The knowledge of antibiotics produced by Archaea (archaeocins) is still limited. So far, only two types of archaeocins are known: (i) sulfolobicins, produced by the extremely thermophilic Sulfolobus spp. and (ii) haloarcheocins, produced by halophilic archaea. Haloarcheocins were first discovered in the 1980s, but most of their characterisation was solely based on supernatant-based assays. In general, the taxonomy of haloarchaea and their typical phenotypic and genotypic characteristics are well studied; however, information regarding their halocins, especially aspects related to genetics, biosynthetic pathways, mechanism of action, and structure-function relationship is very limited. A few studies have demonstrated the potential applications of halocins in the preservation of salted food products and brine-cured hides in leather industries, protecting the myocardium from ischemia and reperfusion injury, as well as from life-threatening diseases such as cardiac arrest and cancers. In recent years, genome mining has been an essential tool to decipher the genetic basis of halocin biosynthesis. Nevertheless, this is likely the tip of the iceberg as genome analyses have revealed many putative halocins in databases waiting for further investigation. Identification and characterization of this source of halocins may lead to antimicrobials for future therapeutics and/or food preservation. Hence, the present review analyzes different aspects of halocins such as biosynthesis, mechanism of action against target cells, and potential biotechnological applications.
Keywords: Haloarchaea, Halocins, Antimicrobials, Production, Mode of action
The use of antibiotics in the last few decades leads to the rise of multi-drug resistant bacteria (MDR), which reduces and nullifies the effect of antibiotics. So, there is a vital requirement to discover novel and effective antimicrobial therapies by exploiting all possible natural and sustainable resources, including extreme environments. Archaeocins are protein antibiotics produced from archaea, and it marks the chronicled beginning in the series of antimicrobial compounds. The term “archaeocin” was used to differentiate the peptide and protein based antibiotics generated from Archaea than those produced by Bacteria (Kumar and Tiwari, 2017). Till date archaeocins (Table 1) have been produced by only two phylogenetic groups, one is euryarchaeal, that are extreme halophiles (haloarchaea) producing “halocins” whereas the other group producing “sulfolobicin” is crenarchaeal genus Sulfolobus that are aerobic hyperthermophile (Quehenberg et al., 2017). Valera et al in 1982 reported the first proteinaceous antimicrobial compounds i.e., halocins, from halophilic members of the Archaeal domain (Valera et al., 1982).
Peptide antibiotics are generally synthesized in two distinguished ways, one is ribosomally using transcripts (gene-encoded) and other is stepwise synthesis hiring either multienzyme complexes or sequential enzyme reactions. H1 and H4 are protein halocins of roughly 30-40 kDa (Mazguene et al., 2018), whereas C8, H6, H7, R1, U1 and S8 and are microhalocins of size smaller than 10kDa. Microhalocins are more vigorous than protein halocins since they are resistant to flexibility in temperature, salinity, exposure to organic solvents, acids and bases (Mazguene et al., 2018). Halocins have a wide ranging of activity against Haloarchaea and members of family Halobacteriaceae (de Castro et al., 2020). Mostly halocin production is prompted during the progression between exponential and stationary phases, with H1 being an exception, which is produced during the exponential phase of the growth cycle (de Castro et al., 2020). Many workers reported the mode of action of halocins that they alter the cell permeability at membrane level and inhibit Na+/H+ antiporter and proton flux ultimately causing cell lysis and death (Karthikeyan et al., 2013). Still till today only the mechanism of halocin H6/H7, produced by Haloferax gibbonsii Ma2.39 was explored. H6/H7 halocin kills delicate cells by inhibiting Na+/H+ antiporter resulting in cell lysis (de Castro et al., 2020).
The most common characteristics, diversity of haloarchaea, taxonomy and their metabolites including halocins have been recently studied by (Kumar et al., 2021; Corral et al., 2020; Ghanmi et al., 2020; Kumar et al., 2016; Kumar and Tiwari, 2017a, 2017b; Kumar and Tiwari, 2019; Oren, 2014). Therefore, in the present review, we will give first a short description of the general properties of haloarchaea and then mainly focus on the molecular basis of halocin biosynthesis, mechanism of action, and their possible applications as antibiotics in food and medicine.
Haloarchaea are salt-dependent archaea (require high content of NaCl to survive) that are typically found in marine solar salterns, natural brines and salt lakes. They were even isolated from a salt sediment dated to the Permian-Triassic period (195–250 million years ago) (Fendrihan et al., 2006). Yet, they can also be found in the human microbiome due to ingestion of salty foods. Some can be merely passengers of the digestive tract, whereas others can thrive in the gut, representing a major fraction of the microbial diversity in obese individuals with microbiota alteration (Seck et al., 2019). Haloarchaeal biocompounds have been studied due to their biotechnological potential, including carotenoids, photosynthetic proteins, enzymes, PHAs/PHBs and halocins (Torregrosa-Crespo et al., 2017), and some of them are already in the market. Carotenoid production had recently gained a lot of attention due to its great biotechnological potential, especially in the medical field (Torregrosa-Crespo et al., 2018; Giani et al., 2019). Bacterioruberin is the main carotenoid produced by haloarchaeal strains and the pigment responsible for their red colour (Torregrosa-Crespo et al., 2018).
