Role of Sphingomyelinase in the Pathogenesis of Bacillus cereus Infection

Abstract

Bacillus cereus is well known as a causative agent of food poisoning but it also causes bacteremia and endophthalmitis in nosocomial infections. However, as an environmental bacterium that lives in soil, it is often treated as simple contamination by hospitals. In recent years, highly pathogenic B. cereus strains that are similar to Bacillus anthracis have been detected in hospitals. The B. cereus sphingomyelinase contributes to its pathogenicity, as do sphingomyelinases produced by Staphylococcus aureus, Pseudomonas aeruginosa, Helicobacter pylori, and B. anthracis. Highly pathogenic B. cereus produces a large amount of sphingomyelinase. In this review, we describe the regulation of sphingomyelinase expression through the PlcR–PapR system, the pathogenicity of bacterial sphingomyelinases, and their potential as therapeutic drug targets.

1. INTRODUCTION

Bacillus cereus is a spore-forming, Gram-positive bacterium found in soil and plants, which in humans causes food poisoning and serious nongastrointestinal tract infections.¹⁾ It is an opportunistic pathogen that causes endophthalmitis, pneumonia, and septicemia. In nongastrointestinal tract infections, B. cereus is often spread via lines and catheters.^2,3)

B. cereus sphingomyelinase (SMase) exhibits both hemolytic and phospholipase C (PLC) activity, hydrolyzing sphingomyelin to phosphocholine and ceramide. Ceramide is an important molecule that is involved in various cellular processes, such as stress responses, growth, and apoptosis.⁴⁾ SMases produced by various bacteria, such as Listeria ivanovii,⁵⁾Staphylococcus aureus,^6,7)Bacillus anthracis,⁸⁾Helicobacter pylori,^9,10)Pseudomonas aeruginosa,¹¹⁾ and B. cereus,^12,13) are associated with both local and systemic infections. These enzymes generally have molecular weights of 27–39 kDa, with the exception of Pseudomonas and Leptospira SMases,^11,14) and their structures are similar to those of mammalian deoxyribonuclease 1 and mammalian neutral SMase, based on the structures of several bacterial SMases (B. cereus Protein Data Bank (PDB) ID: 2DDT, S. aureus beta-toxin PDB ID: 315 V, L. ivanovii PDB ID: 1ZWX, and Streptomyces griseocarneus PDB ID: 3WCX).^15–17)

B. cereus SMase enzymatic activity is dependent on the presence of a divalent metal ion^18–20); it is enhanced by some, e.g., Mg²⁺, but attenuated by others, e.g., Ca²⁺.^15,21–23) In terms of metal ion activation, the order is Co²⁺≥Mn²⁺≥Mg²⁺≫Ca²⁺≥Sr²⁺.¹⁵⁾ Mutagenesis studies showed that glutamic acid (Glu)-53 is essential for binding Mg²⁺.^22,24) The crystal structure revealed that water-bridged double-divalent metal ions at the center of the catalytic cleft in both Mg²⁺-bound forms were required for SMase activity.¹⁵⁾ In contrast, Ca²⁺ bound only one site.¹⁵⁾ A beta-hairpin with aromatic amino acid residues participates in the binding of its substrate, membrane-bound sphingomyelin.¹⁵⁾

2. REGULATION OF SMASE GENE EXPRESSION BY PLCR

B. cereus secretes various toxins, such as cereulide, hemolysin BL, nonhemolytic enterotoxin, cytokine K, phosphatidylinositol–PLC (PI–PLC), phosphatidylcholine–PLC (PC–PLC), and SMase. The levels of many of these toxins are controlled by the transcription factor PlcR, and plcR deletion significantly reduces B. cereus pathogenicity.^25,26) PlcR, a pleiotropic regulator of extracellular virulence factors, is active in B. cereus, Bacillus thuringenesis, and B. anthracis. The plcR gene encodes a 34-kDa protein that activates the transcription of at least 15 genes encoding secreted proteins, including phospholipases, proteases, and enterotoxins.^27,28) A BLAST search of B. cereus PlcR sequences revealed that its N-terminal portion is homologous to HTH XRE-family transcriptional regulators.²⁹⁾ PlcR secondary structure analysis revealed a DNA-binding domain from residues 14 to 45, which contains an HTH motif, with two alpha helices connected by a two-residue beta sheet.^29–31)

