Lipid A structural diversity among members of the genus Leptospira

International audienceLipid A is the hydrophobic component of bacterial lipopolysaccharide and an activator of the host immune system. Bacteria modify their lipid A structure to adapt to the surrounding environment and, in some cases, to evade recognition by host immune cells. In this study, lipid A structural diversity within the Leptospira genus was explored. The individual Leptospira species have dramatically different pathogenic potential that ranges from non-infectious to life-threatening disease (leptospirosis). Ten distinct lipid A profiles, denoted L1-L10, were discovered across 31 Leptospira reference species, laying a foundation for lipid A-based molecular typing. Tandem MS analysis revealed structural features of Leptospira membrane lipids that might alter recognition of its lipid A by the host innate immune receptors. Results of this study will aid development of strategies to improve diagnosis and surveillance of leptospirosis, as well as guide functional studies on Leptospira lipid A activity

Distribution of identified errors in the original TPA Nichols and TPA SS14 genome sequences.

<p>Note that the chromosomal positions of identified errors are highly similar in both genomes as a result of the CGS sequencing approach used for sequencing the SS14 genome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074319#pone.0074319-Matjkov1" target="_blank">[9]</a>.</p

Unrooted trees constructed from whole genome sequences of TPA strains.

<p>Trees were constructed using the Neighbor-Joining method using the Tamura-Nei genetic distance model and 1,000 bootstrap replicates. The numbers above the branches show bootstrap support and the bar scale represents 0.0001 substitutions per target site. A. A tree constructed from the alignment of genomes from TPA strains Chicago (CP001752.1), DAL-1 (CP003115.1) and Mexico A (CP003064.1) with original versions of whole genome sequences of TPA Nichols (AE000520.1) and SS14 (CP000805.1). B. A tree constructed from alignment of genomes from TPA strains Chicago (CP001752.1), DAL-1 (CP003115.1) and Mexico A (CP003064.1) with the improved whole genome sequences of TPA Nichols-RS (CP004010.2) and SS14-RS (CP004011.1).</p

Effects of error correction on the improved TPA Nichols-RS genome with respect to comparisons with other TPA strains.

<p>The left part of the table shows former number of differences between the original TPA Nichols genome (AE000520.1) and other TPA genomes, the right part of the table shows verified number of differences between the improved TPA Nichols genome (CP004010.2) and other TPA genomes. Numbers in brackets show the percentage of verified differences between the improved TPA Nichols genome and other genomes compared to numbers observed during the comparison of the original Nichols genome and other TPA genomes. Because of high sequence diversities, <i>tprD</i> and <i>tprK</i> were excluded from the analyses.</p><p>sub, substitution; in, insertion; del, deletion.</p

Newly identified genomic regions showing intrastrain heterogeneity in the TPA SS14-RS genome.

<p>Underlined variants were used in the improved whole genome sequence of the TPA SS14 strain (CP004011.1). Corresponding amino acid variants are shown in brackets.</p>a<p>variant nucleotides are part of the same codon (therefore cause the same amino acid change).</p

Proteotyping of knockout mouse strains reveals sex- and strain-specific signatures in blood plasma.

We proteotyped blood plasma from 30 mouse knockout strains and corresponding wild-type mice from the International Mouse Phenotyping Consortium. We used targeted proteomics with internal standards to quantify 375 proteins in 218 samples. Our results provide insights into the manifested effects of each gene knockout at the plasma proteome level. We first investigated possible contamination by erythrocytes during sample preparation and labeled, in one case, up to 11 differential proteins as erythrocyte originated. Second, we showed that differences in baseline protein abundance between female and male mice were evident in all mice, emphasizing the necessity to include both sexes in basic research, target discovery, and preclinical effect and safety studies. Next, we identified the protein signature of each gene knockout and performed functional analyses for all knockout strains. Further, to demonstrate how proteome analysis identifies the effect of gene deficiency beyond traditional phenotyping tests, we provide in-depth analysis of two strains, C8a-/- and Npc2+/-. The proteins encoded by these genes are well-characterized providing good validation of our method in homozygous and heterozygous knockout mice. Ig alpha chain C region, a poorly characterized protein, was among the differentiating proteins in C8a-/-. In Npc2+/- mice, where histopathology and traditional tests failed to differentiate heterozygous from wild-type mice, our data showed significant difference in various lysosomal storage disease-related proteins. Our results demonstrate how to combine absolute quantitative proteomics with mouse gene knockout strategies to systematically study the effect of protein absence. The approach used here for blood plasma is applicable to all tissue protein extracts

A schematic representation of errors identified in the original TPA Nichols and TPA SS14 genomes.

<p>The corresponding effects of error corrections are shown. AF stands for authentic frameshift. The group denoted “Other changes” includes in-frame errors; indels in the RNA region and indels in genes with annotated authentic frameshifts (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074319#pone.0074319.s004" target="_blank">Table S4</a>). A. Effects of error corrections on the TPA Nichols genome. B. Effects of error corrections on the TPA SS14 genome. The numbers close to arrows indicate the number of nucleotide changes leading to changes in the proteome.</p

Effects of error correction on the improved TPA SS14-RS genome with respect to other TPA strains.

<p>The left part of the table shows former number of differences between the original TPA SS14 genome (CP000805.1) and other TPA genomes, the right part of the table shows verified number of differences between the improved TPA SS14 genome (CP004011.1) and other TPA genomes. Numbers in brackets show the percentage of verified differences between the improved TPA SS14 genome and other genomes compared to numbers observed during the comparison of the original SS14 genome and other TPA genomes. Because of high sequence diversities, <i>tprD</i> and <i>tprK</i> were excluded from the analyses. Please note that the number of sequence differences between TPA SS14 and Nichols genomes increased after error correction.</p><p>sub, substitution; in, insertion; del, deletion.</p

Calculated nucleotide diversities (π ± standard deviation) between individual TPA strains and the original and resequenced versions of the Nichols and SS14 strains.

<p>Calculated nucleotide diversities (π ± standard deviation) between individual TPA strains and the original and resequenced versions of the Nichols and SS14 strains.</p