It is also sold as a powerful antioxidant that protects cells from the damage of free radicals generated by intense UV radiation (Asgarani et al., 1999). Bacterioruberin extracts from a hyperpigmented Haloferax volcanii have also proved to be useful in the assisted reproduction field, as studies conducted on ram sperm cells revealed that this carotenoid has a protective effect and increases the yield of the cryopreserved sperm cells (Zalazar et al., 2019). Additionally, bacteriorhodopsin inclusion in optical and optoelectronic devices seems a promising application (Jiang and Wen 2015) and proteoglycans, lipids and squalene extracts are applied as bioactive solutions for personal care (Halotek Biomaterials). As for halocins, their study is still far behind in comparison with the antibiotics produced by the other domains. This is mostly due to their relatively recent discovery as well as the establishment of Archaea as a domain. They were first discovered in 1982 (Rodriguez-Valera et al., 1982), in a time where extreme halophilic archaea still belonged to the domain Bacteria. Consequently, halocin term was loosely used to describe any proteinaceous antimicrobial compound produced by halophilic prokaryotes (archaea and bacteria) (O’Connor and Shand, 2002; Atanasova et al., 2013). So, sometimes, the application of the term halocins is unclear. In our opinion, it is useful to have classification schemes based on the producers. So, we propose that the use of haloarcheocins and halobacteriocins to distinguish between halocins produced by haloarchaea from the ones produced by halophilic bacteria, respectively, can be a useful alternative. The discovery of new haloarcheocins goes hand in hand with the discovery of haloarchaeal strains (Kumar and Tiwari, 2017). Their discovery still relies on cross-inhibition tests and their purification being only partial hinders their classification. Not all haloarcheocins are at the same level of characterisation, and to our knowledge, only three of all halocins described have their peptide sequence determined as well as their respective gene sequence: H4, which was the first haloarcheocin described, halocin S8 and halocin C8 (Besse et al., 2015). The haloarcheocins described so far are mainly produced by Haloferax spp. (32%), followed by Natrinema spp. (18%) and Haloterrigena spp. (18%).Also, currently, it is known that some strains are able to produce more than one haloarcheocin (Ghanmi et al., 2019). Their mode of action was only characterised for haloarcheocin H6, which is able to inhibit the archaeal Na+/H+ antiporters (Meseguer et al., 1995). More recently, it was revealed that they are probably secreted, but rather exert their activity while bounded to the producer’s membrane, as it is the case of haloarcheocin H17 (Mazguene et al., 2018). As for the ecological role of haloarcheocins, it has been proposed that they are involved in competition among haloarchaeal strains (Kis-Papo and Oren, 2000).
Halocins are the proteinaceous antimicrobial proteins/peptides (AMPs) secreted by several members of haloarchaea belonging to class Halobacteria. Most of the haloarchaea produce optimum halocin during transition from exponential to stationary phase. The exception is halocin H1 whose production is found to be maximum at mid-exponential phase and remains stable until the stationary phase (Shand and Leyva, 2007; Besse et al., 2015). Few members of haloarchaea, e.g., Haloferax mediterranei, show a decline in their halocin level during the stationary phase. Hence, the production of halocin is found to be growth associated, and it can be used as model to study phase-dependent halocin expression (Cheung et al., 1997; Price and Shand, 2000). Based on an antagonism study, hundreds of different types of halocins are expected to be present in nature but few halocins have been characterized to date (Ghanmi et al., 2016). The biochemical properties of different halocins are summarized in table 2, and the recent updates about halocins (producers belonging to phylum Euryarchaeota) and sulfolobicins (producers belonging to phylum Crenarchaeota) are described.
Halocin H4: Halocin H4 is the first studied halocin produced from Haloferax mediterranei R4 (ATCC33500), isolated from a Spanish solar salt pond in Alicante, Spain (Rodriguez-valera et al. 1981; Meseguer and Rodriguez-valera, 1985). The molecular weight of halocin H4 is 39.6 kDa. The activity was initially detected as the culture began its transition into the stationary phase, which is salt-dependent and sensitive to proteases and high temperature. The antimicrobial activity of halocin H4 disappears at 60°C (for 24 h), 70°C (for 4 h), and 80°C (for 30 min) (O’Connor and Shand, 2002). The halocin activity remains stable at above 15% NaCl (Rodriguez-valera et al., 1982) and exhibits several features of halophilic proteins such as high content of negatively charged amino acids, especially aspartate, low content of lysine, and high content of non-bulky residues (proline, glycine, valine, and threonine). It possesses hydrophobic 32-amino acid fragments (residues 178–209) (Cheung et al., 1997).
