Streptolysin S-like virulence factors: the continuing sagA

Abstract

Streptolysin S (SLS) is a potent cytolytic toxin and virulence factor produced by nearly all Streptococcus pyogenes strains. Despite a 100-year history of research on this toxin, it has only recently been established that SLS represents the archetypal example of an extended family of post-translationally modified virulence factors also produced by some other streptococci and Gram-positive pathogens, such as Listeria monocytogenes and Clostridium botulinum. In this Review we describe the identification, genetics, biochemistry and various functions of SLS. We also discuss the shared features of the virulence-associated SLS-like peptides, as well as their place within the rapidly expanding family of thiazole/oxazole-modified microcins (TOMMs).

Introduction

Humans are the natural host and sole reservoir of group A Streptococcus pyogenes (GAS) (for classification, see ^1-2). The organism can survive and replicate in a variety body locations including the skin, throat and blood ³, and is a common cause of illness, especially in children. The more typical infections include self-limiting skin disorders such as impetigo and respiratory tract infections such as pharyngitis. In rare cases, complications can lead to destructive soft tissue infections including necrotizing fasciitis and the multisystem disorder of streptococcal toxic shock syndrome ³. In recent decades, a dramatic increase in severe invasive GAS infections has been documented worldwide ^4-5. These infections carry a significant risk of mortality ^6-7 with an estimated 500,000 deaths worldwide, most of which are attributed to invasive infection or acute rheumatic fever and subsequent rheumatic heart disease ^8-9.

Although the ability of certain strains of streptococci to haemolyse red blood cells (beta-haemolysis) ^10-12 was first observed as early as 1895 ^13-14, it was not until 1938 that Streptolysin S (SLS) was identified as being one of two distinct toxins responsible for the ability of GAS to lyse mammalian erythrocytes ¹⁵; the other being the unrelated, large cholesterol-dependent, oxygen-sensitive toxin, Streptolysin O (SLO) ^16-17. Although SLS has been subject to rigorous investigation (Box 1), isolation of the mature SLS toxin and elucidation of its molecular structure has proven elusive ¹⁸.

Box 1. History of SLS Research.

The SLS-associated beta-haemolytic phenotype of GAS has been subject to rigorous investigation for over a century.

SLS is a 2.7 kDa ribosomally synthesised, post-translationally modified peptide that is regarded as the prototypical member of a distinct group of Streptococcus-associated toxins whose haemolytic activity is abolished by the addition of trypan blue ¹⁹. It is extensively modified, resulting in the formation of distinctive heterocycles, prior to export ^20-21. In addition to an unusual structure, SLS is cytolytic only when associated with the bacterial cell surface or in the presence of certain “carrier” molecules ^22-24. The cytolytic spectrum of the toxin is broad and includes erythrocytes, leukocytes, platelets and sub-cellular organelles ^25-28, but excludes bacteria with intact cell walls ²⁹. SLS is not immunogenic in the course of natural infection ³⁰, which may reflect both its small size, highly modified nature (which removes proteolytic sites that are critical for antigen digestion/display) and, perhaps most importantly, its potent cytotoxicity against cells involved in both innate and adaptive immunity ^31-32. While the exact mechanism of SLS toxicity is not yet fully understood, it has been suggested that its accumulation in cell membranes results in the formation of transmembrane pores and irreversible osmotic lysis ³³.

It is becoming apparent that SLS represents the founding member of a class of post-translationally modified virulence peptides, with homologous toxins now identified in other Streptococcus species. Furthermore, analysis of genomic data has identified gene clusters that are similar to the SLS-associated cluster in other disease-causing pathogens such as Listeria monocytogenes, Clostridium botulinum and Staphylococcus aureus. In this Review we describe the identification of SLS, the GAS chromosomal locus that encodes SLS and the proteins involved in its production, as well as the initial steps towards characterisation of its structure. We provide a brief overview of the role of SLS in virulence and other putative functions. We discuss the identification of SLS-like loci in non-GAS streptococci and the advances in identification of SLS-like loci in other genera. Finally, we highlight the recent discovery that similar biosynthetic clusters are found in numerous microbial phyla and the subsequent definition of a new family of ribosomally-produced peptides i.e. thiazole/oxazole-modified microcins (TOMMs).

The GAS SLS-associated gene cluster

The chromosomal locus responsible for SLS production was first identified following the characterisation of transposon mutants of GAS that did not produce SLS ^34-35. The transposons disrupted the promoter for a gene, designated sagA (SLS-associated gene), that encodes the 53-amino acid SLS precursor, SagA. Subsequent chromosome-walking studies and genome-sequence data resulted in the identification of a contiguous nine-gene locus (sagA to sagI), which was found to constitute an operon ^36-37 (Figure 1). Further analysis confirmed that the sag operon encodes all the accessory proteins required for proper processing and export of SLS ³⁶.

Figure 1. Overview of the production, processing and export of SLS and microcin B17.

The C-terminal core peptide of SagA (depicted in red; A), produced by GAS, and McbA (depicted in orange; B), produced by E. coli, serve as structural templates that undergo a series of post-translational tailoring reactions by the SagBCD and McbBCD biosynthetic complexes to form biologically active SLS (cytotoxin) and microcin B17 (DNA gyrase inhibitor), respectively. The N-terminal leader (depicted in black) is cleaved from the mature core peptide following modification, resulting in a mature peptide product. Also shown is the ‘leaky’ rho-independent terminator sequence between sagA and sagB, and mcbA and mcbB, that acts as a regulatory mechanism yielding a stoichiometric excess of structural gene transcripts versus a substoichiometric, catalytic amount of transcripts for the modification and transport machinery.

