Comparative proteogenomic analysis of the Leptospira interrogans virulence-attenuated strain IPAV against the pathogenic strain 56601

Abstract

The virulence-attenuated Leptospira interrogans serovar Lai strain IPAV was derived by prolonged laboratory passage from a highly virulent ancestral strain isolated in China. We studied the genetic variations of IPAV that render it avirulent via comparative analysis against the pathogenic L. interrogans serovar Lai strain 56601. The complete genome sequence of the IPAV strain was determined and used to compare with, and then rectify and reannotate the genome sequence of strain 56601. Aside from their highly similar genomic structure and gene order, a total of 33 insertions, 53 deletions and 301 single-nucleotide variations (SNVs) were detected throughout the genome of IPAV directly affecting 101 genes, either in their 5' upstream region or within their coding region. Among them, the majority of the 44 functional genes are involved in signal transduction, stress response, transmembrane transport and nitrogen metabolism. Comparative proteomic analysis based on quantitative liquid chromatography (LC)-MS/MS data revealed that among 1 627 selected pairs of orthologs, 174 genes in the IPAV strain were upregulated, with enrichment mainly in classes of energy production and lipid metabolism. In contrast, 228 genes in strain 56601 were upregulated, with the majority enriched in the categories of protein translation and DNA replication/repair. The combination of genomic and proteomic approaches illustrated that altered expression or mutations in critical genes, such as those encoding a Ser/Thr kinase, carbon-starvation protein CstA, glutamine synthetase, GTP-binding protein BipA, ribonucleotide-diphosphate reductase and phosphate transporter, and alterations in the translational profile of lipoproteins or outer membrane proteins are likely to account for the virulence attenuation in strain IPAV.

Figure 1

Histological sections in the lung, liver and kidney of guinea pigs infected with L. interrogans strain IPAV (A) and strain 56601 (B) (H&E, magnification × 400).

Figure 2

Circular representation of IPAV chromosomes with predicted CDSs. Left, large chromosome (CI); right, small chromosome (CII). The outer scales are numbered in megabases from the origins of replication (Ori). Circles range from 1 (outer circle) to 7 (inner circle) for CI and from I (outer circle) to V (inner circle) for CII. Circle 1, representation of genome structure when compared with L. interrogans strain Fiocruz L1-130 (yellow, the collinear region; blue, the large inverse region). Circle 2/I and 3/II, genes on plus and minus strands (colors representing functional categories according to COGs). Circle 4, tRNA genes (red) and rRNA genes (blue). Circle 5/III, IS elements (both intact and remnant are included; green, ISlin1; black, other types of ISs). Circle 6/IV, GC skew (calculated using a 2 500 bp (CI)/500 bp (CII) window sliding 200 bp (CI)/100 bp (CII) at a time; red, values > 0; green, values < 0). Circle 7/V, GC content.

Figure 3

Functional category of in silico predicted proteins and MS-validated proteins. The colors used in the bars represent different objects: purple, IPAV all predicted proteins; green, IPAV MS-validated proteins; red, 56601 all predicted proteins; blue, 56601 MS-validated proteins; gray, 56601 proteins that were not detected in this work but confirmed by our previous MS data. Proteins were clustered by COG assignment: (J) translation, ribosomal structure and biogenesis; (K) transcription; (L) replication, recombination and repair; (B) chromatin structure and dynamics; (D) cell-cycle control, cell division and chromosome partitioning; (V) defense mechanisms; (T) signal transduction mechanisms; (M) cell wall/membrane/envelope biogenesis; (N) cell motility; (U) intracellular trafficking, secretion and vesicular transport; (O) posttranslational modification, protein turnover and chaperones; (C) energy production and conversion; (G) carbohydrate transport and metabolism; (E) amino-acid transport and metabolism; (F) nucleotide transport and metabolism; (H) coenzyme transport and metabolism; (I) lipid transport and metabolism; (P) inorganic ion transport and metabolism; (Q) secondary metabolites biosynthesis, transport and catabolism; (R) general function prediction only; (S) function unknown.

Figure 4

Distribution of IS elements in large chromosomes of leptospires. The scale is numbered in megabases from the origins of replication. The bars represent the chromosomes. The short vertical lines in each bar indicate the IS elements at different coordinates (red, intact ISs, >1 000 bp; blue, remnant ISs, 100-1 000 bp). The symbols below the genomes are selected IS markers (pathogenic Leptospira specific, sky blue circles; saprophytic Leptospira specific, orange circles; L. borgpetersenii specific, green triangles; L. interrogans specific, magenta triangles; L. interrogans serovar Lai specific, navy blue rhombuses; strain Fiocruz L1-130 specific, brown asterisk; strain 56601 specific, purple asterisk; strain IPAV specific, red asterisk; strain JB197 specific, light yellow asterisk; strain L550 specific, gray asterisk; strain Ames specific, blue asterisk). For the IS markers in each evolutionary level, only one was selected to illustrate in this figure. The colors of the branches in the phylogenic tree consistent with the colors used on the IS markers and the dashed lines indicate that no IS marker is identified at the corresponding levels.

Figure 5

Large Indels in the genomes of L. interrogans strains IPAV or 56601. (A) Loss of 14-kb DNA sequence in strain 56601. (B) Type I R/M gene cluster. Genes are color coded according to their functional categories. A red asterisk is labeled above a gene if its product can be detected by MS.

Figure 6

Functional classification of upregulated orthologs in IPAV and 56601. Green: upregulated genes in IPAV. Blue: upregulated genes in 56601. The upregulated genes include those orthologs that are only expressed in one of the strains. Proteins were clustered according to COG assignment. If a protein had more than one COG category, all were used. A purple/red asterisk is situated in the functional category where the enrichment in IPAV/56601 was discovered by chi-square test.

Figure 7

Comparison of the mutation-affected STPK in IPAV to its orthologs in other species. (A) Multiple sequence alignment of STPK sequences retrieved from NCBI (strain 56601, NP_711603.1; Fiocruz L1-130, YP_002258.1; JB197, YP_801432.1; L550, YP_798524.1; Microscilla marina, ZP_01687505.1). (B) MS-detected peptides mapping on the STPKs in IPAV and 56601. The bars with different colors represent the genes, and the purple dots above the genes represent the detected peptides at their corresponding sites.

Figure 8

Multiple sequence alignments of proton-PPase (A), phosphate sodium symporter (B), GlnA (C) and FliG (D). Each site of substitution is indicated by an asterisk above the sequence. The numbers refer to the sites of amino-acid residues. The GenBank accession numbers of the used sequences are as follows: Streptomyces coelicolor (SC, NP_627745.1), Arabidopsis thaliana (AT, NP_173021.1), Bacteroides vulgatus (BV, YP_001299118.1), Flavobacteriales bacterium (FB, ZP_01105510.1), Porphyromonas gingivalis (PG, NP_906040.1), Escherichia coli (EC, NP_418306.1), Mycobacterium tuberculosis (MT, YP_001283561.1), Treponema pallidum (TP, NP_218840.1) and Bacillus subtilis (BS, NP_389504.1).