Halocin H1: Halocin H1 was purified from Haloferax mediterranei strain M2a (previously Xia3) isolated from salt ponds in Santa Pola (Alicante, Spain) (Rodriguez-Valera et al., 1981; Platas et al., 2002). The molecular weight of halocin H1 is 31 kDa. Optimum production of halocin H1 occurs when the culture enters into the stationary phase grown at 20% NaCl, 37°C, and 220 rpm. The halocin activity remains stable in the stationary phase. Like halocin H4, the antimicrobial activity of halocin H1 is sensitive to higher temperature and salt dependent. Halocin H1 is stable up to 50°C only and requires the minimum 6% NaCl concentration for activity (O’Connor and Shand, 2002; Platas et al., 2002). Dialysis of CFS against lower salt concentrations caused an immediate decrease in its activity and can not be restored by dialysis against the initial saline conditions. The activity was completely reduced within 7 days after dialyzing against water (Platas et al., 2002). The desalting effect irreversibly denatures halocin activity which may be due to perturbation in three-dimensional structure of halocin H1 (Torreblanca et al., 1989; Platas et al., 2002; Pasic et al., 2008; Kavitha et al., 2011). Most of the properties of halocin H1 are common to halocin H4 such as both are produced from Haloferax mediterranei isolated from Spanish salterns and their sizes are above 30 kDa. Both producer strains are not able to inhibit each other. All these properties of both halocins suggested that both of them are related or might even be the same halocin (Shand and Leyva, 2007).
Halocin H6: Halocin H6 is produced from Haloferax gibbonsii Ma2.39 isolated from Spanish salt ponds in Alicante (Torreblanca et al., 1989). A halocin over producing mutant of strain Ma2.39 was named Haloferax gibbonsii Alicante SPH7. The halocin produced by strain SPH7 was designated halocin H7 (Torreblanca et al., 1989; Lequerica et al., 2006). The molecular weight of halocin H6 is 32 kDa. Similar to halocin H4, the production of halocin H6 reached maximum level at the transition from log to stationary phase (Torreblanca et al., 1989). Halocin H6 was heat-resistant and active up to 90°C but activity reduced to 50% at 100°C and completely lost at autoclaving temperature. Trypsin, even at a concentration of 5 mg/ml, does not denature the halocin activity (Torreblanca et al., 1989). Interestingly, halocin H6 is able to inhibit the sodium hydrogen exchanger (NHE) present in the cell membrane of haloarchaea and eukaryotic cell, but it was not tested against bacteria for NHE inhibition (Lequerica et al., 2006). The amino acid sequence of halocin H6 has not been determined (Torreblanca et al., 1989; Platas et al., 2002). Meseguer hypothesizes that mature halocin H6 would be in fact a 3 kDa peptide that is released from a precursor protein, but the experimental data supporting this hypothesis are not available yet (Shand and Leyva, 2007).
Halocin SH10: Halocin SH10 is produced by Natrinema sp. BTSH10 isolated from saltpan of Kanyakumari, Tamil Nadu, India. The molecular weight of halocin SH10 is 20 kDa. The halocin production reaches optimum level during the transition to the stationary phase of growth of producing strain incubated at 42°C, pH 8.0, and 200 rpm in Zobell’s medium containing 3 M NaCl (Karthikeyan et al., 2013).
Halocin S8: Halocin S8 is the first peptide halocin (microhalocin) produced by uncharacterized haloarchaeal strain S8a isolated from the Great Salt Lake in Utah, USA (Price and Shand, 2000). The molecular weight of halocin S8 is 3.58 kDa. Similar to halocin H4 and H6, the production of halocin S8 reaches maximum at transition to the stationary phase of growth (Shand and Leyva, 2007). Halocin S8 activity is thermostable up to 94°C and sensitive to proteinase K but resistant to trypsin. It has salt-independent nature of activity (Price and Shand, 2000). Complete sequence of halocin S8 (36 amino acid residues) was revealed using Edman degradation method. Most of the residues are hydrophobic and consist of four cystine residues along the sequence, which may form disulfide bridges. The information regarding its tertiary structure is unavailable. However, BLAST analysis of the halocin S8 sequence revealed no homology with other sequences available in the database (Shand and Leyva, 2007; Besse et al., 2015).
Halocin R1: Halocin R1 is the second studied microhalocin produced by Halobacterium salinarum GN101, isolated from a solar saltern in Guerrero Negro, Mexico, by Barbara Javor (Ebert et al., 1986). The molecular weight of halocin R1 is 3.8 kDa. The production of halocin R1 reaches maximum during transition to the stationary phase of growth of the producing strain. Halocin R1 activity is found to be resistant up to 60°C for 24 h but lost after treatment at 93°C for 5 min. The halocin R1 activity is resistant to desalting and to various proteolytic enzymes such as papain, trypsin, and thermolysin, but it is sensitive to proteinase K, pronase, and elastase (Besse et al., 2015). The complete sequence of halocin R1 was determined using Edman degradation. It consists of 38 amino acids that are 63% identical and 71% similar to halocin S8 (O’Connor and Shand, 2002; Price and Shand, 2000). The halocin R1 is reported to be archaeostatic as suggested by Rdest and Sturm (1987). The halocin R1 producing strain GN101 is studied for complete sequence of megaplasmid 2 (283 kb) and is available in GenBank database under accession number EU080936. It carries halS8 gene that suggests for the strong relationship between halocins S8 and R1 and might be encoded by same gene or multiple deriving copies of the halocin S8 gene.