Following an in silico analysis of the sag operon, it became apparent that SLS is related to the bacteriocin family of antimicrobial peptides or, more specifically, to a number of post-translationally modified (Class I) bacteriocins ^36,38. Class I bacteriocins are encoded by an operon that contains a structural gene for a precursor peptide with an N-terminal “leader” region and C-terminal “core” peptide ³⁹. The operon also includes genes encoding the machinery required for post-translational modification of the core peptide, as well as leader cleavage and export of the mature form of the bacteriocin. In addition to their antimicrobial activity, in rare cases modified bacteriocins also exhibit broader haemolytic and cytolytic properties ⁴⁰, as is the case with the cytolysin produced by Enterococcus faecalis ⁴¹.

SagA possesses features reminiscent of bacteriocin precursor peptides, such as a potential Gly-Gly leader cleavage site that would yield a 23 aa leader peptide and a 30 amino acid structural peptide ^42-43. This structural peptide contains an abundance of residues that are frequently the target of post-translational modification in bacteriocins, including serines, threonines, cysteines and glycines ⁴³. Site-directed mutagenesis of conserved residues in SagA supports its designation as a bacteriocin-like toxin ²⁰, as does the fact that SagA shares features reminiscent of McbA ³⁶, the precursor of the bacteriocin microcin B17 of Escherichia coli ⁴⁴. Furthermore, in common with many bacteriocin operons, a ‘leaky’ rho-independent terminator sequence after sagA acts as a regulator of transcript abundance ³⁶ (Figure 1A).

The sagA gene is followed by sagBCD, genes that exhibit low sequence identity to mcbBCD located in the E. coli mcb gene cluster ²¹ (Figure 1B). McbBCD form an enzyme complex required for the post-translational modification of McbA ^45-46. The predicted protein product of sagB is a 36 kDa species that shares some similarity with the McbC dehydrogenase (22% identical), SagC (40.3 kDa) is 13% identical to the McbB cyclodehydratase and SagD is a 51.6 kDa “docking” protein (18% identical to McbD) ^21,36. Due to these similarities, it was hypothesised that SagBCD might function in a similar manner to the McbBCD synthetase complex, which converts four serines and four cysteines within McbA into oxazole and thiazole heterocycles, respectively ⁴⁷. These modifications are essential for the activity of mature microcin B17 ⁴⁸. It was subsequently established that SagBCD is also capable of catalysing heterocycle formation when recombinant SagBCD successfully substituted McbBCD to process McbA in vitro ²¹.

Further studies with SagBCD established that the complex is indeed responsible for the conversion of SagA into SLS ²¹. Separate sites within the N-terminal leader peptide provide SagC with a high affinity substrate binding site, leading to efficient modification of the structural peptide by the SagBCD complex ⁴⁹. The modifications involve the conversion, via two distinct steps, of cysteine, serine, and threonine residues to thiazole, oxazole, and methyloxazole heterocycles, respectively (Figure 2) ²¹. SagC removes water from the peptide backbone to produce thiazoline and (methyl)-oxazoline rings, followed by a dehydrogenation reaction catalysed by SagB that formally removes hydrogen to generate aromatic thiazole and (methyl-)oxazole heterocycles ²¹. SagD is proposed to have a role in SagBCD complex formation and the regulation of enzymatic activity ²¹. The incorporation of these heterocycles into the unmodified precursor peptide restricts backbone conformational flexibility and provides the mature SLS with a more rigid structure. Rigidification is essential for bioactivity, as unstructured peptides must pay too large an entropic penalty to bind efficiently to molecular targets ⁵⁰.

Figure 2. Post-translational modification of SagA via the combined activity of a cyclodehydratase (SagC) and dehydrogenase (SagB) from within a three protein complex (SagBCD).

SLS heterocycles are formed via two distinct steps: SagC, a zinc-tetrathiolate containing cyclodehydratase, removes water from cysteine, serine, and threonine residues in the peptide backbone to generate thiazoline and (methyl)-oxazoline rings. Subsequently, a flavin mononucleotide-dependent dehydrogenation reaction catalysed by SagB removes hydrogen to generate the aromatic thiazole and (methyl)-oxazole heterocycles. An example of each heterocyclizable residue is shown for illustrative purposes, with glycine included as a typical residue that is found N-terminal to cyclized residues to facilitate the orbital alignment required for cyclodehydration ^{45,50,153-154}.

Recent research has yielded increased information regarding the structure-activity relationships of SLS, but the precise chemical structure of mature SLS has yet to be fully elucidated. Site-directed mutagenesis approaches with alanine (to reduce backbone rigidity) and/or proline (which retains rigidity) suggest that oxazoles are formed at residues Ser34, Ser39, Ser46, and Ser48, that a thiazole is formed at Cys32 ⁴⁹, and that Cys24 and Cys27 are also important residues with respect to haemolytic activity ^20,49. As the incorporation of thiazoles or (methyl-)oxazoles results in a net loss of 20 Da in peptide mass, LC-MS/MS with in vitro modified SLS has been employed to provide evidence of heterocycle formation by SagBCD and to confirm the incorporation of oxazole moieties at Ser46 and Ser48 ⁵¹.

