Identification and structural characterization of novel bacteriocins by genome-wide screening of hypothetical mini-proteins in Staphylococcus aureus

doi:10.21203/rs.3.rs-5024297/v1

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Identification and structural characterization of novel bacteriocins by genome-wide screening of hypothetical mini-proteins in Staphylococcus aureus

https://doi.org/10.21203/rs.3.rs-5024297/v1

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Bacteriocins are ribosomally synthesized small antimicrobial peptides secreted by bacteria to overcome colonization resistance from closely related competitive microorganisms. Staphylococcus aureus is an opportunistic pathogen, that colonizes the skin and nasal cavity of healthy individuals and causes both superficial and systemic infections. The bacteriocins are the primary tool of S. aureus to compete with other members of the human microbiota for effective colonization. Different S. aureus strains produce various bacteriocins with widely diverse target organisms, which are mostly unexplored. Identification of new bacteriocins and their target organism can help us to understand the ecology of the S. aureus infection and lead to finding better treatment. Moreover, bacteriocins can help in the alternative treatment of antibiotic-resistant infections as well as have major applications in food preservation and agriculture. A large section of the S. aureus genome encodes small proteins consisting of 100 or fewer amino acids called mini-proteins. Analyzing the amino acid sequence of the hypothetical mini-proteins for the co-occurrence of antimicrobial activity and signal for extracellular secretion, seven novel bacteriocins genes are identified from S. aureus NCTC 8325. Newly identified bacteriocin genes are found to have stress and starvation inducible upstream regulatory elements, which upregulate bacteriocin production in nutrient-limited or colonization-resistant induced stress environments. The identified peptides have a high net positive charge, which facilitates interaction with negatively charged bacterial membranes. Ab initio modeling of the peptides, molecular dynamic simulation and structural comparison with known AMPs identified structural elements important for membrane disruption and bactericidal activity.

Staphylococcus aureus

Bacteriocin

Mini-proteins

Antimicrobial peptides

Hypothetical proteins

Staphylococcus aureus is one of the deadliest microorganisms that cause various infections in humans as well as in other animals. Being a commensal and opportunistic pathogen, S. aureus has to combat the host immune system as well as the host microbiota [1]. Healthy microbiota can resist the growth of pathogens, therefore, S. aureus secretes molecules to weaken the protective barrier, which leads to dysbiosis. Small oligopeptides play an important role in S. aureus colonization because of their versatile interacting ability and membrane permeability. The S. aureus is known to secrete bacteriocins, small antimicrobial peptides (AMPs), to get competitive advantages over other microorganisms for colonization in the skin and nasal cavity. The bacteriocins are ribosomally synthesized small anti-microbial peptides that may or may not have posttranslational modification [2], [3]. The producing species are resistant to the bacteriocin, however, it affects closely related species. Various narrow-spectrum bacteriocins are produced by different S. aureus strains with highly diverse antimicrobial specificity. Around 84% of the isolated staphylococcal species and 54% of isolated S. aureus strains from the human nasal cavity show antimicrobial activity through the production and secretion of bacteriocins [1]. The abundance and diversity of bacteriocin production by human nasal S. aureus isolates indicates its importance in shaping nasal microbiota [4]. However, very little is known about the genetic diversity, structure, mode of action and gene expression regulation of these bacteriocins. The identification and characterization of new bacteriocins from S. aureus would lead us to explore the underlying mechanisms of host microbiota modulation by S. aureus and enrich our understanding of the ecology of the staphylococcal infection.

This pool of genes in S. aureus is abundant with small open reading frames (ORF) that encode peptides with lengths no longer than 100 amino acids and are called mini-proteins [5]. In bacteria and archaea, around 11% of all annotated ORFs code for mini-proteins [5]. The low molecular weight of these proteins makes them difficult to work with using standard in vitro techniques, whereas, the short length of the sequence makes it difficult for in silico annotation as well [6]. However, some mini-proteins have already been characterized and identified to have important biological functions. Mini-proteins can be a part of big assemblies like the ribosome, membrane transporter, and cell division machinery as well as can act stand-alone as signaling peptides, protease inhibitors, toxins, and bacteriocins [7]. A few of the extracellular secretory bacterial peptides are the most well-characterized mini-proteins and utmost importance for virulence. In S. aureus, the phenol soluble modules (PSM) consisting of secretory peptides of 20 to 40 amino acid length were involved in biofilm formation, cytotoxicity and many other virulence-associated functions [8]. Despite the importance of mini-proteins in S. aureus colonization and signaling, the majority of the mini-proteins are still uncharacterized. In the present study, we report a methodology of genome-wide screening to identify novel bacteriocins by analyzing amino acid sequences of the hypothetical mini-proteins. There are seven novel bacteriocins have identified and characterized from S. aureus NCTC 8325. The analysis of amino acid sequences and three-dimensional structures of newly identified bacteriocins by sequence alignment and ab initio modeling indicate the presence of structural similarity with known membrane-disrupting AMPs and provide insight into their mode of action.

