Multiple leptospiral sphingomyelinases (or are there?).
ABSTRACT Culture supernatants of leptospiral pathogens have long been known to haemolyse erythrocytes. This property is due, at least in part, to sphingomyelinase activity. Indeed, genome sequencing reveals that pathogenic Leptospira species are richly endowed with sphingomyelinase homologues: five genes have been annotated to encode sphingomyelinases in Leptospira interrogans. Such redundancy suggests that this class of genes is likely to benefit leptospiral pathogens in their interactions with the mammalian host. Surprisingly, sequence comparison with bacterial sphingomyelinases for which the crystal structures are known reveals that only one of the leptospiral homologues has the active site amino acid residues required for enzymic activity. Based on studies of other bacterial toxins, we propose that leptospiral sphingomyelinase homologues, irrespective of their catalytic activity, may possess additional molecular functions that benefit the spirochaete. Potential secretion pathways and roles in pathogenesis are discussed, including nutrient acquisition, dissemination, haemorrhage and immune evasion. Although leptospiral sphingomyelinase-like proteins are best known for their cytolytic properties, we believe that a better understanding of their biological role requires the examination of their sublytic properties as well.
- SourceAvailable from: Joseph M Vinetz
Article: Leptospiral Pathogenomics.[Show abstract] [Hide abstract]
ABSTRACT: Leptospirosis, caused by pathogenic spirochetes belonging to the genus Leptospira, is a zoonosis with important impacts on human and animal health worldwide. Research on the mechanisms of Leptospira pathogenesis has been hindered due to slow growth of infectious strains, poor transformability, and a paucity of genetic tools. As a result of second generation sequencing technologies, there has been an acceleration of leptospiral genome sequencing efforts in the past decade, which has enabled a concomitant increase in functional genomics analyses of Leptospira pathogenesis. A pathogenomics approach, by coupling of pan-genomic analysis of multiple isolates with sequencing of experimentally attenuated highly pathogenic Leptospira, has resulted in the functional inference of virulence factors. The global Leptospira Genome Project supported by the U.S. National Institute of Allergy and Infectious Diseases to which key scientific contributions have been made from the international leptospirosis research community has provided a new roadmap for comprehensive studies of Leptospira and leptospirosis well into the future. This review describes functional genomics approaches to apply the data generated by the Leptospira Genome Project towards deepening our knowledge of virulence factors of Leptospira using the emerging discipline of pathogenomics.Pathogens (Basel, Switzerland). 01/2014; 3(2):280-308.
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ABSTRACT: Leptospirosis is arguably the most widespread zoonosis; it is also a major cause of economic loss in production animals worldwide. At the level of the host animal or human, the progression of infection and the onset of disease are well documented. However, the mechanisms of pathogenesis at the cellular and molecular level remain poorly understood, mainly as a result of the lack of modern genetic tools for mutagenesis of pathogenic Leptospira spp. The recent development of transposon mutagenesis and the construction of a very small number of directed leptospiral mutants have identified a limited number of essential virulence factors. Perhaps surprisingly, many leptospiral proteins with characteristics consistent with a role in virulence have been shown to not be required for virulence in animal models, consistent with a high degree of functional redundancy in pathogenic Leptospira. A large number of putative adhesins has been reported in Leptospira, which interact with a range of host tissue components; however, almost none of these have been genetically confirmed as having an essential role in pathogenesis.Veterinary Microbiology 06/2014; · 2.73 Impact Factor
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ABSTRACT: Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of "core" housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants.PLoS Pathogens 03/2014; 10(3):e1004004. · 8.06 Impact Factor
Multiple leptospiral sphingomyelinases (or are
Suneel A. Narayanavari,1Manjula Sritharan,1David A. Haake2,3,4,5
and James Matsunaga3,6
Suneel A. Narayanavari
1Department of Animal Sciences, University of Hyderabad, Hyderabad, India
2Division of Infectious Diseases, VA Greater Los Angeles Healthcare System, Los Angeles, CA,
3Department of Medicine, University of California at Los Angeles, Los Angeles, CA, USA
4Department of Urology, University of California at Los Angeles, Los Angeles, CA, USA
5Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los
Angeles, Los Angeles, CA, USA
6Research Service, VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA
Culture supernatants of leptospiral pathogens have long been known to haemolyse erythrocytes.
