Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection

Abstract

Background: It has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of L. interrogans allowed identification of a repertoire of putative leptospiral surface proteins.

Results: Here, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis.

Conclusion: Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis.

Figure 1

Analysis of Lsa21 recombinant protein from NaCl-induced E. coli Bl21-SI by SDS-PAGE and Circular dichroism spectrum. In A. SDS-15% PAGE showing lane 1 – molecular weight protein marker (kDa), lane 2 – non-induced culture, lane 3 – induced culture, lanes 4 and 5 – supernatant and inclusion body pellet after bacterial cell lysis and centrifugation, respectively, lane 6 – purified protein eluted from Ni⁺² – charged chelating sepharose column with 1 M imidazole. In B. CD spectrum of Lsa21 protein depicting both α-helical and β-sheets in its secondary structure composition. Far-UV CD spectrum is presented as an average of five scans recorded from 190 to 260 nm.

Figure 2

Distribution and expression of LIC10368 gene in saprophytic and pathogenic leptospires. (A) Genomic DNA from L. biflexa Patoc and from five serovars belonging to the pathogenic species L. interrogans was subjected to PCR analysis with LIC1368 specific primers designed according to L. interrogans serovar Copenhageni genome sequences. The expected size of the PCR product is 540 bp. No DNA was added to the negative control reaction (-). (B) RT-PCR analysis of LIC10368 transcripts in high-passage L. interrogans strains and in the low-passage LO4 (Canicola), Fiocruz L1-130 (Copenhageni), LPF (Pomona) strains. Reactions were performed with the same primer pairs mentioned above. Samples quantity and integrity were verified by amplification of a 1042-bp 16S ribosomal cDNA fragment. RT+: reverse transcriptase present; RT -: reverse transcriptase omitted; M: molecular mass markers.

Figure 3

Regulation of LIC10368 expression by environmental factors. (A) RT-PCR analysis of LIC10368 transcripts upon culture-attenuation. RNA was extracted from low- and high-passage L. interrogans strains. Above, low-passage L. interrogans serovar Pomona strain LPF (passages 1 and 4, P1 and P4) and high-passage L. interrogans serovar Pomona strain Pomona (HP). Below, low-passage L. interrogans serovar Canicola strain LO4 (passages P1 and P3) and high-passage L. interrogans serovar Canicola strain Hond Utrechet IV (HP). (B) Regulation of LIC10368 transcript levels by osmolarity. Cultures of L. interrogans serovar Pomona strain LPF grown in EMJH with 1% rabbit serum were centrifuged and resuspended in fresh EMJH medium or in EMJH containing 120 mM NaCl. Cultures were incubated for 24 h before being harvested for RNA isolation. RNA was also obtained from a culture grown under our standard laboratory conditions (10% rabbit serum supplemented with amino acids and salts). LIC10368 transcripts obtained from cultures with 1% serum (1%), 1% serum + 120 mM NaCl (1% + NaCl) and 10% serum + amino acids and salts referred as enriched medium, EM (10% EM). 16S ribosomal cDNA fragments (1,042-bp) were co-migrated in all lanes. LIC12099 (lipL53 gene) transcripts were included as a positive control (1%, 1% + NaCl). (C) The effect of temperature on LIC10368 transcript levels was assessed by culturing leptospires at 20ºC, 29ºC and 37ºC. Additional cultures grown at 29ºC and at 37ºC were shifted overnight to 37ºC and to 39ºC, respectively, in order to simulate conditions encountered by bacteria upon entry into the host and in a febrile stage. Optical densities of LIC10368 and LIC12099 transcripts were normalized for each sample with corresponding 16S ribosomal densitometry data to obtain the relative expression levels. RT+: reverse transcriptase present in the reaction; RT-: reverse transcriptase omitted; M: molecular mass markers.

Figure 4

Indirect immunofluorescence staining of intact fixed leptospires. L. interrogans serovar Pomona strain LPF and L. biflexa serovar Patoc strain Patoc 1 were incubated with mouse anti-Lsa21 serum, followed by incubation with a fluorescein isothiocyanate-conjugated goat anti-mouse antibody. Magnification × 1,260.

Figure 5

Binding of Lsa21 recombinant protein to ECM components. Wells were coated with 1 μg of laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, and the control proteins BSA and fetuin. Recombinant Lsa21 and Loa22 proteins attachment to those ECM macromolecules was assessed by an ELISA-based assay. One microgram of recombinant protein was added per well. Optical densities were taken at 492 nm. Data represent the mean ± standard error of three independent experiments. For statistical analysis, the attachment of Lsa21 to ECM macromolecules was compared to its binding to BSA by the Student's two tailed t- test (*, P < 0.01).

Figure 6

(A) Lsa21 binds to ECM components in a dose-dependent manner. Binding of Lsa21 to laminin, collagen IV and plasma fibronectin, as a function of protein concentration (0 to 2,000 nM). Each point represents the mean absorbance value at 492 nm ± the standard deviation of three independent experiments. BSA was included as a negative control. (B) Sugar moiety contribution to the laminin-Lsa21 interaction. Immobilized laminin was treated with sodium metaperiodate (5 to 100 mM) for 15 min at 4°C in the dark. Each point represents the mean absorbance value at 492 nm ± the standard deviation of three independent experiments. Reduction in attachment compared to the level of attachment to untreated laminin was statistically significant: in each case (*), the P value, as measured by the Student two-tailed t -test, was ≤ 0.004.

Figure 7

Sequence comparison between Lsa21 and leptospiral proteins. Unrooted phylogenetic tree of predicted amino acid sequences of the Len family, LigA/LigB and Lsa21 proteins. The tree was generated by Clustal X program and displayed by NJ plot. Branch lengths are depicted.

Figure 8

(A) Leptospirosis, human liver: Lsa21 is present as red deposits in the cytoplasm of isolated macrophages along the sinusoidal lining (arrow heads). Similar deposits are observed focally on the apical sinusoidal plasma membrane of hepatocytes and as small granules in the underneath cytoplasm (arrows). IHC and light hematoxylin counterstain; (B) Leptospirosis, human kidney: Lsa21 antigen is present over the luminal side of distal tubules. Red granules are also seen in the cell cytoplasm below. Notice the absence of Lsa21 antigen in the proximal tubules. Glomerulus present is apparently normal. IHC and light hematoxylin counterstain; (C) Leptospirosis, human kidney: Lsa21 antigen is present at the luminal side of collecting ducts as red granules over the epithelial cells and in the cell cytoplasm below; IHC and light hematoxylin counterstain. In A, B, and C: original magnification ×300.