The outer membrane glycolipids of bacteria from cold environments: isolation, characterization, and biological activity

ABSTRACT

Lipopolysaccharides (LPSs) are the main components of the external leaflet of the outer membrane of Gram-negative bacteria. Microorganisms that colonize permanently or transiently cold habitats have evolved an array of structural adaptations, some of which involve components of bacterial membranes. These adaptations assure the perfect functionality of the membrane even at freezing or sub-freezing growth temperatures. This review summarizes the state-of-the-art information concerning the structural features of the LPSs produced by cold-adapted bacteria. The LPS structure has recently been elucidated from species mainly belonging to Gammaproteobacteria and Flavobacteriaceae. Although the reported structural heterogeneity may arise from the phylogenetic diversity of the analyzed source strains, some generalized trends can be deduced. For instance, it is clear that only a small portion of LPSs displays the O-chain. In addition, the biological activity of the lipid A portion from several cold-adapted strains is reported.

Rough-LPS, smooth-LPS, structure, lipid A, outer membrane, cold adaptation, isolation and characterization

Abbreviations

 
• D,D-Hep

  d-glycero-d-manno-heptose

 
• DMSO

  Dimethyl sulfoxide

 
• DOC

  Sodium deoxycholate

 
• Fuc

  6-deoxygalactose

 
• GalNAc

  2-acetamido-2-deoxy-galactose

 
• GC-MS

  Gas chromatography–mass spectrometry

 
• GlcNAc

  2-acetamido-2-deoxy-glucose

 
• GroP

  Glycerol-phosphate

 
• IL-6

  Interleukin 6

 
• Kdo

  3-deoxy-d-manno-oct-2-ulosonic acid

 
• L,D-Hep

  l-glycero-d-manno-heptose

 
• LOS

  Lipooligosaccharide

 
• LPS

  Lipopolysaccharide

 
• MD-2

  Myeloid differentiation protein-2

 
• MS

  Mass spectrometry

 
• NMR

  Nuclear magnetic resonance

 
• OM

  Outer membrane

 
• PAGE

  Polyacrylamide gel electrophoresis

 
• PCP

  Phenol/chloroform/petroleum ether

 
• PMAA

  Partially methylated acetylated alditol

 
• PW

  Phenol/water

 
• Rha

  6-deoxymannose

 
• SDS

  Sodium dodecyl sulfate

 
• THP-1

  Human monocytic cells

 
• TLR4

  Toll-like receptor 4

 
• TNF-α

  Tumor necrosis factor-α

INTRODUCTION

For a long time, ‘extreme’ environments have been considered by scientists as a terra incognita since the harsh conditions characterizing these habitats were a priori regarded as incompatible with life. When researchers started to establish the physical and chemical boundaries for life, it became increasingly clear that these environments are not only copiously populated but they may also represent the habitats of the first living organisms on the Earth and perhaps on other planets.

It is frequently overlooked that environments permanently exposed to temperatures below 5°C cover more than 80% of the Earth's biosphere (Rodrigues and Tiedje 2008). Today, the cryosphere covers about one-fifth of the surface of the Earth, including sea ice in the Arctic and Antarctic oceans, mountain glaciers and terrestrial ice sheets, with substantial seasonal variations and a long-term trend of losses in its area and volume due to climate warming (Fountain et al. 2012).

All these low-temperature environments have been successfully colonized by cold-adapted organisms, which include a large range of representatives from all three domains of life: Bacteria, Archaea, and Eukarya.

These cold-adapted organisms are generally referred to as psychrophiles or cold-loving as they do not just tolerate cold and inhospitable conditions but are perfectly adapted to these environments. Some of these microorganisms have a large growth temperature range, from low to mild temperatures, and are named psychrotrophs or psychrotolerants. Nevertheless, in the absence of clear metabolic or physico-chemical parameters to differentiate them, any organism thriving and dividing actively at a low temperature is presently defined as a psychrophile (Feller 2017).

The capability of microorganisms to live in cold conditions is the result of a vast array of adaptive features at nearly all levels of the cell architecture and function, as cold temperatures impose severe physico-chemical constraints on several cellular functions by negatively influencing cell integrity, water viscosity, solute diffusion rates, membrane fluidity, enzyme kinetics and macromolecular interactions (Rodrigues and Tiedje 2008; Tribelli and López 2018).

The mechanisms of adaptation to cold stress have received considerable attention in the last few decades, particularly in the light of the perceived biotechnology application potential of these organisms and their biomolecules (Margesin and Feller 2010; Balcerzak et al. 2014; Bar Dolev, Braslavsky and Davies 2016; Deller et al. 2016; Caruso et al. 2018a; Caruso et al. 2018b). Several adaptation strategies, including the higher flexibility and lower thermal stability of their enzymes, the up-regulation of genes encoding for cold-shock proteins, and the production of anti-freeze proteins and glyco-proteins, have been reported (Moyer and Morita 2007; De Maayer et al. 2014; Collins and Margesin 2019). A common physiological adaptation strategy is related to the cellular membrane composition, providing a mechanism to compensate for the kinetic and thermodynamic effects of the cold. Indeed, one of the main responses to the lower temperatures concerns the cellular membrane. The maintenance of its functionality, and consequently of the cell integrity, during changing environmental conditions are related to the ability of the microorganism to rapidly adjust the lipid composition. Generally, a reduction in temperature leads to changes in lipid class composition, in terms of the content of unsaturated, polyunsaturated and methyl-branched fatty acids, and/or shorter acyl-chain lengths (Beales 2004; Chattopadhyay 2006; D'Amico et al. 2006), which affect phospholipid packing, and the conversion of trans- to cis-isomeric fatty acids (Chintalapati, Kiran and Shivaji 2004; Margesin and Miteva 2011).

The Gram-negative bacteria display on their surface the presence of amphiphilic molecules termed lipopolysaccharides (LPSs). Here we will discuss only the variations of LPSs since it is reasonable to assume that structural changes could be present in these macromolecules isolated from cold-adapted bacteria. In addition, the physiological role of the LPSs’ structure in cold adaptation and the potential biomedical applications of these molecules will be discussed.