PlcR activates the transcription of its target genes by binding to a consensus sequence defined as TAT GnAnnnnTnCAT A.^25,32) PlcR activity is controlled by the 48-amino acid signalling polypeptide PapR.³³⁾ PapR is secreted and then reimported through the oligopeptide permease system.³⁴⁾ The papR gene belongs to the PlcR regulon and is located 70 bp downstream from plcR, and at least four classes of PlcR–PapR pairs were reported^33,35) (Table 1). The crystal structure of a group 1 PlcR in complex with the pentapeptude PapR₅ (LPFEF) was published in 2007.³⁶⁾ Three-dimensional analysis suggests that PlcR–PapR complexes bind PlcR-boxes on the DNA.³¹⁾ Bouillaut et al. reported that the active form of PapR in culture medium was 7 amino acids (PapR₇) as shown by LC-MS/MS and matrix assisted laser desorption/ionization (MALDI)-MS/MS.³⁷⁾ The expression levels of 45 genes, including three phospholipases (plcA, plcB, and smase), are controlled by the PlcR–PapR system (Fig. 1).

Table 1. PlcR–PapR Group

Group	1	2	3	4
PapR	LPFE(F/Y)	MPFEF	VP(F/Y)E(F/Y)	LPFEH

Fig. 1. Transcription Regulation by PlcR–PapR

(Color figure can be accessed in the online version.)

3. PATHOGENICITY OF B. CEREUS SMASE

B. cereus SMase enhances bacterial growth in the peritoneal cavities of mice, resulting in death. In addition, overexpression of B. cereus SMase in Bacillus subtilis, an avirulent bacteria, induces its growth in mice. SMase nonproducing B. cereus (ATC C21928) has no effect on mouse death. The administration of SMase nonproducing B. cereus with exogenous SMase to mice resulted in death, while administering PI–PLC and PC–PLC with the bacteria did not.¹²⁾ The administration of anti-B. cereus SMase antibodies or immunization with B. cereus SMase protected mice from the lethality induced by high-toxicity B. cereus from clinical isolates.¹²⁾ Furthermore, the SMase C inhibitor SMY-540 significantly reduced the lethality of infection with a B. cereus SMase-producing strain.^12,13) In addition, B. cereus SMase decreases the fluidity of the macrophage cell membrane and impairs phagocytic function.¹²⁾ Callegan et al. reported that intraocular infection with wild-type B. cereus or isogenic mutants specifically deficient in PI–PLC or PC–PLC resulted in similar degrees of retinal architecture destruction and a complete loss of retinal function.^38,39) L. ivanovii SMase disrupts the phagosome membrane and escapes into the cytosol, where it promotes intracellular survival and replication.⁴⁰⁾ A SMase-knockout strain of L. ivanovii had impaired capacity for intracelluar proliferation and was less virulent in mice than the wild-type strain.⁴⁰⁾

S. aureus hlb encodes a SMase C also known as beta-toxin, which is expressed in large quantities in clinical isolates and induces bovine mastitis.^41,42) Beta-toxin is cytotoxic to human keratinocytes and could thus promote skin colonization.⁴³⁾ It also inhibits interleukin-8 expression by endothelial cells, which decreases neutrophil transendothelial migration.⁴⁴⁾ Furthermore, beta-toxin is cytotoxic to polymorphonuclear leukocytes and proliferating T lymphocytes, contributes to the phagosomal escape of S. aureus,⁴⁵⁾ and induces biofilm formation.⁶⁾ The hemolytic and lymphotoxic activities of beta-toxin are linked to its SMase C activity, but its capacity to induce biofilm formation is independent of its catalytic activity.⁶⁾ S. aureus mutant strains lacking hlb have reduced virulence in murine models of pneumonia and skin infection,^43,46) and a mutant strain expressing biofilm formation-deficient beta-toxin had diminished pathogenicity in a rabbit model of endocarditis.⁶⁾ Purified beta-toxin is cytotoxic to bovine mammary epithelial cells, and nonbeta-toxin-producing strains had reduced virulence in a mouse model of intramammary infection.⁴⁷⁾ Beta-toxin is also required for S. aureus supernatants to kill silkworm larvae.⁴⁸⁾