This property is due, at least in part, to sphingomyelinase activity. Indeed, genome sequencing
reveals that pathogenic Leptospira species are richly endowed with sphingomyelinase
homologues: five genes have been annotated to encode sphingomyelinases in Leptospira
interrogans. Such redundancy suggests that this class of genes is likely to benefit leptospiral
pathogens in their interactions with the mammalian host. Surprisingly, sequence comparison with
bacterial sphingomyelinases for which the crystal structures are known reveals that only one of the
leptospiral homologues has the active site amino acid residues required for enzymic activity.
Based on studies of other bacterial toxins, we propose that leptospiral sphingomyelinase
homologues, irrespective of their catalytic activity, may possess additional molecular functions that
benefit the spirochaete. Potential secretion pathways and roles in pathogenesis are discussed,
including nutrient acquisition, dissemination, haemorrhage and immune evasion. Although
leptospiral sphingomyelinase-like proteins are best known for their cytolytic properties, we believe
that a better understanding of their biological role requires the examination of their sublytic
properties as well.
Sphingomyelinases are of great interest because of their
potential to mediate key aspects of leptospiral pathogen-
esis. Leptospirosis is most prevalent in tropical countries
where moist conditions favour environmental survival of
pathogenic Leptospira species excreted by animal carriers of
the spirochaete. Transmission occurs when contaminated
soil or water comes into contact with cutaneous lacerations
or mucous membranes of the mouth, eyes and nose (WHO,
2003). Leptospirosis is an invasive infection manifested by a
broad spectrum of symptoms that are often mistaken for
other infections. The disease is usually self-limiting but can
progress to a severe form characterized by renal failure,
haemorrhagic diathesis and jaundice. Pulmonary haem-
orrhage is a feared complication caused by damage to the
endothelial lining of blood vessels (Dolhnikoff et al., 2007),
possibly caused by a toxin as leptospires are often not
detected at the site of the lesion (Miller et al., 1974). Another
occasional complication is haemolytic anaemia (Feiginet al.,
1975). Through their action on host cell membranes,
leptospiral sphingomyelinases are potentially involved in
aspects of pathogenesis, including tissue invasion, endothe-
lial damage, immune evasion and nutrient acquisition.
Sphingomyelinases are enzymes that catalyse the hydrolysis
of sphingomyelin into ceramide and phosphorylcholine.
Biochemically, sphingomyelinases are classified as either
acidic, neutral or alkaline, depending on their pH optimum
for activation. Most of the neutral sphingomyelinases of
bacteria and mammals form a family defined by a set of con-
served catalytic core residues and overall sequence relatedness
(Clarke et al., 2011). Mammalian members of the neutral
sphingomyelinase family are membrane-associated, whereas
the bacterial members are secreted. Mammalian sphingomye-
linases act on the sphingomyelin present on the membranes
and release ceramide, which controls cellular functions by
acting as a signalling molecule and by altering the biophysical
Microbiology (2012), 158, 1137–1146
057737Printed in Great Britain1137
properties of the membrane (Hannun & Obeid, 2008).
Ceramide is also the central hub of the sphingolipid
signalling network, which includes other bioactive sphingo-
lipids such as sphingosine and sphingosine-1-phosphate.
The levels of ceramide and other sphingolipids are therefore
tightly controlled (Breslow & Weissman, 2010), and their
dysregulation contributes to the patho-biology of numerous
infectious and non-infectious disease processes (Zeidan &
Hannun, 2007). For example, cellular infection by diverse
pathogens, including Neisseria gonorrhoea, rhinovirus and
Cryptosporidium parvum, involves activation of the host acid
sphingomyelinase by translocation of the enzyme from the
endolysosomal to the plasma membrane (Grassme ´ et al.,
2005; Zeidan & Hannun, 2007). Hydrolysis of sphingomye-
lin in the plasma membrane by acid sphingomyelinase leads
to assembly of ceramide-enriched membrane platforms,
which may be necessary to concentrate receptors to facilitate
intracellular signal transduction and microbial internaliza-
tion (Lafont & van der Goot, 2005).