THE LPS MOLECULE

The LPSs represent the major and essential component of the OM of almost all Gram-negative bacteria and of some cyanobacteria (Wilkinson 1977; Westphal et al. 1986; Carillo et al. 2014), constituting approximately 75% of the outer surface. LPSs are heat-stable, amphiphilic endotoxin molecules indispensable for the viability and survival of Gram-negative bacteria, as they contribute significantly to the cell homeostasis by affecting cellular integrity and OM permeability (Alexander and Rietschel 2001). The colony morphology of Gram-negative bacteria can appear as smooth or rough, as a consequence of the different structures of the LPSs, named smooth (S-LPS) or rough (R-LPS), respectively. The structure of an S-LPS molecule can be divided into three covalently linked domains: (i) the glycolipid anchor, called lipid A; (ii) the intermediate core oligosaccharide (the core); and (iii) the O-specific polysaccharide (the O-chain) (Fig. 1) (Caroff and Karibian 2003). Instead, the R-LPSs (also called lipooligosaccharides, LOSs) are completely devoid of the O-specific polysaccharide chain, either due to a genetic mutation or due to the inherent nature of the bacteria (Luderitz et al. 1966).

Figure 1.

General scheme of a lipopolysaccharide structure. The structure of an S-LPS is composed of three regions, named lipid A, core, and O-antigen (or O-chain). Lipid A consists of a typical structure characterized by an amino sugar disaccharidic backbone that, in most cases, comprises two glucosamines. Usually, the lipid A is phosphorylated at position 1 of the proximal glucosamine (GlcN I) and 4′ of the distal glucosamine (GlcN II). The core moiety consists of a linear or branched oligosaccharide that joins up the lipid A to the O-chain moiety. This latter portion represents the outermost and most variable portion of the LPSs. The O-antigen is absent in the R-LPSs.

The LPSs of Gram-negative bacteria are constituted by a family of glycoforms, due to the structural variations that occur in each portion of the entire molecule. The lipid A anchors the macromolecule into the OM and is the most conserved region even among different species belonging to the same genus (Holst et al. 1996). The phosphate or pyrophosphate groups, usually located on the lipid A, serve to maintain the integrity of the OM, through the bridging action of divalent cations, such as Ca²⁺ and Mg²⁺ (Nikaido 2003). The core oligosaccharide can be divided into two regions, the inner core, which substitutes the lipid A, and the outer core (Raetz and Whitfield 2002). In S-LPSs, this latter provides the attachment site for the O-chain. The inner core is a quite conserved structure within a genus or family, and is usually characterized by the presence of the Kdo (3-deoxy-d-manno-oct-2-ulosonic acid), which can be in turn phosphorylated or substituted by another Kdo residue. Another typical component of the inner core, proximal to the Kdo unit, is the l-glycero-d-manno-heptose (l,d-Hep), even if some bacteria contain its diastereoisomer, d-glycero-d-manno-heptose (d,d-Hep) (Alexander and Rietschel 2001; Caroff and Karibian 2003; Holst 2007). More uncommon is the substitution of heptoses for other sugars, such as glucose or mannose. The outer core displays structural variations within the same genus or family, due to the external position prone to reflect the different environmental conditions that the bacterial cells experience. Finally, the O-chain polysaccharide consists of repeating oligosaccharide units, the structure and composition of which can be different for different bacteria. It is noteworthy that the number of repeating units contributes to the extremely complex mixture of the S-LPS glycoforms (Alexander and Rietschel 2001; Caroff and Karibian 2003).

METHODS FOR LIPOPOLYSACCHARIDE CHARACTERIZATION

The isolation of S- or R-type LPSs from dried bacterial cells is usually performed by following one of two different standardized procedures, the hot phenol/water (PW) extraction procedure (Westphal, Luderitz and Bister 1952) or the phenol/chloroform/petroleum ether (PCP) method (Galanos, Luderitz and Westphal 1969). The macromolecule can be recovered in the aqueous, phenol, or organic phase, depending on the polarity of the glycolipid. Both methods require additional purification steps (Nguyen et al. 2019). The PW procedure usually involves the extraction also of nucleic acids and proteins that are selectively removed by enzymatic treatments with nuclease and protease. The PCP method is more selective than the PW, allowing the obtainment of a sample almost free from nucleic acid contaminations. Both methods suffer from phospholipid contamination, which can be partially removed by cell washing with chloroform. Electrophoresis analysis is the first step in the analysis of an unknown LPS, because it can rapidly show the size of the extracted LPS molecule and the type of the extracted material. Typically, the electrophoresis profile of an S-type LPS shows a ladder-like pattern, whereas bands exclusively at the bottom of the gel indicate an R-LPS. As LPSs are amphiphilic molecules, able to form large micelles, it is necessary to denaturate the samples before and during the gel electrophoretic run. The denaturing agents generally used to perform the analysis are sodium dodecyl sulfate and sodium deoxycholate (DOC). The latter gives a better resolution for low-molecular-mass molecules (Komuro and Galanos 1988). Nevertheless, the best resolution in this range can be achieved by using tricine-sodium dodecyl sulfate-PAGE. For a visualization of LPSs, the most frequently used method is silver staining (Tsai and Frasch 1982).

Chemical and spectroscopic methods are currently used for the glycolipid characterization. The chemical methods also employ the use of gas chromatography–mass spectrometry (GC–MS). Usually, the qualitative and quantitative sugar analysis is carried out by a derivatization of monosaccharides as acetylated methyl glycosides or as alditol acetates. They are identified both by the GC column retention time and by the fragmentation pattern (Kenne and Lindberg 1983).