The role of the remaining Sag proteins is less clear. SagF (26.2 kDa) is predicted to be membrane-associated ^21,52 and SagE (25.4 kDa) is expected to be a membrane-spanning peptidase ²¹. Although it is reasonable to suggest that SagE might be the enzyme responsible for leader cleavage, it has also been noted that intact sagE is required for viability and that the corresponding protein shares weak homology with a candidate bacteriocin immunity (self-protection) protein, PlnP, of Lactobacillus plantarum ²⁰. SagG (41.7 kDa), SagH (42.2 kDa), and SagI (41.7 kDa) are thought to be membrane proteins that form an (ABC)-type transporter ^20-21 similar to those frequently involved in bacteriocin export ^36,53, with SagG displaying the signature ATP-binding pocket motifs ²⁰.

SLS-like loci in other streptococci

The production of SLS-like cytolysins is not an exclusively GAS-associated trait (Table 1). Such activity is also associated with invasive human isolates of the beta-haemolytic group C and G streptococci, which belong to the streptococcal species S. dysgalactiae subsp. equisimilis. Homologues are also present in the animal pathogens Streptococcus iniae and Streptococcus equi.

Table 1. TOMMs referred to in this review.

Organism	Disease associated with organism	TOMM	Genes	TOMM function	References
TOMMs in pathogenic bacterial species
Streptococcus pyogenes	respiratory tract infections, skin diseases, invasive infections	Streptolysin S	sagA-I	haemolytic exotoxin	15,34,36
S. dysgalactiae subsp. equisimilis	pharyngitis, invasive infections	Streptolysin S	sagA-I	haemolytic exotoxin	57
S. iniae	fish pathogen, rare human pathogen causing invasive infection	Streptolysin S	sagA-I	haemolytic exotoxin	62
S. equi	horse pathogen	Streptolysin S	ND	haemolytic exotoxin	65
Listeria monocytogenes	gastroenteritis, septicaemia, meningitis	Listeriolysin S	llsA-X	haemolytic exotoxin	21,111
Clostridium botulinum, (Clostridium sporogenes)	botulism poisoning	Clostridiolysin S	btsA-I	haemolytic exotoxin	21,51,111
Staphylococcus aureus RF122	bovine mastisis	Staphylysin S	stsA-Z	haemolytic exotoxin	21,49,111
TOMMs in non-pathogenic bacterial species
Escherichia coli	NA (gut commensal / pathogen)	Microcin B17	mcbA-G	antibacterial: DNA gyrase inhibitor	44,46-47
Prochloron sp.	NA (photosynthetic endosymbiont of the ascidian Lissoclinum patella)	Patellamide D	ND	reversal of multiple drug resistance in a human leukaemia cell line	147-148
Streptomyces sp. TP-A0584	NA (soil inhabitant)	Goadsporin	godH-I, godR	antibiotic activity, promotion of secondary metabolism and morphogenesis	120,142-143
B. amyloliquefaciens FZB42	NA (plant growth-promoting saprophyte)	Plantazolicin	pznA-L	narrow-spectrum antibacterial compound	21,150

Group C Streptococcus (GCS) is a relatively common cause of acute pharyngitis among young adults ^54-55. The normally commensal group G Streptococcus (GGS) can also cause acute pharyngitis ⁵⁶ and has been associated with necrotising soft tissue infections in patients with underlying medical conditions ⁵⁷. Both GCS and GGS display a hallmark beta-haemolytic phenotype on blood agar ⁵⁸, which has been linked to the action of SLS-like cytolysins ⁵⁷. The GCS and GGS sagA genes are identical to each other and the corresponding peptides share 89% identity with the GAS SagA ⁵⁷. Other conserved features include the sag operon promoter, a rho-independent attenuator located downstream of sagA, as well as eight genes resembling the remaining SLS associated genes ⁵⁷. Targeted mutagenesis of the GGS sagA gene has been found to abolish beta-haemolytic activity, a phenotype that is partially restored upon transformation of the mutant with the GAS homologue ⁵⁷.

S. iniae is a beta-haemolytic pathogen of commercial fish species that can also be a rare human pathogen ^59-61. Characterisation of the genomic region responsible for the haemolytic phenotype of S. iniae identified a 9-gene locus that has 73% sequence similarity to that in GAS ⁶². Furthermore, heterologous expression of sagA from S. iniae restored haemolytic activity to GAS ΔsagA ⁶³. Finally, S. equi is the causative agent of “strangles”, a prevalent and highly contagious disease of horses ⁶⁴. While an associated sag cluster has not yet been identified in this species, the haemolytic activity of S. equi has been characterized and found to be caused by a SLS-like toxin ⁶⁵.

Functions of SLS

A recent genetic screen of clinical GAS samples has shown that 99% of all isolates are SLS-positive, while the remaining 1% are not predicted to produce SLS and are assumed to be non-haemolytic (Lowry-type) strains ⁶⁶. Although these atypical strains have occasionally been associated with human infection ^66-68, there is little doubt that SLS has an important role in Streptococcus pathogenicity. Unsurprisingly, the specific mechanisms by which SLS contributes to virulence have been the subject of much investigation. These proposed mechanisms fall into three categories, namely soft tissue damage, an impact on host phagocytes and a contribution to GAS translocation across the epithelial barrier. SLS also functions as a signalling molecule, and it has been speculated that SLS may contribute to iron acquisition (Figure 3). In the latter case it has been suggested that SLS production could provide a means via which GAS could gain access to intracellular iron through the lysis of host red blood cells ^69-70.

