Isolation and identification of the antimicrobial producing strain
Approximately one thousand colonies were isolated from 13 samples grown on different media. From these, seven colonies were chosen as potential producers of antimicrobial molecules active against at least one of the tested pathogenic strains. Following purification of the isolates and confirmation of antimicrobial production, it was found that one isolate (BGNM1), that originated from soil, showed a markedly larger zone of inhibition against the Gram-positive indicator strain Listeria monocytogenes ATCC19111, (20 mm zone) (Fig. 1) and was chosen for further analysis. 16S rRNA gene sequence (BLAST) analysis of the antimicrobial producing isolate revealed that Bacillus cereus BGNM1 showed 100% identity with many Bacillus cereus strains including A1, M13, FDAARGOS_798, WPySW2, DLOU. According to the PubMLST scheme, B. cereus BGNM1 belongs to ST1465, clonal complex ST-142. Representatives of B. cereus ST1465 were isolated from primary cutaneous anthrax-like infection in newborn infants in India. However, their role in the development of cutaneous lesions has been considered arguable since strains belonging to ST-142 clonal complex are generally regarded as foodborne with potential to cause foodborne illness (Saikia et al. 2019). In this study, the number of antimicrobial producing colonies was very low, which is in contrast to other surveys where up to 68% of tested bacterial isolates produced antimicrobial substances (Ahmed Sheikh 2010; Motta et al. 2004). The choice of indicator strains, especially significant human pathogens that are not closely related to the producers of antimicrobial molecules, may have impacted the positivity rate.
Bacteria from the genus Bacillus and related genera produce many substances with antimicrobial activity, including non-ribosomally synthesized lipopeptides and peptides (Fischbach and Walsh 2006; Marahiel and Essen 2009), polyketide compounds (Weissman and Leadlay 2005) and bacteriocins (Le Marrec 2000; Kiss 2008). To determine the nature of the antimicrobial activity produced by BGNM1, it was treated with a crystal of the proteolytic enzyme, pronase E. Following incubation, no antimicrobial activity was detected in the vicinity of the crystal on all tested indicator strains, thereby confirming its proteinaceous nature (Fig. 1).
Spectrum of activity of Bacillus cereus BGNM1 cell free supernatant
The antimicrobial activity of B. cereus BGNM1 CFS was tested against various Gram-positive and Gram-negative spoilage and pathogenic bacteria (Table 2). The CFS of strain BGNM1 inhibited the growth of most Gram positive tested, and just three Gram negative bacteria (Ralstonia solanacearum, Xanthomonas oryzae and Erwinia amylovora) (Table 2). During testing, it was noticed that some isolates of S. agalactiae species were sensitive while others were resistant to the CFS of Bacillus cereus BGNM1 (Table 2).
Table 2
Antimicrobial activity of BGNM1 CFS
Indicator strains | Activity |
Staphylococcus aureus ATCC25923 | + |
Listeria monocytogenes ATCC19111 | + |
Streptococcus agalactiae B782 | + |
Streptococcus agalactiae B761 | - |
Lactococcus lactis BGMN1-596 | + |
Bacillus cereus ATCC11778 | + |
Bacillus subtilis subsp. subtilis ATCC23857 | + |
Ralstonia solanacearum | + |
Xanthomonas oryzae | + |
Erwinia amylovora | + |
Acinetobacter baumanii 6077/12 | - |
Burkholderia cenocepacia ST856 | - |
Salmonella Enteritidis ATCC13076 | - |
Escherichia coli ATCC 25922 | - |
Klebsiella pneumoniae Ni9 | - |
Pseudomonas aeruginosa MMA83 | - |
Genome sequence analysis shows that Bacillus cereus BGNM1 strain encodes, the two-peptide lantibiotic, thusin
The genome of B. cereus BGNM1 was sequenced and summary statistics for genome assemblies is presented in Supplementary Table S1. The draft genome sequence of isolate BGNM1 was deposited in the NCBI GenBank database under accession number GCA_013303115.
