Catecholamine-Modulated Novel Surface-Exposed Adhesin LIC20035 of Leptospira spp. Binds Host Extracellular Matrix Components and Is Recognized by the Host during Infection

Abstract

In this study, the effect of the host stress hormone catecholamine on Leptospira gene transcripts encoding outer membrane proteins was investigated. There was no impact of catecholamine supplementation on the in vitro growth pattern of Leptospira interrogans; however, 7 genes out of 41 were differentially transcribed, and the effect was reversed to the basal level in the presence of the antagonist propranolol. Comprehensive analysis of one of the differentially regulated proteins, LIC20035 (in serovar Copenhageni)/LB047 (in serovar Lai) (due to catecholamine supplementation), revealed immunogenicity and ability to adhere to host extracellular matrices. Protease accessibility assay and phase partition of integral membrane proteins of Leptospira showed LIC20035/LB047 to be an outer membrane surface-exposed protein. The recombinant LIC20035 protein can be serologically detected using human/bovine sera positive for leptospirosis. Moreover, the recombinant LIC20035 can bind to diverse host extracellular matrices, with a higher affinity toward collagen and chondroitin sulfate.IMPORTANCE Leptospirosis is a neglected tropical disease of global importance. This study aimed to identify outer membrane proteins of pathogenic Leptospira responding to host chemical signals like catecholamines, with the potential to serve as virulence factors, new serodiagnostic antigens, and vaccine candidates. This study mimicked the plausible means by which Leptospira during infection and hormonal stress intercepts host catecholamines to disseminate in host tissues.

Keywords: LB047; LIC20035; Leptospira; catecholamines; differential transcription; qRT-PCR; stress; stress hormone.

FIG 1

Effects of catecholamines and inhibitor on growth of Leptospira. L. interrogans Lai cultures were incubated and grown in the presence of Epi, NE, the β-antagonist propranolol (PO), or the catecholamines and PO combined. Spirochete cultures were monitored daily, and spirochete numbers were determined by counting under 40× phase-contrast microscopy. Each treatment showed growth similar to those of untreated control cultures, and at no time point was a significant difference detected among any group. Results are indicative of those from two independent experiments.

FIG 2

Effects of catecholamines and inhibitor on the selective gene transcripts of Leptospira. Transcript analyses of the 7 genes encoding OMPs out of 41 by qRT-PCR of the cDNA synthesized from Leptospira grown in the presence of catecholamines and inhibitors (500 μM) were conducted. The gene transcription was calculated based on threshold cycle (C_T) values by use of the 2^−ΔΔCT method and normalized against 16S rRNA values. (A) The genes of L. interrogans serovar Lai that showed significant differential transcription. The differential transcription of seven genes in the presence of catecholamines was restored to the basal level on addition of its inhibitor propranolol describing the specific role of the catecholamines in modulating gene transcription. (B) The differential transcription of 7 orthologous genes of L. interrogans serovar Copenhageni. The patterns of differential transcription of 7 orthologs of Copenhageni to serovar Lai are very close. (C and D) The fold change in gene transcription of L. interrogans Lai and L. interrogans Copenhageni, responding significantly to catecholamines. Each gene is represented by a unique colored bar along with fold change value at the apex of each bar. Error bars indicate the SDs from 2 independent qRT-PCR analyses.

FIG 3

Characterization of hypothetical protein LIC20035/LB047. (A) PCR for LIC20035 gene using genomic DNA of pathogenic (Copenhageni, Lai, and Canicola) and nonpathogenic (Patoc) serovars of Leptospira. An amplicon size of 1,275 bp by agarose gel electrophoresis confirmed the existence of the LIC20035 gene in pathogenic serovars (lanes 1, 3, and 5). In contrast, no amplification of LIC20035 was observed in the nonpathogenic serovar of Leptospira (lane 7). Primers of the constitutive flaB gene were used as a positive control for determining the quality of DNA (lanes 2, 4, 6, and 8). (B) Induction and purification of recombinant LIC20035. The CDS of LIC20035 of L. interrogans Copenhageni L1-130 was cloned without its signal peptide sequence in the pET28a vector and expressed in BL21 E. coli. Induction of r-LIC20035 expression was done using 0.5 mM IPTG, and its purification was performed using Ni-NTA column chromatography under hybrid conditions. The uninduced and induced lysates of E. coli BL21 along with the purified recombinant LIC20035 are shown on an 12% SDS-PAGE gel stained with Coomassie. (C) Generated polyclonal antibody titer raised against r-LIC20035. The pooled mouse immune serum obtained 10 days after a second booster dose was used to calculate the titer of polyclonal antibodies generated against r-LIC20035 using ELISA. Serum obtained before the immunization of r-LIC20035 antigen was used as a control for evaluation of antibody titer, and data are presented as means ± SEMs from two independent experiments. (D) Immunoblot demonstrating the differential expression of LB047 in L. interrogans serovar Lai grown in the presence of catecholamines and inhibitor. There was a repression of LB047 in the presence of Epi alone in comparison to control and NE, which was restored to the basal levels when the organism was grown along with its inhibitor, propranolol. Anti-LipL31 was used as a control to demonstrate equal loading of Leptospira lysates. (E) Relative densitometry of the immunoblot shown in panel D was calculated from two independent experiments and normalized with the band intensity of LipL31 using Image Lab software. The repression of LB047 was evident in the presence of Epi, and the effect was restored to the basal level using its inhibitor, PO. (F) Far-UV circular dichroism (CD) of r-LIC20035. The CD spectra are depicted in the range of 190- to 240-nm wavelengths showing a predominant signal of β-strand. CD spectra are shown as averages from 3 scans with a scanning speed of 100 nm/min.