Figure 3. Summary of the functions of SLS.

The mechanisms via which SLS contributes to the virulence of GAS and other functions.

Role of SLS in tissue injury

In vivo studies have demonstrated that SLS is an important virulence factor in skin and soft tissue infection, where it contributes to tissue injury ²⁰. The virulence of a SLS-negative GAS mutant is substantially reduced in a murine soft-tissue infection model ³⁵. Further studies confirming the importance of SLS with respect to GAS and GGS pathogenesis ^57,71-73 have revealed that even a single point mutation predicted to interfere with heterocycle formation can render a strain of GAS avirulent in a murine model of skin infection ⁴⁹. The direct toxicity of SLS toward cells of the deep soft tissues and feeding vessels, leading to cell death and provoking neutrophil influx, is thought to promote the development of necrotising fasciitis ⁵⁷. It has been suggested that other virulence factors, such as SLO ²⁰, the anti-phagocytic M surface protein ^20,74-75, as well as host neutrophil-derived oxidants and proteases ^24,76-78, interact synergistically with SLS to accelerate necrosis. It has also been established that expression of SLS is required for S. iniae-induced local tissue necrosis ^62-63.

Role of SLS in phagocytic clearance

The role of SLS in the resistance of GAS to phagocytic clearance was first uncovered when it was revealed that a ΔsagA mutant did not survive as well as wild-type GAS in killing assays with human whole blood and purified neutrophils ²⁰. It was subsequently revealed that cell-associated SLS actively destroyed neutrophils recruited to the site of infection ⁷⁹. The elimination of neutrophils may be a specific virulence mechanism that effectively inactivates the phagocytic cells that are primarily responsible for the ingestion and killing of GAS, thus allowing them to evade the innate immune system ⁷⁹. In support of this hypothesis, a paucity or lack of neutrophils is regarded as an unfavourable prognostic sign in patients suffering from necrotizing fasciitis ⁸⁰ and in primates who fail to survive GAS-associated necrotizing fasciitis and myositis ⁷⁸. Macrophages constitute another crucial component of the host phagocytic defence against GAS infection. It has been established that GAS can kill macrophages, through the activation of an inflammatory programmed cell death pathway mediated by SLS and SLO ⁸¹. It is important to note, however, that the relative contribution of SLS to phagocyte resistance is subject to both species and strain variation ^62-63,72-73, and as such, other factors may also play a prominent role.

GAS is known to produce virulence factors that diminish the host's immune response to infection ^82-84. SLS is thought to contribute by affecting the host cell's ability to produce signals that are chemotactic for neutrophils ⁸⁵. Using a zebrafish model, it was shown that, in contrast to wild-type GAS, an SLS- mutant was significantly less virulent and was associated with a more robust recruitment of neutrophils ⁸⁵.

Finally, little is known about the entry and subsequent multiplication of S. equi following the exposure of a susceptible equine host ⁸⁶, but data that indicates that virulence is as a result of a potent anti-phagocytic effect and the failure of innate immune defences ⁸⁷ suggests that its SLS-like toxin may play an important role.

Role of SLS in GAS translocation

Systemic dissemination of GAS involves bacterial colonisation of the pharynx or damaged skin, followed by penetration of the epithelial barrier. The mechanisms underlying the adherence of GAS to epithelial cells and subsequent internalization have been extensively studied ^88-91. Most internalized GAS are eliminated by intracellular killing. However, it has been speculated that the programmed cell death of epithelial cells induced by massive adherence or internalization of GAS may reduce the barrier function of the epithelium, providing GAS with access to deeper tissues via an intracellular route ^92-94. GAS tissue invasion via a paracellular route has been also been noted ⁹⁵ and, significantly, recent research has identified SLS as a critical factor in this process ⁹⁶. SLS does this by recruiting the host cysteine protease calpain to the plasma membrane by an as yet uncharacterised mechanism, and then utilises proteolytic activity to degrade intercellular junctions to allow invasion via a paracellular route ⁹⁶.

SLS as a signalling molecule

Many species of bacteria use a cell density-dependent response system, or quorum sensing, to regulate gene expression ⁹⁷. Indeed, several virulence genes in GAS are modulated by quorum sensing ^98-99. luxS is essential for the production of AI-2 of the autoinducer II (AI-2) pathway in a diverse range of bacterial species ¹⁰⁰ and, after the identification of a luxS homologue in GAS ¹⁰¹, a GAS luxS mutant was found display a number of altered phenotypes ¹⁰². Notably, the SLS activity of the mutant was found to be enhanced as a result of increased sagA transcription. As expression of sagA is normally induced at high cell density, these results suggested that the AI-2 pathway was not the only element of population-dependent control but rather indicates that luxS interacts with a second regulatory pathway to influence its sensitivity ¹⁰².

Indeed, since it was first established that SLS expression increases with cell density ³⁵, it was suspected that SLS itself might act as a quorum sensing molecule ¹⁰³. Several bacteriocins are known to be regulated by quorum sensing ³⁸ and in some instances, as is the case for nisin, the structural peptides also function as signalling molecules and induce their own expression upon activation of the density-dependent autoinduction loop ¹⁰⁴. Significantly, it has since been established that expression of sagA is up-regulated by exposure to SLS ¹⁰³. Furthermore, global regulatory functions have been attributed to the sagA gene itself, resulting in the designation of pel, for ‘pleiotropic effects locus’, to the ORF ^36,105-109. More specifically, it has been suggested that sagA mRNA is involved in the pre- and post-translational control of other virulence factors of the human pathogen, including M proteins, the expression of capsule (SpeB), and streptokinase ^108-110. In contrast to these results, Datta et al. found that elimination of sagA does not produce significant pleiotrophic effects ²⁰, suggesting that the role of sagA/SagA/SLS in the regulation of other virulence factors may differ in a strain variable manner.