Sphingomyelinases produced by Bacillus cereus, Staphylo-
coccus aureus and Listeria (List.) ivanovii are the best
characterized among the bacterial sphingomyelinases. As
most bacteria do not synthesize sphingomyelin, bacterial
sphingomyelinases probably target the sphingomyelin in
the external leaflet of the host cell’s plasma membrane.
Their inactivation in S. aureus and List. ivanovii diminished
their infectivity in animal models (Bramley et al., 1989;
Gonza ´lez-Zorn et al., 1999). List. ivanovii sphingomyeli-
nase enables the intracellular pathogen to escape from
phagocytic vacuoles in epithelial cells by rupturing the
membrane of the vacuole (Gonza ´lez-Zorn et al., 1999). The
sphingomyelinase activity of S. aureus b-toxin promotes
excessive inflammation and vascular leakage in the lungs by
inducing shedding of the ectodomain of the proteoglycan
syndecan-1 in a mouse model of pneumonia (Hayashida
et al., 2009). The response does not occur when the
catalytic residues of b-toxin are altered, highlighting the
importance of the enzymic activity of the toxin in
triggering uncontrolled inflammation. In this review, we
examine the evidence that sphingomyelinase-like proteins
are involved in mechanisms of leptospiral pathogenesis.
Discovery of many leptospiral genes encoding
Sphingomyelinase activity was first detected in Leptospira
cultures in the 1960s (Ka ˘sarov & Addamiano, 1969), yet
cloning of a sphingomyelinase gene was not reported until
1989 (del Real et al., 1989), when a genomic expression
library of Leptospira (Lept.) borgpetersenii serovar Hardjo
was screened for haemolytic activity. Haemolytic and
sphingomyelinase activities were expressed from a single
gene that was later designated sphA (del Real et al., 1989;
Segers et al., 1992). The sphingomyelinase encoded by sphA
shared significant similarity to those found in S. aureus and
Bacillus subtilis (Segers et al., 1990). Multiple sphingomye-
linase sequences were detected in pathogenic members of
Leptospira by low stringency Southern hybridization using
Lept. borgpetersenii sphA as a probe (Segers et al., 1992).
SphH, one of the sphingomyelinase homologues in the
genome of serovarLai,wasidentified froma genomiclibrary
using sphA as the probe (Lee et al., 2000). The protein
showed 75% similarity to SphA. However, the clone failed
to express sphingomyelinase (or phospholipase) activity,
although the partially purified recombinant protein lysed
sheep erythrocytes (Lee et al., 2000, 2002). The haemolytic
activity of SphH was neutralized with rabbit antiserum
raised against SphH, eliminating the possibility that
haemolysis was due to the cryptic haemolysin of E. coli.
Transmission electron microscopy of sheep erythrocytes
incubated with the SphH preparation revealed pores in the
membrane, suggesting that the haemolytic activity of SphH
was due to pore-forming ability (Lee et al., 2002). However,
another group was unable to confirm the haemolytic activity
of a purified preparation of rSphH (Carvalho et al., 2010),
possibly due to improper refolding of the insoluble re-
Genome sequencing uncovered the multiple sphingomyeli-
nase-like proteins encoded in several pathogenic Leptospira.
The Lai, Copenhageni, Manilae and Pomona strains each
carried genes annotated as sph1, sph2, sph3, sph4 and sphH
(Bulach et al., 2006b; Nascimento et al., 2004; Ren et al.,
2003) (B. Adler, personal communication). In contrast,
the genomes of two Lept. borgpetersenii strains harboured
only sphA, sphB and sph4 (Bulach et al., 2006b). The non-
pathogen Leptospira biflexa lacks sph coding sequences
(Picardeau et al., 2008).
