Bacillus cereus phospholipases, enterotoxins, and other hemolysins

Bacillus cereus phospholipases, enterotoxins, and other hemolysins

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Toril Lindbäck and Per Einar Granum Norwegian University of Life Sciences, NMBU—School of Veterinary Science, Department of Food Safety and Infection Biology, Oslo, Norway

Introduction Bacillus cereus sensu lato is a group of seven Bacillus species, displaying a wideranging virulence spectrum. The group comprises Bacillus cereus sensu stricto, Bacillus thuringiensis, Bacillus anthracis, Bacillus weihenstephanensis, Bacillus mycoides, Bacillus pseudomycoides, and the most recent member Bacillus cytotoxicus [1]. B. anthracis, the causative agent of anthrax, is highly monomorphic, nonmobile, nonhemolytic and sensitive to penicillin. These phenotypic features are used to differentiate B. anthracis from the other members of the group [2]. Three different proteins are necessary for the pathogenic potential of B. anthracis: the edema factor (EF) and lethal factor [LF), and protective antigen (PA). The PA associates with one of the two other factors (EF or LF) to form the edema and the lethal toxins, respectively, leading to the specific syndrome of anthrax [3]. The genes encoding EF, LE, and PA are located on the large plasmids pXO1 and pXO2 and have probably been acquired through horizontal gene transfer [4,5]. B. cereus strains causing anthrax-like symptoms have been isolated [6,7], and the presence of pXO1 and pXO2 do not principally separate B. anthracis from other bacilli of the B. cereus group. However, B. anthracis carries a mutation in the global regulator PlcR; hence, the expression of the virulence factors and causes of disease described in this chapter are mainly restricted to B. cereus, B. thuringiensis, and B. cytotoxicus. The animal pathogen in the B. cereus group, B. thuringiensis, is active against the larvae of lepidopteran, dipteran, and coleopteran insects [8]. The major virulence factors of B. thuringiensis are the Cry delta-endotoxins. They are produced during sporulation and form protein crystals inside the sporangium, a feature restricted to this species [9]. Upon ingestion by the insect larvae, the crystal is dissolved in the insect intestine due to the high pH and the protoxin is subsequently activated by the insect proteases. The free toxins specifically recognize and lyse the epithelial cells of the insect, leading to a drop in pH, germination of the bacterial spores, and the development of septicemia, which is lethal for the larvae [8]. The Comprehensive Sourcebook of Bacterial Protein Toxins. DOI: http://dx.doi.org/10.1016/B978-0-12-800188-2.00029-X © 2015 Elsevier Ltd. All rights reserved.

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B. weihenstephanensis is distinguished from the other B. cereus group members by its psychrotolerant character, the genetic specificity of its 16S rDNA and by harboring the heat shock protein gene cspA [10]. Based on 16S rDNA sequence similarity and multilocus sequence typing (MLST) data, the newest of the species, B. cytotoxicus, shows a robust and well-separated cluster and constitutes the most distant cluster within the B. cereus group. It is also separated from the other species of the group by its thermotolerant growth profile [1]. The last two members of the B. cereus s.l. group are B. mycoides and B. pseudomycoides. These species, characterized by their rhizoid growth on agar plates, can be distinguished by their fatty acid composition [11]. So far, no pathogenic or opportunistic activities have been associated with the three latter bacteria, although many of them carry nhe, hbl, or cytK genes [12–14]. In spite of their variable virulence potential, members of the B. cereus s.l. share extremely close rDNA [15–17] and therefore could be considered as the same species [18]. Moreover, the genes coding for the entomopathogenic determinants of B. thuringiensis are on plasmids, which can be exchanged among the different B. cereus members [19]; e.g., transforming a B. cereus into a B. thuringiensis by plasmid acquisition [20]. However, for simplicity and for socioeconomical reasons, it is preferable to maintain this identification system in order to point out the specific virulence of each species. B. cereus sensu stricto, the archetype of the B. cereus s.l. group, is frequently linked to foodborne infections, with symptoms of gastroenteritis [21]. Foodstuffs potentially in contact with soil are particularly prone to B. cereus s.s. contamination: vegetables, fruits, milk, dehydrated milk, spices, and powdery products [21]. The symptoms caused by B. cereus s.s. are divided in two types: diarrheal and emetic syndromes. The diarrheal syndrome was first described in Norway in 1948 [21] as being the result of vanilla sauce contamination in a hospital, while the emetic syndrome was noted some 20 years later following rice intoxication in London [22]. However, the first case of food intoxication with acute gastroenteritis symptoms by B. cereus or a close bacterium was reported in 1906, when 300 people in a hospital became ill after the ingestion of meatballs in which aerobic spore-forming bacteria were found [22]. The diarrheal syndrome appears 8 to 16 h after ingestion of the contaminated food, with an infective dose of between 105 to 107 CFU. The symptoms consist of abdominal pain, diarrhea, and often nausea, and sometimes also vomiting, which can last from 12 to 24 h. For the emetic syndrome, the symptoms appear 0.5 to 5 h after ingestion and last from 6 to 24 h [23]. The disease is characterised by nausea, vomiting, malaise, and occasionally diarrhea [23]. The causative agent of emesis is the cereulide [24], a dodecadepsipeptide synthesized by a nonribosomal peptide synthetase (NRPS) [25]. It is a K+-ionophoretic channel, which is highly resistant to pH values between 2 and 11, to heat, and to proteolytic cleavage. Since 1997, four cases of children and young adults dying of cereulide intoxication after ingestion of reheated pasta or rice dishes have been reported [26–28]. Postmortem findings in these cases have revealed liver damage and brain edema, suggesting that cereulide can cause acute encephalopathy and liver failure [27,29]. Three commercial immunoassays are available for detection of B. cereus enterotoxins. The BCET-RPLA Toxin Detection Kit from Oxoid measures the presence of the L2 component of the HBL enterotoxin whereas the 3M Tecra Bacillus Diarrhoeal