SLS-like loci in other bacterial genera

Until recently, post-translationally modified virulence peptides have rarely been reported and SLS-like cytolysins have been exclusively associated with the genus Streptococcus. As noted above, however, many similarities exist between the SLS and the MccB17 biosynthetic operons present in some E. coli. This observation prompted a search of public genomic databases in the belief that other prokaryotes might use related machinery to introduce Ser/Thr/Cys-derived heterocycles into a wider variety of ribosomally produced peptides ^21,111. From this approach, it became evident that a number of Sag-like gene clusters are present in some of the most notorious Gram-positive pathogens, including L. monocytogenes, C. botulinum and S. aureus ^21,111 (Table 1 & Figure 4). In addition, more distantly related clusters were identified in an even more diverse collection of bacteria ²¹. This lead to the definition of a new class of compounds, characterised by a biosynthetic gene cluster that encodes a small precursor peptide and three adjacent synthetase proteins that serve to introduce thiazole and (methyl-)oxazole heterocycles onto the ribosomally-produced protoxin scaffold. These bioactive natural products and their biosynthetic gene clusters are now referred to as thiazole/oxazole-modified microcins (TOMMs) ^21,50. Although the biological purposes of the majority of newly identified TOMMs have not been uncovered, it is likely, based on the similarity to McbA and SagA, that some will act as either DNA gyrase inhibitors or membrane-damaging agents ¹¹².

Figure 4. Top Panel: Amino acid sequence of the unmodified precursors of Staphylysin S (StsA), Listeriolysin S (LlsA), Streptolysin S (SagA) and Clostridolysin (BtsA).

The (predicted) leader regions are to the left and terminate in (putative) Gly-Gly/Ala-Gly leader cleavage sites (blue). Residues potentially involved in modification of the precursor peptides are indicated in red (cysteine), orange (serine), green (threonine) and blue (glycine). Based on the sequence of sagA and assuming cleavage after the Gly-Gly site, the molecular weight of modified SagA is estimated to be approximately 2.7 kDa. (although this does not preclude the possibility that mature SLS is an assemblage of modified SagA peptides). Bottom Panel: SLS-like gene clusters in S. aureus, L. monocytogenes, S. pyogenes and C. botulinum are depicted. Related genes are indicated by colour. A: structural gene; B: dehydrogenase; C: cyclodehydratase; D: “docking” protein; E/P: CaaX protease; G, H, I: ABC-type transporter components; Z, X, F: proteins of unknown function. In the case of RF122, the genomic map appears to be fragmented, giving multiple ORFs for several biosynthetic proteins.

Within TOMM-associated gene clusters, the genes encoding the SagB-like dehydrogenase, SagC-like cyclodehydratase, and SagD-like “docking” protein are often found as adjacent open reading frames (ORFs). It is probable that this will aid in the identification of additional orthologous clusters as more genome sequences become available. However, the associated TOMM structural genes are frequently overlooked during annotation because of their small and hyper-variable nature ^111,113. Furthermore, although short ORFs encoding proteins of 50–70 residues that are rich in Cys/Ser/Thr are usually found in organisms with sagBCD-like genes as adjacent ORFs ^21,111, TOMM peptides are sometimes situated far from the genes encoding the thiazole/(methyl-)oxazole-forming machinery and in such cases it can be difficult to assign the substrate for a particular TOMM pathway ¹¹⁴. The situation is further complicated by the observation that numerous substitutions in the C-terminal core sequence of the propeptide can often be tolerated ⁴⁹. This can result in the production of numerous similar peptide products ^50,115-117 by “natural combinatorial biosynthesis”. In addition, new families of more unusual modifying enzymes are often not sufficiently related to characterised relatives to be identified by BLAST searching ^21,118-119.

Several factors can increase confidence in the designation of an ORF as a TOMM precursor, including sequence similarity to previously identified TOMM precursors, the presence of a suitable leader peptide cleavage motif and a C-terminal core region rich in heterocyclizable residues ^21,49 (Figure 4). The detection of genes encoding enzymes involved in thiazole/oxazole synthesis, dehydroalanine production ¹²⁰ and peptide macrocyclization ^{113,115,119,121} provides further support when annotating post-translationally modified peptide biosynthetic clusters. The identification of a TOMM cluster is also facilitated by the tendency of the modification enzymes to cluster with other genes associated with the cleavage and export of the final product ^21,36,46.

It is clear that the extent of structural variation and biological impact of TOMM clusters is gaining greater appreciation. Thus, it is likely that novel bioinformatics-based approaches such as those reported by Haft et al., whereby multiple highly sensitive profile-based search models are used to analyse large numbers of sequenced genomes ^50,114, will be applied to uncover additional members of this family of modified peptides, as well as helping to identify cognate modification and export genes.

Listeria monocytogenes: Listeriolysin S

L. monocytogenes is an intracellular pathogen that is usually transmitted to humans through contaminated food products ¹²². It has a high mortality rate in pregnant women, neonates and individuals with a compromised immune system ¹²³. Strains of L. monocytogenes are divided into three evolutionary lineages ¹²⁴, with lineage I (which consists of strains of serotype 1/2b and the notorious ‘epidemic’ serotype 4b) contributing to the majority of sporadic cases and epidemics associated with this often fatal pathogen ^125-126.

