Gamma Delta TCR and the WC1 Co-Receptor Interactions in Response to Leptospira Using Imaging Flow Cytometry and STORM.

The WC1 cell surface family of molecules function as hybrid gamma delta (γδ) TCR co-receptors, augmenting cellular responses when cross-linked with the TCR, and as pattern recognition receptors, binding pathogens. It is known that following activation, key tyrosines are phosphorylated in the intracytoplasmic domains of WC1 molecules and that the cells fail to respond when WC1 is knocked down or, as shown here, when physically separated from the TCR. Based on these results we hypothesized that the colocalization of WC1 and TCR will occur following cellular activation thereby allowing signaling to ensue. We evaluated the spatio-temporal dynamics of their interaction using imaging flow cytometry and stochastic optical reconstruction microscopy. We found that in quiescent γδ T cells both WC1 and TCR existed in separate and spatially stable protein domains (protein islands) but after activation using Leptospira, our model system, that they concatenated. The association between WC1 and TCR was close enough for fluorescence resonance energy transfer. Prior to concatenating with the WC1 co-receptor, γδ T cells had clustering of TCR-CD3 complexes and exclusion of CD45. γδ T cells may individually express more than one variant of the WC1 family of molecules and we found that individual WC1 variants are clustered in separate protein islands in quiescent cells. However, the islands containing different variants merged following cell activation and before merging with the TCR islands. While WC1 was previously shown to bind Leptospira in solution, here we showed that Leptospira bound WC1 proteins on the surface of γδ T cells and that this could be blocked by anti-WC1 antibodies. In conclusion, γδ TCR, WC1 and Leptospira interact directly on the γδ T cell surface, further supporting the role of WC1 in γδ T cell pathogen recognition and cellular activation.

Measurement of the Cell-Body Rotation of Leptospira.

Leptospira spp. swim in liquid and crawl on surfaces with two periplasmic flagella. The periplasmic flagella attach to the protoplasmic cylinder via basal rotary motors (flagellar motors) and transform the ends of the cell body into spiral or hook shape. The rotations of the periplasmic flagella are thought to gyrate the cell body and rotate the protoplasmic cylinder for propelling the cell; however, the motility mechanism has not been fully elucidated. Since the motility is a critical virulence factor for pathogenic leptospires, the kinematic insight is valuable to understand the mechanism of infection. This chapter describes microscopic methodologies to measure the motility of Leptospira, focusing on rotation of the helical cell body.

FcpB Is a Surface Filament Protein of the Endoflagellum Required for the Motility of the Spirochete Leptospira.

The spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have recently reported that FcpA is a novel flagellar protein and a major component of the sheath of the filament of the spirochete Leptospira. By screening a library of random transposon mutants in the spirochete Leptospira biflexa, we found a motility-deficient mutant harboring a disruption in a hypothetical gene of unknown function. Here, we show that this gene encodes a surface component of the endoflagellar filament and is required for typical hook- and spiral-shaped ends of the cell body, coiled structure of the endoflagella, and high velocity phenotype. We therefore named the gene fcpB for flagellar-coiling protein B. fcpB is conserved in all members of the Leptospira genus, but not present in other organisms including other spirochetes. Complementation of the fcpB- mutant restored the wild-type morphology and motility phenotypes. Immunoblotting with anti-FcpA and anti-FcpB antisera and cryo-electron microscopy of the filament indicated that FcpB assembled onto the surface of the sheath of the filament and mostly located on the outer (convex) side of the coiled filament. We provide evidence that FcpB, together with FcpA, are Leptospira-specific novel components of the sheath of the filament, key determinants of the coiled and asymmetric structure of the endoflagella and are essential for high velocity. Defining the components of the endoflagella and their functions in these atypical bacteria should greatly enhance our understanding of the mechanisms by which these bacteria produce motility.

Virulence of the zoonotic agent of leptospirosis: still terra incognita?