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Enterotoxin Visual Immunoassay detects the NheA component. The new Duopath Cereus Enterotoxins from Merck KGaA/EMD Chemicals is an immunological screening and confirmation test for simultaneous detection of both Hbl and Nhe. No commercial kit is available for detection of CytK. However, since the toxins detected by these kits are also found in other B. cereus s.l. members, the detection specificity of these kits is not strictly limited to B. cereus s.s. Additional tests, therefore, are required for a more appropriate diagnostic [30]. B. cereus s.s. is also a potential contaminant in hospitals because of the resistance of its spores to heat, radiation, and certain disinfectants such as ethylic alcohol [31]. B. cereus has lately been recognized as an important opportunistic pathogen causing severe nosocomial infections in immunocompromised persons, and may occasionally cause periodontitis, endocarditis, septicemia, meningitis, or pneumonia [32]. These cases are rare, but the evolution of the disease is fulminating and often lethal. In addition, B. cereus may cause endophthalmitis, often with a catastrophic result [33]. In each of these cases, the toxins involved are not yet precisely known; however, the contribution of the Hbl enterotoxin, Hemolysin II, and Sphingomyelinase (SMase), have been evoked and will be discussed in this chapter.

Toxins of B. cereus s.l. In addition to the specific virulence factors of B. thuringiensis and B. anthracis, most B. cereus group members possess other virulence factors involved in the invasion and survival of the bacteria in the host. Among these factors, some, such as phospholipases, destroy the cell membrane by enzymatic activity while others, such as certain hemolysins and enterotoxins, lyse the cells by pore formation [34]. The virulence factors described in this section are the protein toxins expressed in both B. cereus and B. thuringiensis (Table 29.1), identified either by experimental evidence or by similarity to other known virulence factors.

Phospholipases There are three types of phospholipases with different specificity recognized in the B. cereus s.l. group. SMase cleaves sphingolipids and phosphatidylcholine phospholipase (PC-PLC) cleaves phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine [35]. In addition, phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves phosphatidylinositol and derived glycolipids [36]. Unlike most hemolysins, which act by pore formation, these phospholipases cause cell lysis by enzymatic activity. The enzymatic reaction involves hydrolysis between the glycerol and the lipidic head groups.

SMase Sphingomyelin consists of a phosphocholine head group, a sphingosine, and a fatty acid tail. It is one of the few membrane phospholipids that are not synthesized from