Halocin C8: Halocin C8 is the largest member of the microhalocin family produced by haloarchaeon Natrinema sp. AS7092 (formerly Halobacterium strain AS7092), isolated from the Great Chaidan Salt Lake, China (Li et al., 2003; Sun et al., 2005). The molecular weight of halocin C8 is 7.44 kDa. It has been reported that several strains of Natrinema are able to produce halocin C8. The gene responsible for halocin production is reported in five other strains of Natrinema species isolated from the Ichekaben salterns in Algeria (Imadalou-Idres et al., 2013). The halocin production began at the transition from the exponential to the stationary phase of growth, and activity remains stable throughout the stationary phase (Sun et al., 2005). The halocin C8 is thermostable up to 100°C for 1 h and salt-independent in nature. The desalted halocin may be stored for more than 1 year at 20°C without losing the activity. The activity remains unaffected after treatment with various organic solvents such as methanol, ethanol, and acetonitrile. It is sensitive to proteinase K and papain but resistant to trypsin. These properties make halocin C8 a quite stable peptide and robust in nature (Li et al., 2003). The halocin C8 sequence is cysteine-rich and contains ten cysteine residues which may form disulfide bridges responsible for higher stability of halocin C8 activity in different conditions (Li et al., 2003; Sun et al., 2005).
Halocin A4: Halocin A4 is produced by an uncharacterized haloarchaeon strain TuA4 isolated from Tunisian saltern (Shand and Leyva, 2008). Previously, halocin A4 was also designated halocin U1. The molecular weight of halocin A4 is 7.4 kDa. The halocin A4 activity is salt-independent and thermostable. It is resistant to boiling temperature up to a week (Shand and Leyva, 2007; Shand and Leyva, 2008). Like halocin C8, it has same molecular mass and similar stability pattern. N-terminal sequencing of halocin A4 revealed 75% identity to halocin C8, thus suggesting it is a variant of halocin C8 (Shand and Leyva, 2008).
Halocin Sech7a: Halocin Sech7a is produced by Haloferax mediterranei Sech7a, isolated from Adriatic solar saltern in Slovenia. The molecular weight of halocin Sech7a is 10.7 kDa. Halocin production starts when cells enter into the exponential phase of growth (30 h) and reaches maximum level at the beginning of the stationary phase (40 h) grown at 45°C and pH 7.0–7.5 and 20% NaCl (Pasic et al., 2008). It is quite stable in a wide range of pH 2.0–10.0 and thermostable up to 80°C. The activity remained unaffected at 6% NaCl, but further desalting reduces the activity. Its activity can be restored up to ~40% of initial halocin activity by dialysis against the initial saline conditions (Pasic et al., 2008; Besse et al., 2015).
Halocin KPS1: Halocin KPS1 is produced by Haloferax volcanii KPSl, isolated from Kovalam saltern, Kanyakumari, India. The growth and halocin production have been monitored at 25% NaCl, 40°C, and pH 7.0 (Kavitha et al., 2011). The production of halocin KPS1 starts during mid-log phase at 48 h, reaches maximum (120 AU/mL) at the stationary phase of growth at 72 h, and persists up to 120 h; thereafter, activity was declined (40 AU/ml) at 144 h (Kavitha et al., 2011). Halocin KPS1 activity is thermolabile and reduced at >80°C and stable over a wide range of pH 3.0–9.0. The activity disappears after treatment with proteolytic enzymes such as proteinase K and trypsin indicating the proteinaceous nature of the inhibitory compound.
Halocin HA1 and HA3: Halocin HA1 and HA3 were produced by Haloferax larsenii HA1 and HA3 isolated from Pachpadra salt lake in Rajasthan, India. Halocin HA1 and HA3 were purified using ultrafiltration, anion-exchange chromatography (AEC), gel-filtration chromatography (GFC), and reversephase high-performance liquid chromatography (RP-HPLC). It was observed that purified halocin HA1 was stable up to 80 °C and lost 50% activity at 100 and 121°C, whereas halocin HA3 activity was reduced up to 88% at 100°C and 94% at 121°C. Halocin HA1 and HA3 were active at pH 4.0–12.0 and pH 2.0–10.0, respectively, suggesting for their stability under acidic to basic pH range. Purified halocin HA1 and HA3 were unaffected with methanol, ethanol, isopropanol, Tween-80 and Triton X-100 but showed complete loss of activity after treatment with trypsin and proteinase K. Both halocins showed the salt-dependent nature of antimicrobial activity and required minimum 10% NaCl for activity. The molecular weights of purified halocin HA1 and HA3 were found to be ~14 and ~13 kDa, respectively, using tricine SDS-PAGE. MALDI-TOF MS/MS analysis of halocin HA1 and HA3 showed no homology with known halocins in NCBI database. The N-terminal sequences of halocin HA1 and HA3 were found to be MIDREILEVN and MNLGIILETN, respectively. Blastp (NCBI) analysis of N-terminal sequence of halocin HA1 showed no significant homology with known proteins, whereas halocin HA3 showed 100% identity with N-terminal of hypothetical protein of Halogeometricum pallidum (Kumar et al., 2016; Kumar and Tiwari, 2017a, b).
