A novel family of non-secreted tridecaptin lipopeptide produced by Paenibacillus elgii.

Bacteria from the genus Paenibacillus make a variety of antimicrobial compounds, including lipopeptides produced by a non-ribosomal synthesis mechanism (NRPS). In the present study, we show the genomic and phenotypical characterization of Paenibacillus elgii AC13 which makes three groups of small molecules: the antimicrobial pelgipeptins and two other families of peptides that have not been described in P. elgii. A family of lipopeptides with [M + H]+ 1664, 1678, 1702, and 1717 m/z was purified from the culture cell fraction. Partial characterization revealed that they are similar to tridecaptin from P. terrae. However, they present amino acid chain modifications in positions 3, 7, and 10. These new variants were named tridecaptin G1, G2, G3, and G4. Furthermore, a gene cluster was identified in P. elgii AC13 genome, revealing high similarity to the tridecaptin-NRPS gene cluster from P. terrae. Tridecaptin G1 and G2 showed in vitro antimicrobial activity against Escherichia coli, Klebsiella pneumonia (including a multidrug-resistant strain), Staphylococcus aureus, and Candida albicans. Tri G3 did not show antimicrobial activity against S. aureus and C. albicans at all tested concentrations. An intriguing feature of this family of lipopeptides is that it was only observed in the cell fraction of the P. elgii AC13 culture, which could be a result of the amino acid sequence modifications presented in these variants.

Co-occurrence of linear and cyclic pelgipeptins in broth cultures of Paenibacillus elgii AC13.

Paenibacillus elgii AC13 produces antimicrobial lipopeptides of agricultural and pharmaceutical importance. It secretes four cyclic lipopeptides named pelgipeptins, previously characterized in P. elgii B69. These lipopeptides result from the expression of a nonribosomal peptide gene cluster. P. elgii AC13 also produced two linear lipopeptides with ratios of [M + H] + 1105 and 1119 m/z. These compounds were previously observed in Paenibacillus sp. strain OSY-N, but due to purification difficulties, their characterization was executed using synthetically produced linear pelgipeptins. In the present study, purification was achieved from the supernatants of cultures from three complex media by high-performance liquid chromatography. The partial characterization of linear pelgipeptins revealed the similar antimicrobial activity and cytotoxicity of their synthetically produced counterparts, known as paenipeptins. Cyclic forms were highly stable to changes in pH, temperature, and organic extraction with n-butanol as shown by mass spectrometry (MALDI-TOF); therefore, these steps did not cause the hydrolysis of pelgipeptins. A low-activity thioesterase could also generate the linear isoforms observed; this enzyme catalyzes the cyclization process and is coded in the same gene cluster. Alternatively, the cyclic forms were hydrolyzed by an unknown protease produced during growth in the complex medium used in the present study. Although culture conditions are known to produce pelgipeptins with different yields and amino acid compositions, the occurrence of linear and cyclic forms simultaneously has not yet been reported. A mixture of cyclic and linear pelgipeptins presents a potential advantage of the higher antimicrobial activity of cyclic forms combined with the lower cytotoxicity of linear isoforms.

In vitro antifungal activity of pelgipeptins against human pathogenic fungi and Candida albicans biofilms.

Systemic mycoses have become a major cause of morbidity and mortality, particularly among immunocompromised hosts and long-term hospitalized patients. Conventional antifungal agents are limited because of not only their costs and toxicity but also the rise of resistant strains. Lipopeptides from Paenibacillus species exhibit antimicrobial activity against a wide range of human and plant bacterial pathogens. However, the antifungal potential of these compounds against important human pathogens has not yet been fully evaluated, except for Candida albicans. Paenibacillus elgii produces a family of lipopeptides named pelgipeptins, which are synthesized by a non-ribosomal pathway, such as polymyxin. The present study aimed to evaluate the activity of pelgipeptins produced by P. elgii AC13 against Cryptococcus neoformans, Paracoccidioides brasiliensis, and Candida spp. Pelgipeptins were purified from P. elgii AC13 cultures and characterized by high-performance liquid chromatography (HPLC) and mass spectrometry (MALDI-TOF MS). The in vitro antifugal activity of pelgipeptins was evaluated against C. neoformans H99, P. brasiliensis PB18, C. albicans SC 5314, Candida glabrata ATCC 90030, and C. albicans biofilms. Furthermore, the minimal inhibitory concentration (MIC) was determined according to the CLSI microdilution method. Fluconazole and amphotericin B were also used as a positive control. Pelgipeptins A to D inhibited the formation and development of C. albicans biofilms and presented activity against all tested microorganisms. The minimum inhibitory concentration values ranged from 4 to 64 µg/mL, which are in the same range as fluconazole MICs. These results highlight the potential of pelgipeptins not only as antimicrobials against pathogenic fungi that cause systemic mycoses but also as coating agents to prevent biofilm formation on medical devices.

Draft Genome Sequence of the Antimicrobial-Producing Strain Paenibacillus elgii AC13.

A Paenibacillus elgii strain isolated from soil samples from Cerrado, Brazil, showed antimicrobial activity. Its genome sequence was acquired (GS20 FLX Titanium 454 platform) and comprises 108 contigs (N50, 198,427 bp) and 6,810 predicted sequences. Here, we shed some light on the antimicrobial genes of the strain, including a nonribosomal peptide synthetase (NRPS) module identified as part of a pelgipeptin gene cluster.