length of C. albicans cells after the treatment. * Significant at p value < 0.05; L: Width of C. albicans cells after the treatment. * * Significant at p value < 0.001.

Fig. 4

Electron-microscopic structure of Candida auris and Bacillus amyloliquefaciens MR14M3 after antifungal susceptibility of MR14M3 on C. auris. A and B: Electron-microscopic structure of C. auris from the edge of the zone of inhibition [T1]. B: C. auris cell membranes disrupted by Bacillus amyloliquefaciens MR14M3; C, D, and E: Electron-microscopic structure of C. auris from the zone of inhibition [T2]; F and G: Electron-microscopic structure of C. auris from normal fungal growth region [T3]; H: B. amyloliquefaciens MR14M3 from bacterial disc [T4].

Fig. 5

Cell viability of sterile broth control and 24-hour-old Bacillus amyloliquefaciens MR14M3 broth devoid of cells against human foreskin fibroblasts (HFF-1). A: Representatives of human foreskin fibroblasts after exposure to [1:2.5, 1:5] dilutions of Bacillus amyloliquefaciens MR14M3 broth compared to the sterile broth control. Scale bar: 200 µm. B: The percentage of human foreskin fibroblast viability. Three sets of experiments were performed, and the average cell viability was used for analysis.

Fig. 6

Circular genome map of Bacillus amyloliquefaciens MR14M3 for genome information. The zoomed region indicates the genes of biosynthesis-related gene clusters identified in the genome of B. amyloliquefaciens MR14M3. Phylogenetic-based confirmation of MR14M3 as Bacillus amyloliquefaciens based on 16 S rRNA from the MR14M3 genome.

Fig. 7

Phylogenetic tree of MR14M3 using the whole genome specifically with 68 single-copy genes aligned with 82 genomes from the genus. Bacillus. A phylogenetic tree of the MR14M3 genome was constructed on PATRIC using 53082 aligned nucleotides and 17694 aligned amino acids. Details of tree analysis statistics, genome statistics and gene family statistics are presented in Supplementary Data 1.

Fig. 8

Representative of the genetic organization of biosynthesis-related gene clusters identified in the genome of Bacillus amyloliquefaciens MR14M3. Conservation of three biosynthesis-related gene clusters in the genome of Bacillus amyloliquefaciens MR14M3 with closely related Bacillus species in the same genus. The forward and reverse orientations of the gene in BGCs are denoted with the direction of the arrow.

Fig. 9

Natural antifungal compounds were predicted through genome mining from the genome of Bacillus amyloliquefaciens MR14M3.