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Bacterial Protein Toxins Active on the Surface of Target Cells

glycerol. Sphingomyelin is involved in signal transduction, cellular proliferation, differentiation, and apoptosis [37,38] by hydrolyzing sphingomyelin in the plasma membrane to phosphocholine and ceramide. B. cereus SMase is structurally related to Staphylococcus aureus beta toxin, Clostridium perfringens alpha toxin and to SMase of the intracellular pathogen Listeria ivanovii [39,40]. SMase is a metal iondependent phospholipase and act at neutral pH. SMase (34 kDa) is known to be neither lethal nor cytotoxic, however, recent reports indicate that the contribution of SMase to B. cereus virulence may have been underestimated in the past [41,42]. B. cereus strains displaying SMase activity are able to grow in vivo in mice after intraperitoneal injection, in contrast to strains without SMase activity [42]. Oda et  al. [42] have shown that formation of ceramide in macrophages treated with SMase resulted in functional alterations of the membranes, eventually leading to inhibition of H2O2 production and phagocytosis induced by peptidoglycan. Thus, SMase may play a crucial role for B. cereus in dodging macrophages during early stages of infection. Doll et  al. [41] used epithelial cells and the in vivo Galleria larvae model to demonstrate that B. cereus SMase contributes, together with nonhemolytic enterotoxin (Nhe), to both in vitro cytotoxicity and in vivo pathogenicity.

PC-PLC B. cereus PC-PLC is a small, monomeric enzyme (28.5 kDa), with three Zn2+ atoms in the active site that are probably involved in the binding of substrate and essential for enzymatic activity and protein conformational stability [43]. The PC-PLC encoding gene lies upstream of that of SMase. This 245-aa Zn + 2 metalloprotein provides the bacteria with a lecithinase activity but not a hemolytic activity. However, SMase and PC-PLC can together form an effective hemolytic complex, named cereolysin AB (CerAB) [39]. B. cereus PC-PLC hydrolyzes phospholipids to give diacylglycerol and an alkyl phosphate in the following order of preference: phosphatidylcholine (PC) > phosphatidylethanolamine (PE) > phosphatidylserine (PS) [35]. In mammalian cells, the diacylglycerol, one of the products formed by PC-PLC action, acts as a second messenger in signal transduction cascade serving as an endogenous activator of protein kinase C [44]. The B. cereus PC-PLC mimics this function when applied to eukaryotic cells [45] and expression of this toxin in fibroblasts leads to oncogenic transformation of the cells [46,47]. In addition, it has been shown that PC-PLC contributes, together with HBL, to virulence of B. cereus endophthalmitis [48].

PI-PLC PI-PLC is a ubiquitous protein found in both eukaryotic and prokaryotic organisms. Indeed, in eukaryotes, degradation products of PI-PLC (diacyglycerol and inositol triphosphate) are signalling molecules, important in metabolic pathways [49] such as activation of protein kinase C or intracellular calcium mobilisation. The PI-PLC of B. cereus is frequently used as a model because it is smaller than its eukaryotic equivalent [50]. However, the bacterial form of the enzyme, containing only the catalytic lipase domain,

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produces cyclic intermediates exclusively, whereas the mammalian isoforms generate predominantly acyclic product. Both proteins act by a general acid-base mechanism, but the mammalian enzyme uses Ca2+, while the B. cereus PI-PLC does not use ions. PI-PLC enzymes catalyze the specific cleavage of the sn-3-phosphodiester bond in phosphatidylinositol (PI) and phosphoinositides [51]. PI hydrolysis occurs in two steps: the first produces diacylglycerol and water-soluble 1,2-cyclic inositol phosphate (cIP), and the second is a phosphodiesterase reaction where water-soluble cIP is hydrolyzed to inositol-1-phosphate. Physiologically, PI-PLC cleaves glycosylphosphatidylinositol (GPI)–anchored proteins on the outer leaflet of eukaryotic cell plasma membranes contributing to bacterial virulence [52–54]. The crystal structure of B. cereus PI-PLC was first solved by [55] in a complex with its substrate-like inhibitor myo-inositol to identify the active site. PI-PLC catalytic domains are members of the triosephosphate isomerase (TIM) barrel (α/β)8 superfamily [55,56]. The conserved active site is located at the C-terminal end of the β-strands in the barrel [55,57], but most of the residues that interact with membranes and control lipid binding are located in the less conserved and more mobile surface helices and loops. Little is known about the role of PI-PLC in Bacillus pathogenesis; however, it has been shown that B. anthracis PI-PLC may hamper the immune response in mice by downmodulating dendritic cell function and T cell response, possibly by cleaving GPI-anchored proteins important for toll-like-receptor-mediated activation of dendritic cells [58]. Finally, it should be noted that the PI-PLC gene is not grouped with the other two phospholipase genes and does not share a similarity with them [50].