Chemical analysis can help in the determination of the ring size and the glycosylation sites of the monosaccharides. To obtain these data, LPS is extensively methylated with CH₃I in DMSO in strong alkaline conditions following the Ciucanu and Kerek procedure (Ciucanu and Kerek 1984). Next, the sample is hydrolysed in acidic conditions and purified by gel chromatography. A reduction with a marked reagent (NaBD₄) allows the obtainment of alditols with free hydroxyl groups at the positions previously involved in glycosidic linkage and cyclization, which can be acetylated forming partially methylated acetylated alditols (PMAAs).

The analysis of PMAAs is always performed by GC–MS, where the mass spectrometry (MS) fragmentation patterns and the relative retention times unambiguously reveal the positions of the attachment and the ring size of the residues in the carbohydrate chain. Finally, the GC–MS analysis of acetylated (R)-2-butyl (Gerwig, Kamerling and Vliegenthart 1979) or (R)-2-octyl glycoside derivatives (Leontein, Lindberg and Lönngren 1978) provides the absolute configurations of the sugars.

NMR spectroscopy is fundamental for carbohydrate backbone characterization. Carbon chemical shift values for each residue are highly conserved, whereas there are small differences in the proton chemical shifts with respect to the reference methyl glycosides, due to the influence of the neighboring residues (Bock and Pedersen 1983). Proton chemical shifts and proton–proton coupling constants also give precious information on the residue type (Agrawal 1992; Duus, Gotfredsen and Bock 2000; Bubb 2003). The sugar anomeric configuration can usually be deduced by the chemical shift of the anomeric protons and carbons and by the ³J_H1,H2 and ¹J_C1,H1 values.

Due to the amphiphilic nature of the LPSs, it is convenient to separate the lipidic and saccharidic portions to achieve a better solubility. Two different approaches can be used, one of which exploits acid hydrolysis, while the other utilizes alkaline conditions. The Kdo monosaccharide, usually substituting the O-6 position of GlcN II, is a key residue for the obtainment of the lipid A moiety separated from the oligo-/polysaccharide portion. In fact, the Kdo, being a ketose, possesses a glycosidic linkage sufficiently labile to be easily hydrolyzed with a mild treatment. The reaction conditions lead to the formation of artifacts (Volk, Salomonsky and Hunt 1972), thus increasing the sample heterogeneity. This can complicate the interpretation of the NMR spectra, because the artifacts can be present in a not negligible amount. Sometimes, an additional Kdo can be found in the outer core; in such cases, combined data obtained from different methodologies are mandatory (Pieretti et al. 2010).

The second approach implies two consecutive treatments. The first is a mild alkaline lysis using anhydrous hydrazine, which removes all the ester-linked fatty acids. The de-O-acylated LPS obtained is subjected to a successive de-N-acylation with KOH 4M, which leads to the isolation of an oligo-/polysaccharide still containing the glycosidic portion of the lipid A (Holst 2000). The increased hydrophilic nature of the molecule sometimes is adequate to obtain some structural information by NMR and does not imply the loss of basic-labile substituents. However, a loss of information about the presence of acetyl groups and pyrophosphates substituents can occur.

A purification step is necessary after both the acidic and alkaline treatments. Indeed, this work in some cases can be very difficult, due to the strong similarity of the oligosaccharide glycoforms (Pieretti et al. 2009, 2012). Finally, the purified oligo-/polysaccharide chain is completely characterized by ¹H/¹³C mono- and two-dimensional NMR spectroscopy, and by MS (Khan et al. 2018).

The structural determination of lipid A follows a different methodology. After the mild acid hydrolysis of the S- or R-type LPS, the glycolipid is recovered by centrifugation as a precipitate. The structural approach makes extensive use of MS analysis by Matrix-Assisted Laser Desorption/Ionization-Time of Flight MS (MALDI-TOF MS) and Electrospray Ionization MS (Que et al. 2000; Corsaro et al. 2004a). These techniques provide insights into the number of glycoforms constituting the fraction and the presence of polar heads on the glycolipid. Instead, the actual distribution of acyl residues on the GlcN residues is achieved by a study of the negative ion mass spectra and positive ion MS/MS spectra of the intact lipid A and NH₄OH treatment product (Silipo et al. 2002).

STRUCTURAL FEATURES OF LPSs FROM COLD-ADAPTED BACTERIA

In the following sections, the structural features of LPSs produced by cold-adapted bacteria will be reported. As a growing number of molecules are described, it is clear that only a portion of them display the O-chain (R-LPSs). The LPS structure has recently been elucidated from species mainly belonging to gammaproteobacteria (representatives of the Alteromonadaceae, Colwelliaceae, Idiomarinaceae, Moraxellaceae, Moritellaceae, Pseudomonadaceae, Oceanospirillaceae, Pseudoalteromonadaceae, Psychromonadaceae, and Shewanellaceae families), and from Flavobacteriaceae.

Acinetobacter

The genus Acinetobacter belongs to the family of Moraxellaceae, and comprises bacteria isolated from soil participating in the mineralization of various organic compounds. Nevertheless, the Acinetobacter genus also comprises important nosocomial pathogens infecting debilitated patients (Gerischer 2008). Acinetobacter sp. VS-15, Acinetobacter lwoffii EK67S (Mindlin et al. 2009), and Acinetobacter lwoffii EK30A (Arbatsky et al. 2010a) have been isolated from 1.6 million- to 3 million-year-old Siberian permafrost samples, and grown at 25°C to test if ancient Gram-negative bacteria may have some unusual features not found in more recent bacteria. These three strains were found to produce an S-LPS, the first two (VS-15 and EK67S) showing an identical O-chain (Fig. 2a) (Arbatsky et al. 2010b). The polysaccharide is characterized by the presence of monosaccharides already reported for the Acinetobacter genus, such as GalNAc and GlcNAc (Fig. 2a) (Fregolino et al. 2010).

Figure 2.