Domains of leptospiral sphingomyelinase-like
Multi-sequence alignment of all available leptospiral sphin-
gomyelinase-like sequences reveals the modular nature of
the proteins (Fig. 1). In addition to signal sequences, there
are N-terminal and C-terminal extensions flanking the
central enzymic domain. The region of sequence similarity
among the proteins comprises the enzymic domain and C-
The crystal structures of the sphingomyelinases of List.
ivanovii (Openshaw et al., 2005), B. cereus (Ago et al., 2006)
and S. aureus (Huseby et al., 2007) have been determined.
These structures revealed the active site configuration of
the conserved residues shown to be crucial for sphingo-
myelinase activity in mutagenesis studies (Huseby et al.,
2007; Obama et al., 2003a, b). The active site of B. cereus
sphingomyelinase contained the divalent metal cation
necessary for catalytic activity (Ago et al., 2006). Using the
numbering for B. cereus sphingomyelinase, essential residues
include Glu-53, His-151, Asp-195 and His-296, the metal-
binding and catalytic functions of which are shown in Fig.
2(a). Surprisingly, the multi-sequence alignment shows that
S. A. Narayanavari and others
only Lept. borgpetersenii SphA and Leptospira interrogans
Sph2 possess these four amino acid residues (Fig. 2b). In
contrast, Sph1 and Sph3 of Lept. interrogans and SphB of
Lept. borgpetersenii have non-conservative amino acid
substitutions for three or all four of these critical residues.
This raises the possibility that these latter Sph proteins are
not true sphingomyelinases, despite their overall sequence
similarity with other bacterial sphingomyelinases. This
observation is consistent with the finding that SphH lacks
sphingomyelinase activity (Lee et al., 2002). Although one
study reported sphingomyelinase activity for recombinant
Sph1, Sph3 and Sph4 expressed in E. coli (Zhang et al.,
2005), their conclusions are in doubt for several reasons.
First of all, Sph4 lacks the entire enzymic domain (Fig. 1)
and therefore should not have exhibited any sphingomye-
because data from the negative control experiment were not
presented. Thirdly, the observed reduction of the sphingo-
myelinase peak as measured by HPLC could have resulted
from the activity of E. coli lipases in the extract. This is
possible because of the high protein concentrations of the
crude extracts (100 mg ml21) in their assays (Zhang et al.,
2005). In conclusion, we propose that pathogenic Leptospira
and that all of the sphingomyelinase-like proteins may
possess additional molecular functions.
What, then, could be the additional functions of the
‘enzymic’ domain of the leptospiral sphingomyelinase-like
proteins? Their non-catalytic function may target host
sphingomyelin on membrane surfaces for attachment of
the protein. For example, the Helicobacter pylori toxin
VacA uses sphingomyelin as a receptor to enter the target
cell (Gupta et al., 2008). The domain may also possess
surfaces that bind other host receptors. This is reminiscent
of the leptospiral haemolysin-like protein TlyC, which
lacks haemolytic activity yet binds to extracellular matrix
proteins fibronectin, collagen IV and laminin (Carvalho
et al., 2009). A novel role for sphingomyelinase has been
described for the S. aureus b-toxin. In the process of
b-toxin covalently interacts with
extracellular DNA, forming insoluble nucleoprotein com-
plexes. Biofilm assembly occurred even when the two
histidine residues responsible for catalytic activity were
altered by mutation, indicating that the residues involved
in biofilm formation are distinct from the ones involved in
catalysis (Huseby et al., 2010).