FIG 4

Cellular localization of LIC20035/LB047. (A) Triton-X-114 phase partitioning of spirochete proteins. Spirochete lysates were subjected to Triton X-114 phase partition of aqueous (A) and detergent (D) phases. The aqueous- and detergent-phase fractions were resolved by 12% SDS-PAGE and immunoblotted with anti-LIC20035 serum. The major portion of LIC20035 was in detergent phase and partly in aqueous phase in both serovars Lai and Copenhageni. Whole-cell lysate of serovar Copenhageni in lane 1 was used as a molecular marker for LIC20035. (B) Immunoblot to validate the Triton X-114 phase partition analysis. LipL32 is a known outer membrane lipoprotein and is exclusively present only in the detergent phase of Triton X-114. Anti-LipL32 (1:500) detected LipL32 only in the detergent phase (D) fraction of serovars Lai and Copenhageni. Whole-cell lysate of serovar Copenhageni in lane 1 was used as a molecular marker for LipL32. (C) Proteinase K accessibility assay of L. interrogans Lai for surface-exposed LB047. Spirochetes were incubated with 25 μg of proteinase K at various time intervals up to 5 h. The spirochete suspensions were washed with PBS and used to coat a microtiter plate. Using ELISA, a drastic decrease in the signal for LB047 reactivity with its antiserum was observed after 1 to 5 h of proteinase K treatment, similar to the case with OmpL54, a known outer membrane protein. LipL31 was used as a control to check the cellular integrity of spirochetes during treatment with proteinase K. Error bars represent SDs from the three replicates. Statistical analysis was performed by Student's t test by comparing the signals obtained for 0 h and other time points of treatment with proteinase K (P < 0.05).

FIG 5

Recombinant LIC20035 is recognized by leptospirosis-positive sera. Enzyme-linked immunosorbent assay (ELISA) was performed to detect r-LIC20035 or r-LIC10191 using the sera of humans (1:100) and bovines (1:100) testing MAT positive (n = 50) and control serum testing MAT negative for leptospirosis. The cutoff value of the assay was derived from the mean of the control group plus 2 SDs for each recombinant antigens (dotted black lines). The mean of each group is represented by black horizontal lines. (A) ELISA to detect r-LIC20035 (400 ng/well) using human sera testing positive for leptospirosis. The sensitivity and specificity of the assay were 98% and 100%, respectively. (B) ELISA to detect r-LIC10191 (400 ng/well) using human sera testing positive for leptospirosis. Recombinant LIC10191 (r-Loa22) (400 ng/well, an amount equivalent to r-LIC20035) was used to scale the recognition capacity of human sera. (C) ELISA to detect r-LIC20035 (400 ng/well) with bovine sera testing positive for leptospirosis. The sensitivity and specificity of the serological assay were both 100%. (D) ELISA to detect r-Loa22 with bovine sera testing positive for leptospirosis. Recombinant Loa22 (400 ng/well, an amount equivalent to r-LIC20035) was used to scale the recognition capacity of bovine sera.

FIG 6

Recombinant LIC20035 binds to host extracellular matrix components. (A) ELISA depicts r-LIC20035 interacts with extracellular matrix (ECM) components, laminin, fibronectin, collagen (calf skin), hyaluronic acid, chondroitin sulfate A, chondroitin sulfate B, and elastin. Bovine serum albumin (BSA) and the highly glycosylated serum protein fetuin were used as controls for nonspecific binding. Loa22 was included as a negative control for nonspecific binding with the ECM components. Recombinant LIC20035 exhibited significant binding with all ECM components compared to fetuin or BSA (P < 0.001) and with a higher affinity toward chondroitin sulfate and collagen. In contrast, Loa22 showed moderate binding with all the ECM components used in this study. Results are indicative of those from two independent experiments. (B) Dose-dependent binding of the LIC20035 to ECM components. A microtiter plate coated with 1 μg of a specific ECM was incubated with increasing concentrations (0 to 8 μM) of r-LIC20035. Binding of LIC20035 was measured using anti-LIC20035 serum at an appropriate dilution. The mean absorbance values of r-LIC20035 binding to ECM in two experiments at 450 nm are shown.