Enterotoxins B. cereus produces at least three different cytotoxins that can be involved in food poisoning (Table 29.1). The three toxins are Hemolysin BL (HBL) [59–61], nonhemolytic enterotoxin (Nhe) [62,63], and cytotoxin K (CytK) [64]. A fourth putative toxin candidate, enterotoxin FM (EntFM), has been suggested [65]. EntFM is detected in B. cereus secretome, show sequence homology to cell-wall hydrolases and peptidoglycan-binding proteins in the Bacillus subtilis group, and are not potential members of the PlcR regulon [66]. No enterotoxin activity of EntFM has been shown. In addition, published data regarding the cytotoxicity of EntFM are limited [67], and more studies to confirm cytotoxic activity are needed. Substantial work has been carried out on the occurrence of the different enterotoxin genes in strains of B. cereus. Probably all strains (detectable in about 99%), contain nhe, 40%–60% contain hbl and about 40% contain cytK [21]. It was recently shown that the three different B. cereus enterotoxins exhibit distinct cytotoxicity to different human cell lines [75]. Vero cells and primary endothelial cells (HUVEC) are highly susceptible to Nhe, whereas Hep-G2, Vero, and A549 cells react most sensitive to Nhe plus HBL. For CytK, the highest toxicity is observed on CaCo-2 cells. Overall, Nhe and HBL account for more than 90% of the total toxicity of B. cereus [75]. Although HBL has been suggested to be a primary virulence factor in B. cereus

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Table 29.1 

Phospholipases, enterotoxins, and hemolysins from B. cereus

Toxins

Genetic organization

Comments

References

SMase

sph in the plcB–sph gene cluster, regulated by PlcR plcB in the plcB–sph gene cluster, regulated by PlcR plcA, regulated by PlcR hblCDA operon, encoding L2, L1 and B, respectively (ratio 1:1:1), regulated by PlcR

Component B of cereolysin AB

[68]

Component A of cereolysin AB

[35]

PC-PLC

PI-PLC Hbl

Nhe

CytK

nheABC operon, encoding NheABC, respectively (optimal ratio 10:10: 1; frequently much less NheC is produced), regulated by PlcR cytK, regulated by PlcR

Cereolysin O (CLO)/ Hemolysin I (Hly-I)

clo/hly-I, regulated by PlcR

Hemolysin II (Hly-II)

hly-II

Hemolysin III (Hly-III)

hly-III

Components bind independently and then constitute a membrane-attacking complex, resulting in lysis of erythrocytes Similarities with Hbl components Specific binding order of the components required for maximum toxicity Two forms exist: CytK-1 (B. cytotoxicus) and CytK-2, which both belong to the β-barrel PFT family. Belongs to the CBC family. Homologues are TLO and anthrolysin O (ALO). Belongs to the β-barrel PFT family

[50] [60,61,69]

[62,70]

[64,71]

[72]

[73] [74]

diarrhea [76], it should be stressed that about half of the food poisoning strains do not produce this enterotoxin [21]. A mixture of the three toxins described here has been shown not to survive the stomach acid and proteolytic enzymes in the duodenum [23,77]. Thus, it seems likely that only toxins produced and secreted in the small intestine during vegetative growth are able to cause diarrhea, and this is supported by an incubation time of more than 8 h [78].

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Figure 29.1  Organization of the genes encoding the components of the three enterotoxins HBL (B. cereus ATCC 14579), Nhe (B. cereus NVH 0075-95), and CytK (B. cytotoxicus NVH 391-98). The name of the genes, protein components, and size of the components (both with and without signal peptide) are indicated. The stem loop–type structure indicates the inverted repeat between nheB and nheC that is probably involved in the regulation of expression of NheC. Putative promoters, direction of transcription, and PlcR binding sites are indicated.

