Expression of sialic acids and other nonulosonic acids in Leptospira
© Ricaldi et al.; licensee BioMed Central Ltd. 2012
Received: 6 February 2012
Accepted: 19 July 2012
Published: 1 August 2012
Sialic acids are negatively charged nine carbon backbone sugars expressed on mammalian cell surfaces. Sialic acids are part of a larger family of nonulosonic acid (NulO) molecules that includes pseudaminic and legionaminic acids. Microbial expression of sialic acids and other nonulosonic acids has been shown to contribute to host-microbe interactions in a variety of contexts, including participation in colonization, immune subversion, and behaviors such as biofilm formation, autoagglutination and motility. Previous research has suggested that some spirochetes may also express these molecules.
Here we use a combination of molecular tools to investigate the presence of NulO biosynthetic gene clusters among clinical and saprophytic isolates of the genus Leptospira. Polymerase chain reaction and Southern blotting suggested that a variety of leptospires encoded NulO biosynthetic pathways. High performance liquid chromatography and mass spectrometry analyses provided biochemical evidence that di-N-acetylated NulO molecules are expressed at relatively high levels by L. interrogans serovar Lai strain 55601, and at lower levels by L. alexanderi serovar Manhao and L. fainei serovar Hurstbridge. Endogenous expression of N-acetylneuraminic acid (Neu5Ac, the most common sialic acid) was documented in L. interrogans serovar Copenhageni strain L1-130. Neu5Ac biosynthesis is also supported by a unique gene fusion event resulting in an enzyme with an N-terminal N-acetylneuraminic acid synthase domain and a C-terminal phosphatase domain. This gene fusion suggests that L. interrogans uses a Neu5Ac biosynthetic pathway more similar to animals than to other bacteria. Analysis of the composition and phylogeny of putative NulO biosynthetic gene clusters in L. interrogans serovar Lai and serovar Copenhageni revealed that both strains have complete biosynthetic pathways for legionamimic acid synthesis, a molecule with the same stereochemistry as sialic acid. Lectin-based affinity purification of NulO-modified molecules, followed by mass spectrometric identification suggests post-translational modification of surface lipoproteins, including Loa22.
Leptospira species encode NulO biosynthetic pathways and synthesize multiple NulO molecules including sialic acid. Additional studies are needed to clarify the exact context and functional significance of NulO expression. These findings have implications for immune evasion during systemic leptospirosis.
Leptospirosis, the most common zoonotic illness affecting humans, is caused by spirochetes of the genus Leptospira[1, 2]. Some Leptospira species live exclusively in water or soil, while others cycle between environmental and mammalian reservoirs. Leptospira can colonize/infect renal tubules of a wide variety of wild and domesticated mammals. Human disease follows exposure to water or soil contaminated with urine of infected animals. Leptospirosis can be asymptomatic, or manifest as a mild flu-like illness. In another subset of individuals (5-10 % of patients) Leptospira can produce more serious systemic infections resulting in pulmonary hemorrhage, jaundice, renal failure, refractory shock, myocarditis, and/or aseptic meningitis.
Despite its medical importance, few virulence determinants of pathogenic Leptospira have been characterized in any detail. Investigation of the organism is hampered by its fastidiousness, slow growth in culture and the lack of available genetic tools. To date, only Omp-A like lipoprotein Loa22 has been demonstrated to be necessary for virulence, appearing to be cytotoxic and capable of inducing apoptosis. [3–5] LipL32, a major outer membrane protein of pathogenic Leptospira, is expressed in vivo and, although it has been shown to bind to host extra-cellular membrane, LipL32 does not seem to be required for acute or chronic infection in vivo in animal models. [6, 7] Other potential virulence leptospiral factors include LigA and LigB that contain immunoglobulin-like repeats associated with adhesion to host cells in other gram-negative bacteria. Other proteins shown to have laminin binding activity in-vitro include LenA/LfhA/Lsf24 and related proteins LenBCDEF. LenA seems to also bind factor H of complement, so it might have more than one role in virulence. [8, 9]. Leptospiral LPS, although not characterized in detail, has some unique characteristics which could explain why it is poorly recognized by the TLR4- MD2 complex. This diminished recognition could contribute to leptospiral survival in the bloodstream and dissemination. Other potential virulence factors for which more evidence remains to be published include mediators of motility and chemotaxis, including chemotaxis towards hemoglobin .