Sequence retrieval

The amino acid sequences of mini-proteins from Staphylococcus aureus NCTC 8325 [9] were retrieved from the KEGG organism database [10]. The criteria for choosing the mini-protein was the sequence length of the gene should not be more than 100 amino acids long. Based on the above criteria, the sequences were extracted manually in FASTA format and used for further analysis.

Anti-microbial activity prediction

The initial screening for the Anti-microbial activity was performed in Antimicrobial Peptide Scanner vr.2 servers [11] and the criteria of selection was the prediction probability equal to 0.9 or higher. A total of 51 mini-proteins were initially identified to have predicted high antimicrobial activity. The possible function of the mini-proteins was predicted by sequence-based motif prediction using the 'MOTIF Search server using both the pfam database [12] and NCBI Conserved Domain Database (NCBI-CDD) [13]. Peptides with any predicted known motif were removed from the final analysis. Only hypothetical proteins were chosen for further study. The sequence alignment of the identified peptides with known AMPs was done using the APD3 Antimicrobial peptide database [14, p. 3].

Sub-cellular localization prediction

The subcellular localization of the bacterial peptides was predicted using its amino acid sequence, as the cell targets peptides to a particular location based on the signal present in the amino acid sequence or the characteristic of the entire polypeptide. The sub-cellular localization of the bacteriocins was analysed in PSORTb [15], and CELLO [16] servers.

Physicochemical characterization

The detailed physicochemical properties of the peptides were computed based on the amino acid composition of the peptide. The analysis of physicochemical properties was done in the ProtParam tool of the Expasy server [17]. Parameters like theoretical pI, Instability index [18], Aliphatic index [19] and Grand average of hydropathicity (GRAVY) [20] of each peptide were recorded. The data set for physiochemical properties of the bacteriocins was then further used to predict if the peptides are globular or membrane-associated in nature, their thermal stability and in vivo/ in vitro half-life.

Gene expression analysis

The sequence of 200 nucleotides upstream to the ORF of the staphylococcal bacteriocin genes was retrieved from the KEGG organism database and used for bacterial promoter prediction analysis with the BPROM server [21]. The sigma (σ) factor binding sites and the presence of any known transcription factor (TF) binding site in the promoter region were analyzed to identify the influence of environmental factors in gene expression regulation of the bacteriocin genes.

Structural analysis of the bacteriocins

The secondary structure prediction using the amino acid sequence was done with the PROTEUS2 web server [22]. The 3D structures of the identified bacteriocins were modeled using the ab initio method by AlphaFold Protein Structure Database server (ebi.ac.uk) [23] or Robetta or RoseTTAFold by Rosetta server [24], [25] and threading-based modeling server I-TASSER [26]. The quality of the modeled structures was validated using Ramachandran plot analysis in PROCHECK [27]. The modeled structures of the Staphylococcal bacteriocins were compared with the experimentally determined structures of known bacteriocins from PDB. All structural figures are generated using PyMol (https://pymol.org/2/).

Molecular dynamic simulation

Molecular dynamic simulations are performed in WEBGRO server (https://simlab.uams.edu/) using GROMOS96 54a7 force field [28, p. 6]. The structures were solvated in a triclinic box with SPC water. To neutralize charged residues in protein, 0.15 M NaCl was added. The structures were subjected to a 5000 step energy minimization and then equilibrated with NVT and NPT. The production simulations were run for 50 nS at 300K temperature and 1 bar pressure. The final data were analyzed and figures were prepared in PyMol and Excel.