Hemolysin BL (HBL) HBL is one of the two tripartite enterotoxins produced by B. cereus, consisting of the B, L1, and L2 components [69,79]. HBL is shown to be dermonecrotic, exhibit vascular permeability activities, and can cause fluid accumulation in ligated rabbit ileal loops [76]. In addition, HBL is probably the most important endophthalmitis virulence factor because of its high toxicity toward retinal tissue both in vitro and in vivo [80]. All three HBL components are necessary for maximal enterotoxin activity, and a 1:1:1 ratio of the three components seems to give maximum biological activity [76], and two-dimensional (2D) protein-gel studies have confirmed that the three proteins are expressed in a 1:1:1 ratio [81]. It was first suggested that HBL-B was the binding component, and that HBL-L1 and HBL-L2 exhibited lytic functions [59]. However, it was later shown that the three components bind to target cells independently and then constitute a membrane-attacking complex, resulting in a colloid osmotic lysis mechanism [69]. The organization of the hbl operon is given in Figure 29.1. The first to be sequenced was hblA, encoding the B-component [60], while hblC and hblD encoding the components L2 and L1, respectively, were sequenced some years later [61]. Until 2010, hblB (Figure 29.1) was considered a pseudogene (similar to hblA), however, Clair et al. [66] demonstrated that hblB is transcribed, translated, and exported at detectable levels in the early secretome of B. cereus. The hblB is transcribed as a monocistronic gene independent of the PlcR-regulated hblCDA polycistron [81], and whether the hblB gene product is actually involved in cytotoxicity has yet to be determined. The X-ray crystal structure of HBL-B was solved in 2008, and the structure implies that it may form pores similar to other soluble channel forming proteins [82]. The

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Bacterial Protein Toxins Active on the Surface of Target Cells

Figure 29.2  Superposition of the structures of HBL-B and NheA. The tail and the head domains are indicated.

structure of HBL-B is predominantly α-helical, consisting of two subdomains, the tail domain made up of a long helical bundle and a small α/β head domain (Figure 29.2). The head domain is packed against the five long helices of the tail domain, forming an elongated shaped helical bundle molecule reminiscent to other water-soluble, channel-forming proteins, such as colicin, aerolysin, and the translocation domain of botulinum neurotoxin [82]. Despite low sequence homology, HBL-B shows strong structural similarity to the single-component hemolytic enterotoxins HlyE, ClyA, and SheA of Escherichia coli, Salmonella enterica, and Shigella flexneri, respectively [82], and to NheA of B. cereus [83].

The nonhemolytic enterotoxin (Nhe) A nonhemolytic three-component enterotoxin (Nhe) was characterized after a foodpoisoning outbreak involving 152 people caused by an hbl-negative B. cereus strain [63]. Nhe is different from HBL; however, they are genetically closely related. Although there are structural and sequence similarities between HBL and Nhe (Table 29.2), components from one complex cannot substitute for components from the other one [84,85]. The most pronounced similarities are found between NheB and NheC, HBL-L1 and NheB, and HBL-L1 and NheC [86]. A combination of the NheA and NheB components possesses some biological activity about 5% of maximal), but the third component, NheC, is necessary for optimal cytotoxicity [84,87]. Under natural conditions, NheB and NheC form a complex that seem to be necessary to induce cytotoxic activity, and to reach maximum cytotoxicity, the binding of the components follows a specific order on the cell membrane

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Table 29.2 

Percentage identity between the Nhe and HBL components from B. cereus ATCC 14579 Hbl L2 Hbl L1 Hbl B NheA NheB

Hbl L1

Hbl B

NheA

NheB

NheC

18%

23% 25%

23% 18% 20%

21% 40% 27% 22%

19% 32% 25% 22% 44%

Source: [87].

[70]. Due to the complex formation with NheB in solution, free NheC have never been detected in natural culture supernatants of B. cereus strains [66,70,81,87]. The optimal ratio between the NheA, NheB, and NheC components appear to be about 10:10:1, respectively [87]. A ratio lower than 5:1 and higher than 50:1 between NheB and NheC strongly reduces cytotoxicity [70,86]. The complex formation between NheB and NheC is directly correlated with the relative concentrations. An equimolar ratio between NheB and NheC or excess of NheC gives maximum complex formation, and no free NheB will be able to bind to Vero cells. These data indicate that a defined level of NheB-NheC complexes, as well as a sufficient amount of free NheB, are necessary for efficient cell binding and toxicity. The intergenic region between nheB and nheC in most B. cereus strains contains an inverted repeat causing a secondary structure that probably ensures a decreased translation of nheC mRNA (Figure 29.1). The X-ray crystal structure of NheA was solved in 2012 [83,88]. The NheA structure is folded similar to HBL-B (Figure 29.2) and E. coli Cly, and is therefore included in the ClyA superfamily of α-helical PFTs (α-PFTs), although its head domain is significantly enlarged compared with those of ClyA or HBL-B. In contrast to the characteristic hydrophobic β-hairpin structure of HBL-B and ClyA, NheA has an amphipathic β-hairpin connected to the main structure via a β-latch that is reminiscent of a similar structure in the β-PFT S. aureus α-hemolysin, and it has been suggested that NheA may form a β rather than an α pore [83].