Mycobacterium tuberculosis is a facultative intracellular pathogen and the most common etiological agent of tuberculosis, a chronic infection that typically becomes active in immunosuppressed patients, destroying their lungs and sometimes other tissues.⁴⁹⁾M. tuberculosis SMase is an important hemolytic determinant, enabling it to use sphingomyelin for nutrients.⁴⁹⁾ Studies on B. cereus SMase-induced hemolysis revealed that sheep erythrocytes treated with B. cereus SMase had rigid (ceramide-rich) and fluid (ceramide-poor) domains. Eventually, the coalescence of ceramide-rich domains and formation of an interface between rigid and fluid domains resulted in the disruption of erythrocyte membranes.^50–54) As described above, bacterial SMase exhibits various actions depending on sphingomyelin metabolism (Fig. 2).

Fig. 2. Role of Bacterial Sphingomyelinase

(Color figure can be accessed in the online version.)

4. CLINICAL ISOLATES OF B. CEREUS

Multilocus sequence typing of B. cereus clinical isolates in Japan (Tokyo, Tochigi, Tottori, and Kochi) revealed that B. cereus ST1420, a novel strain, was the major strain detected in nosocomial infections and bacteremia cases in different hospitals over different years.⁵⁵⁾ ST1420 was classified into the Cereus III lineage, which is more closely related to the Anthracis lineage than to the Cereus I and II lineages. Additionally, most strains isolated from patients with bacteremia belonged to the Cereus III lineage.⁵⁵⁾ The Zambian B. cereus strains LZ77-2 and LZ78-8, which are highly pathogenic, are classified in the Anthracis lineage.⁵⁶⁾ It is suggested that highly pathogenic bacteria are spreading throughout the world. B. cereus clinical isolates produce significant amounts of SMase, and in mice invade the bloodstream after intraperitoneal injection and cause death, which can be prevented by specific antibodies against SMase.¹²⁾ B. cereus SMase plays a crucial role in avoiding the host innate immune response during the early stages of infection.

5. CONCLUSION

Increasing evidence indicates that the transcription of B. cereus SMase is regulated by the PlcR–PapR system. However, the relationship between SMase production and PlcR–PapR remains unclear.