Identification of bacteriocins by S. aureus genome-wide screening

The genome of S. aureus NCTC 8325 contains 487 small ORF-encoding mini-proteins with an amino acid sequence length of 100 or less. The bacteriocins are small peptides with antimicrobial activity as well as have signal sequences for extracellular secretion. Therefore, amino acid sequences of the mini-proteins were screened for antimicrobial activity as well as for subcellular localization using in silico methods. The screening of the amino acid sequence of the mini-protein in Antimicrobial Peptide Scanner vr.2 servers identified 51 peptides with predicted antimicrobial activity. Amino acid sequence base analysis of sub-cellular localization indicates only 13 out of the 51 antimicrobial peptides are secreted to extracellular environments like bacteriocins. Further, motif prediction indicates many of the extracellular peptides have known functional motifs, which are different from bacteriocin and, therefore, removed from the list. The final list of bacteriocin contains seven hypothetical mini-proteins (Table 1). Analysis of orthologues genes in the sequence database indicates only three of the seven bacteriocins, SAOUHSC_01507, SAOUHSC_00977 and SAOUHSC_A02794 are conserved among eight, ten and nine different other S. aureus strains respectively; whereas, rest of the bacteriocins, like SAOUHSC_01111, SAOUHSC_01544, SAOUHSC_01118 and SAOUHSC_00839 are almost unique for S. aureus NCTC 8325 strain only.

Table 1

List of bacteriocins identified by screening *S. aureus* NCTC8325 mini-protein library and their physicochemical properties.
Kegg Accession No.	NCBI Acc No	UniProt	amino acid sequence	sequence length	Molecular Weight (Da)	pI	Net Charges	Grand average of hydropathicity (GRAVY)	instability index	Aliphatic index
SAOUHSC_01111	3920753	Q2FZC1	MLKTVKSTRLLGIKKHTELNSCTPNVRKQC	30	3428.13	10.21	6.25	-0.57	31.24	84.33
SAOUHSC_01544	3920587	Q2FYC0	MNLMQHWKQTGLNPTARKERGVVSFVASMLHHQK	34	3961.64	11.1	4.75	-0.632	7.76	65.88
SAOUHSC_00839	3918951	Q2FZZ5	MLGPRQLAHYCKLTYHQLLCWGPAIIEKFVIGVFSFG	37	4238.1	8.8	2.5	0.562	44.2	105.41
SAOUHSC_01507	3919049	Q2FYE6	MTIFTQLSDRIKKAISKINQANNIPNNARKSPMYHSPIT	39	4443.16	10.56	5.25	-0.626	39.22	77.69
SAOUHSC_00977	3920122	Q2FZM3	MRYVTMRQFIKRTVKTILVGYVIKFIRNKLSGKSSHPTDNKHN	43	5107.08	11.17	9.5	-0.551	28	81.4
SAOUHSC_01118	3920719	Q2FZB6	MGWGPTQKLAKSQHTKMCKLAGPQQREIGSPIPTNNASWRGPQHRSWRKVSLQKCASWRGPNIEKLEPQFLQTMQVGVGHR	81	9188.61	11.04	10.75	-0.941	45.29	54.2
SAOUHSC_A02794	3921713	Q2FUZ5	MIIMRGYNGRYCSWTNTAIQCDYFNKVRQVKKDGKSDRLSNNINSNIIYRVLEKCITLYNVCSRCKRALILSEHSKKNIFNIKKAV	86	10095.84	9.89	12.25	-0.433	29.33	88.37

The amino acid sequence-based physicochemical characterization of the bacteriocins is reported in Table 1. All the bacteriocins have a net positive charge. The majority of the known bacteriocins are reported to have a net positive charge, which is important for their function to disrupt negatively charged microbial cell envelopes. The hydrophobicity of the bacteriocins was predicted with the Grand average of hydropathicity index (GRAVY). A negative value of GRAVY indicates the hydrophilic and globular nature of any peptide, whereas, a positive GRAVY value indicates the hydrophobic and membrane-associated nature of a peptide. The GRAVY analysis indicates that only SAOUHSC_00839 is membrane associated with a low positive GRAVY value, rest of all bacteriocins are globular with negative GRAVY value. The in vitro as well as in vivo half-life of the bacteriocins are predicted using the instability index. All of the bacteriocins have longer half-life as the instability index is less or close to a value of 40 [18]. Like the instability index, another physicochemical parameter, the aliphatic index indicates the thermo-stability of globular proteins. The aliphatic index of a peptide is calculated as the volume occupied by amino acids with an aliphatic side chain. Among all the bacteriocins, SAOUHSC_01118 and SAOUHSC_01544 have an aliphatic index lower than 70, which indicates they are comparatively less thermostable [19]. The rest of the bacteriocins are comparatively thermostable with a high aliphatic index.