The crystal structures of the sphingomyelinases of B. cereus,
List. ivanovii and S. aureus revealed a protruding hydro-
phobic b-hairpin and a second external hydrophobic loop
adjacent to the active site. The surface hydrophobic loops
relation to the sphingomyelin substrate in the target
membrane. Replacement of the hydrophobic residues in
the b-hairpin with alanine in B. cereus sphingomyelinase
impaired its binding to sphingomyelin liposomes and
disrupted its sphingomyelin hydrolytic activity (Ago et al.,
2006; Narayanavari et al., 2012). The leptospiral sphingo-
2005). Hence the initial interaction of the leptospiral
sphingomyelinase-like proteins with the target membrane
may involve sequences located outside of the enzymic
The leptospiral sphingomyelinase-like proteins and Pseu-
domonas strain TK4 sphingomyelinase have a carboxy-
the other bacterial sphingomyelinases (Narayanavari et al.,
2012; Sueyoshi et al., 2002). The role of the C-terminal
extension in the Pseudomonas sphingomyelinase has been
examined. Deletion of 186 aa from the C-terminal end of
Fig. 1. Schematic representation of leptospiral
sphingomyelinase-like proteins. NTRs, N-ter-
minal repetitive sequences; S, signal peptide;
EEPD, exo-endo phosphatase domain; CTE,
A . Listeria
B . Bacillus
C . LA1029–Sph2
D . LA1027–Sph1
E . LA4004–Sph3
F . LA3540–SphH
G . LBJ0291–SphA
H . LBJ0527–SphB
I . Staphylococcus
J . Pseudomonas
K . HnSMase1
L . HnSMase2
Fig. 2. Catalytic site functions and multi-sequence alignment of the active-site amino acid residues required for
sphingomyelinase activity. (a) The proposed function of amino acids at the catalytic site of B. cereus sphingomyelinase
(adapted from Obama et al., 2003a). Asn-197 interacts with the phosphate group of sphingomyelin, and Glu-53 and Asp-295
coordinate a divalent cation. His-296 and His-151 function as the acid-base catalytic residues; His-296 and the metal ion
activate the water molecule that attacks the phosphorus of sphingomyelin, resulting in its hydrolysis to phosphocholine and
ceramide. Asp-195 maintains the appropriate spatial arrangement of the catalytic histidine residues. (b) Multi-sequence
alignment showing six of the amino acids (highlighted in red) conserved in all members of the extended neutral
sphingomyelinase family, including the two human neutral sphingomyelinases. Note that Glu-53, His-151, Asp-195 and/or
His-296 are not conserved (highlighted in grey) in Sph1, Sph3, SphH and Sph4.
S. A. Narayanavari and others
1140 Microbiology 158
Pseudomonas sphingomyelinase completely abolished the
haemolytic activity without affecting the sphingomyelinase
activity, indicating that the C-terminal extension is indis-
pensable for haemolytic activity (Sueyoshi et al., 2002). This
observation suggests that the function of the C-terminal
extension is to interact with the target host membrane to
position the enzymic domain near the sphingomyelin
substrate (Sueyoshi et al., 2002).
Export and secretion signals
Sphingomyelinase activity has been detected in the culture
fluids of several strains of pathogenic Leptospira (Bernheimer
& Bey,1986).The secreted sphingomyelinaseismostlikely to
be SphA or Sph2 because only these enzymes possess the
essential catalytic residues. Sph2 has been detected in the
culture supernatant with specific antiserum (Carvalho et al.,
2010; Matsunaga et al., 2007). However, the mechanism by
which Sph2 is secreted is unknown because the protein
appears to lack an amino-terminal signal peptide (Fig. 1). In
contrast, Sph1, Sph3, SphB and SphH are predicted to have
a cleavable amino-terminal signal peptide, suggesting that
they are exported out of the cytoplasm to an unknown
destination. Lept. interrogans also releases sphingomyelinase
in membrane vesicles under some culture conditions
(Velineni et al., 2009).
Transport of Sph2 and SphA out of the leptospiral cell
could involve either the type I or type II secretion pathway
(Bulach et al., 2006a). Recently a 63 kDa TolC homologue
(LA0957) was immunoprecipitated from an outer mem-
brane preparation of Lept. interrogans with antiserum
raised against the enzymic domain of Sph3 (Velineni et al.,
2009). Although further experimentation is necessary to
confirm the association of the proteins, this observation
suggests that at least one of the sphingomyelinase-like
proteins is secreted via the TolC-based type I secretory
pathway (Jenewein et al., 2009). Another TolC homologue
(LA3927/LIC13135) was also noted as potentially function-
ing in sphingomyelinase secretion (Louvel et al., 2006).