B. cereus strains with genetic characteristics similar to B. anthracis are being increasingly detected in hospital settings. These highly pathogenic strains produce many secretory toxins, including SMase. As SMase antibodies and inhibitors are effective against infections caused by high SMase-producing B. cereus strains in mice, the development of therapeutic methods to target SMase in humans, including inhibitors and vaccines, may be effective in treating infections with pathogenic bacteria that produce SMase. In addition, the evidence suggests that bacterial SMase levels are important factors in bacterial pathogenicity and could be used to measure infection severity.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin. Microbiol. Rev., 23, 382–398 (2010).
• 2) Yamada K, Shigemi H, Suzuki K, Yasutomi M, Iwasaki H, Ohshima Y. Successful management of a Bacillus cereus catheter-related bloodstream infection outbreak in the pediatric ward of our facility. J. Infect. Chemother., 25, 873–879 (2019).
• 3) Sasahara T, Hayashi S, Morisawa Y, Sakihama T, Yoshimura A, Hirai Y. Bacillus cereus bacteremia outbreak due to contaminated hospital linens. Eur. J. Clin. Microbiol. Infect. Dis., 30, 219–226 (2011).
• 4) Hannun YA, Obeid LM. The Ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J. Biol. Chem., 277, 25847–25850 (2002).
• 5) Gonzalez-Zorn B, Dominguez-Bernal G, Suarez M, Ripio MT, Vega Y, Novella S, Vazquez-Boland JA. The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole. Mol. Microbiol., 33, 510–523 (1999).
• 6) Huseby MJ, Kruse AC, Digre J, Kohler PL, Vocke JA, Mann EE, Bayles KW, Bohach GA, Schlievert PM, Ohlendorf DH, Earhart CA. Beta toxin catalyzes formation of nucleoprotein matrix in staphylococcal biofilms. Proc. Natl. Acad. Sci. U.S.A., 107, 14407–14412 (2010).
• 7) Boe DM, Curtis BJ, Chen MM, Ippolito JA, Kovacs EJ. Extracellular traps and macrophages: new roles for the versatile phagocyte. J. Leukoc. Biol., 97, 1023–1035 (2015).
• 8) Heffernan BJ, Thomason B, Herring-Palmer A, Shaughnessy L, McDonald R, Fisher N, Huffnagle GB, Hanna P. Bacillus anthracis phospholipases C facilitate macrophage-associated growth and contribute to virulence in a murine model of inhalation anthrax. Infect. Immun., 74, 3756–3764 (2006).
• 9) Chan EC, Chang CC, Li YS, Chang CA, Chiou CC, Wu TZ. Purification and characterization of neutral sphingomyelinase from Helicobacter pylori. Biochemistry, 39, 4838–4845 (2000).
• 10) Tseng HJ, Chan CC, Chan EC. Sphingomyelinase of Helicobacter pylori-induced cytotoxicity in AGS gastric epithelial cells via activation of JNK kinase. Biochem. Biophys. Res. Commun., 314, 513–518 (2004).
• 11) Montes LR, Ibarguren M, Goni FM, Stonehouse M, Vasil ML, Alonso A. Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR(2), a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa. Biochim. Biophys. Acta, 1768, 2365–2372 (2007).
• 12) Oda M, Hashimoto M, Takahashi M, Ohmae Y, Seike S, Kato R, Fujita A, Tsuge H, Nagahama M, Ochi S, Sasahara T, Hayashi S, Hirai Y, Sakurai J. Role of sphingomyelinase in infectious diseases caused by Bacillus cereus. PLOS ONE, 7, e38054 (2012).
• 13) Oda M, Imagawa H, Kato R, Yabiku K, Yoshikawa T, Takemoto T, Takahashi H, Yamamoto H, Nishizawa M, Sakurai J, Nagahama M. Novel inhibitor of bacterial sphingomyelinase, SMY-540, developed based on three-dimensional structure analysis. J. Enzyme Inhib. Med. Chem., 29, 303–310 (2014).
• 14) del Real G, Segers RP, van der Zeijst BA, Gaastra W. Cloning of a hemolysin gene from Leptospira interrogans serovar hardjo. Infect. Immun., 57, 2588–2590 (1989).
• 15) Ago H, Oda M, Takahashi M, Tsuge H, Ochi S, Katunuma N, Miyano M, Sakurai J. Structural basis of the sphingomyelin phosphodiesterase activity in neutral sphingomyelinase from Bacillus cereus. J. Biol. Chem., 281, 16157–16167 (2006).
• 16) Openshaw AE, Race PR, Monzo HJ, Vazquez-Boland JA, Banfield MJ. Crystal structure of SmcL, a bacterial neutral sphingomyelinase C from Listeria. J. Biol. Chem., 280, 35011–35017 (2005).