Since it was first established that Listeriolysin O (LLO)-negative mutants of L. monocytogenes did not lyse blood cells, it was believed that this cholesterol-dependent virulence factor was the only cytotoxin produced by L. monocytogenes ^127-128. Recent in silico analysis identified a gene cluster in a number of L. monocytogenes strains that resembles the sag operon and was designated the Listeriolysin S (LLS) gene cluster ¹¹¹. The LLS structural gene within this cluster, llsA, encodes a peptide consisting of a N-terminal leader region and C-terminal core region with an extreme predominance of cysteine, serine and threonine residues, as well as a putative Ala-Gly leader cleavage motif ¹¹¹. As the predicted llsA promoter (PllsA) was found to be induced only under oxidative stress conditions, constitutive expression of the operon was used to establish that these genes did indeed encode a SLS-like cytolysin ¹¹¹. Like SLS, LLS was found to be active in a cell-associated form but inactive in cell-free situations in the absence of a stabilizer ¹¹¹. It is now apparent that previous detection of LLS haemolysis was hindered by the absence of the LLS cluster from the majority of the most frequently used laboratory strains, coupled with the inducible nature of PllsA and the masking effect of LLO activity.

With respect to the rest of the LLS operon, the products of llsB (42 % similarity to SagB), llsY (37 % similarity to SagC) and llsD (46 % similarity to SagD) are predicted to form a synthetase complex necessary for the production of mature, active LLS ^111,129. LLS is believed to be post-translationally modified in a similar manner to SLS, supported by the fact that a SagA-LlsA chimera (SagA leader sequence fused to LlsA core peptide) is converted into a cytolytic entity by SagBCD in vitro ⁴⁹. Other proteins encoded by the operon include LlsGH, two components of an ABC transporter (represented by three ORFs, sagGHI, in the sag operon), and LlsP, which has been annotated as a CaaX protease with 36% similarity to SagE ^111,129-130. The llsX gene is not homologous to any known gene and, as such, is used as a target for a real-time PCR assay designed to identify LLS-positive strains of L. monocytogenes ¹²⁹. Deletion mutagenesis has established that of the seven genes located downstream of llsA in the lls operon (i.e., llsG-llsP), all except llsP are essential for LLS activity ^111,129.

LLS contributes to pathogenesis as evidenced by significantly reduced levels of a LLS-negative mutant in the livers and spleens of mice following intraperitoneal inoculation ¹¹¹. Furthermore, wild-type L. monocytogenes survives significantly better than the LLS-negative mutant in purified human polymorphonuclear neutrophils (PMNs). Notably, PMNs are crucial for the resolution of L. monocytogenes infections ¹³¹. Given the oxidative-stress inducible nature of PllsA and the contribution of LLS to neutrophil endurance, enhancement of the survival of cells still retained in the phagosome upon phago-lysosomal fusion has been proposed as a mechanism by which LLS contributes to virulence ¹¹¹. As a consequence of the contribution of the LLS-encoding TOMM cluster to the virulence potential of producer strains, it was named Listeria pathogenicity island 3 (LIPI-3) in order to distinguish it from previously identified Listeria-associated pathogenicity islands ^132-133. Given that LIPI-3 is consistently absent from all lineage II and III L. monocytogenes and that relatively few lineage I outbreak-associated strains lack LIPI-3, it has been postulated that this discovery may well provide the long sought-after explanation for the enhanced virulence of a proportion of lineage I L. monocytogenes ¹¹¹.

Staphylococcus aureus RF122: Stapholysin S

It is noteworthy that a sag-like cluster is also present in the genome of S. aureus RF122 ^21,111, a pathogenic strain responsible for bovine mastitis ¹³⁴. Of the known sag-like clusters, it is most closely related to the lls cluster ^21,49,111. The putative structural gene again encodes a peptide consisting of a proposed N-terminal leader region and C-terminal precursor peptide with an abundance of cysteine, serine and threonine residues, as well as an Ala-Gly suspected leader cleavage site ^21,111. Thus, it was predicted that mature “Stapholysin S” would act as a cytolysin ²¹. This hypothesis was experimentally validated when a chimeric substrate comprised of the SagA leader peptide fused to the StaphA C-terminus was converted into a cytolytic entity by SagBCD in vitro ⁴⁹.

Clostridium botulinum/sporogenes: Clostridiolysin S

Using comparative genomic analysis, a nine-gene SLS-type cluster was identified in the genomes of clostridia, including the biological warfare-associated pathogen C. botulinum and food pathogen C. sporogenes ⁵¹. C. botulinum is an anaerobic, spore-forming rod that causes the potentially fatal neuro-paralytic diseases of food-borne-, infant-, wound- and inhalation-botulism as well as other invasive infections through the elaboration of potent neurotoxins ¹³⁵. C. botulinum strains that do not produce botulinum toxin are referred to as C. sporogenes ¹³⁶.