Analysis of the sequences attached to the N-termini of the
enzymic domain using RADAR (Heger & Holm, 2000)
revealed between two and seven short N-terminal imper-
fect repeats (NTRs) in Sph1, Sph2 and SphB (Table 1).
The repeats are enriched in disorder-promoting amino
acids (Tompa, 2005). Based on the known functions of
intrinsically disordered sequences, the NTRs may harbour
proteolytic sites, function as a flexible linker between the
signal peptide and the enzymic domain, or bind macro-
molecules or small ligands (Tompa, 2005).
Phylogenetic analysis of leptospiral sphingomyelinase-
A phylogenetic tree was constructed from a multi-sequence
alignment of the amino acid sequences of the leptospiral
sphingomyelinase-like proteins from four strains of Lept.
interrogans and two strains of Lept. borgpetersenii (Fig. 3).
Sph4 was excluded from the analysis because it lacks the
enzymic domain. The dendrogram shows that the leptos-
piral sphingomyelinase-like proteins can be grouped into
six clusters. The Lept. interrogans and Lept. borgpetersenii
proteins form separate clusters. The genes encoding Sph1
and Sph2 in Lept. interrogans appear to have arisen from a
relatively recent duplication event, consistent with sph1 and
sph2 being located next to each other on the Lept.
interrogans chromosome. In contrast, only one copy of
sphA is present in the same genomic position in Lept.
Expression of leptospiral sphingomyelinase-like
proteins during infection
Clear evidence for expression of a sphingomyelinase-like
protein during a natural leptospiral infection came from a
study of equine leptospirosis. Sera from mares infected
with Lept. interrogans serovar Pomona strongly recognized
recombinant Sph2 protein (Artiushin et al., 2004). A more
recent study showed that IgG antibodies present in the sera
of leptospirosis patients recognized recombinant Sph2 but
not Sph1, Sph4 or SphH (Carvalho et al., 2010). Moreover
anti-Sph2 and anti-SphH antisera reacted with renal
tubular epithelium of laboratory hamsters infected with
Lept. interrogans. These results indicate that Sph2 and
possibly SphH are expressed during infection.
The expression of sph2 can be regulated by simulating host-
like conditions. Except in several strains of serovar Pomona,
Sph2 was not detected by Western blot analysis in Lept.
interrogans strains cultivated in the standard leptospiral
culture medium EMJH (Artiushin et al., 2004; Carvalho
et al., 2010; Matsunaga et al., 2007). When sodium chloride
or sucrose was added to raise the osmolarity of the culture
medium to equal that found in the mammalian host, Sph2
was detected in the Lept. interrogans strain Fiocruz L1-130
cell lysates and in a processed form in the culture
supernatant fluid, suggesting that the increase in osmolarity
experienced by leptospires entering the host triggers sph2
expression (Matsunaga et al., 2007).
Possible roles of leptospiral sphingomyelinase-
like proteins in leptospirosis
A role in nutrient acquisition has been proposed for
the leptospiral sphingomyelinases (Bulach et al., 2006a).
Leptospira depend on b-oxidation of fatty acid to meet
their carbon and energy needs in vitro (Henneberry & Cox,
1970). Inside the host, cell membranes could provide a rich
source of fatty acids as nutrients. However, sphingomye-
linase would seem to be an inefficient means for obtaining
fatty acid. Since the genomes of pathogenic Leptospira do
not encode a ceramidase homologue, a host ceramidase
would be necessary to release fatty acid molecules from
ceramide for utilization by Leptospira. Leptospira also
express phospholipases that yield fatty acid from abundant
glycerophospholipids directly, seemingly rendering sphin-
gomyelinases unnecessary for acquisition of fatty acid
(Kasa ˘rov, 1970).