Sialic acids are a diverse family of acidic nine-carbon backbone (nonulosonic) monosaccharides found in abundance on the surfaces of mammalian cells and are sometimes expressed by microbial pathogens. The most common sialic acid in nature is N-acetylneuraminic acid (Neu5Ac). Expression of Neu5Ac by pathogenic bacteria has been linked mechanistically to complement and neutrophil evasion in disseminated infections with Streptococcus and Neisseria and with the induction of autoimmune neuropathy following infection with Campylobacter. Sialic acids are part of an even wider family of di-N-acetylated nonulosonic acid (NulO) sugars, which also includes pseudaminic and legionaminic acids. Legionaminic acid was first described as part of the Legionella lipopolysaccharide O-antigen , which is thought to have roles in environmental and host associations . Legionaminic and pseudaminic acids are also found as post-translational modifications of flagellin, best studied in Campylobacter and Helicobacter[13, 14]. Even further, recent data suggest that in Helicobacter proteins other than flagellins may also undergo glycosylation . Our recent genomic and phylogenetic analyses indicated the presence of NulO biosynthetic gene clusters in the available genomes of L. interrogans. In this study, we sought to investigate the presence of NulO biosynthetic gene clusters in other Leptospira species and to determine whether these genes produced functional biosynthetic pathways. Here we define the presence of putative nonulosonic acid biosynthetic gene clusters in a variety of Leptospira species. Further biochemical investigations show that some Leptospira are capable of endogenous synthesis of nonulosonic acids, including sialic acids.
Results and discussion
Nonulosonic acid biosynthetic gene clusters are present among pathogenic and some intermediately pathogenic Leptospira species
DMB-derivatization and HPLC-MS analysis reveals multiple varieties of nonulosonic acids expressed by Leptospira
Composition and phylogenetic analysis of NulO biosynthetic gene clusters and enzymes
L. interrogans encodes a complete pathway for legionaminic acid synthesis
C. jejuni Pathway number (Figure 5)
L. interrogans L1-130 & 56601 NCBI accession numbers
Predicted L. interrogans Pathway number (Figure 5)
Predicted enzymatic Function
LegB (cj 1319)
Aminotransferase in legionaminic acid synthesis (Figure 6A)
4, 13, or ?
2-epimerase/NDP sugar hydrolase in legionamimic acid synthesis
Legionaminic acid synthase (Figure 6B)
Legionaminic or neuraminic acid synthase (Figures 6B & 7)
8 or 11
CMP-Legionaminic acid or neuraminic acid synthetases (Figure 6C)
8 or 11
Phylogenetic comparisons were performed to provide additional insights into the potential functions of Leptospira nonulosonic acid biosynthesis enzymes. We included in the phylogenetic analysis the well-characterized enzymes of Campylobacter jejuni that participate in parallel pathways of legionamimic, pseudaminic, and neuraminic acid synthesis [14, 17–21]. A schematic of these biosynthetic pathways is shown in Figure 5, noting the structural differences between neuraminic (sialic), legionamimic, and pseudaminic acids. These different NulOs are used by C. jejuni to modify a variety of different surface structures including the O-antigen of lipooligosaccharides, flagellin, and other surface proteins. To add further resolution to our phylogenetic analysis, we also included NulO biosynthetic enzymes from two Photobacterium profundum genome strains (3TCK and SS9), previously demonstrated to synthesize legionamimic and pseudaminic acids respectively . In addition, homologous enzymes from other Leptospira genomes (L. noguchii str. 2006001870, L. biflexa serovar Patoc, L. santarosai str. 2000030832, L. borgpetersenii serovar Hardjo-bovis str. L550) were included in the phylogenetic analysis to better place the L. interrogans NulO enzymes into context with other putative leptospiral NulO biosynthetic enzymes.
Nonulosonic acids are elaborated on Leptospira surface lipoproteins
The affinity-purified material was subjected to DMB-derivatization and HPLC analysis, which showed the Neu5Ac peak, but not the Kdo peak (data not shown), strongly suggesting that this material was free of LPS-components. This does not rule-out that LPS may be modified with NulOs, just that LPS was not present in this affinity-purified preparation. We performed mass spectrometry to identify protein components in the affinity-purified material. Three proteins were identified by mass spectrometry (Figure 8B): Loa22, LipL32, and LipL41, all of which have been described in previous publications as surface-exposed lipid-linked outer membrane proteins of L. interrogans[23–27]. Indeed, Loa22 and LipL31 are among the most abundant proteins expressed on the Leptospira cell surface . Loa22 was identified with the highest number of peptide matches. Loa22 is an outer membrane protein encoded within all Leptospira genomes sequenced to date. It has been observed to be upregulated in vivo and it is one of very few leptospiral proteins so far that has been shown to be necessary for virulence . Additional studies are needed to define the precise context of NulO expression on L. interrogans and understand its potential significance in virulence.
