Isolation and toxicity assays
A total of 300 bacterial isolates from 30 soil samples were screened by Gram staining and CBB staining. Of which, 75 crystal-forming isolates in the sporulating culture were selected for the toxicity experiments against A. castellanii trophozoites. In the initial screening, proteins extracted from 14 isolates showed activity against trophozoites. However, after testing with different concentrations of the protein (ranging from 0.045-100 µg/mL), some of the isolates did not demonstrate antiprotozoal activity, while others only showed activity after 48 h of interaction (Table 1). Only three isolates MA8, CAB7, and AB5 demonstrated activity after 24 and 48 h of incubation with IC50 values of 4.9 µg/mL, 7.19 µg/mL, and 20.4 µg/mL after 24 h, respectively. Furthermore, a significant improvement in the antiparasitic activity after 48 h (IC50 values of 4.1 µg/mL, 4.35 µg/mL, and 8.3 µg/mL, respectively). These three isolates were selected for further examination by SEM to analyze the spore and parasporal inclusion structure. Morphological analyses demonstrated that AB5 and CAB7 have ellipsoidal spores and round parasporal inclusions, while MA8 spores are more rounded with small round inclusions (Fig. 1).
Biotechnological findings have explored the use of parasporal proteins primarily for their cytotoxic effects against human cancer cells (Okassov et al., 2015; E. N. Santos et al., 2022; Souissi et al., 2022), and as stated before, their effect on protozoan cells have been poorly investigated. In a study by (Kondo et al., 2002), bipyramidal parasporal inclusions of B. thuringiensis were used against the protozoan Trichomonas vaginalis. The extracted protein was found to cause 100% cell mortality after 48 h of interaction with the extracted protein at a concentration of 100 µg/mL. Our results also surpass those described in the only patent found in the databases (EP0461799A2) with the efficacy of parasporal inclusions against protozoans.
Table 1
Soil sample collection sites from rural and urban areas from different regions of Brazil and the inhibitory concentration (IC50) of extracted crystal proteins from soil isolates after 24 and 48 h against Acanthamoeba castelanii trophozoites.
Sample name / Location | City / State | Coordinates | IC50 (µg/mL) 24h | IC50 (µg/mL) 48h |
M.A. 8 / Atlantic Forest soil | Paraíba do Sul, Rio de Janeiro | 22°18'10.7"S 43°12'01.8"W | 4.9 | 4.1 |
CAB 7 / Soil with goat manure | São Cristóvão, Sergipe | 10°55'50.7"S 37°06'18.0"W | 7.19 | 4.35 |
AB 5 / Sandy soil from Abais beach | Estância, Sergipe | 11°16’25.0”S 37°15’18.0”W | 20.4 | 8.3 |
CD 1.1 / Chapada Diamantina National park | Mucugê, Bahia | 12°59’35.0”S 41°21’11.0”W | - | 40.1 |
S.AN 1.8 / Grota do Angico Natural reserve | Canindé de São Francisco, Sergipe | 9°37”49.1”S 37°43’42.5”W | - | 80.8 |
A.V. 7 / Organic aloe vera plantation | São Cristóvão, Sergipe | 10°55'51.4"S 37°06'16.1"W | - | 12.1 |
SF 9 / Riverbank soil | Propriá, Sergipe | 10°12'32.2"S 36°49'58.2"W | - | 72.4 |
CAC 1 / Soil from the edge of a waterfall | Petrópolis, Rio de Janeiro | 22°21’11.5”S 43°11’31.0”W | - | 57.4 |
(-) no antiprotozoal activity was observed until 24 h
The patent suggests that concentrations below 100 µg/mL of crystal proteins from B. thuringiensis are ineffective against Giardia lamblia trophozoites and require an interaction time of up to one week. The lack of patents for the use of parasporal proteins effective against Acanthamoeba sp. demonstrates a largely unexplored potential for the biotechnological application of these isolates.