The analysis of sequence alignment of the bacteriocins in the APD3 AMP database identified similarity with known bacteriocins or AMPs. The SAOUHSC_00977 shows 35.5% similarity with toxin counterpart PepG1 of the staphylococcal type-1 toxin-antitoxin system [29]. The SAOUHSC_01111 and SAOUHSC_01544 show sequence similarity with Ranatuerin-1C (40.6%) and Hymenochirin-4B (35.3%) respectively found in skin secretions of frogs [30], [31]. Whereas, SAOUHSC_00839 shows 34.1% similarities with Mutacin, a bacteriocin secreted by Streptococcus rattus BHT. The SAOUHSC_01507 has 33.1% similarity with andropin, a male-specific AMP from fruit flies [32]. The SAOUHSC_A02794 has around 30% sequence similarity with uberolysin, a cyclic bacteriocin from Streptococcus uberis [33]. SAOUHSC_01118 shows 29% sequence similarity with bacteriocin Microcin M secreted by gram-negative bacteria like E. coli MC4100.

Analysis of upstream promoter sequence indicates stress and starvation-induced gene expression of new bacteriocins

The expression of the bacteriocin genes are majorly regulated by quorum sensing (QS) systems, as well as induced by stress or starvation [34]. The upstream region of the newly identified bacteriocin genes are analyzed to predict the presence of any regulatory elements. Upstream 200 bp DNA sequences were analyzed to identify regular − 35 and − 10 base pair AT-rich promoter sequences and associated regulatory elements or transcription factor binding sites. Several transcription regulator binding sites in the upstream promoter-operator region of the staphylococcal bacteriocins genes are identified, which indicates their active role in gene expression regulation (Table 2). LexA is a key regulator of DNA damage response. The LexA binding site is identified in the upstream promoter-operator region of the SAOUHSC_00839 gene, which indicates LexA regulated gene expression. The LexA is previously reported to regulate gene expression of known bacteriocins like colicins in E. coli [35], [36]. The upstream promoter-operator region of SAOUHSC_00839 has also been found to consist of binding sites of RpoH2 (σ32) and TyrR. The RpoH2 (σ32) is an alternative sigma factor associated with heat shock response, whereas, the TyrR is a transcription regulator of aromatic amino acid biosynthesis and transport. The binding site of RpoS (σ38), a starvation/stationary phase responsive sigma factor identified in the promoter-operator region of SAOUHSC_00977. The binding site of transcription regulator ArgR is identified in the promoter-operator region of SAOUHSC_00839, SAOUHSC_01507, and SAOUHSC_A02794 genes. The ArgR regulates the metabolism and transport of L-arginine and is well known for its role in acid-induced stress [37] and oxidative stress [38] adaptation. The promoter-operator region of SAOUHSC_A02794 and SAOUHSC_01111 contains the binding site of PhoB, which is a key player in phosphate homeostasis as well as reported to regulate gene expression under nutrient starvation and stress [39]. The binding sequence of MetR and GcvA are identified in the promoter-operator region of SAOUHSC_01118 and SAOUHSC_01544 respectively. The MetR and GcvA are LysR family transcriptional regulators also involved in starvation and stress response [40]. The MetR regulates the expression of genes associated with the mitigation of nitric oxide stress [41]. Therefore, expression of all the identified bacteriocin genes are found to be regulated by stress or starvation-associated transcription regulators.