Core and O-antigen structures from cold-adapted bacteria. O-antigens obtained from the S-LPS of (A) Acinetobacter sp. VS-15 and Acinetobacter lwoffii EK67S, and (B) Acinetobacter lwoffii EK30A. (C) Core oligosaccharide from the R-LPS of Colwellia psychrerythraea 34H. The O-antigen from the S-LPS of (D) Idiomarina zobellii KMM 231T and (E) Moritella viscosa strain M2–226. Core oligosaccaride structures from the R-LPS of Pseudoalteromonas haloplanktis strains (F) TAC 125 and (G) TAB 23. (H) Core obtained from the R-LPS of Psychrobacter arcticus 273–4. Chemical structures of O-chains from the S-LPS of (I) Psychrobacter cryohalentis K5T, (L) Psychrobacter muricolla 2pST, and (M) Psychrobacter maritimus 3pS. (N) Core region obtained from the R-LPS of Psychromonas arctica. (O) Core oligosaccharide from the R-LPS of Shewanella sp. HM13. (P) O-chain from the S-LPS of Flavobacterium psychrophilum.

Instead, a different O-chain structure has been found for the LPS from Acinetobacter lwoffii EK30A (Arbatsky et al. 2010a), where the unusual features are represented by the novel derivatives of 4-amino-4,6-dideoxy-d-glucose with N-acetyl-d-homoserine and N-[(S)-3-hydroxybutanoyl]-d-homoserine (Fig. 2b).

All these structures are neutral, with rather hydrophobic residues, revealing the quite distinct features that characterize LPSs isolated from permafrost soil sediment bacteria compared to marine and sea-ice isolates (see below).

Colwellia

At the time of writing, the genus Colwellia comprises 21 species, of which 15 are psychrophiles. Among these, Colwellia psychrerythraea 34H (Cp34H) is the best characterized (Deming et al. 1988; Huston, Krieger-Brockett and Deming 2000; Methé et al. 2005; Nunn et al. 2015). Cp34H was isolated from enriched Arctic marine sediments at –1°C and it is considered a model organism for cold-adaptation mechanisms (Boetius et al. 2015). Cp34H produces an R-LPS and in silico analysis of its genome (Methé et al. 2005) revealed the absence of the waaL gene, which is essential for the assembly of an S-LPS. Indeed, the waaL gene encodes the lipid A-core surface polymer ligase, which is the only enzyme known to be able to mediate the ligation of a pre-assembled O-antigen to a lipid A-core. Obviously, the absence of the waaL gene prevents the biosynthesis of an S-LPS molecule (Heinrichs et al. 1998).

The LPSs produced by Cp34H at 4°C have been elucidated in terms of their lipid A and core structures. In particular, the core structure does not show any hetereogeneity relative to the carbohydrate backbone and consists of only one glycoform, where the more frequently found heptose linked to the inner core Kdo is substituted by a mannose (Carillo et al. 2013). This structural feature is commonly found in the Rhizobiaceae family, but has never been identified previously in extremophiles. In addition, the carbohydrate backbone displays the unusual substitution of a phosphoglycerol moiety (GroP), which has sometimes been found in O-chain structures (Fig. 2c). Unlike the core, the lipid A structure is highly heterogenous, displaying a lysophosphatidic moiety that contributes to the increase in the mixture complexity (Fig. 3a) (Casillo et al. 2017a). The Cp34H lipid A structure, displaying many glycoforms with unsaturated fatty acids, clearly indicates that the LOS contributes with the lipid moiety to the enhancement of the membrane fluidity. A comparison of the Cp34H lipid A with the structures of the lipid A from C. piezophila and C. hornerae revealed substantial differences, of which the most important is the absence of any unsaturated fatty acids and the phosphoglycerol moiety (Sweet et al. 2015). Instead, C. piezophila and C. horneraea lipid A structures (Fig. 3b and c, respectively) share the same pattern of primary fatty acids, showing 3-hydroxyundecanoic [C11:0(3-OH)] linked through amide bonds and 3-hydroxytetradecanoic acids linked as esters. The difference between the two structures consists in the secondary acylation pattern: P. piezophila displays a decanoic acid, whereas the P. hornerae species a dodecanoic one.

Figure 3.

Lipid A structures from cold-adapted bacteria: (A) Colwellia psychrerythraea 34H, (B) Colwellia piezophila and (C) Colwellia horneraea, (D) Marinomonas vaga ATCC 27119T, (E) Marinomonas communis 27118T, (F) Pseudoalteromonas haloplanktis TAC 125, and (G) Pseudoalteromonas haloplanktis TAB 23, (H) Pseudomonas sp. PAMC 28 618, (I) Psychrobacter cryohalolentis, (L) Psychrobacter arcticus 273–4, and (M) Psychromonas marina.

Idiomarina

The psychrotolerant and halophilic Idiomarina zobellii KMM 231^T was isolated from a seawater sample taken at a depth of 4000 m in the Pacific Ocean. The O-antigen obtained from the S-LPS contains quite unusual monosaccharides, such as 2-amino-2-deoxy-l-guluronic acid and 4-amino-4,6-dideoxy-d-glucose. Indeed, the pentasaccharidic repeating unit is distinguished by the presence of both uronic acids and amino sugars. In addition, differently from the Acinetobacter and Flavobacterium species, the absence of acyl substituents on two amino sugars of the repeating unit confers both positive and negative charges to the entire polysaccharide (Fig. 2d) (Kilcoyne et al. 2004).

Marinomonas

Marinomonas vaga ATCC 27119^T and Marinomonas communis 27118^T are members of γ-subclass of Proteobacteria, both displaying psychrophilic and moderate halophilic properties. The LPSs from both strains were recovered from the cells after phenol/water extraction and were hydrolyzed to obtain lipid A fractions. Both structures are characterized by the presence of short chain fatty acids, with an unusual lack of the phosphate group at position 4′ of GlcN II. In particular, the monophosphorylated lipid A moiety of M. vaga ATCC 27119^T displays five fatty acid residues, of which four are 10:0(3-OH) (Fig. 3d) (Krasikova et al. 2004). Interestingly, two 10:0(3-OH) units are located as two N-linked acyl chains, whereas only one is linked as an acyl ester at position 3 of GlcN I. The fourth C10:0(3-OH) is localized as a secondary fatty acid at position 3′ of GlcN II. Finally, the remaining secondary fatty acids were found to be a C12:0 or C12:1, linked to the amide-linked primary 10:0(3-OH) unit on the reducing glucosamine. M. communis ATCC 27118^T monophosphorylated lipid A was found to bear five short acyl chains, three primary, C10:0(3-OH), and two secondary, C10:0 (replaceable with one C12:0) and C10:0(3-OH) (Fig. 3e). It is worth noting that no unsaturated acyl chains were recovered in this case (Vorob'eva et al. 2005).