Sulfolobicins: These are the archaeocins produced from several members of genus Sulfolobus belonging to phylum Crenarchaeota. The members of genus Sulfolobus grow in entirely different environments such as high temperature and acidic conditions. Sulfolobicins are entirely different from halocins and their activity is due to cells and not the supernatant (Besse et al., 2015). Sulfolobicin is the proteinaceous antimicrobial agent, first characterized from Sulfolobus islandicus. It is not the secretory proteins but remains bound to the cell membranes or cell-derived S-layer-coated membrane vesicles (Prangishvili et al., 2000). The host range of sulfolobicin was found to be restricted to other members of the Sulfolobales. The sulfolobicin from Sulfolobus islandicus inhibited Sulfolobus solfataricus P1, Sulfolobus shibatae B12, and six non producing strains of Sulfolobus islandicus but did not inhibit Sulfolobus acidocaldarius DSM639. The purified sulfolobicin from strain HEN2/2 did not inhibit Halobacterium salinarum R1 or Escherichia coli. Sulfolobicin was purified from the late stationary phase culture using sonication high-speed centrifugation and separated with Triton X-100. The molecular weight of sulfolobicin was 20 kDa tested on SDS-PAGE (Prangishvili et al., 2000; Besse et al., 2015). Its activity remained stable up to 6 months. Enzymatic treatment with α-amylase, α- and β-glucosidases, phospholipase C, and lipoprotein lipase did not affect the sulfolobicin activity, whereas treatment with proteolytic enzymes such as pronase E, proteinase K, and trypsin showed complete loss of activity (Shand and Leyva, 2007). The study of genes responsible for sulfolobicin synthesis and resistance would be useful to select as genetic markers that are still scarce in Sulfolobus (Ellen et al., 2011).
ANTIMICROBIAL SPECTRUM OF HALOCINS
Halocins usually inhibit haloarchaea species closely related to the halocin producers, however, few have shown cross-domain inhibition including bacteria and several archaea belonging to different clades as summarized in table 3 (Besse et al., 2015; Atanasova et al., 2013; Shand and Leyva, 2007). The antimicrobial spectrum of halocins varies against different target strains. Halocin H1 showed an antimicrobial spectrum against various haloarchaea strains but was unable to inhibit the tested non-haloarchaea microorganisms (Platas et al., 2002). Halocin H6 inhibited the growth of different haloarchaea members belonging to Halobacteriales (Meseguer et al., 1995; Torreblanca et al., 1989). Halocin H4 inhibited related haloarchaea members such as H. salinarium CCM2090. Halocin S8 showed a wider target range (within targeted haloarchaea microorganisms) inhibiting the growth of Halobacterium sp. strain GRB, H. salinarum NRC817, H. gibbonsii, and Sulfolobus sp. (Haseltine et al., 2001; Price and Shand, 2000). Halocin R1 of Halobacterium sp. GN101 is archaeostatic and showed inhibition against H. salinarum (formerly Halobacterium halobium), Sulfolobus sp., and Methanosarcina thermophila (Haseltine et al., 2001). Like microhalocin S8 and R1, halocin A4 also inhibited Sulfolobus solfataricus (crenarchaeal hyperthermophile) (Haseltine et al., 2001; Shand and Leyva, 2007). Halocin C8 showed a wide target range inhibiting different haloarchaea strains such as Natronobacterium magadii, Natronobacterium gregoryi, Natronomonas pharaonis, Sulfolobus sp., and Methanosarcina thermophile however, it was unable to inhibit the tested bacteria (Li et al., 2003). Halocin KPS1 has been reported for its broad antimicrobial spectrum as it showed inhibitory action against various bacteria, such as Streptococcus mutans MTCC896, Bacillus subtilis MTCC1134, Escherichia coli MTCC1671, Staphylococcus aureus MTCC916, Pseudomonas aeruginosa MTCC6538, and related haloarchaea strains including Halorubrum sodomense S2 (Kavitha et al., 2011). H. salinarum ETD5, a strain that produces multiple halocins, has been reported for cross-domain inhibition (Ghanmi et al., 2016). It has activity against different genera particularly Haloterrigena, Halobacterium, Haloarcula, and to a lesser extent against Natrinema and halophilic bacteria, such as Lentibacillus halophilus and Salinibacter ruber. The cell-free supernatants of strain ETD5 showed inhibition against P. aeruginosa, suggesting the need for further investigation on other halocins for their potential applications against problematic bacteria to control infectious diseases (Ghanmi et al., 2020; Ghanmi et al., 2016).