Table 2

Analysis of upstream promoter sequence of the bacteriocin genes
Kegg Accession No.	Transcriptional regulator	Expression triggering factor
SAOUHSC_01111	narL, phoB3,	extracellular phosphate / nutrient starvation
SAOUHSC_01544	gcvA	Lack of Glycine/nutrient starvation
SAOUHSC_00839	lexA, argR2, tyrR rpoH2	heat shock, acid induced stress, lack of aromatic amino acid/nutrient starvation
SAOUHSC_01507	rpoD17, argR2	acid induced stress
SAOUHSC_00977	rpoS17	starvation/stationary phase
SAOUHSC_01118	metR	Nitric oxide stress
SAOUHSC_A02794	argR, ihf, phoB	acid induced stress, extracellular phosphate / nutrient starvation

Secondary structure prediction and ab initio modeling show an amphiphilic helix-rich structure of the bacteriocins

To analyze the individual structures of the identified bacteriocins, amino acid sequence-based secondary structure prediction identified an abundance of α-helices (Fig. 1). The three-dimensional structure models of the peptides were predicted using ab initio modeling server AlphaFold [23] and Rosetta server [24] or template-guided modeled using I-TASSER server [26]. The structures were validated with the Ramachandran plot [27]. The modeled structures have around 80% of all the residues in the most favorable region in the Ramachandran plot and no outlier, which indicates good model quality.

The SAOUHSC_01544 structure obtained from AlphaFold server has two α-helices connected with a hinge region (Fig. 2A). The flexible hinge ¹⁰TGLN¹³ in SAOUHSC_01544 connects the two helices. The structure of many well-known AMPs consists of two amphipathic α-helices connected with a glycine-rich flexible hinge. An example is ceropins, which show antimicrobial activity against gram-positive, and gram-negative bacteria as well as fungus. The mode of action of cecropins is found to be permeabilizing cell membrane by channel-like pores formation [42], which results in osmotic imbalance across the membrane. Moreover, the amphiphilicity of the helices enhances the antimicrobial ability of the cecropins [43]. Both the N-terminal and C-terminal helices in SAOUHSC_01544 are found to be amphiphilic with hydrophobic moment (µH) of around 0.47 and 0.27 respectively. A partially modeled structure of SAOUHSC_01507 in AlphaFold shows an N-terminal helix and a partially built C-terminal helix connected with a loop (Fig. 2B). The N-terminal α-helical part of SAOUHSC_01507 is amphiphilic with a hydrophobic moment (µH) of 0.47 and a net positively charged, which may be important for its membrane interference. The secondary structure prediction also shows coil structure in the C-terminal part of SAOUHSC_01507 (Fig. 1). The SAOUHSC_01507 has amino acid sequence similarity with Andropin, a male-specific AMP from fruit flies [32]. The Andropin is active against gram-positive bacteria only. Although, no experimentally determined structure of Andropin is available, however, AlphaFold driven structure of Andropin shows two α-helices connected with a flexible loop. Therefore, both SAOUHSC_01544 and SAOUHSC_01507 consist of helix-turn-helix structures which may get further stabilized in the lipid membrane environment and cause membrane permeabilization by toroidal pores formation.

The SAOUHSC_00977 appears α-helical when modeled with AlphaFold server with unstructured C-terminal parts (Fig. 2C). The SAOUHSC_00977 has sequence similarity with PepG1, a toxin part of type-I toxin-antitoxin system from S. aureus. The NMR structure of S. aureus PepG1 (PDB ID: 7NS1) is comprised of a long transmembrane α-helix and a cytosolic C-terminal part with positively charged residues [44]. The structural alignment of PepG1 and the SAOUHSC_00977 indicates a similar structure. The N-terminal helical part of SAOUHSC_00977 is amphiphilic with a hydrophobic moment (µH) of 0.31 and has a high net positive charge. The C-terminal of SAOUHSC_00977 is predicted to be unstructured by secondary structure prediction as well as in modeling. The twelve amino acid long C-terminal unstructured part of SAOUHSC_00977 contains positively charged residues at the end similar to PepG1, which are important for the anchoring of the peptide to the negatively charged membrane [45, p. 1].