• 17) Clarke CJ, Truong TG, Hannun YA. Role for neutral sphingomyelinase-2 in tumor necrosis factor alpha-stimulated expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM) in lung epithelial cells: p38 MAPK is an upstream regulator of nSMase2. J. Biol. Chem., 282, 1384–1396 (2007).
• 18) Ikezawa H, Mori M, Ohyabu T, Taguchi R. Studies on sphingomyelinase of Bacillus cereus. I. Purification and properties. Biochim. Biophys. Acta, 528, 247–256 (1978).
• 19) Ikezawa H, Mori M, Taguchi R. Studies on sphingomyelinase of Bacillus cereus: hydrolytic and hemolytic actions on erythrocyte membranes. Arch. Biochem. Biophys., 199, 572–578 (1980).
• 20) Ikezawa H, Matsushita M, Tomita M, Taguchi R. Effects of metal ions on sphingomyelinase activity of Bacillus cereus. Arch. Biochem. Biophys., 249, 588–595 (1986).
• 21) Tomita M, Taguchi R, Ikezawa H. The action of sphingomyelinase of Bacillus cereus on bovine erythrocyte membrane and liposomes. Specific adsorption onto these membranes. J. Biochem., 93, 1221–1230 (1983).
• 22) Obama T, Kan Y, Ikezawa H, Imagawa M, Tsukamoto K. Glu-53 of Bacillus cereus sphingomyelinase acts as an indispensable ligand of Mg²⁺ essential for catalytic activity. J. Biochem., 133, 279–286 (2003).
• 23) Fujii S, Inoue B, Yamamoto H, Ogata K, Shinki T, Inoue S, Tomita M, Tamura H, Tsukamoto K, Ikezawa H, Ikeda K. Mg²⁺ binding and catalytic function of sphingomyelinase from Bacillus cereus. J. Biochem., 124, 1178–1187 (1998).
• 24) Oda M, Takahashi M, Tsuge H, Nagahama M, Sakurai J. Role of side-edge site of sphingomyelinase from Bacillus cereus. Biochem. Biophys. Res. Commun., 422, 128–132 (2012).
• 25) Gohar M, Faegri K, Perchat S, Ravnum S, Okstad OA, Gominet M, Kolsto AB, Lereclus D. The PlcR virulence regulon of Bacillus cereus. PLoS ONE, 3, e2793 (2008).
• 26) Salamitou S, Ramisse F, Brehelin M, Bourguet D, Gilois N, Gominet M, Hernandez E, Lereclus D. The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects. Microbiology, 146, 2825–2832 (2000).
• 27) Gohar M, Økstad OA, Gilois N, Sanchis V, Kolsto AB, Lereclus D. Two-dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon. Proteomics, 2, 784–791 (2002).
• 28) Pomerantsev AP, Kalnin KV, Osorio M, Leppla SH. Phosphatidylcholine-specific phospholipase C and sphingomyelinase activities in bacteria of the Bacillus cereus group. Infect. Immun., 71, 6591–6606 (2003).
• 29) Pomerantsev AP, Pomerantseva OM, Leppla SH. A spontaneous translational fusion of Bacillus cereus PlcR and PapR activates transcription of PlcR-dependent genes in Bacillus anthracis via binding with a specific palindromic sequence. Infect. Immun., 72, 5814–5823 (2004).
• 30) Luscombe NM, Austin SE, Berman HM, Thornton JM. An overview of the structures of protein–DNA complexes. Genome Biol., 1, reviews001.1–001.37 (2000).
• 31) Grenha R, Slamti L, Nicaise M, Refes Y, Lereclus D, Nessler S. Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR. Proc. Natl. Acad. Sci. U.S.A., 110, 1047–1052 (2013).
• 32) Agaisse H, Gominet M, Okstad OA, Kolsto AB, Lereclus D. PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol. Microbiol., 32, 1043–1053 (1999).
• 33) Slamti L, Lereclus D. A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group. EMBO J., 21, 4550–4559 (2002).
• 34) Gominet M, Slamti L, Gilois N, Rose M, Lereclus D. Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence. Mol. Microbiol., 40, 963–975 (2001).
• 35) Slamti L, Lereclus D. Specificity and polymorphism of the PlcR-PapR quorum-sensing system in the Bacillus cereus group. J. Bacteriol., 187, 1182–1187 (2005).
• 36) Declerck N, Bouillaut L, Chaix D, Rugani N, Slamti L, Hoh F, Lereclus D, Arold ST. Structure of PlcR: insights into virulence regulation and evolution of quorum sensing in Gram-positive bacteria. Proc. Natl. Acad. Sci. U.S.A., 104, 18490–18495 (2007).
• 37) Bouillaut L, Perchat S, Arold S, Zorrilla S, Slamti L, Henry C, Gohar M, Declerck N, Lereclus D. Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides. Nucleic Acids Res., 36, 3791–3801 (2008).
