Fungal infections have a significant impact on the world population, with estimates of over 1.6 million deaths a year. One contributing factor is the increasing number of fungi resistant to the current clinical treatments, including the last approved family of antifungal compounds introduced into the market over a decade ago. This is driving the search for new antifungals with different biological targets. A new potential antifungal occidiofungin, is a cyclic glycolipopeptide isolated from the soil bacterium Burkholderia contaminans MS14 with a broad spectrum of activity against both human and plant pathogens. Kill kinetics demonstrated that this compound is fungicidal and activates the cell wall integrity pathway at suboptimal dosing as determined by Mkc1 MAPK phosphorylation studies. As three of the four classes of currently available antifungals target ergosterol or ergosterol biosynthesis, the bioactivity of occidiofungin was assayed in the presence of ergosterol containing DOPC vesicles and was shown to retain antifungal properties. Occidiofungin was also found to have a similar activity profile against the S. cerevisiae -1,3-glucan synthesis mutant, indicating that it does not share a target with the fourth class of antifungals. Stability testing showed occidiofungin retained in vitro potency in the presence of human serum, across a broad range of pH and temperature conditions, and was resistant to gastric proteases. Based on cell morphology, occidiofungin did not target a specific stage of the yeast cell cycle, however cells were smaller in size and acquired ‘dancing bodies’, both properties of apoptosis. This was confirmed with data showing concentration dependent increases in DNA fragmentation, reactive oxygen species, and extracellular localization of phosphatidylserine. In addition to these findings, cells deleted for the yeast caspase gene exhibit a 2old resistance to occidiofungin. While SEM showed no morphological differences between treated and untreated cells, TEM did identify a thinning of the cell wall and inclusion bodies in cells treated with occidiofungin. As a stable fungicidal compound that induces apoptosis in yeast, occidiofungin has a great potential to become a new candidate drug for clinical use in treating fungal infections, including those resistant to current antifungals.

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. Occidiofungin produced by strain MS14 is an octapeptide dedicated to a broad range of antifungal activities of the bacterium. The 58.2-kb genomic fragment containing 18 open reading frames (ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin production. Putative proteins encoded by five nonribosomal peptide synthetase genes (occA – occE) of the gene cluster were predicted to contain the catalytic modules responsible for the biosynthesis of occidiofungin. Transcription of all the ORFs identified in the region except ORF1 and ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory genes located at the left border of the cluster. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1::nptII). Sequence analysis revealed that the occ gene cluster shared high similarity (99% nucleotide coverage and 91% identity) to an uncharacterized DNA region of B. ambifaria strain AMMD. The gene cluster was not found in other Burkholderia strains available in GenBank (nucleotide coverage < 24%). Analysis of G+C composition and prediction using “IslandPick” indicate that the occ gene cluster has possibly been horizontally transferred between bacteria. In addition, the absence of the gene cluster in clinical strains of Burkholderia indicates that occidiofungin is not required for potential human pathogenesis. The findings have provided insights into the development of antifungal medicines and agricultural fungicides based on occidiofungin.

Burkholderia contaminans MS14 shows excellent antimicrobial activities against a wide range of pathogens. Complete sequence analysis reveals that the MS14 genome harbors multiple gene loci that contribute to its antimicrobial activities and lacks key virulence features commonly found in pathogenic Burkholderia species. A mutagenesis study identified the genes required for MS14 antibacterial activities and gene expression profiling targeted a polyketide synthase (PKS) gene cluster. Site-specific mutagenesis confirmed the PKS gene cluster is directly related to MS14 antibacterial activities and the PKS gene product is predicted to be the MS14 antibacterial compound. Strain UFB2 isolated from Mississippi shows significant antifungal and antibacterial activities. UFB2 was classified to be Pseudomonas chlororaphis and its complete genome sequence was reported in this study. Green house trails showed P. chlororaphis strain UFB2 could efficiently reduce the disease severity of bacterial canker of tomato, by significantly inhibiting the growth of the pathogen Clavibacter michiganensis subsp. michiganensis. The research findings of B. contaminans MS14 and P. chlororaphis UFB2 have provided insights into the development of MS14 antibacterial compound for agricultural application and potential use of strain UFB2 as a biocontrol agent.