AntiSMASH and BAGEL4 analyses of BGNM1 genome sequence revealed the presence of a ~ 10 kb gene cluster that is 100% identical to the gene cluster responsible for the production of two-peptide lantibiotic thusin in Bacillus thuringiensis BGSC 4BT1 (Xin et al. 2016) (GenBank Accession number KT454399.1). There was no evidence of additional genetic determinants in BGNM1 genome that were likely to encode other antimicrobial compounds, suggesting that the antimicrobial activity of the BGNM1 strain could be entirely attributable to the production of thusin.
Purification of thusin produced by Bacillus cereus BGNM1
The antimicrobial molecule(s) produced by BGNM1 was purified by Amberlite XAD16N, C18 SPE and RP-HPLC and the molecular mass of the active peptides confirmed by MALDI-TOF mass spectrometry. HPLC fractions collected at 30 second intervals were assayed against Lactococcus lactis BGMN1-596 and activity detected in fractions 75–77 (Fig. 2A) corresponding to a single peak eluting at 37.5 minutes on the HPLC chromatogram (Fig. 2A). MALDI TOF mass spectrometry analysis revealed that fraction 76 contained masses of 3926 Da and 2908 Da (Fig. 2B), which correspond with the theoretical masses of the individual thusin peptides, Thsα (3928 Da) and Thsβ (2908 Da), respectively (Xin et al. 2016). This suggests that the peptides were co-eluting. Further attempts to separate the peptides using an analytical RP-HPLC column and shallower acetonitrile gradients were unsuccessful. However, the successful purification of thusin from BGNM1 CFS and the lack of evidence of production of other antimicrobials is in agreement with the genome sequence data.
Selected thusin-resistant mutants of the thusin-sensitive strain Streptococcus agalactiae B782 show higher level thusin resistance than naturally resistant strains
The spectrum of activity of BGNM1 CFS showed that some Streptococcus agalactiae strains were resistant to thusin while others were naturally sensitive, suggesting differences in the cell envelope of natural isolates. To identify the target molecule/structure for thusin on the cell membrane, random mutagenesis was used to generate thusin resistant mutants from an S. agalactiae sensitive strain, with a view to comparing induced mutations in thusin resistant derivatives with naturally occurring resistant isolates. MNNG (200 µg/ml) was used to generate random mutations in wild-type Streptococcus agalactiae B782, a strain naturally sensitive to thusin, with a survival rate of about 2%. Thusin-resistant mutants were selected on BHI agar plates containing 2x concentrated cell-free culture supernatant of the thusin-producing strain, BGNM1. Twenty-six colonies grew on the selective plates after incubation for 48 h at 37°C in an atmosphere of 5% CO2. All mutants showed a thusin-resistant phenotype in repeated antimicrobial tests. Comparative analysis of MIC values, obtained by broth microdilution method, of the thusin-resistant mutants, named B782R1, B782R2 and B782R3 (0.368 µM), with the thusin sensitive parental strain, S. agalactiae B782 (0.046 µM), showed that the resistant mutants have eight times higher MIC values. Furthermore, comparison with S. agalactiae B761, the naturally resistant strain, showed that mutants express higher level of resistance to thusin (0.368 µM) comparing to naturally resistant isolates (0.184 µM). Similar results of MIC determination were obtained by spot-on-the-lawn inhibition assay on Petri dish (Fig. 3). Since different lantibiotics, both single-peptide and two-peptides, have been found to target the same receptor on the cell membrane (Islam et al. 2012; Morgan et al. 2005), thusin resistant S. agalactiae mutants were tested for nisin sensitivity. A wide range of nisin concentrations of double dilutions were used to determine MIC values for thusin sensitive and resistant derivatives. Nisin sensitivity results showed that there is no difference in nisin sensitivity between thushin sensitive and resistant derivatives (250 IU/ml) indicating that there is no cross resistance for these two lantibiotics, ie that these two lantibiotics use different receptors to interact with sensitive cells and achieve antimicrobial effect.