Pathogenic leptospires are the bacterial agents of leptospirosis, which is an emerging zoonotic disease that affects animals and humans worldwide. The success of leptospires as pathogens is explained by their spiral shape and endoflagellar motility (which enable these spirochetes to rapidly cross connective tissues and barriers), as well as by their ability to escape or hijack the host immune system. However, the basic biology and virulence factors of leptospires remain poorly characterized. In this Review, we discuss the recent advances in our understanding of the epidemiology, taxonomy, genomics and the molecular basis of virulence in leptospires, and how these properties contribute to the mechanism of pathogenesis of leptospirosis.

Comparative analysis of lipopolysaccharides of pathogenic and intermediately pathogenic Leptospira species.

BACKGROUND: Lipopolysaccharides (LPS) are complex, amphipathic biomolecules that constitute the major surface component of Gram-negative bacteria. Leptospira, unlike other human-pathogenic spirochetes, produce LPS, which is fundamental to the taxonomy of the genus, involved in host-adaption and also the target of diagnostic antibodies. Despite its significance, little is known of Leptospira LPS composition and carbohydrate structure among different serovars. RESULTS: LPS from Leptospira interrogans serovar Copenhageni strain L1-130, a pathogenic species, and L. licerasiae serovar Varillal strain VAR 010, an intermediately pathogenic species, were studied. LPS prepared from aqueous and phenol phases were analyzed separately. L. interrogans serovar Copenhageni has additional sugars not found in L. licerasiae serovar Varillal, including fucose (2.7%), a high amount of GlcNAc (12.3%), and two different types of dideoxy HexNAc. SDS-PAGE indicated that L. interrogans serovar Copenhageni LPS had a far higher molecular weight and complexity than that of L. licerasiae serovar Varillal. Chemical composition showed that L. interrogans serovar Copenhageni LPS has an extended O-antigenic polysaccharide consisting of sugars, not present in L. licerasiae serovar Varillal. Arabinose, xylose, mannose, galactose and L-glycero-D-mannoheptose were detected in both the species. Fatty acid analysis by gas chromatography-mass spectrometry (GC-MS) showed the presence of hydroxypalmitate (3-OH-C16:0) only in L. interrogans serovar Copenhageni. Negative staining electron microscopic examination of LPS showed different filamentous morphologies in L. interrogans serovar Copenhageni vs. L. licerasiae serovar Varillal. CONCLUSIONS: This comparative biochemical analysis of pathogenic and intermediately pathogenic Leptospira LPS reveals important carbohydrate and lipid differences that underlie future work in understanding the mechanisms of host-adaptation, pathogenicity and vaccine development in leptospirosis.

The leptospiral outer membrane.

The outer membrane (OM) is the front line of leptospiral interactions with their environment and the mammalian host. Unlike most invasive spirochetes, pathogenic leptospires must be able to survive in both free-living and host-adapted states. As organisms move from one set of environmental conditions to another, the OM must cope with a series of conflicting challenges. For example, the OM must be porous enough to allow nutrient uptake, yet robust enough to defend the cell against noxious substances. In the host, the OM presents a surface decorated with adhesins and receptors for attaching to, and acquiring, desirable host molecules such as the complement regulator, Factor H.Factor H. On the other hand, the OM must enable leptospires to evade detection by the host's immune system on their way from sites of invasion through the bloodstream to the protected niche of the proximal tubule. The picture that is emerging of the leptospiral OM is that, while it shares many of the characteristics of the OMs of spirochetes and Gram-negative bacteria, it is also unique and different in ways that make it of general interest to microbiologists. For example, unlike most other pathogenic spirochetes, the leptospiral OM is rich in lipopolysaccharide (LPS). Leptospiral LPS is similar to that of Gram-negative bacteria but has a number of unique structural features that may explain why it is not recognized by the LPS-specific Toll-like receptor 4 of humans. As in other spirochetes, lipoproteins are major components of the leptospiral OM, though their roles are poorly understood. The functions of transmembrane outer membrane proteins (OMPs) in many cases are better understood, thanks to homologies with their Gram-negative counterparts and the emergence of improved genetic techniques. This chapter will review recent discoveries involving the leptospiral OM and its role in leptospiral physiology and pathogenesis.