Before the functionality of the C. botulinum SLS-like gene cluster had been confirmed, in vitro production of the bioactive natural product via SagBCD modification supported its designation as a TOMM, as did investigations revealing the haemolytic nature of the biotoxin generated ²¹. This is not unexpected in light of the organization and amino acid sequence of proteins present in the C. botulinum cluster (closA-I), which corresponds closely with sagA-I. In fact, C. botulinum harbours the most highly related sag-like genetic cluster known outside of the streptococci ⁴⁹. The cytolytic gene product of closA has been named Clostridiolysin S, or CLS ⁵¹. The functional equivalence of the clostridial SLS-like gene clusters to the SLS biosynthetic pathway was confirmed by the complementation of targeted GAS SLS-operon knockouts with the corresponding Clostridium genes ⁵¹. Furthermore, the availability of the complete genome sequence of C. sporogenes ¹³⁷ and the identification of a CLS cluster led to the use of the safer C. sporogenes for mutagenesis studies, which established that this gene cluster was indeed responsible for the biogenesis of a haemolytic toxin ⁵¹.

A more in-depth investigation of the clos genes further highlights the TOMM-associated features. The first gene in the operon, closA, encodes the inactive precursor peptide that is post-translationally modified by the synthetase complex, ClosBCD, to form the mature biotoxin ⁵¹. ClosG, H, and I are ABC transporters and therefore likely constitute the system required for exporting the mature haemolytic product. ClosE has been annotated as an immunity protein but, like SagE, is similar to the CaaX protease super family ¹³⁰ and so might be responsible for leader cleavage from the post-translationally modified propeptide ⁵¹. ClosF is a protein of unknown function.

The in vitro reconstitution of CLS activity allowed direct confirmation by mass spectroscopy that the non-toxic precursor, ClosA, is post-translationally modified by the synthetase enzymes to contain heterocyclic moieties ⁵¹. A heterocycle was identified at position Thr46 within ClosA and the dramatically negative impact of substitution of an alanine at Thr46 on haemolytic activity confirmed the importance of heterocyclic conversion at this site ⁵¹. These investigations represent the first step towards the elucidation of the structure of mature CLS.

Other related clusters

The research of Lee et al. first highlighted the fact that in addition to the Gram-positive pathogens discussed previously, similar TOMM clusters are widely disseminated in the genomes of a diverse group of microorganisms spanning six phyla ²¹. Indeed, even very distantly related microbes, such the thermophilic archaeon Pyrococcus furiosus, can contain heterocycle-forming synthetases ²¹. In addition to the SLS-like toxins with established or suspected roles in virulence described above, some examples of other TOMM clusters of note are mentioned (Table 1). As a consequence of the recent expansion of the TOMM family, it now encompasses such diverse bacterial products as the microcins ¹³⁸, thiazolylpeptides ^139-140 and cyanobactins ¹⁴¹. Emerging TOMM subfamilies include Bacillus-associated putative thiazole-containing heterocyclic bacteriocins ¹¹⁴, as well as a group of nitrile hydratase and Nif11-related precursor peptides ⁵⁰.

A gene cluster identical in arrangement to that responsible for microcin B17 production has been found in the plant symbiont Pseudomonas putida KT2440 ¹³⁸ and it is speculated that it too functions as an inhibitor of DNA gyrase ²¹. Indeed, many other prokaryotes harbour a sag-like genetic cluster that is not associated with the production of a cytolysin, such as the goadsporin-producing microorganism, Streptomyces sp. TP-A0584 ¹⁴². The molecular targets of this secondary metabolite have yet to be elucidated, but it is known that goadsporin promotes secondary metabolism and sporulation in actinomycetes, as well as exhibiting antibiotic activity ^112,120,143. Other diverse functions encoded by TOMM peptides include the inhibition of ribosomal protein synthesis ¹⁴⁴ by the thiazolylpeptides, a family of more than fifty bactericidal antibiotics ^145-146, and reversal of multiple drug resistance in a human leukaemia cell line by the cyanobactin, patellamide D ^147-148.

Lastly, the genome of B. amyloliquefaciens FZB42, a Gram-positive, plant-growth promoting saprophyte that can suppress the growth of bacterial and fungal plant pathogens ¹⁴⁹, was recently shown to contain a novel TOMM cluster ²¹. Subsequent investigations have revealed that this unique TOMM, plantazolicin, functions as a narrow-spectrum antibacterial compound ¹⁵⁰. It has been suggested that the biological role of this natural product might be to suppress the growth of closely related competitors within the plant rhizosphere ¹⁵⁰.

It is apparent that these ribosomally-produced peptide natural products represent a largely hidden arsenal of active small molecules, which, although grouped as a consequence of possessing the same general modifications, can perform a wide variety of functions other than contributing to pathogenicity.

Conclusion

With the ongoing discovery and analysis of biosynthetic genes associated with the production of SLS-like cytolytic peptides, evidence is rapidly mounting that these pathways are both more common and more diverse than previously suspected ⁵⁰ and can contribute substantially to the virulence potential of pathogenic bacteria ¹⁵¹. Of the myriad of novel putative TOMM-encoding clusters identified, those associated with L. monocytogenes, C. botulinum/sporogenes and S. aureus are particularly noteworthy as a consequence of their similarity to the sag cluster and the notoriety of the associated pathogens.

The number and variety of potential producers of SLS-like toxins, and by extension TOMMs, coupled with the fact that secondary metabolites produced by similar biosynthetic clusters have been an abundant source of pharmaceuticals in the past, suggests that further research into this group of peptides will lead to the identification of novel targets for antibiotic and vaccine development. Vaccine or chemotherapeutic strategies designed to neutralize SLS activity could be of benefit as adjuncts to surgical and antibiotic management of human streptococcal infection. The observations that synthetic peptides corresponding to the SLS precursor retain sufficient features of the mature toxin to raise neutralizing antibodies ^33,152, suggest that subunits or inactivated toxoids of each haemolysin could have significant immunogenicity. Insights gained through the in vitro application of the SagBCD biosynthetic machinery and the SagA peptide ⁴⁹ could be also used to guide the design of artificial toxins with a view to structure-based vaccines. Furthermore, because targeting virulence factors is becoming an attractive anti-infective strategy, selective inhibition of the family of TOMM synthetase enzymes may be of value.

