Transcriptional responses of Leptospira interrogans to host innate immunity: significant changes in metabolism, oxygen tolerance, and outer membrane

Abstract

Background: Leptospira interrogans is the major causative agent of leptospirosis. Phagocytosis plays important roles in the innate immune responses to L. interrogans infection, and L. interrogans can evade the killing of phagocytes. However, little is known about the adaptation of L. interrogans during this process.

Methodology/principal findings: To better understand the interaction of pathogenic Leptospira and innate immunity, we employed microarray and comparative genomics analyzing the responses of L. interrogans to macrophage-derived cells. During this process, L. interrogans altered expressions of many genes involved in carbohydrate and lipid metabolism, energy production, signal transduction, transcription and translation, oxygen tolerance, and outer membrane proteins. Among them, the catalase gene expression was significantly up-regulated, suggesting it may contribute to resisting the oxidative pressure of the macrophages. The expressions of several major outer membrane protein (OMP) genes (e.g., ompL1, lipL32, lipL41, lipL48 and ompL47) were dramatically down-regulated (10-50 folds), consistent with previous observations that the major OMPs are differentially regulated in vivo. The persistent down-regulations of these major OMPs were validated by immunoblotting. Furthermore, to gain initial insight into the gene regulation mechanisms in L. interrogans, we re-defined the transcription factors (TFs) in the genome and identified the major OmpR TF gene (LB333) that is concurrently regulated with the major OMP genes, suggesting a potential role of LB333 in OMPs regulation.

Conclusions/significance: This is the first report on global responses of pathogenic Leptospira to innate immunity, which revealed that the down-regulation of the major OMPs may be an immune evasion strategy of L. interrogans, and a putative TF may be involved in governing these down-regulations. Alterations of the leptospiral OMPs up interaction with host antigen-presenting cells (APCs) provide critical information for selection of vaccine candidates. In addition, genome-wide annotation and comparative analysis of TFs set a foundation for further studying regulatory networks in Leptospira spp.

Figure 1. Schematic representation of the macrophage infection models (A) and search tactics of specific transcription factors from the leptospiral genomes (B).

Figure 2. Genome-wide transcriptional changes of the L. interrogans Serovar Lai Strain Lai 56601 in the infection models.

Cluster analysis (Euclidean distance) revealed several distinct subclades in the whole transcriptomics (A). The subgroup of most highly down-regulated genes was defined as Clade 1, which included several major outer membrane protein genes, such as ompL47 (LA0505), lipL41 (LA0616), lipL48 (LA3240), ompL1 (LA3138), and lipL32 (LA2637), etc. The most significantly up-regulated genes were included in Clade 3. The Clades 2,4,5 and 6 included the moderately down-regulated genes (B).

Figure 3. Validation of microarray data using quantitative real-time RT-PCR.

The transcriptional levels for the randomly selected 6 genes (Table S1) were determined by quantitative real-time RT-PCR using new batch of RNA samples (A). M: the mRNA change folds from normalized microarray data; Q: the mRNA change folds from normalized qRT-PCR data; a, b, c, d, e, and f: the mRNA change folds of M45, J45, T45, M90, J90 and T90. No PCR amplification was detected in negative controls. The quantitative real-time RT-PCR values were plotted against the microarray data values. The high correlation coefficient values (R²) indicated that the microarray signal represented by multiple oligonucleotide probes was valid for transcriptomics research (B).

Figure 4. Statistic analysis of the leptospiral transcriptional regulation based on KEGG pathway.

The percentage of differentially regulated genes was calculated by dividing the number of up-regulated or down-regulated genes by the total number of genes in each category, respectively. A, Biosynthesis of Polyketides and Nonribosomal Peptides (9 genes); B, Biosynthesis of Secondary Metabolites (28 genes); C, Carbohydrate Metabolism (224 genes); D, Cell Motility (78 genes); E, Energy Metabolism (78 genes); F, Folding, Sorting and Degradation (34 genes); G, Glycan Biosynthesis and Metabolism (43 genes); H, Lipid Metabolism (132 genes); I, Membrane Transport (36 genes); J, Metabolism of Cofactors and Vitamins (117 genes); K, Replication and Repair (72 genes); L, Signal Transduction (45 genes); M, Transcription (3 genes); N, Translation (76 genes). M-up, J-up, and T-up: the percentages of up-regulated genes in M, J and T samples; M-down, J-down, and T-down: the percentages of down-regulated genes in M, J and T samples. A gene regulated either at a time-point or at two time-points was included in this statistics analysis. If a gene was up-regulated at a time-point but down-regulated at another time-point, it was included both in up-regulation and in down-regulation.

Figure 5. Verification of the leptospiral protein changes by Western blotting.

The leptospiral samples at 1, 2 and 4 hour in the infection models [J774A.1 cell model (A) and THP-1 cell model (B)] were harvested for semi-quantitative protein assay. The protein expression levels of LipL41 (LA0616), LipL32 (LA2637), Mce (LA2055), OmpA (LB328), OmpL1 (LA3138), FliH (LA2589), FliI (LA2592), FliY (LA2613) and FliN (LA2069) were estimated by Western blotting band intensities.

Figure 6. Sequential changes of the predicted leptospiral OMP genes.

The balance between up-regulation (red) and down-regulation (green) indicated that L. interrogans altered its membrane in the infection models. The highly down-regulated transmembrane OMP and lipoprotein genes were clustered into two distinct subclades. The most highly down-regulated subclade included the genes of LipL41 (LA0616), LipL48 (LA3240), a putative OMPs (LA1538), OmpL1 (LA3138) and LipL32 (LA2637). Another highly down-regulated subclade included the genes of LipL21 (LA0011), LipL46 (LA2024), two putative outer membrane proteins (LA0100 and LA2066), LipL45 (LA2295), putative lipoprotein qlp42 (LA0136), Loa22 (LA0222), LruB (LA3469) and LruA/LipL71 (LA3097).

Figure 7. Domain structures of all predicted leptospiral specific transcription factors.

Based on protein domain similarity, all specific TFs from the six released Leptospira genomes were classified into 18 TF families. The total number of TFs in each family was shown behind the structure model. The detailed TF catalog and evolutionary analysis can be inquired in Table S3.

Figure 8. Molecular evolution and gene regulation of leptospiral OmpR transcription factors.

The molecular evolutionary tree was constructed using the Neighbor-Joining method implemented in the MEGA 4.0 program with confidences of topology summarized from 1000 bootstrap replications based on the whole sequences of OmpRs. Only the bootstrap values larger than 50% were shown on the branches. Orthologous OmpRs sharing among all of the six Leptospira genomes syntenies were marked in a yellow green. lil: L. interrogans Serovar Lai Strain Lai 56601; lic: L. interrogans Serovar Copenhageni Strain Fiocruz L1-130; lbj: L. borgpetersenii Serovar Hardjo-bovis Strain JB197; lbl: L. borgpetersenii Serovar Hardjo-bovis Strain L550; lbf: L. biflexa Serovar Patoc Strain Patoc I (Ames); lbi: L. biflexa Serovar Patoc Strain Patoc I (Paris) (A). Gene regulation analysis of the OmpR TFs showed that LB333 was the unique OmpR TF gene which was highly-expressed in EMJH and RPMI 1640 medium (E0, M45 and M90), but significantly down-regulated in infection models (J45, J90, T45 and T90) (B).