Cytotoxin K (CytK) The latest discovered enterotoxin cytotoxin K (CytK) was the cause of the symptoms in a severe outbreak of B. cereus foodborne illness in France in 1998 [64]. In this outbreak, several people developed bloody diarrhea, and there were three fatalities. It would be fair to call this an outbreak of B. cereus necrotic enteritis, although it is not nearly as severe as the C. perfringens type C food poisoning [89]. In 2013, the strain causing the French outbreak (NVH 391-98) and four other similar strains were validated as members of a novel species of the B. cereus group, Bacillus cytotoxicus sp. nov. [1]. CytK (CytK-1) produced by these five strains are more effective in lysing erythrocytes than B. cereus produced CytK (CytK-2) [71]. It has been shown that in B. cereus type strain ATCC 14579, only a small subpopulation is responsible for CytK

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(CytK-2) production in a homogeneous monoculture [90]. Whether this phenomenon is restricted to expression of CytK, the B. cereus type strain, or both has yet to be determined. Several proteins have been described as putative virulence factors in B. cereus, and in 2000, Beecher et al. [48] partially characterized a novel hemolysin designated Hemolysin IV. This toxin appeared to be one of the most rapidly acting or one of the most abundant hemolysins in crude culture supernatants from B. cereus. Amino acid sequencing showed that 28 of 30 amino acids in the N-terminal region were identical to those of CytK, and Hemolysin IV and CytK are probably the same protein [48,64]. The amino acid sequence of CytK is homologous (about 30% identity) to that of α-hemolysin, leucocidins, and γ-hemolysin from S. aureus and β-toxin from C. perfringens, which all belong to the family of β-barrel pore-forming toxins (PFTs) [91–93]. The ability to form pores in planar bilayers [94] is consistent with CytK being a member of this family of pore-forming proteins. CytK has been shown to be weakly anion selective and to exhibit an open channel probability close to 1 [94]. The predicted minimum pore diameter is approximately 7 Å. CytK, like other β-barrel PFTs, spontaneously forms oligomers (probably heptamers), which are resistant to SDS, but not to boiling [94]. These oligomers are able to form pores in planar bilayers, and to lyse erythrocytes, but show no cytotoxicity toward epithelial cells [71].

Hemolysins Cereolysin AB (CerAB) The phospholipases PC-PLC and SMase can act together to induce hemolytic activity e.g., against human erythrocytes [39]. This complex is named cereolysin AB.

Cereolysin O/Hemolysin I (CLO/Hly-I) Cereolysin O [CLO, named thuringiolysin O (TLO) in B. thuringiensis and anthrolysin O (ALO) in B. anthracis] [95] is a member of the cholesterol-binding cytolysins (CBC). CLO consists of a single polypeptide chain with a molecular weight of 52 kDa [96]. The CBC group comprises cytolysins of several pathogens such as Streptococcus pyogenes (streptolysin O, or SLO), C. perfringens (perfringolysin O, or PFO), and Listeria monocytogenes (listeriolysin O, or LLO). The relatively high aa sequence homology between members of this family suggest that they all have similar activities and 3D structures. In addition, the toxins contain a highly conserved tryptophan-rich sequence (ECTGLAWEWWR) of 11 residues close to the C-terminus, which participates in the binding of some CDCs to cholesterol-rich membranes [97]. As the name indicates, CBC bind to cholesterol at the cell surface [72] and disrupt the cell membrane by formation of large pores, up to 150 Å, which is about 50 monomeres [98]. Purified CLO exhibits a LD50 in mice of about 1 μg, and is hemolytic at concentrations as low as 1 ng/mL [96], indicating that CLO contributes significantly to the B. cereus virulence potential. Distribution on the CLO encoding gene is ubiquitous in all B. cereus s.l. members. The gene seems to be highly conserved within the B. cereus group (more than 95% of

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identity) and does not differentiate the species; however, B. cytotoxicus NVH 391-98 do not carry the CLO encoding gene. Transcription of clo (and tlo in B. thuringiensis) is under the control of PlcR. Here, clo is coexpressed with a small PlcR-regulated multicopy gene, suggesting an unknown relationship between this peptide and the cholesterol-dependent cytolysin in bacteria belonging to the B.cereus group [99]. Deletion of the ALO encoding gene in B. anthracis, together with those of the three B. anthracis phospholipases, resulted in a clear attenuation of bacterial virulence, growth, and survival in the phagocytes [100]. Therefore, it has been suggested that ALO can be one of the tools used by B. anthracis to destabilize and break the phagosomal membrane [101]. The alo gene is preceded by the PlcR binding sequence [95]; however, the plcR gene of B. anthracis encodes a nonfunctional protein and the regulatory mechanism promoting ALO expression is still unknown.

