The passive diffusion of Leptospira interrogans.

Motivated by recent experimental measurements, the passive diffusion of the bacterium Leptospira interrogans is investigated theoretically. By approximating the cell shape as a straight helix and using the slender-body-theory approximation of Stokesian hydrodynamics, the resistance matrix of Leptospira is first determined numerically. The passive diffusion of the helical cell is then obtained computationally using a Langevin formulation which is sampled in time in a manner consistent with the experimental procedure. Our results are in excellent quantitative agreement with the experimental results with no adjustable parameters.

Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans.

Mass-spectrometry-based methods for relative proteome quantification have broadly affected life science research. However, important research directions, particularly those involving mathematical modelling and simulation of biological processes, also critically depend on absolutely quantitative data--that is, knowledge of the concentration of the expressed proteins as a function of cellular state. Until now, absolute protein concentration measurements of a considerable fraction of the proteome (73%) have only been derived from genetically altered Saccharomyces cerevisiae cells, a technique that is not directly portable from yeast to other species. Here we present a mass-spectrometry-based strategy to determine the absolute quantity, that is, the average number of protein copies per cell in a cell population, for a large fraction of the proteome in genetically unperturbed cells. Applying the technology to the human pathogen Leptospira interrogans, a spirochete responsible for leptospirosis, we generated an absolute protein abundance scale for 83% of the mass-spectrometry-detectable proteome, from cells at different states. Taking advantage of the unique cellular dimensions of L. interrogans, we used cryo-electron tomography morphological measurements to verify, at the single-cell level, the average absolute abundance values of selected proteins determined by mass spectrometry on a population of cells. Because the strategy is relatively fast and applicable to any cell type, we expect that it will become a cornerstone of quantitative biology and systems biology.

Repeated domains of leptospira immunoglobulin-like proteins interact with elastin and tropoelastin.

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7'-8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17-27HTE) as determined by ELISA (LigBCon4, K(D) = 0.50 microm; LigBCen7'-8, K(D) = 0.82 microm; LigBCen9, K(D) = 1.54 microm; and LigBCen12, K(D) = 0.73 microm). The interaction of LigBCon4 and 17-27HTE was further confirmed by steady state fluorescence spectroscopy (K(D) = 0.49 microm) and ITC (K(D) = 0.54 microm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17-27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.

Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing.

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum and Borrelia burgdorferi, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.

Correlation of virulence, susceptibility to leptospiricidal activity test mediated by antiserum plus complement and colonial morphology of five lines of a strain of Leptospira interrogans serovar copenhageni.

Correlation of virulence, susceptibility to leptospiricidal activity test mediated by antiserum plus complement (LAT) and colonial morphology was investigated using five lines of Leptospira interrogans serovar copenhageni Shibaura. Line No. 2, a moderately virulent strain, passaged in guinea pigs for 24 successive times caused occasional death of guinea pigs. Leptospiras recovered from deceased animals were characterized by decreased susceptibility to LAT and predominance of hazy colonies upon culture. Leptospiras recovered from surviving guinea pigs were of increased susceptibility to LAT and grew predominantly in non-hazy colonies. Virulent line No. 1 subjected to passage through liquid medium for 20 successive times resulted in a slight decrease in virulence and slight increase in susceptibility to LAT, but the predominance of hazy colonies upon culture was invariable. The other three lines, Nos. 3, 4 and 5 which have been maintained for more than 10 years by passage through different media respectively, were avirulent, susceptible to LAT and produced only non-hazy colonies.

Helix handedness of Leptospira interrogans as determined by scanning electron microscopy.

Representative serovars and strains of the seven genetic groups of Leptospira interrogans, and two previously studied serovars, were all found to form exclusively right-handed helices as determined by scanning electron microscopy. No change in handedness occurred in cells grown in a minimal medium (Tween-80 albumin) compared to cells grown in a rich medium (rabbit serum). The right-handedness of the organisms was related to the evolution, cell wall structure, and the mechanism of motility of L. interrogans.

The effects of alkalinity and hypertonicity on the morphology and motility of Leptospira interrogans (biflexa) strain B16.

Effects of alkalinity and hypertonicity on the motile behaviour of Leptospira interrogans (biflexa) B16 were observed, quantified, and compared with effects previously shown by similar factors on the motility of eubacteria. Leptospira interrogans tolerated relatively high concentrations of hydroxide ions. Motility similar to that in controls was observed at pH values up to 9-8; but at pH 10-0 motility declined sharply with time of exposure, and there was structural alteration, visible as a blebbing of the cell envelope. Unlike the behaviour of eubacteria, immobilization of L. interrogans induced by hydroxide ions could not be reversed by lowering the pH. It is suggested that by restricting entry of hydroxide ions, the cell envelope protects its motility apparatus from adverse effects. Leptospira interrogans was completely immobilized in 0-5 M and 1-0 M-sucrose solutions. Unlike the eubacteria, leptospires were incapable of spontaneous reversion to motile forms and resumption of motility was dependent on both concentration and time of exposure to sucrose. Deuterium oxide did not affect movement, suggesting that even though leptospire endoflagella and the exoflagella of eubacteria are analogous, the motile behaviour of L. interrogans is significantly different from that of eubacteria.