Moritella

The Moritellaceae family includes the genera Moritella and Paramoritella. All species have been isolated from marine environments and have been characterized as halophilic anaerobes. In addition, the genus Moritella consists solely of a psychrophilic species (Hidetoshi 2014). The Moritella viscosa strain M2–226 (Benediktsdottir et al. 2000), earlier called Vibrio viscosus (Lunder et al. 1995), is a psychrophilic bacterium considered responsible for the salmon winter ulcer. It has been isolated first not only from Atlantic salmon, but also from a plaice caught in the wild (Lunder et al. 2000). The strain was grown at 12°C but only a small amount of S-LPS was produced under these conditions. Interestingly, mild acid hydrolysis gave, after purification, an O-polysaccharide with a trisaccharide repeating unit, containing d-GlcNAc, d-glucuronic acid (d-GlcA), and l-Fuc (Fig. 2e) (Hoffman et al. 2012).

Pseudoalteromonas

Bacteria belonging to the genus Pseudoalteromonas (Bosi et al. 2015) are a group of marine gamma-proteobacteria microorganisms colonizing seawater, that was frequently isolated from an extreme environment. In particular, their adaptation ability (Parrilli et al. 2019) leads this species to successfully colonize even hostile marine habitats as Polar Seas or deep oceanic waters (Bowman et al. 1997).

The marine Gammaproteobacteria Pseudoalteromonas haloplanktis strains TAC 125 (PhTAC125) (Birolo et al. 2000) and TAB 23 (PhTAB23) (Feller et al. 1992) were isolated from Antarctic coastal sea water samples. The LPSs isolated from both bacteria grown at 15°C were investigated. Both molecules are rough (in silico analysis of both genomes (Médigue et al. 2005; Bosi et al. 2015) revealed the absence of the waaL gene), and display a very similar oligosaccharidic skeleton (Fig. 2) and lipid A (Fig. 3) (Corsaro et al. 2001; Carillo et al. 2011). This is not surprising, as they are phylogenetically related (Bosi et al. 2015). While the non-reducing end monosaccharide of the TAC125 core structure is a 2-amino-2-deoxy-d-mannose (d-ManN) (Fig.   2f), it is a galactose in TAB23 (Fig. 2g) (Corsaro et al. 2001; Carillo et al. 2011). Another interesting difference between the two strains is the phosphorylation pattern, as the structure from PhTAB23 shows up to five phosphate groups, whereas in PhTAC125 only three were found. In consideration of the role that phosphorylation plays in cold adaptation (Ray, Kumar and Shivaji 1994; Kumar, Jagannadham and Ray 2002), the influence of the growth temperature on the structure of the R-LPS from PhTAC125 has also been investigated (Corsaro et al. 2004b). In this study, it was demonstrated that growth at high temperatures was associated with an increased phosphorylation profile; instead, growth at low temperatures influenced the lipid portion, with an increased unsaturation and hydroxylation and more branched and shorter acyl chains (Corsaro et al. 2004b).

The lipid As of the strains TAC125 (Corsaro et al. 2002) and TAB23 (Carillo et al. 2011) are the only structures from Pseudoalteromonas haloplanktis up to now characterized. The structures display the presence of 3-hydroxydodecanoyl residues linked both as esters and amides at 2′ and 3′ (GlcN II), and 2 and 3 positions (GlcN I) of the sugar backbone, respectively (Fig. 3). The only difference between the main structures of P. haloplanktis TAB23 and TAC125 is the position of the secondary fatty acid, constituted by a dodecanoyl residue. In fact, in both cases it is localized as a secondary fatty acid on GlcN II, being acyloxyamide and acyloxyacyl linked in TAC125 (Fig. 3f) and TAB23 (Fig. 3g) structures, respectively.

Pseudomonas

The Pseudomonas sp. strain PAMC 28618 is a new Gram-negative bacterium isolated from thawing Arctic soils located in the glacier foreland of Midtre Lovenbreen, Svalbard (Park et al. 2017). The cells were grown at 30°C, and the lipid A was recovered as a precipitate after LPS acetic acid hydrolysis and centrifugation. The strain exhibited a mixture of mono- and diphosphorylated penta-acylated and hexa-acylated lipid A species bearing C12:0(3-OH) units in the amide linkage, and C10:0(3-OH) units in the ester linkage. In addition, a C12:0(2-OH) and a C12:0 were found to be an acyl oxacylamide and an acyloxacyl ester on GlcN I and GlcN II, respectively. Finally, the penta-acylated species was characterized by the absence of a C12:0(3-OH) at position 3′ (Fig.   3h). Interestingly, Park et al. reported that this microorganism possesses a lipid A identical to that of the mesophilic Pseudomonas cichorii, which can cause rot disease in endives (Cichorium endivia).

Psychrobacter

The Psychrobacter genus comprises psychrophilic, mesophilic, halotolerant, aerobic, non-motile, and Gram-negative coccobacilli (Bowman 2006). These bacteria live in extremely cold habitats, such as Antarctic ice, soil, and sediments, as well as in deep sea environments (Maruyama et al. 2000; Romanenko et al. 2002). It has been demonstrated that species belonging to this genus display a high longevity within permafrost, which is not constrained by chromosomal DNA damage resulting from ionizing radiation or entropic degradation over geological time (Amato et al. 2010).