Parasporal proteins including anticancer parasporins and antihelmintic Cry toxin (Cry5B) are being extensively researched for their potential use in human therapies. Their reported activity is comparable to the results obtained in this study. Parasporin, particularly parasporin-3 (Cry41Aa), which has been shown to be effective at targeting human leukemia and liver cancer cells (Souissi et al. 2022). These proteins work via a pore-forming mechanism that leads to membrane damage and cell death in cancer cells and require proteolytic cleavage for their activation, mainly at the N-terminal, which is critical for their cytotoxic properties (Domanska 2016). However, further studies are needed to assess their in vivo efficacy and safety, particularly in animal models (Okassov et al. 2015; Souissi et al. 2022). After six days of experimentation, Hu et al. (2013) and Kho et al. (2011) working with Cry5B crystal proteins against the helminth Caenorhabditis elegans reported a mean lethal concentration (LC50) and a median lethal dose (LD50) of approximately 7–9 µg/mL. The same protein Cry5B demonstrated to be even more lethal, achieving a mean effective dose (LD50) of 1.1 µg/mL by the fourth day of interaction with the L4 stage of the helminth larvae of Ascaris suum (Urban Jr et al. 2013).
Figure 1 Scanning electron microscopy images of Bacillus sp. spores (SP) and their parasporal inclusions (PCI). Bacterial isolates and their characteristic parasporal inclusions are shown for (A) AB5, (B) CAB7, and (C) MA8 isolates.
Genome characterization of CAB7 and MA8 strains
To enhance our understanding of the genomic characteristics, genome sequencing, annotation, and phylogenetic analysis were conducted on the CAB7 and MA8 isolates, which demonstrated superior antiparasitic activity. The draft genome of strain CAB7 presented a total genome size of 5,199,906 bp, with an average GC content of 35.6%. The reads were assembled into three large contigs, with a mean sequence size of 1,733,302 bp. The genome presented 5,493 predicted genes, of which 5,347 were protein-coding genes and 146 were RNA genes. In addition, 3,749 (68.25%) genes were annotated with COG functional categories.
The draft genome sequence of strain MA8 exhibited a total length of 5,292,368 bp, with an average GC content of 35.7%. The sequencing reads were assembled into a single large contig, revealing 5,634 predicted genes, comprising 5,487 protein-coding genes and 147 RNA genes. In addition, 4,566 genes (equivalent to 81.04%) were assigned COG functional classifications. The genomic features of the CAB7 and MA8 strains are listed in Table 2. The complete genome sequences of CAB7 and MA8 were deposited in GenBank with the accession numbers CP156026 and CP155804, respectively.
Table 2
Genomic features of MA8 and CAB7 isolates.
Features | Value |
MA8 | CAB7 |
Genome size (bp) | 5,292,368 | 5,199,906 |
Number of contigs | 1 | 3 |
G + C content (%) | 35.7 | 35.6 |
Shortest contig size | - | 7,542 |
Median sequence size | - | 10,299 |
Mean sequence size | - | 1,733,302 |
Longest contig size | - | 5,182,065 |
Total genes | 5,634 | 5,493 |
Protein-coding genes | 5,487 | 5,347 |
RNA genes | 147 | 146 |
rRNAs | 42 | 42 |
tRNAs | 105 | 104 |
CRISPR repeats | 0 | 1 |
Genes with no function prediction | 921 | 1,598 |
Genes with function prediction | 4,566 | 3,749 |
Phylogenetic analyses were conducted on eight representative genomes of Bacillus species, as well as strains CAB7 and MA8, using OAT (Fig. 2). The representative genomes used were Bacillus anthracis (AE017334), Bacillus paranthracis (CP101135), Bacillus cereus (AE017334), Bacillus thuringiensis (CM000753), Bacillus toyonensis ( CM000753), Bacillus mycoides (CP035997), and Bacillus subtilis (AL009126). The results revealed that the genomes of CAB7 and MA8 shared the highest identities with Bacillus paranthracis (97.80% and 92.06%, respectively). Moreover, MA8 genome shared 92.02% identity with Bacillus anthracis, 91.52% with Bacillus cereus, 91.35% with Bacillus thuringiensis, and 91.32% with Bacillus toyonensis. According to the current bacterial taxonomy, a possible novel species within the Bacillus genus is indicated by an average nucleotide identity (ANI) of less than 95% (Kim et al., 2014). Therefore, MA8 may potentially be a new species of Bacillus. The Bacillus cereus group include at least 21 related species, known for their innumerous biotechnological functions. Among these, Bacillus paranthracis stands out as a Gram-positive, facultatively anaerobic, non-motile, rod-shaped bacterium found in various environments. Despite its ecological presence, information about this organism remains limited, as it was only officially classified in 2017. This classification was based on Average Nucleotide Identity (ANI) values and the physiological and biochemical characteristics of the strains. To date, few papers have provided a comprehensive analysis of Bacillus paranthracis (Baev et al 2024).