Hemolysin II (Hly-II) The 42-kDa Hly-II protein displays about 30% identity to a toxin family called β-barrel PFTs, comprising the α-hemolysin of S. aureus or CytK of B. cereus (3.2) [73,102]. This family of heptameric toxins is characterized by two antiparallel transmembrane and glycin-rich strands [103,104]. Each monomer inserts a glycine-rich segment into the membrane to form a transmembrane pore, and since Hly-II also contains a glycine-rich segment, a similar mechanism is suggested for this toxin. Hly-II has been shown to form anion-selective channels with an inner pore diameter of 1.5–2 nm in liposomes [105]. Hly-II possesses 94 additional amino acids at the C-terminus, in comparison to other members of the toxin family. These residues do not seem to be essential to the activity and the addition of this tail to the other toxins of the family does not cause problems. However, the specific activity of hemolysin II seems higher than for other β-barrel PFTs [74,104,105]. Hly-II displays an important Arrhenius effect (regain of activity after a short increase of temperature up to 90–100°C), and a long lag period before appearance of hemolysis in the activity tests with blood containing media [102]. Distribution studies of Hly-II suggest that the toxin is most frequently found in B. thuringiensis and its distribution is limited among B. cereus strains [106]. Hly-II is not regulated by the pleiotropic transcriptional regulator PlcR but is controlled by two negative regulators, HlyIIR and Fur [107,108]. Glucose 6P binds and activates HlyIIR (Guillemet et al., 2013), while iron binds and activates Fur [108], to repress B. cereus hemolysin hlyII gene expression. Therefore, so long as glucose and iron is abundant in the bacterial environment, hlyII will not be expressed. Hly-II is unlikely to contribute to food poisoning since there is a lysine residue at the end of the β-barrel loop penetrating the cell membrane. This will readily be cut by trypsin in the small intestine (if not inhibited by trypsin inhibitors or low trypsin secretion), and the toxin will be inactivated such as the C. perfringens β − toxin [71]. However, Hly-II has been shown to provoke macrophage cell death by apoptosis through its pore-forming activity [109]. Hly-II is expressed in a high amount in clinical isolates of human origin [110] and has been suggested to play an important role in opportunistic infections.

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Hemolysin III (Hly-III) Hemolysin III is the least characterized hemolytic toxin from the B. cereus group. The hly-III encoding gene has been cloned and characterized in E. coli [111]. The toxin (24 kDa) acts by oligomeric pore formation in three steps: the protein first binds to the erythrocyte surface, and monomers are then assembled to form the transmembrane pore, leading to erythrocyte lysis. While the first two steps are temperature dependent, the final lysis is not [74]. Hly-III of B. cereus shows 47% aa sequence homology to Hly-III of the highly virulent marine bacterium Vibrio vulnificus, the causative agent of serious wound infections and fatal septicemia. When Hly-III of V. vulnificus was expressed in E. coli, crude extracts exhibited hemolytic activity similar to that of hemolysin III from B. cereus [112]. A hly-III deficient mutant showed an attenuated virulence when injected intraperitoneally in mice. The role of B. cereus Hly-III in virulence has not been investigated in vivo and remains a matter of speculation.

Regulation of transcription and secretion of toxins Transcription of nearly all protein toxins encoding genes in the B. cereus group, except for B. anthracis, is regulated by the quorum-sensing regulator PlcR [113]. PlcR is a master transcriptional regulator that activates the transcription of 45 genes, including important virulence factors, such as Nhe, HBL, CytK, and hemolysins [81,114]. PlcR activates transcription of its target genes by binding to a pseudopalindromic consensus sequence, the PlcR box, defined as wTATGnAwwwwTnCATAw [81,114]. The PlcR box is located at various distances upstream of the transcriptional start site [114], and the sequence may show some divergence without drastically affecting the binding of PlcR, as is the case for cytK [99], and for inhA2 encoding the InhA2 metalloprotease [115]. Inactivation of the plcR gene causes drastic reduction of the pathogenic potential of B. cereus and B. thuringiensis, both in mice and insects [116]. Directly downstream of the plcR gene is a short open reading frame (ORF) encoding a 48-amino-acid peptide. This peptide, PapR for peptide activating PlcR, is positively regulated by PlcR itself. The 48-aa PapR peptide is secreted by the bacteria and then extracellularly cleaved into a heptapeptide that is reimported into the cell by an oligopeptide permeation system (Opp A, B, C, D, and E) essential for PlcR regulation [117,118]. PlcR is a helix-turn-helix (HTH)–type transcription factor, and the X-ray crystal structure of the apoform of PlcR from B. thuringiensis strain 407 has recently been solved [119]. Apo PlcR is an obligate biological dimer, and during PlcR activation, apo PlcR binds PapR, which induces several small molecular reorientations. These reorientations result in significant structural changes allowing proper positioning of the HTH domains in the major groove of the two half-sites of the pseudopalindromic PlcR-box [119]. During exponential growth, low concentrations of PapR do not allow the expression of PlcR regulons, while at the start of the stationary phase, the increase in extracellular PapR leads to activation of PlcR [113,117,118]. An opposite regulation