The antimicrobial activity of the different extracts obtained from two new haloarchaeal strains was assayed against a collection of typical fish and human pathogenic bacteria, against representative microalgae and yeast species and over other haloarchaea typically found in hypersaline environments, by using the agar diffusion method (Gómez-Villegas et al., 2020). This screening revealed that only the acetone extracts showed considerable antimicrobial activity. Acetone extracts from the two tested haloarchaeal strains, H. hispanica HM1 and H. salinarum HM2, showed to be active against bacteria, microalgae, and archaea, but not on yeasts. (Table 4). In this study, it have untapped novel unexplored bioactivities of haloarchaea, which can be the source of potential new therapeutic compounds. However, many archaea species are still undiscovered, many potential bioactivities remain to be studied, and the isolation and identification of the compounds responsible for these activities is still a pending issue. Further work will be carried out to fully characterize the composition of the active extracts and their efficacy in vivo assays.
Halocins may have also potential application in food industry as preservative agents (Charlesworth and Burns, 2016). Halocins such as HA1, HA3 (Kumar and Tiwari, 2017a, b), Sech7a (Pasic et al. 2008), H4 (Meseguer and Rodriguez-valera, 1985), H6 (Torreblanca et al., 1989), C8 (Li et al., 2003), and SH10 (Karthikeyan et al., 2013) have been reported which suggest that these halocins killed the indicator organisms by cell swelling followed by cell lysis (O’Connor and Shand, 2002; Sun et al., 2005; Pasic et al., 2008; Karthikeyan et al., 2013). The halocin-treated sensitive cells demonstrated change in internal pH, membrane potential, proton motive force, and sodium and proton flux. Currently, halocin H6 is the only archaeocin that exerts a specific inhibitory effect on Na+ /H+ exchanger (NHE) in both haloarchaean and mammalian cells (Lequerica et al., 2006).
Moreover, Aponte et al., 2010 studied the effects of halophilic archaea on the quality and safety of salted anchovies (Engraulis encrasicolus), which are traditional foods in the Mediterranean region. The results of this study, comparing the addition of Halobacterium salinarium CER6a that produces protease versus Haloarcula marismortui 1R strain that has no protease activity, showed that according to a preference survey conducted through sensory test, people preferred salted anchovies with addition of haloarchaea more than the control group despite having no significant differences in the hydrolytic rate of muscle sarcoplasmic protein. The most interesting fact is that in the case of salted anchovies with addition of haloarchaea, production of histamine, which is an anti-nutritional factor, was inhibited during the initial ripening process. Histamine, which is a representative bionic amine, found much in fish such as mackerels, sauries, and sardines, is an important factor that must be inhibited in fermented foods. Intake of histamine causes scombroid poisoning leading to various toxicities such as rash, hives, nausea, vomiting, diarrhea, flushing, among others (Bang et al., 2009), and so the US FDA regulates the concentration of histamine to be below 500 ppm, over which would lead to toxicity in humans. Development of methods for reducing histamine is in much need because such histamine is not only produced by fermenting bacteria but is also very stable and hardly broken down except through gamma radiation. Study results of Tapingkae et al., 2010, in which strains with histamine breakdown activity were examined among the 156 strains of halophilic archaea isolated from anchovy fish sauce, showed that the histamine breakdown activity was present in 60 strains and that the strain (HDS1-1) with the strongest activity was Natrinema gari. Accordingly, it is considered that using halophilic archaea as a starter for fermented foods would not only shorten the fermenting period and improve on flavor but also increase the safety of the food product. Among the proteinous substances produced by halophilic archaea, halocin has the highest potential of uses. Halocin is an antibiotic substance in the form of a small-molecule protein that is produced by most of the rod-type haloarchaea, and until now, not many of its physical and chemical properties characterized from purification have been elucidated; the characteristics of only three halocins have been cloned and studied (Cheung et al., 1997; Price and Shand, 2000; Sun et al., 2005). However, as NGS technology is being developed and the genomic sequence of halophilic archaea is continuously being reported, orthologs with a similar amino acid sequence to halocin are receiving attention as the new halocin. Although the antibiotic spectrum of halocin has been not studied in detail, the potential for its application can be said to be great since there are no antibiotic substances that can selectively kill archaea for now.