In summary, the structural diversity and biological impact of both the SLS-like peptide group and the wider TOMM family of natural products is just beginning to be fully appreciated ⁵⁰. The proven success of in silico analyses suggests that further studies of this type will help to uncover novel TOMM (and other natural product) secondary metabolite biosynthetic systems and will aid in the definition of the evolutionary routes and inter-relationships of the rapidly expanding TOMM ribosomal peptide group. Such studies inform structural investigations into natural products and pave the way for combinatorial biosynthetic studies and rational engineering.

Glossary

Streptococcal pharyngitis

Inflammation of the pharynx also known as “strep throat”.

Impetigo

An acute and highly contagious infection of the surface layers of the skin characterised by blisters, pustules and yellowish crusts

Necrotizing fasciitis

A rare but very severe type of soft tissue infection, which can be caused by GAS, that can destroy the muscles, skin, and underlying tissue. It develops when the bacteria enter the body, usually through a minor cut or as a complication of surgery. The mortality rate is high, even with aggressive treatment and powerful antibiotics

Streptococcal toxic shock syndrome

A rare but extremely severe infection that usually presents in people with pre-existing skin infections with GAS and has a very high mortality rate. It is characterised by hypotension and shock. Other symptoms can include kidney impairment, abnormality in blood clotting ability, acute respiratory distress syndrome, rash and local tissue destruction

Beta-haemolysis

Complete red blood cell lysis that appears as yellowing and transparency around and under colonies grown on blood agar media, a phenotype which is routinely used as a diagnostic tool for GAS identification. Beta haemolysis is primarily dependent on SLS, with SLO making a minimal contribution

Streptolysin O

SLO is a thiol-activated, approximately 57 kDa cytolysin produced by group A, C, and G streptococci that is inhibited by small amounts of cholesterol. As a result of its oxygen-labile nature, SLO is most often responsible for haemolysis under the surface of blood agar, while the oxygen-stable SLS results in the zone of clearing surrounding colonies on the surface of blood agar. SLO is antigenic, resulting in anti-SLO antibodies that are useful for documenting recent exposure to GAS

Trypan blue

A defining feature of SLS-like peptides is the fact that they are completely inactivated by trypan blue, a vital stain that is usually used to selectively colour dead tissues, or cells, blue.

Heterocyclic compound

A compound that has atoms of at least two different elements within its ring stucture(s). With respect to bioorganic chemistry, heterocycles contain one or more carbon atoms and at least one ring member other than carbon.

SLS “carrier” molecules

The addition of certain high molecular weight “carrier” molecules, such as non-ionic detergents, albumin, α-lipoprotein, lipoteichoic acid and the RNAase-resistant fraction of yeast RNA (RNA core), can stabilize the haemolytic activity from a SLS-producing growing culture or resting cell suspension. SLS is irreversibly inactivated on separation from the carrier or on destruction of the carrier

Chromosome-walking

A technique used for identifying and characterising regions of DNA by the sequential isolation of overlapping sequences of DNA, starting with a known fragment of DNA.

Bacteriocin

A small, ribosomally synthesised, heat-stable peptide produced by one bacterium that is active against other bacteria, either in the same species (narrow spectrum), or across genera (broad spectrum).

Class I bacteriocins

Antimicrobial peptides that undergo extensive post-translational modification to yield their active form. Although this term is most frequently applied to the lantibiotic (i.e. lanthioine-containing) family of bacteriocins, it can be extended to include other post-translationally modified bacteriocins, including microcin B17.

Microcin B17

A ribosomally synthesized antibacterial peptide produced by strains of Escherichia coli that is active, after post-translational modification, against closely related bacterial species. The intracellular target of this natural product is the essential enzyme DNA gyrase.

Lowry-type strains of GAS

In 1971, James and McFarland reported the isolation of a completely non-haemolytic strain of GAS from an outbreak of rheumatic fever at Lowry Air Force Base, Colorado. As a result, these atypical strains are often referred to as “Lowry-type”

Intra- and Para-cellular Invasion

Translocation of pathogens across an epithelial barrier can occur via an intracellular route (within the host cells) or via a paracellular route (between the host cells).

Quorum Sensing

A mechanism of communication between bacteria that requires the production and secretion of a signalling molecule, which, when present at or above a critical threshold concentration, induces changes in gene expression in neighbouring cells.

Thiazole/Oxazole-ModifiedMicrocins (TOMMs)

Structurally and functionally diverse family of ribosomally produced peptides with post-translationally installed heterocycles derived from cysteine, serine and threonine residues. These modifications rigidify the precursor peptide to endow biological function on the mature natural product

Natural combinatorial biosynthesis

Natural combinatorial biosynthesis is a phenomenon whereby Nature's existing biosynthetic machinery has been repurposed by a particular organism for the production of a library of related compounds. In the case of some TOMM biosynthetic pathways, a single enzyme complex processes numerous substrates that have a common recognition motif, but variable structural C-termini. One striking example is the cyanobactin family of natural products.