SAOUHSC_01111 has sequence similarity with the antimicrobial peptide Ranatuerin. The only available structure rantuerin-2csa (PDB ID: 2K10) shows a helix-turn-helix structure [46]. The ab initio model of SAOUHSC_01111 shows a helix-turn-helix like structure (Fig. 3A), which is similar to rantuerin-2csa (Fig. 3B). In a polar environment, these peptide lacks any secondary structure, however, in a nonpolar environment like in a membrane it adopts helix–turn–helix structure. Moreover, the ranatuerin-2 family of peptides show a C-terminal six amino acid cyclic peptide structure connected with a disulfide bridge. A similar C-terminal disulfide bridge mediated by nine amino acids cyclic peptide structure is indicated by the presence of two Cystine residues (Cys22 and Cys30) in SAOUHSC_01111. The mode of action of the ranatuerin-2 family peptides is pore formation by a ‘Carpet-like model’, where, the peptides arrange themselves parallel to the membrane plane and then destabilize the membrane in a similar way as detergent. The high net positive charge and amphiphilic nature of ranatuerin-2 family peptide facilitates carpet-like membrane interaction. The SAOUHSC_01111 is also found to be amphiphilic with an N-terminal helix having a hydrophobic moment (µH) of 0.23 and the C-terminal part having a hydrophobic moment (µH) of 0.41 with an overall net positive charge.

The AlphaFold generated model structure of SAOUHSC_00839 contains N-terminal beta-hairpin and a C-terminal α-helix (Fig. 4A). The antiparallel stands of the beta-hairpin in SAOUHSC_00839 are cross-linked with an intramolecular disulphide bond formed between Cys 11 and Cys 20. The C-terminal α-helix is amphiphilic (µH = 0.35). Pediocin-like bacteriocins Leucocin-A (PDB ID: 1CW6) have a similar structure with N-terminal antiparallel β-sheet stabilized by disulfide bridge and C-terminal amphiphilic α-helix (Fig. 4B) [47]. The type-IIa bacteriocins have a conserved N-terminal YGNGV/L motif which is completely absent in SAOUHSC_00839 and thus SAOUHSC_00839 may belong to a novel sub-group (Fig. 4C). However, the interface of β-sheet and α-helix contains a conserved motif WGXA in both Leucocin-A and SAOUHSC_00839. The Pediocin-like bacteriocins show antimicrobial activity by permeabilizing the membrane of the target cells. The positively charged N-terminal β-sheet electrostatically interacts with the negatively charged cell membrane of the target cell. The C-terminal amphiphilic α-helix perforates the hydrophobic core of the membrane and causes leakage. The amino acid sequence of the C-terminal α-helical part is highly variable among Pediocin-like bacteriocins and therefore, is believed to be important for target specificity.

The SAOUHSC_01118 and SAOUHSC_A02794 have sequence similarities with type II circular or leaderless bacteriocins like microcins and Uberolysin respectively. Ab initio modeling of SAOUHSC_01118 in the Rosetta server shows α-helical bundle structure (Fig. 5A). Many of the known circular or leaderless bacteriocins show conserved structural features called saposin-like folds found in bacteriocins like AS-48 (PDB ID: 1E68) (Fig. 5B). Saposin D is a human peptide that interacts with anionic lipids. The saposin-like fold containing proteins possess a globular structure consisting of four or five helices. A characteristic feature of a saposin-like fold is a ‘v-shape’ formed by two helices and a third helix perpendicular to the ‘v-shape’. [48]. The SAOUHSC_A02794 modeled in Rosetta shows a unique structure consist both α-helices and β-sheet. The three β-stands from the N-terminal and one β-stand from the C-terminal form an antiparallel β-sheet in SAOUHSC_A02794 (Fig. 5C). The N-terminal β-sheet and C-terminal helix SAOUHSC_A02794 form intramolecular disulfide bridges as presumed from the presence of two and three Cysteine residues respectively, which would provide thermal stability to the bacteriocin and important for its function in extreme extracellular environment.

The leaderless and circular bacteriocins may interact with the membrane directly or through membrane-bound receptors. Moreover, unlike other globular proteins, leaderless and circular bacteriocins have solvent-exposed aromatic residues in N- or C-termini ends, which play an important role in membrane interaction [48], [49]. The SAOUHSC_01118 has four surface exposed Trp3, Trp39, Trp47 and Trp58 residues and SAOUHSC_A02794 has N-terminal surface exposed Tyr7, Tyr11 Trp14 and Tyr23 (Fig. 5A and 5C). These aromatic amino acids facilitate the interaction between the bacteriocin and lipid membrane and may have an important role in membrane disruption.