Psychrobacter arcticus 273–4 is a Gram-negative bacterium isolated from the Kolyma region of the Siberian permafrost core (Vishnivetskaya et al. 2006). It is considered a psychro-tolerant microorganism as it can grow at temperatures ranging from −10 to 28°C (Ayala-del-Río et al. 2010). The strain has been chosen as a model for cold-adaptation mechanisms in permafrost, due to its growth at sub-zero temperatures and widespread prevalence (Ayala-del-Río et al. 2010). Psychrobacter arcticus 273–4 produces R-LPSs since its genome is devoid of the waaL gene (Ayala-del-Río et al. 2010). The R-LPSs isolated from the cells of this bacterium grown at 4°C have been characterized after acetic acid hydrolysis and removal of lipid A (Casillo et al. 2015). Interestingly, the core OS terminated with a muramic acid, a typical component of the bacterial cell-wall peptidoglycan (Fig. 2h).

A comparison of this structure with those of marine cold-adapted LOSs revealed the quite unusual substitution of the first Kdo with a second unit of Kdo instead of a phosphate group, which is characteristic of enteric bacteria. In addition, the observed substitution of the heptose with a glucose unit in the inner core has been found so far only in the Moraxellaceae (Masoud et al. 1994) and Rhizobiaceae families (Carlson and Krishnaiah 1992; Forsberg and Carlson 1998).

The chemical structures of three O-chains of other species belonging to the Psychrobacter genus, namely P. cryohalentis K5^T, P. muricolla 2pST and P. maritimus 3pS, were characterized. All these strains display very particular O-chain structures (Fig. 2).

Psychrobacter cryohalolentis K5^T was recovered from permafrost samples within the Kolyma lowland region of Siberia (Gilichinsky et al. 1992), and grown at 24°C. The polysaccharide was found to contain d-Gal and l-Rha, together with several di-amino and tri-amino sugars, including 2,4-diamino-2,4,6-trideoxy-glucose (Qui2,4N), 2,3-diamino-2,3-dideoxy-glucuronic acid (Glc2,3NA), and 2,3,4-triamino-2,3,4-trideoxy-arabinose (Ara2,3,4N), that had not previously been reported in natural carbohydrates. This peculiar structure is neutral, since the only acidic monosaccharide, Glc2,3NA, occurs in the amide form (Fig. 2i) (Glc2,3NAN). Another peculiar feature of this polysaccharide is the presence of a novel unique tri-amino sugar, the 2,3,4-triamino-2,3,4-trideoxy-arabinose, occupying the non-reducing terminal position of the O-chain repeating unit. The terminal monosaccharides of carbohydrate antigens are those most accessible to environmental factors, such as the immune system and bacteriophages. Then, as reported by Kondakova et al. (2012a), their diversity provides specificity to the bacterial cell surface and is believed to be important for bacterial survival and niche adaptation.

P. muricolla 2pS(T) expressed instead an acidic polysaccharide consisting of a disaccharide repeating unit featuring an amide of 2-acetamido-2-deoxy-l-guluronic acid with a glycine residue (l-GulNAcA6Gly), which had not previously been found in nature (Fig. 2l) (Kondakova et al. 2012b).

Interestingly, P. maritimus 3pS, isolated from cooled water brines within permafrost in the same region of the P. cryohalolentis first isolation (Gilichinsky et al. 2005), revealed an acidic tetrasaccharide repeating unit characterized by the presence of a bacillosamine derivative, namely 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutanoyl]amino-d-glucose (d-QuiNAc4NHb) (Fig.   2m) (Kondakova et al. 2012c). This unusual structure shares a →2)-α-l-Rhap-(1→4)-α-d-GalpNAcA-(1→3)-α-d-QuipNAc4NHb- trisaccharide sequence with the Pseudomonas fluorescens IMV 24 7¹¹ LPS O-chain moiety (Shashkov et al. 1998).

Lipid As isolated from the Psychrobacter genus have been characterized from only two strains, Psychrobacter cryohalolentis (Fig. 3i) (Sweet et al. 2015) and Psychrobacter arcticus 273–4 (Fig. 3l) (Korneev et al. 2014; Casillo et al. 2018). These two strains seem to display the same acylation pattern on their glucosamine disaccharide, even if some differences can be noticed. The most abundant species in P. chryohalolentis is the hexa-acylated species, which displays four 3-hydroxydodecanoyl residues [C14:0(3-OH)] as primary fatty acids, and two decanoyl residues (C10:0) as secondary fatty acids at the positions 2′ and 3′ of GlcN II (Fig.   3i). Instead, the mass spectra of the P. arcticus lipid A indicated the presence of additional penta-acylated and tetra-acylated lipid A species (Fig. 3l). Besides these, two hexa-acylated species were revealed, of which one had the same composition as that of P. chryohalolentis, while the other contained a supplementary phosphoethanolamine residue (Casillo et al. 2018). Moreover, a comparison between the lipid A produced by the microorganism grown at 4 and 25°C, respectively, revealed that at 4°C the bacterium adjusted the membrane fluidity by synthesizing lipid A structures containing fatty acids chains shorter than those at 25°C (Korneev et al. 2014; Casillo et al. 2018).

Psychromonas

At the time of writing, the Psychromonas genus consists of 15 species and all are Gram-negative rods exhibiting a great phenotypic diversity, ranging in degrees of piezophily and temperature range of growth (Auman et al. 2010). Despite the well-known presence of unsaturated fatty acids in the Psychromonas phospholipid membrane (Auman et al. 2010 and references therein), only one lipid A structure has been thoroughly characterized. In particular, Psychromonas marina, grown at 15°C, principally produces two glycoforms, a penta-acylated one and a hexa-acylated one (Sweet et al. 2014). Both structures show C14:0(3-OH) as the primary fatty acid and the unsaturated C14:2 as a secondary acyl chain, with an additional C12:0 as an acyloxacyl at the 2′ position for the hexa-acylated form (Fig.   3m).