Cell lysis by sphingomyelinase or the pore-forming activity
of SphH may also be important in iron acquisition. Haem
released from damaged erythrocytes is a potential source of
iron for Leptospira during infection. Expression of the
Table 1. N-terminal repeats
Protein Locus tag Species/serovarSignal
Sph1 LA1027List. interrogans serovar Lai Yes (39–40)2 60–70 (NVNEKIEDSTN)
LIC12632 List. interrogans serovar CopenhageniYes (38–39)2
LIP0979List. interrogans serovar PomonaYes (38–39)2
LiL49501006List. interrogans serovar ManilaeNo 2
Sph2LA1029 List. interrogans serovar Lai No3
LIC12631List. interrogans serovar CopenhageniNo3
LIP0980 List. interrogans serovar PomonaNo4
LiL49501008List. interrogans serovar ManilaeNo3
List. interrogans serovar Lai
List. interrogans serovar Copenhageni
List. interrogans serovar Pomona
List. interrogans serovar Manilae
List. interrogans serovar Lai
List. interrogans serovar Copenhageni
List. interrogans serovar Pomona
List. interrogans serovar Manilae
Lept. borpetersenii serovar Hardjobovis
LBL 2552 Lept. borpetersenii serovar Hardjobovis
SphA LBJ 0291 Lept. borpetersenii serovar
Hardjobovis strain JB197
Lept. borpetersenii serovar
Hardjobovis strain L550
LBL 2785Yes (26–27)––
*The number in parentheses represents the amino acids flanking the putative signal peptidase cleavage site.
DThe number represents the amino acid position in the protein sequence. Lower case characters are used for amino acid residues that are not
aligned. Gaps are represented by –.
S. A. Narayanavari and others
haemin-binding protein HbpA, identified in Lept. inter-
rogans (Sritharan et al., 2005) is induced upon iron
limitation and acquires iron from haemin (Asuthkar et al.,
2007). Although the expression and release of a 42 kDa
sphingomyelinase-like protein in outer membrane vesicles
in the presence of the chelator EDDA may support a role
for an Sph protein in iron acquisition by Lept. interrogans
serovar Lai (Velineni et al., 2009), microarray analysis with
a strain of serovar Manilae failed to show changes in sph
transcript levels when iron was depleted with 2,29-dipyridyl
(Lo et al., 2010). The different strains or chelators selected
for the studies may account for the discrepancies in the
Another case where membrane damage may be critical to
leptospiral survival is immune evasion. Although Leptospira
is primarily an extracellular pathogen, it is able to escape
from the phagosome of cultured mouse macrophages (Toma
et al., 2011). As observed for several Listeria species, escape
from the phagocytic vacuole may require the cooperation of
lipases and pore-forming activities (Gonza ´lez-Zorn et al.,
1999; Schnupf & Portnoy, 2007), which may be provided by
the sphingomyelinase activity of Sph2 and the pore-former
Sphingomyelinasesmayalsohavea roleincytotoxicity aspart
of the pathogenesis of leptospirosis. Recombinant Sph2 was
cytotoxic towards mouse lymphocytes and macrophages
(Zhang etal., 2008).Someevidence suggeststhat the immune
cells undergo a proinflammatory form of apoptosis when
exposed to Sph2 in vitro (Zhang et al., 2008). Additionally,
damage to the vascular endothelium may be responsible for
the haemorrhage observed during severe disease (Carvalho &
Bethlem, 2002). Recombinant Sph2 (Lk73.5) from a Pomona
strain of Lept. interrogans was cytotoxic to equine pulmonary
endothelial cells (Artiushin et al., 2004). However, disruption
of endothelial cell layer integrity by Lept. interrogans crossing
the monolayer did not affect the viability of the cells
(Martinez-Lopez et al., 2010). Thus, the evidence accumu-
lated to date does not support a cytotoxic role for sphin-
gomyelinases in leptospiral dissemination or haemorrhage.