A molecular dynamics simulation study identified the structural plasticity of the helix-turn-helix bacteriocins

The ab initio models of the bacteriocins are subjected to molecular dynamic simulation to validate structural stability in the water environment and examine the structural dynamic. The RMSD value of all the simulations was below 2Å, which indicates the accuracy of the initial model (Fig. 6A). All the modeled structures of the bacteriocins are found to be stable in a water environment and equilibrated well by 2 to 4 ns of simulation. Other than the helix-turn-helix bacteriocins, all the other structures maintained stable RMSD for up to 50 ns. The amphiphilic helix-turn-helix bacteriocins, SAOUHSC_00977, SAOUHSC_01507, SAOUHSC_01544, and SAOUHSC_01111 show comparatively higher structural changes as apparent from the fluctuating RMSD values. Furthermore, no abrupt change in the radius of gyration (Rg) in any of the model structures of bacteriocins was identified during the simulation (Fig. 6B). The Rg plot of the peptides indicates that the globular nature of the structure has been maintained throughout the simulation time course. During simulation, the amphiphilic helix-turn-helix peptides are found to go through cycles of partially unfolding and refolding, which is reflected in their Rg polt as fluctuations. Solvent accessible surface area and overall H-bonding of all the peptides were mostly stable during a simulation run, which is also in argument with the Rg plot indicating structural interiority of the bacteriocin models (supplementary Figure S1 and S2). Analyzing the structural flexibility using RMSF value identified that bacteriocins with longer polypeptide chains like SAOUHSC_A02794 and SAOUHSC_01118 have less structurally disordered regions in their structure (Fig. 6C). Structural flexible regions in most of the helix-turn-helix bacteriocin structures are identified in the N- and C-terminal regions. The hinge regions in the helix-turn-helix bacteriocin structures are found to have comparatively higher RMSF values indicating structural flexibility.

The initial starting models of the bacteriocins are superimposed with the simulated energy-minimized structure for structural comparison (Fig. 7). The bacteriocins SAOUHSC_00977, SAOUHSC_00839, SAOUHSC_01544, SAOUHSC_01507, and SAOUHSC_01111 with amphiphilic helix-turn-helix are found to have gone through a significant structural change during simulation, which indicates their conformational plasticity. Amphiphilic helical bacteriocines or antimicrobial peptides are reported to be unstructured in a water environment, and adopt the helical or helix-turn-helix structure only in the water-lipid interface [50]. The hinges and C-terminal helix in SAOUHSC_00839 are found to have comparatively higher structural flexibility. The loop Thr14 to leu18 connecting the β-hairpin structure shows the highest RMSF value. However, the disulfide bond between the two stands stabilizes the β-hairpin structure. The structural stability of the β-hairpin indicates its importance in the functional activity of SAOUHSC_00839. The SAOUHSC_01118 and SAOUHSC_A02794 hold the globular shape during the simulation and the saposin-like characteristic fold remains intact. The N-terminal α-helix in SAOUHSC_01118 is found to become more structured and elongated out words by increasing helix length including four N-terminal amino acid residues. With an elongated N-terminal helix, the Trp3 residue in SAOUHSC_01118 seems important for interaction with the lipid membrane extended out of the globular structure (Fig. 7).

Being a commensal and opportunistic pathogen, S. aureus is found to colonize the skin and nasal cavity of 20–30% of healthy individuals [51] and for that, it has to overcome the colonization resistance posed by the members of the human microbiota. Lantibiotics, bacteriocins, and bacteriocin-like inhibitory substances (BLIS) are the well-known AMPs that are secreted by S. aureus and have antimicrobial activity against closely related gram-positive bacteria [52], [53] [54]. The bacteriocins are ribosomally synthesized small peptides, which are produced by S. aureus in the polymicrobial niche to inhibit the growth of the competitor. The S. aureus NCTC8325 genome-wide screening identified 487 mini-proteins with amino acid lengths lower or equal to 100 amino acids. The mini-protein library has been screened to identify novel bacteriocins. Our search for bacteriocins among mini-proteins resulted in the identification of seven novel bacteriocins. Only less than half of the bacteriocins identified in this study are conserved among a handful of other S. aureus strains, the majority are unique for the S. aureus NCTC8325. Identification of a significant number of unique bacteriocins from a single strain like S. aureus NCTC8325 indicates the extent of diversity of the S. aureus bacteriocin arsenal. In addition, the target organism specificity of the bacteriocins is also highly specific and mostly narrow spectrum. The bacteriocins are known to play an important role in shaping human microbiota. Therefore, to understand the ecology of S. aureus infection and to identify effective treatment, it is important to explore, identify and characterize different bacteriocins present in S. aureus.