The 3-hydroxydodecanoic acids are also the primary acyl chains on both the glucosamine residues of the lipid A portion of the Psychromonas arctica R-LPS (Corsaro et al. 2008). P. arctica is an eury-psychrophilic microorganism, i.e. capable of growth in a broad growth range of temperatures (from −15 to 20°C) (Feller and Gerday 2003). This strain has been grown at three different temperatures, namely at 4, 10, and 20°C, in order to establish variations in the structure of the LPSs, if any. Indeed, the lipid A portion of the LPS isolated at 20°C showed an additional dodecanoic acid, generating a hepta-acylated species. All the lipid A species display a cyclopropanoid tetradecanoic acid as a secondary acyl chain, which can be considered a cold adaptation in the same way as a double linkage.

The complete structure of the carbohydrate backbone of the R-LPS isolated from this strain was obtained by analysing both products of the two alkaline degradations (Corsaro et al. 2008). The KOH product was constituted by a highly phosphorylated octasaccharide (Fig. 2n). Intriguingly, a terminal β-fructofuranose residue decorates the oligosaccharide structure, which until then had only been found in the core of LPSs from Vibrio cholerae strains (Vinogradov et al. 1995; Knirel et al. 1997).

Shewanella

The genus Shewanella comprises more than 60 species (Gai et al. 2017; Nogi 2017), of which most are widely spread amongst the marine environments. Some members of this genus are piezopsychrophiles, such as S. benthica, S. piezotolerans, S. psychrophila, and S. violacea. Shewanella sp. HM13 is a cold-adapted Gram-negative bacterium isolated from the intestine of a horse mackerel that produces outer membrane vesicles carrying a single major cargo protein named P49. To study the reasons for which this protein is associated with the surface of outer membrane vesicles, the LPS characterization of Shewanella sp. HM13 was undertaken. The strain was grown at 4°C, and the isolated LOS was characterized (Casillo et al. 2019). After an alkaline degradation, the characterized product indicated the presence of the Shewanella hallmark LPS, which is the 8-amino-3,8-dideoxy-manno-oct-2-ulosonic acid (Kdo8N) (Fig. 2o). In addition, another typical feature of Shewanella core oligosaccharide, a d,d-heptose, was found in this strain (Casillo et al. 2019).

Flavobacterium

Flavobacterium psychrophilum (formerly known as Cytophaga psychrophilia as well as Flexibacter psychrophilus) is the etiological agent of bacterial cold water disease in salmonids and rainbow trout fry syndrome. Several immunogenic cell surface molecules that may be involved in the pathogenesis of these diseases, including LPSs, were identified as potential vaccine candidates (Crump et al. 2001). LPSs from several strains of F. psychrophilum were identified, and all of them showed immunogenic low-molecular-mass carbohydrate antigens, although only strain 259–93 has so far been characterized (MacLean 2001). This strain has been grown at 15°C, and the isolated LPS released the O-chain after mild acetic acid hydrolysis. It is an unbranched polymer of trisaccharide repeating units composed of l-Rha, 2,6-dideoxy-2-acetamido-l-galactose (l-FucNAc) and 2,4,6-trideoxy-2,4-diamino-d-glucose (d-Qui2,4N), where R is 3S,5S-3,5-dihydroxyhexanoyl (Fig. 2p) (MacLean 2001). It is important to note that also for this strain the O-chain is composed of hydrophobic monosaccharides.

IMPLICATIONS OF LPSs STRUCTURAL FEATURES IN MICROBIAL COLD ADAPTATION

To live under extreme conditions, microorganisms established numerous specific mechanisms that made them unique. The psychrophiles colonized cold environments in different areas of the Earth (poles, deep sea, and alpine regions) (De Maayer et al. 2014). The low temperature characterizing these environments is the main challenge for life as this parameter heavily affects biological processes; indeed at low-temperature solute transport and diffusion occur at lower rates likewise biochemical reactions, and cell integrity and membrane fluidity are impaired. Moreover, low-temperature environments often are characterized by low nutrient availability, low pH, increased water viscosity, high osmotic pressures, and ice crystal formation (D'Amico et al. 2006; Garcia-Lopez and Cid 2017, Collins and Margesin 2019; Dhaulaniya et al. 2019). All these conditions shaped the molecular and physiological features of cold-adapted bacteria.

Although it is reasonable to suppose that outer membrane glycolipids have a key role in adaptation mechanisms evolved by cold-adapted bacteria, studies about established relationships between the LPS structure and the cold adaptation are still in their infancy. Since the LPS structure consists of three different domains, each of them can give its contribution to the cold adaptation. Starting from the lipid A, it has been demonstrated that the obligate psychrophiles Psychromonas marina (Sweet et al. 2014) and Colwellia psychrerythraea (Casillo et al. 2017a) show in their LPSs structure unsaturated acyl chains both as primary and/or secondary fatty acids. This fact is consistent with the request of augmenting the outer membrane fluidity at low temperature, as already reported for phospholipids (Beales 2004; Chattopadhyay 2006; D'Amico et al. 2006). More difficult is to find out how the microbial growth temperature influences the production of S-LPS and/or R-LPS molecules. To date, all the characterized S-LPSs have been isolated from cold adapted bacteria grown at temperatures above 20°C. In these studies, no results about the LPS structure obtained from growths at a lower temperature are reported. In contrast, the identified R-LPSs come from microbial growths at a temperature from 4 to 25°C. To understand the role of the O-chain in cold adaptation and acclimation, molecular biology experiments should be performed.

Two different cold-adapted microorganisms have been analyzed for the impact of a mutation in the LPS's genetic loci, namely Photobacterium profundum SS9 (Lauro et al. 2008) and Pseudomonas extremaustralis (Benforte et al. 2018). Photobacterium profundum FL26 mutant obtained by transposon mutagenesis is not able to express O-antigen ligase whereas the mutant FL25 has a disruption in a putative LPS glycosyltransferase (Lauro et al. 2008). Both mutants have been demonstrated to be cold-sensitive phenotypes and are not able to produce S-LPS. In contrast, Pseudomonas extremaustralis wapH mutant is able to produce a higher amount of S-LPSs (Benforte et al. 2018). Nevertheless, the mutant showed to be incapable to grow after a cold shock, thus demonstrating that alteration in the LPS structure is crucial for cold growth (Benforte et al. 2018). Further studies concerning the role of the LPSs when cold-adapted bacteria are grown at different temperatures will be necessary.