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mechanism involves Spo0A, the key regulator for sporulation. Two Spo0A-binding sites of are found upstream of the plcR gene. Under sporulation conditions, Spo0A is activated and binds to DNA, inhibiting the transcription of plcR. So, under poor conditions, PlcR expression is blocked and virulence genes are not expressed. In a rich medium, the transitory phase is long and the expression of PlcR is effective during this period, before the activation of Spo0A [120]. The activation mechanism of PlcR by PapR has been shown to be strain specific, and four classes of PlcR-PapR pairs, defining four distinct phenotypes in the B. cereus group, have been identified [118,121]. A recent microarray study on B. licheniformis biofilm revealed that SinR, a member of the transition state regulator family, controls HBL expression, together with PlcR [122]. HBL was expressed only in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses HBL. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. Since B. thuringiensis and B. cereus are enteric bacteria that possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms, therefore, could be delivered directly to the target tissue [122]. B. anthracis possesses an arsenal of virulence genes similar to those found in the other members of the B. cereus group. Yet, B. anthracis has a truncated a plcR gene [114]. So, the PlcR regulon is not expressed but can be reactivated by incorporation of active PlcR [123–125]. Although B. anthracis is considered nonhemolytic, it has been reported that growth in a rich medium [95] or growth under strictly anaerobic conditions [126] leads to hemolysis caused by ALO; therefore, a plcR papR–independent expression of ALO has been suggested [127]. Sec-type signal peptides are identified in all toxin components produced by B. cereus, and HBL, Nhe, and CytK appear to be secreted using the Sec pathway [128]. Lack of flagella and reduced toxin secretion in a flagellar export apparatus (FEA)–deficient strain implied that HBL was secreted using the FEA, despite the presence of Sectype signal peptides [129]. However, the concurrent lack of flagella and reduced toxin secretion in the FEA deficient strain may point toward the presence of a regulatory link between motility and virulence genes rather than FEA-dependent toxin secretion.

Conclusion The species belonging to the B. cereus s.l. group are genetically closely related (their 16S rRNA are almost identical), and they display a broad spectrum of virulence factors. The contribution of each particular virulence factor is not well understood. The main contributors to gastrointestinal disease, the three enterotoxins (HBL, Nhe and CytK), are probably the most explored B. cererus toxins. However, recent research has revealed a much wider spectrum of B. cereus virulence potential. SMase seem to be an important factor for B. cereus virulence both in vitro and in vivo, and SMase and Nhe seem to display a concerted action together regarding hemolytic and cytotoxic activity. SMase plays a crucial role in the evasion from macrophage response during the early stages of infections of B. cereus by preventing phagocytosis. Hly-II has been

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shown to provoke macrophage cell death by apoptosis through its pore-forming activity, and it may be of special importance in opportunistic infections. B. thuringiensis is the most important biopesticide used for several decades in the control of insect pests or vector of human and animal diseases. B. thuringiensis harbors and expresses many of the genes encoding the virulence factors described here; however, whether this bacterium is a potential threat human health is still debated. B. anthracis represents another important issue because of the severity of the disease it causes. The inactivation of its transcriptional regulator PlcR is now well documented, and alternative regulatory pathways have been suggested. Recent studies indicate that a Bacillus species other than B. anthracis can cause anthrax-like disease, and plasmid analogs to the B. anthracis virulence plasmids pXO1 and pXO2 have been isolated from B. cereus. These strains give rise to a borderline group between B. anthracis and B. cereus.

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