The upstream promoter regions of all the identified bacteriocin genes are found to possess binding sites of transcription regulators that are associated with stress and starvation. The bacteriocin expression is upregulated in stress and starvation; which is reported to be a universal characteristic of bacteriocin genes [34]. Healthy microbiota prevent the growth of pathogenic bacteria by different mechanisms like AMP secretion, change in pH of the environment and competing for nutrition and binding surface. The S. aureus can sense stress associated with nutrition starvation, acidic environment, cell membrane damaging chemicals, etc, using surface receptors and reciprocate to activate defense mechanisms. Once S. aureus senses stress and starvation, it upregulates the expression of the bacteriocins as a response to overcome the competing microbes. Therefore, the identification of stress and starvation-induced regulatory elements in identified genes are in good agreement with their proposed function as bacteriocins.

The ab initio three-dimensional model building and structural analysis shows all the bacteriocins have amphiphilic helix-rich structures. The mode of action of amphiphilic α-helical AMPs is known to disrupt microbial membranes [55]. Bacterial membranes are negatively charged due to a high abundance of anionic lipid components like dipalmitoyl phosphatidyl glycerol (DPPG) and dipalmitoyl phosphatidic acid (DPPA). The high net positive charge of the bacteriocins increases their selectivity towards the negatively charged lipid plasma membrane of the microbes. There are two major ways in which bacteriocin disrupts cell membranes, either by pore formation or by disruption of the membrane structure. The SAOUHSC_01111, SAOUHSC_01544, SAOUHSC_00839, SAOUHSC_01507, and SAOUHSC_00977 have mostly helix-turn-helix or helix-turn-β-sheet structures ideal for pore formation in membrane. The helix-turn-helix peptides with sharp turns are generally associated with toroidal pores formation [56]. A single amphiphilic helical structure like SAOUHSC_00977 is good for barrel-shaped pore formation. Furthermore, a molecular dynamic simulation study indicates that the amphiphilic helices in helix-turn-helix bacteriocins have high structural plasticity and alter between unstructured to helical structures in the transition from water to lipid environment. The SAOUHSC_01118 and SAOUHSC_A02794 also have stable globular structures with amphiphilic α-helix barrel structures. The surface-exposed aromatic amino acids and high positively charged C-terminal residues of the SAOUHSC_01118 and SAOUHSC_A02794 seem to facilitate interaction with the membrane.

In summary, using in silico screening, seven novel S. aureus bacteriocins are identified. The physicochemical properties, promoter analysis and structural characterization validate the antimicrobial function of the predicted bacteriocins. Identified bacteriocins are mostly unique for the S. aureus NCTC8325 strain, which indicates the diversity of bacteriocins in S. aureus. Further identification and characterization of staphylococcal bacteriocins would improve our understanding of the detailed mechanism of S. aureus mediated dysbiosis, which is important for effective treatment. The reported in silico methodology can be used to predict and identify novel bacteriocin genes from any other organisms for their application in the treatment of antibiotic-resistant infection, food preservation, agriculture, and other relevant fields.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding statement

This work is supported by the Science and Engineering Research Board, Government of India (SRG/2022/000707).

Author Contribution

A.D. initiated the project, conceptualize the idea, collected data, analyzed results, and wrote the manuscript. S.D., B.D., A.K., A.M. and T.K. performed data collection and analysis. All authors reviewed the manuscript

Data availability

All the data described in the article are available from the corresponding author on request.

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Identification and structural characterization of novel bacteriocins by genome-wide screening of hypothetical mini-proteins in Staphylococcus aureus

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Abstract

Figures

Introduction

Materials and methods

Sequence retrieval

Anti-microbial activity prediction

Sub-cellular localization prediction

Physicochemical characterization

Gene expression analysis

Structural analysis of the bacteriocins

Molecular dynamic simulation

Results

Analysis of upstream promoter sequence indicates stress and starvation-induced gene expression of new bacteriocins

A molecular dynamics simulation study identified the structural plasticity of the helix-turn-helix bacteriocins

Discussion

Declarations

Declaration of competing interest

Funding statement

Author Contribution

Data availability

References

Additional Declarations

Supplementary Files

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