Halocin production is found to be a growth-associated phenomenon where optimal production depends on culture conditions like media components, temperature, aeration, pH, etc. (Platas et al., 2002; Price and Shand, 2000; Cheung et al., 1997). Halocin production usually starts at the beginning of the exponential growth phase and reaches the optimal level during the transition from exponential to stationary phase (Besse et al., 2015; Shand and Leyva, 2007). During the stationary growth phase, the level of halocin activity varies in different members of haloarchaea and it may either remain constant (H1, S8, and C8) or decline (H4, H6, KPS1, HA1, HA3, and H17) (Mazguene et al., 2018; Kumar and Tiwari, 2017a; Kumar et al., 2016; Kavitha et al., 2011; Li et al., 2003; Platas et al., 2002; Price and Shand, 2000; Cheung et al., 1997; Torreblanca et al., 1989). The decline in halocin activity may be due to the release of proteolytic enzymes from lysed cells during the late stationary phase (DasSarma et al., 2009; Cheung et al., 1997; Kamekura and Seno, 1990). The production of halocin H4 by H. mediterranei R4 was initiated during the mid-exponential growth phase and reached a maximum level between the exponential and the stationary phase. Thereafter, within 2 h, the production was rapidly declined and remained unchanged for 12 h (Cheung et al., 1997). Halocin H4 transcript was first detected when OD600 was around 0.26–0.73; however, antimicrobial activity was not detectable during this period. During the optimal halocin production, the transcript level was increased six-fold from its basal level during active production and then returned to the basal level during the stationary phase (Cheung et al., 1997). For halocin S8 from the haloarchaeal strain S8a, the transcript level was very low when halocin activity was not detectable and then increased nine-fold during active halocin production (Price and Shand, 2000). During the entry into the stationary phase, the halocin S8 transcript reached a maximum level and remained unchanged for 13 h before it rapidly returned to the basal level during the late stationary phase. However, halocin activity remained unchanged throughout the stationary phase after attaining the maximum level (Price and Shand, 2000). Similarly, in the case of halocin C8 produced by Natrinema sp. strain AS7092, the transcript followed parallelly the halocin activity profile from mid-exponential to early stationary phase. However, during the stationary phase, the transcript level started to decline and halocin activity plateaued (Sun et al., 2005). Therefore, the activity profile of halocin C8 resembled halocin S8 but not H4. In the case of halocin HA1, HA3, and H17 produced by Haloferax larsenii HA1, H. larsenii HA3, and H. alexandrines SWI17, respectively, the optimal production was reported during the mid-exponential phase and then declined rapidly (Mazguene et al., 2018; Kumar and Tiwari, 2017a; Kumar et al., 2016).
MECHANISM OF ACTION OF HALOCINS
Halocins usually inhibit (cytostatic) or kill (cytocidal) related strains but few are reported to be active across different genera, phyla, and domains (Mazguene et al., 2018; Besse et al., 2015; Atanasova et al., 2013; Shand and Leyva, 2007). Halocin-treated cells may undergo changes in the internal pH, Na+/H+ flux, membrane potential, proton motive force (PMF), due to either cell membrane deformation or inhibition of Na+/H+ antiporter. Such modifications are responsible for membrane disruption leading to cell death (Charlesworth and Burns, 2015; Karthikeyan et al., 2013; Bakkal et al., 2012; Pasic et al., 2008; Sun et al., 2005; O’Connor and Shand, 2002). Many small AMPs from bacteria and eukaryotes are reported to be cationic in nature and form amphipathic α-helices or β-sheets that interact with anionic membrane lipids of target strains leading to membrane disruption (O’Connor and Shand, 2002). However, halocins are generally neutral or slightly anionic and are not able to fold into amphipathic helices and therefore, fail to interact with the bacterial membranes, which are negatively charged. The mechanism of action of halocin H4, H6, C8, A4, HA1, HA3, Sech7a, and SH10 has been demonstrated either by observing the treated cells under electron/phase-contrast microscopes or by performing various tests that suggest drastic changes in permeability across the cell membrane, likely due to halocin-created channels responsible for the killing of sensitive cells (Kumar and Tiwari, 2017a, 2017b; Karthikeyan et al., 2013; Pasic et al., 2008; Li et al., 2003; Torreblanca et al., 1989; Meseguer and Rodriguez-Valera, 1985). Most of the reported halocins such as halocin H4, Sech7a, SH10, HA1, and HA3 kill the target cells by swelling followed by cell lysis (Pasic et al., 2008; Sun et al., 2005; O’Connor and Shand, 2002). Halocins S8 and R1 are cytostatic whereas, halocins A4 and C8 are cytocidal (Haseltine et al., 2001). The effect of halocin C8 was observed against Halorubrum saccharovorum under a transmission electron microscope (Li et al., 2003). Halocin C8-treated cells showed morphological changes by swelling and forming elliptic or spherical shapes. Thereafter, the cell wall became disintegrated causing leakage of the cellular contents and cell lysis after 24 h (Li et al., 2003). This indicates that halocin C8 acts on the cell wall rather than the cell membrane of the target strain. To date, halocin H6 is the only halocin whose mode of action has been studied in detail (Lequerica et al., 2006; Meseguer et al., 1995). Halocin H6 is reported to be archaeolytic with “single-hit kinetics” i.e., the killing correlates with exponential-type kinetics (Torreblanca et al., 1989). Halocin H6 has been