The true relevance of sphingomyelinase in leptospiral
pathogenesis may lie in sublytic effects that do not damage
Fig. 3. Phylogenetic analysis of leptospiral sphingomyelinase-like proteins. Multi-sequence alignment was performed using
Geneious software utilizing the BLOSUM62 score matrix. The phylogenetic tree was constructed using MEGA tool version 5
utilizing the neighbour-joining method. The robustness of the tree was determined using bootstrapping with 500 replicates. The
tree was rooted with the Pseudomonas species TK4 sphingomyelinase (GenBank accession no. BAB69072.1).
the host cell membrane. For example, alteration of vascular
permeability is caused in part by generation of ceramide by
acid sphingomyelinase (Go ¨ggel et al., 2004), which may
explain the ability of sphingomyelinase-producing Lep-
tospira to cross the endothelial layer without cytolytic
effects. Excessive ceramide production induced by leptos-
piral sphingomyelinase could also explain the pulmonary
oedema observed in some cases of severe leptospirosis.
Alterations of sphingolipid homeostasis and lipid rafts have
also been linked to altered renal function (Zager, 2000).
The activity of the renal Na+/H+NH3transporter, whose
levels are diminished in the proximal tubule of severe
leptospirosis patients, depends on formation of lipid rafts
(Araujo et al., 2010; Murtazina et al., 2006). Finally, the
novel non-catalytic role of S. aureus sphingomyelinase in
biofilm formation described earlier may also be an
important function of leptospiral sphingomyelinase-like
proteins during infection (Huseby et al., 2010). b-Toxin
also promoted biofilm formation in vivo in a rabbit model
of S. aureus endocarditis (Huseby et al., 2010). Pathogenic
leptospires have been shown to form biofilms in vitro
(Ristow et al., 2008), and biofilm formation may be
essential for long-term leptospiral survival in the renal
tubules of the reservoir host.
The pore-forming activity of SphH may also have profound
biological consequences. The pore-forming proteins a-toxin
of S. aureus and pneumolysin of Streptococcus pneumoniae
activate the metalloprotease ADAM10, which cleaves E-
cadherin, an intercellular protein essential for epithelial
barrier function (Inoshima et al., 2011). ADAM10 is
required by a-toxin to disturb the alveolar barrier function
in the mouse model of pneumonia (Inoshima et al., 2011).
These results raise the possibility that SphH promotes the
acute lung injury that is observed in many cases of severe
In this review, we have examined a number of potential
roles for sphingomyelinase and its non-enzymic homo-
logues in leptospirosis. In Lept. interrogans, only Sph2
retains all of the active-site amino acid residues essential
for catalysis. Because the other sphingomyelinase homo-
logues lack at least three of the residues, experimental
studies are still needed to settle the fundamental issue of
whether Sph1, Sph3 and SphH have sphingomyelinase
activity. Irrespective of their catalytic activity, the proteins
may dock onto sphingomyelin or some other host
molecule as a prelude to performing their effector function,
which may include the type of pore-forming activity
described for SphH. Even in the case of Sph2, sphingo-
myelin hydrolysis is likely to be relevant to pathogenesis in
ways that go beyond mere host cell membrane damage.
Previous studies that addressed the biological functions of
leptospiral Sph2 have focused on its cytotoxic potential.
However, disruption of sphingolipid homeostasis by
leptospiral sphingomyelinase activity also has the potential
to alter cellular functions in ways that do not necessarily
kill the host cell. Future studies should therefore also seek
non-cytotoxic effects of Sph2 on host cells. We hope that
by broadening our view of the potential biological activities
of the Sph proteins, we can acquire the evidence we need to
truly understand the role of leptospiral sphingomyelinases
and sphingomyelinase-like proteins in leptospiral patho-
S.A.N. would like to acknowledge the United States India Educa-
tional Foundation (USIEF) for financial support in the form of
Fulbright Nehru Doctoral and Professional Research Fellowship. This
study was supported by Public Health Service National Institute of
Allergy and Infectious Diseases grant AI-034431 (to D.A.H.) and VA
Medical Research Funds (to J.M. and D.A.H.). We thank Ben Adler
(Monash University) for providing the sphingomyelinase sequences
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