Based on a combination of experimentation and in silico genomic analysis, we have demonstrated the function of NulO biosynthetic gene clusters in pathogenic and intermediately pathogenic species of Leptospira, several of which are capable of synthesizing di-N-acetylated NulO species, as well as the true sialic acid, N-acetyneuraminic acid, a finding of considerable consequence for the leptospirosis field. This finding expands the number of important human pathogens that utilize endogenous biosynthetic pathways to elaborate surface structures containing sialic acids and related NulO molecules . Sialic acids have proven roles in complement evasion, intracellular survival, and biofilm formation , and evidence is emerging that some human pathogens with Neu5Ac on their surfaces can engage sialic acid-binding receptors (Siglecs) on leukocyte cell surfaces, resulting in phagocytosis or dampening of bactericidal activities [30–32]. The roles of other NulO molecules such as legionaminic and pseudaminic acids are less well defined, but these molecules have been shown to play roles in behaviors such as autoagglutination, motility, and host colonization [33–37]. Curiously, disease caused by L. interrogans includes bacteremia and meningitis as components of the clinical disease spectrum, similar to the well-characterized Neu5Ac-expressing human bacterial pathogens Group B Streptococcus Neisseria meningitidis E. coli K1, and Haemophilus influenzae. As genetic tools and small animal infection systems for study of Leptospira are further refined, analysis of the contribution of NulO biosynthesis to the virulence of this neglected disease can be further elucidated.
Strains and culture conditions
Intermediately pathogenic strains L. licerasiae serovar Varillal strains MMD3731, MMD4847 and CEH008 (isolated from rodents in Peru), L. fainei serovar Hurstbridge strain BUT 6T and the saprophyte L. biflexa serovar Patoc were used for these experiments. Pathogenic Leptospira used in this study included L. interrogans serovar Copenhageni strain L1-130, L. interrogans serovar Lai strain 55601, and L. interrogans serovar Icterohaemorrhagiae wild rodent isolate MMD 3731 that were passaged fewer than 5 times in vitro after re-isolation from hamster liver to maintain virulence. L. santarosai and L. alexanderi serovar Manhao were originally isolated from clinical cases of leptospirosis and now serve as reference laboratory strains. Generally, Leptospira were cultivated at 30°C in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (catalog #279510, Becton Dickinson, Sparks, Maryland). Chemically-defined, sialic acid-free medium, prepared as previously described and verified by HPLC to be sialic acid free, was used to cultivate Leptospira in experiments where the lack of exogenous sialic acids was a necessary condition .
PCR of sialic acid cluster genes
Primers based on the genome of L. interrogans L1-130 were designed for the detection of genes in the sialic acid cluster as follows: sasfrontF (5′- TCC GGA AAT GCG AAT GAT G-3′), sasfrontR (5′- CAC CGG GCA AAA GAC TAA CCT - 3′), sasendF (5′- CGG ATA TAG CGG ACG ATG TAA - 3′), sasendR (5′- CGC CAA AAA GCC AAG GAA - 3′), neuA2F (5′- TGA AGC GGC AAA AAG AGC - 3′), neuA2R (5′- TGA AAT AAC ATG CCG ACA AAT A - 3′), neuCfrontF (5′- CGC TAC GGG AAT GCA TCT GTC TC - 3′), neuCfrontR (5′- CCC ATT CCC CCA ACC AAA AA - 3′), neuCendF (5′- GGC GAG GAT CCT TCT AAT GTT TTT - 3′) and neuCendR (5′- ACT CGC TCC GCC TTC ACC A - 3′). PCR reactions were prepared using 0.2 mM of each primer in a 20 μL reaction with DNA from the pathogens L. interrogans Lai, L. interrogans L1-130, the intermediates L. licerasiae and L. fainei and the saprophyte L. biflexa serovar Patoc. NeuA2 and neuBfront reactions used an annealing temperature of 52°C. NeuCfront, neuCend, sasfront and sasend were run using an annealing temperature of 58°C. A 16 S gene PCR reaction using previously published primers fLIP and rLIP1 was used as a control for integrity of DNA.