BIOMEDICAL APPLICATIONS: PSYCHROPHILIC MICROORGANISMS AS A SOURCE OF VACCINE ADJUVANTS AND PHARMACEUTICAL AGENTS

In mammals, LPSs trigger both innate and adaptative immune systems through the lipid A portion, that is recognized by the innate immunity receptor complex composed of Toll-like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) (Fig. 4) (Triantafilou and Triantafilou 2002; Akira, Uematsu and Takeuchi 2006). Fine-tuning of the lipid A structure is responsible for the immunopotential of an LPS (Golenbock et al. 1991; Park et al. 2009). In addition, the research has focused on the potential applications of new lipid As, obtained from either engineered bacterial extracts (Needham et al. 2013) or synthetic (Maiti et al. 2010; Shimoyama et al. 2011) and semisynthetic approaches (Pieretti et al. 2014; D'Alonzo et al. 2016). Furthermore, the natural lipid As from psychrophiles may be a promising source of non-toxic and/or immunomodulating molecules since the psychrophilic biodiversity is largely underexplored.

Figure 4.

General scheme of transduction pathways LPS-mediated. LPSs binding the TLR4/MD-2 complex lead to the activation of different signaling transduction pathway. The main determinants that influence the toxicity of lipid A are the number and the distribution of acyl chains, and the phosphorylation pattern. The hexa-acylated bisphosphorylated lipid A from Escherichia coli triggers the biosynthesis of inflammatory cytokines. Instead, the tetra-acylated lipid A is considered an antagonist in human cells. The deletion of a phosphate group in Salmonella enterica sv. Minnesota lipid A results in a decreased endotoxic activity, making it a good immunoadjuvant in vaccines.

Many lipid As reported in this review have been tested for their biological activity. The lipid A isolated from PhTAB23 displays a good antagonistic activity in THP-1 human monocytic cells. Indeed, when the cells were stimulated with only the PhTAB23 lipid A, the treatment induced the production of low content of both IL-6 and TNF-α. Instead, when tested together with E. coli LPS, the PhTAB23 lipid A was able to almost completely inhibit the LPS induced up-regulation of both TNF-α and IL-6 (Carillo et al. 2011).

The overacylated lipid A from Colwellia psychrerythraea 34H has neither an agonistic effect nor an antagonistic effect on the LPS-induced TNF production on human macrophages (Casillo et al. 2017a). Clearly, in this case, some significant alterations in the lipid A conformation would be expected, due to the presence of the bulky lysophosphatidic acid group, with consequent modification of the interaction with the MD-2 pocket (Park et al. 2009).

Similarly to the hexa-acylated E. coli glycoforms, the lipid A from P. cryohalolentis, showing a predominant hexa-acylated form, induced a stronger TNF-α release, whereas the P. arcticus lipid A displayed only a weaker agonist activity, consistent with the lower content of the hexa-acylated species (Korneev et al. 2014; Casillo et al. 2018).

Instead, the deep-sea M. communis ATCC 27118T, possessing a short chain lipid A, was unable to induce TNF-α release from peripheral human blood cells, whereas it was capable of inhibiting the TNF-α release induced by the E. coli LPSs (Vorobeva, Krasikova and Solov'eva 2006).

Finally, the finding of a toxic lipid A in the Pseudomonas sp. strain PAMC 28618 isolated from permafrost is more intriguing (Park et al. 2017). Since this structure is identical to that of the mesophilic and plant pathogenic P. cichorii, serious questions have been raised concerning the effects of global warming on cold environments microbiology.

CONCLUDING REMARKS AND OUTLOOK

LPSs from psychrophilic Gram-negative bacteria are involved in life adaptation at low temperatures. Indeed, at least for the membrane-buried domain (the lipid A structure), shorter fatty acid chains than those of mesophiles, as well as unsaturated ones, have been discovered. This finding is in line with the psychrophilic outer membrane's need to maintain the correct fluidity, a membrane that allows good solute exchange rates even when the temperature approaches the freezing point of water. More intriguingly, looking at the O-chain domain for all the considered bacteria, most of the structures share two key features, namely the presence of hydrophobic monosaccharides and a peculiar distribution of anionic charges. It is tempting to speculate that, of these structural features, the former is able to counteract the ice crystal formation around the cell, as demonstrated for some capsular and extracellular polysaccharides (Carillo et al. 2015; Casillo et al. 2017b). Moreover, the presence of anionic groups sequestering mono-and/or divalent cations such as Na⁺ or Ca²⁺ furnishes an additional strength to the membrane outer layer (Alexander and Rietschel 2001), thus providing a decreased ion permeability.

More fascinating is the fact that the presence of chaotropic agents, such as glycerol and MgCl₂, this latter abundant in marine environments, facilitates life at low temperatures (Chin et al. 2010). In addition, it has been demonstrated that glycerol promotes macromolecular interactions around or below 0°C (Chin et al. 2010). Therefore, the presence of two residues of glycerol, together with acidic monosaccharides and phosphate groups in the Cp34H LPS structure, can be considered an emblematic adaptation to counteract the cold stress (Carillo et al. 2013; Casillo et al. 2017a).

Bacterial LPSs from isolates coming from cold environments may also be a source of potential immunostimulant molecules. Indeed, lipid As isolated from several psychrophiles have been reported to antagonize E. coli LPS toxicity, while others have proved to be able to act as weak agonists. Both types are interesting, due to the possibility of their use as a treatment for inflammatory conditions and as immunoadjuvants, respectively.

Finally, knowledge of the chemical and biological aspects of cold-adapted LPSs is a key strategic point for an in-depth comprehension of the functionality of membranes at freezing or sub-freezing growth temperatures. The quasi-infinite source of bioactive products arising from these extreme environments hints at a bright future in the identification of new molecules that may prove to have important and beneficial effects on human well-being.

Conflicts of interest. None declared.