Identification of toxin genes in the genomes of CAB7 and MA8 strains
For the identification of toxin genes in the genomes of CAB7 and MA8, we conducted a BLAST search, along with SVM and HMM machine-learning techniques, using the comprehensive toxin protein database (https://www.bpprc.org). The focus of this annotation step was to identify the genes responsible for synthesizing parasporal crystalline inclusions. The genome annotation results indicated that neither CAB7 nor MA8 contains any typical parasporal inclusion genes (Cry or Cyt) reported in Bacillus or related species. The genome of the CAB7 strain revealed the presence of the Spp1Aa1 toxin gene, with 79.08% identity and 99.21% coverage based on BLAST searches and HMM. Additionally, an S-layer protein was identified with 99% query coverage, an E-value = 9e-41, and 81.98% identity based on the BLAST search. A BLAST search also identified ten other toxins genes, including Zwa6, ChitinaseC, Zwa5A, InhA1, Bmp1, and InhA2.
BLAST and HMM analyses of the genome of the MA8 strain revealed the presence of two Spp1Aa1 toxin genes, with identities ranging from 80.28–90.77%, and coverage ranging from 99.21–100%. Additionally, two Vpb toxin genes and ten other toxin genes, namely Zwa6, Zwa5A, InhA1, and InhA2 were also identified via the HMM approach. However, the only gene associated with parasporal crystals that was identified was that of SLP. The SLP gene was identified through a BLAST search, with 82% query coverage, an E-value = 6e-43, and 82.66% identity. SLPs are found in both gram-negative and gram-positive bacteria, including Bacillus (Sará and Sleytr 2000). Like delta-endotoxins, SLPs assemble at parasporal positions, and exhibit different morphological features such as oblique, square, or hexagonal shapes (Rubio et al. 2017). They typically have a molecular mass ranging from 40 to 170 kDa (Sleytr et al. 1993, 2014) and play crucial roles in the growth, survival, and maintenance of cell integrity (Gang et al. 2008). Additionally, reports indicate their antiviral and antibacterial properties, as well as their anti-inflammatory effects (Sleytr et al. 1993, 2014). SLPs derived from the B. thuringiensis strain have been reported to possess insecticidal activity (Pena et al. 2006) as well as highly selective in vitro cytotoxicity against the MDA-MB-231 breast cancer cell line (Rubio et al., 2017). Pena et al. (2006) reported parasporal crystals produced by the GP1 strain of B. thuringiensis with insecticidal activity against the coleopteran pest E. varivestis. The gene responsible for encoding this protein was cloned and sequenced, revealing that it is an S-layer protein highly similar to those previously described in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). Phylogenetic analysis revealed that SLPs from several bacteria, including Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis, are grouped in the same main cluster, suggesting a common evolutionary origin.