NeuA2 southern blot
Genomic DNA samples of Salmonella enterica, L. interrogans serovar Lai str. 56601, L. interrogans serovar Copenhageni str. L1-130, L. biflexa serovar Patoc, L. licerasiae strains CEH008 and MMD4847, L. interrogans serovar Icterohaemorrhagiae str.MMD3731 and L. fainei serovar Hurstbridge were prepared into plugs using 1 % agarose and 0.5x TBE. These were subjected to depurination and denaturing conditions. DNA was then transferred to a positively charged membrane via overnight capillary transfer with 20x SSC. Finally the DNA was cross-linked to the membrane using short wave DNA for 15 min. 10 mL of pre-hybridization solution (QuikHyb, Stratagene) were warmed to 40°C prior to hybridization. Hybridization was done overnight at 40-42°C using the same solution and adding 10 mL of DIG-labeled PCR product of primer neuA2F (5′ - TGA AGC GGC AAA AAG AGC - 3′) and neuA2R (5 ′- TGA AAT AAC ATG CCG ACA AAT A - 3′). 2xSSC at room temperature and 1x SSC at 68°C were used for stringency washes. A chemiluminescent substrate and an alkaline phosphatase conjugated anti-DIG antibody were used to demonstrate binding of the probe.
Mild acid hydrolysis and DMB-derivatization of nonulosonic acids
Mild (2 N) acetic acid hydrolysis was performed to release surface nonulosonic acids from Leptospira. 4 N acetic acid was added to an equal volume of extensively washed and resuspended pellets followed by 3 h of incubation at 80°C. The resulting soluble fraction was filtered to remove large molecular weight components and derivatized with DMB (1,2-diamino-4,5-methylene dioxybenzene), a reagent that reacts with the α-keto acid portion of nonulosonic acids. Final reaction conditions were 7 mM DMB, 18 mM sodium hydrosulfite, 1.4 M acetic acid, and 0.7 M 2-mercaptoethanol. Derivatization was carried out for 2 hours at 50°C in the dark.
High performance liquid chromatography and mass spectrometry
DMB-NulO derivatives were resolved by HPLC using a reverse phase C18 column (Varian) eluted isocratically at a rate of 0.9 ml/min over 50 minutes using 85 % MQ-water, 7 % methanol, 8 % acetonitrile as previously described [16, 39, 40]. In some experiments, HPLC was performed without online mass spectrometry and detection of fluorescently labeled NulO sugars was achieved using an online fluorescence detection using excitation and emission wavelengths of 373 nm and 448 nm respectively. In other experiments HPLC was combined with online mass spectrometry using a Thermo-Finnigan model LCQ ion trap mass spectrometer system. When mass spectrometry was performed, the mobile phase also included 0.1 % formic acid, and online UV detection of DMB-NulO molecules preceded mass spectrometric analysis. We note that similar HPLC-MS analyses have been described previously DMB-derivatized α-keto acids [39–41].
We performed BLAST searches (blastp) against the NCBI genome database using as seeds the sequences of 1) proteins encoded by Campylobacter jejuni pseudaminic, legionaminic, and neuraminic acid biosynthetic pathways or 2) enzymes encoded in the Leptospira interrogans NulO biosynthetic gene cluster (Figure 1A). NCBI accession numbers are provided in Table 1 and a schematic of the biosynthetic pathways is illustrated in Figure 5. Complete protein sequences of homologous amino acids were aligned using ClustalW in MacVector 11.1.1 software and alignments were checked manually. The Neighbor Joining (NJ) method was utilized for phylogenetic tree construction using MacVector 11.1.1 software, including 1000 Bootstrap replications to obtain confidence values for branches of the NJ trees.
Solid-phase lectin binding
Whole cell lysates were prepared using three cycles of freeze-thawing of PBS washed L. interrogans serovar Copenhageni strain L1-130. In order to probe the abundance and nature of the sialylated molecules on L. interrogans, these lysates were fractionated using a lectin-based solid phase assay (Q Proteome Sialic Acid kit, Qiagen) using three immobilized sialic acid binding lectins: wheat germ agglutinin (WGA), Sambucus nigra agglutinin (SNA), and Maackia amurensis lectin (MAL), according to manufacturer’s instructions. Molecules captured by each of these lectins were eluted according to the manufacturers instructions. then analyzed by SDS-PAGE followed by silver staining (SilverQuest Silver Staining Kit, Invitrogen).
To determine whether L. interrogans uses nonulosonic acids for post-translational modification of proteins, pooled affinity-purified material from above mentioned experiment was subjected to denaturation, reduction, and alkylation, followed by trypsin digestion and MS/MS analysis using a nano-flow LC- tandem mass spectrometer. Peptide mass fingerprints identified in the affinity-purified material were used to identify L. interrogans proteins by searching against the NCBInr bacterial genome database.
We thank Ajit Varki and Victor Nizet for valuable advice and Sandra Diaz for technical assistance with HPLC-MS. The Scripps Research Institute’s Center for Mass Spectrometry performed nano-flow MS/MS data and provided results of the NCBInr database search (http://masspec.scripps.edu/). This work was supported in part by U.S. Public Health Service grants from the National Institutes of Health 1D43TW007120 and 1RO1TW05860.
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