Comparative analysis of the genome sequences of Streptococcus agalactiae B782 mutants reveals that the C-protein α antigen is involved in thusin resistance
A genome sequencing approach was used to identify gene(s) involved in the thusin resistance phenotype. Specifically, the genomes of three of the 26 thusin-resistant mutants (S. agalactiae B782R1, B782R2 and B782R3), and one naturally thusin-resistant clinical isolate, S. agalactiae B761, were sequenced and compared to S. agalactiae B782WT, the wild type sensitive strain. Genome alignment of S. agalactiae B782WT and resistant mutants is presented in Fig. 4.
Sequence analysis showed that all three newly generated thusin-resistant mutants (B782R1-B782R3) possessed a common duplication of a region encoding 79 amino acids of the repeat domains in the C-protein α antigen compared to those of the sensitive WT strain, S. agalactiae B782WT. No other common gene mutations were identified. This suggests that an intact C-protein α antigen, which is an important cell surface protein contributing to the virulence and immunity of S. agalactiae (Lindahl et al. 2005; Maeland et al. 2015; Michel et al. 1991), is necessary for the activity of thusin against S. agalactiae. Specifically, genome analysis of S. agalactiae B782WT showed that it possesses two genes annotated as the C-protein α antigen; one is a truncated gene encoding a 109 amino acid protein and the second complete, encoding a protein with 79 amino acid repeat regions. Thusin induced resistant mutants S. agalactiae B782R1, B782R2 and B782R3 also have two such genes, with the truncated gene unchanged, but the second containing additional duplications of the region encoding 79 amino acids potentially resulting in thusin resistance. Genome analysis of the naturally resistant S. agalactiae B761 strain, revealed deletions within both genes encoding the C-protein α antigen containing only the C-terminal part of C protein II, thus confirming its involvement in thusin resistance. Taken together, the results strongly suggest that the C-protein α antigen is critical to the activity of thusin against S. agalactiae and that resistance evolves through multiplication of the region encoding repeatable 79 aa domains, most likely changing the 3D structure and preventing direct interaction, or deletion mutations of the gene. It is interesting that both types of changes in the C-protein α antigen, through either prolongation of the protein or its deletion, lead to the resistance to thusin (Fig. 3), indicating that in both cases the ability of thusin to inhibit is reduced. Species other than S. agalactiae are also sensitive to thusin, but do not have C-protein α antigen or similar protein. In addition, structural changes in C-protein α antigen lead to an increase in MIC values, only. Thus C protein α antigen is probably not a receptor protein for thusin, but is definitely involved in modulation of the thusin-receptor interaction. Variations in the number of repeating domains within cell-surface proteins of streptococci have previously been shown to impact significantly on their biological functions. More specifically, changes in the number of tandem repeats within the C-antigen proteins of streptococci have been shown to affect their immune evasion and pathogenicity in immunized hosts, where proteins with lower number of repeats were probably hidden by capsular polysaccharide and other cell surface proteins resulting in decrease in antigen size (Gravekamp et al. 1996). Furthermore, changes in a number of R28 protein tandem repeats, among alpha-like proteins of streptococci, resulted in changes in transcriptional levels of the gene encoding the given protein and, in turn, alternations of virulence and global transcriptomic changes (Eraso et al. 2020). Variability in Rib (resistance to proteases, immunity, group B) domain number of cell-surface proteins has also been shown to result in differential projection of key host-colonization domains affecting immune evasion of streptococci (Whelan et al. 2019). With this in mind, we speculate that the increase in 79 amino acid repeats within C-protein of group B streptococci could be an adaptive trait that enables survival in the presence of thusin as a selective agent. By multiplication of the 79 amino acids domain in C-protein α antigen S. agalactiae achieves a higher level of resistance to thusin, which indicates that in this way C-protein α antigen masks the thusin binding site on the receptor. Since S. agalactiae modulates the number of 79 aa repeats of the C-protein α antigen as an adaptation to the host immune response, this modulation could be successfully used to influence the thusin-receptor interaction, indicating an even more significant role of this surface protein in survival and infection. Further research is needed to elucidate all relevant participants for thusin activity, mutual positions of C-protein α antigen and membrane receptor as well as whether direct interaction between C-protein α antigen and thusin occurs and, if so, which domains are involved.