• 38) Callegan MC, Cochran DC, Kane ST, Gilmore MS, Gominet M, Lereclus D. Contribution of membrane-damaging toxins to Bacillus endophthalmitis pathogenesis. Infect. Immun., 70, 5381–5389 (2002).
• 39) Callegan MC, Booth MC, Jett BD, Gilmore MS. Pathogenesis of Gram-positive bacterial endophthalmitis. Infect. Immun., 67, 3348–3356 (1999).
• 40) González-Zorn B, Dominguez-Bernal G, Suarez M, Ripio MT, Vega Y, Novella S, Rodriguez A, Chico I, Tierrez A, Vazquez-Boland JA. SmcL, a novel membrane-damaging virulence factor in Listeria. Int. J. Med. Microbiol., 290, 369–374 (2000).
• 41) Yang FL, Li XS, Liang XW, Zhang XF, Qin GS, Yang BZ. Detection of virulence-associated genes in Staphylococcus aureus isolated from bovine clinical mastitis milk samples in Guangxi. Trop. Anim. Health Prod., 44, 1821–1826 (2012).
• 42) Delgado S, Garcia P, Fernandez L, Jimenez E, Rodriguez-Banos M, del Campo R, Rodriguez JM. Characterization of Staphylococcus aureus strains involved in human and bovine mastitis. FEMS Immunol. Med. Microbiol., 62, 225–235 (2011).
• 43) Katayama Y, Baba T, Sekine M, Fukuda M, Hiramatsu K. Beta-hemolysin promotes skin colonization by Staphylococcus aureus. J. Bacteriol., 195, 1194–1203 (2013).
• 44) Tajima A, Iwase T, Shinji H, Seki K, Mizunoe Y. Inhibition of endothelial interleukin-8 production and neutrophil transmigration by Staphylococcus aureus beta-hemolysin. Infect. Immun., 77, 327–334 (2009).
• 45) Giese B, Glowinski F, Paprotka K, Dittmann S, Steiner T, Sinha B, Fraunholz MJ. Expression of delta-toxin by Staphylococcus aureus mediates escape from phago-endosomes of human epithelial and endothelial cells in the presence of beta-toxin. Cell. Microbiol., 13, 316–329 (2011).
• 46) Hayashida A, Bartlett AH, Foster TJ, Park PW. Staphylococcus aureus beta-toxin induces lung injury through syndecan-1. Am. J. Pathol., 174, 509–518 (2009).
• 47) Bramley AJ, Patel AH, O’Reilly M, Foster R, Foster TJ. Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus aureus for the mouse mammary gland. Infect. Immun., 57, 2489–2494 (1989).
• 48) Hossain MS, Hamamoto H, Matsumoto Y, Razanajatovo IM, Larranaga J, Kaito C, Kasuga H, Sekimizu K. Use of silkworm larvae to study pathogenic bacterial toxins. J. Biochem., 140, 439–444 (2006).
• 49) Speer A, Sun J, Danilchanka O, Meikle V, Rowland JL, Walter K, Buck BR, Pavlenok M, Holscher C, Ehrt S, Niederweis M. Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages. Mol. Microbiol., 97, 881–897 (2015).
• 50) Oda M, Takahashi M, Matsuno T, Uoo K, Nagahama M, Sakurai J. Hemolysis induced by Bacillus cereus sphingomyelinase. Biochim. Biophys. Acta, 1798, 1073–1080 (2010).
• 51) Montes LR, Ruiz-Arguello MB, Goni FM, Alonso A. Membrane restructuring via ceramide results in enhanced solute efflux. J. Biol. Chem., 277, 11788–11794 (2002).
• 52) Popov J, Vobornik D, Coban O, Keating E, Miller D, Francis J, Petersen NO, Johnston LJ. Chemical mapping of ceramide distribution in sphingomyelin-rich domains in monolayers. Langmuir, 24, 13502–13508 (2008).
• 53) Goñi FM, Alonso A. Effects of ceramide and other simple sphingolipids on membrane lateral structure. Biochim. Biophys. Acta, 1788, 169–177 (2009).
• 54) Montes LR, Lopez DJ, Sot J, Bagatolli LA, Stonehouse MJ, Vasil ML, Wu BX, Hannun YA, Goni FM, Alonso A. Ceramide-enriched membrane domains in red blood cells and the mechanism of sphingomyelinase-induced hot–cold hemolysis. Biochemistry, 47, 11222–11230 (2008).
• 55) Akamatsu R, Suzuki M, Okinaka K, Sasahara T, Yamane K, Suzuki S, Fujikura D, Furuta Y, Ohnishi N, Esaki M, Shibayama K, Higashi H. Novel Sequence type in Bacillus cereus strains associated with nosocomial infections and bacteremia, Japan. Emerg. Infect. Dis., 25, 883–890 (2019).
• 56) Ogawa H, Ohnuma M, Squarre D, Mweene AS, Ezaki T, Fujikura D, Ohnishi N, Thomas Y, Hang’ombe BM, Higashi H. Bacillus cereus from the environment is genetically related to the highly pathogenic B. cereus in Zambia. J. Vet. Med. Sci., 77, 993–995 (2015).