demonstrated as Na+/H+ exchanger (NHE) inhibitor in both haloarchaeal and mammalian cells (Lequerica et al., 2006; Meseguer et al., 1995). It adsorbs firmly to both target cells and membrane vesicles of H. halobium NRC 817 (Meseguer et al., 1995). It causes uptake inhibition and enhancement of the release of α-aminoisobutyric acid (a non-metabolizable amino acid) and alters light-induced pH changes mediated by bacteriorhodopsin. The effect of halocin H6 has been observed to be quite similar to dicyclohexylcarbodiimide (DCCD), an ATPase and Na+/H+ inhibitor in H. halobium (Meseguer et al., 1991). Both intensify the light-dependent acidification of the medium. The increase in acidification and inhibition of light-induced Na+ out-flow in the membrane suggested that it significantly affects Na+/H+ antiporter or Na+ ion-dependent H+ ion influx activity (Meseguer et al., 1995). Halocin H6 treatment also showed additional effects such as changes in intracellular volume, cytosolic pH, proton motive force, membrane potential and ionic flux in the target strain (Meseguer et al., 1995). Further, Lequerica et al. (2006) performed an in vitro study to demonstrate the effect of halocin H6 as NHE inhibitor in a dose-dependent manner in various mammalian cell lines including HEK293 (human embryonic kidney cell line), NIH3T3 (fibroblast cell line), Jurkat E6-1 cells (human T-leukemia cell line), cardiomyocyte HL-1 cell line, as well as human muscle myocytes and fibroblasts. NHE is an antiporter transmembrane protein present in all living organisms from bacteria to higher eukaryotes and regulates cytosolic pH by electroneutral exchange of H+ ion from inside by Na+ ion from outside (Padan et al., 2001). It also plays a very significant role in adaptation to a hypersaline environment for different members of haloarchaea (Paulino et al., 2014; Meseguer et al., 1995). Lequerica et al. (2006) also performed an in vivo study to evaluate the cardio-protective efficacy of halocin H6 on the reperfused and ischemic myocardium in the animal model, dogs, and it was found that halocin H6 significantly reduced the infarct size and premature ventricular ectopic beats but did not affect blood pressure and heart rate. Like halocin H6, halocin H4 induces morphological changes and lysis of sensitive cells, affects light-induced pH change, and inhibits α-aminoisobutyric acid transport (Meseguer and Rodriguez-Valera, 1986). Therefore, these halocins target the membrane and affect the maintenance of the electrochemical gradients. The effect of halocins produced by H. larsenii HA1 and HA3 was studied in terms of cell viability and morphology of sensitive cells of H. larsenii HA10 (Kumar and Tiwari, 2017a, 2017b). Halocins HA1 and HA3 displayed cytocidal activity as halocins treated cells were poorly Gram-stained compared to untreated cells. This was further supported by scanning electron microscopy (SEM), which showed changes in the morphology from round to rod, disc, spherical shapes with swollen appearance leading to cell lysis after treatment with these halocins. Most of the halocin-treated cells were elongated suggesting inhibition of cell division (Kumar and Tiwari, 2017b). To find out the exact mechanism of action of different halocins against sensitive cells, further investigation at the molecular level is required as it may discover novel mechanisms of action.
CONCLUSION AND PERSPECTIVES
The metabolite diversity among haloarchaea has attracted immense interest during the last decade. Studies carried out in recent years have increased our knowledge of different aspects of halophilic Archaea, such as systemic and phylogenetic relationships, ecology and to a lesser extent physiology and genetics. Many of them have their origin in man-made hypersaline environment developed for various purposes, e.g., commercial solar salt, salted food, etc. Future research is necessary to address how the halophilic microorganisms originated during the early stage in the evolution of life and how they diversified and are distributed throughout the world.
Their adaptiveness to both high salt concentration and temperature makes them sources of industrially valuable enzymes. Their biotechnological potential for producing compatible solutes, biopolymers, and other compounds is of industrial interest. Moreover, haloarchaea constitute an excellent model for study of adaptive mechanisms that permit them to tolerate multiple extreme conditions. Haloarchaea have developed strategies to survive under harsh conditions of pH, temperature, or ionic strength. The knowledge on halocins and understanding how they act in such harsh environments represent an emerging domain of research. This field has been seldom explored until now. Halophilic bacteria and archaea generally use disulfide bond formation and ionic interactions as a main strategy to specifically balance and localize particular amino acids at the surface of the protein to stabilize their three dimensional structures. Therefore, it would be interesting to investigate the specific properties of halocins that make them different from others that usually denatured under harsh conditions. Future research may clarify the different aspects of the production and roles of these halocins, biosynthesis, and maturation pathways, detailed structures and mechanisms of action, as well as their ecological roles. In order to realize their full clinical potential, further studies need to focus on their physical structure and modes of action so that clinicians will be able to predict which halocins have the desired pharmaceutical effect. It will also reveal its potential applications for mankind in the coming years.
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