[Leptospirosis now-the centennial of the discovery of Weil's disease pathogen].

This year, 2014, marks the centennial of the discovery of Leptospira as the Weil's disease pathogen by Ryokichi Inada, Yutaka Ido and their colleagues. Leptospirosis is a worldwide zoonosis especially in countries with tropical and subtropical climates. Recently, the number of patients with leptospirosis dramatically decreased in Japan because public hygiene has greatly improved, the mechanization of agriculture was introduced, and farmers started using rubber boots and gloves while working in the field. The opportunities for percutaneous infection with Leptospira was reduced, but have not been totally eliminated in Japan.We previously reported a novel combination of five antimicrobial agents for selective isolation of Leptospira from contaminated samples. This cocktail, after being incorporated into Leptospira growth medium, inhibited the growth of contaminants and allowed successful detection of leptospires in environmental samples. We collected soil and environmental water and cultured them using this selective medium. It was revealed that not only saprophytic Leptospira but also pathogenic Leptospira are widely living in the environment. We hypothesized that soil serves as reservoir for Leptospira and infectious source for leptospirosis. In this review, we also discuss the Leptospira-rat-human relationship in the Philippines, natural defense of host against oral and percutaneous infection by Leptospira, the mechanism of jaundice in leptospirosis, and the development of immunochromatography-based methods for detection of leptospiral antigen in urine.

The characteristics of ubiquitous and unique Leptospira strains from the collection of Russian centre for leptospirosis.

BACKGROUND AND AIM: Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity of Leptospira in natural and anthropurgic foci was not clearly defined. METHODS: The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS). RESULTS: The most wide spread and prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmental Leptospira strain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species. CONCLUSIONS: Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions.

A filtration based technique for simultaneous SEM and TEM sample preparation for the rapid detection of pathogens.

Diagnostic electron microscopy for infectious diseases has the advantage that "everything" in the specimen can be observed, without a priori knowledge of the likely identity of the microorganisms present in the sample. The classical specimen preparation method used employs a droplet of sample, which allows particles to adsorb to a support film, and is subsequently negative stained. This "grid on drop" procedure has a sensitivity range of approximately 106 viruses per mL if no enrichment procedures are used. In the current investigation we present a novel use of filtration that allows us to detect viruses at concentrations as low as 102 viruses per mL. We present here methods based on filtration, in which total virus, and not virus concentration, is the limiting factor for detection. We show that filtration is more sensitive than conventional negative staining and can detect as few as 5 × 103 particles per sample.

Three-dimensional structures of pathogenic and saprophytic Leptospira species revealed by cryo-electron tomography.

Leptospira interrogans is the primary causative agent of the most widespread zoonotic disease, leptospirosis. An in-depth structural characterization of L. interrogans is needed to understand its biology and pathogenesis. In this study, cryo-electron tomography (cryo-ET) was used to compare pathogenic and saprophytic species and examine the unique morphological features of this group of bacteria. Specifically, our study revealed a structural difference between the cell envelopes of L. interrogans and Leptospira biflexa involving variations in the lipopolysaccharide (LPS) layer. Through cryo-ET and subvolume averaging, we determined the first three-dimensional (3-D) structure of the flagellar motor of leptospira, with novel features in the flagellar C ring, export apparatus, and stator. Together with direct visualization of chemoreceptor arrays, DNA packing, periplasmic filaments, spherical cytoplasmic bodies, and a unique "cap" at the cell end, this report provides structural insights into these fascinating Leptospira species.

Electron microscopic study of the effects of antimicrobial agents on the cellular architecture of Leptospira.

Morphological changes of Leptospira induced by ampicillin, streptomycin, or ciprofloxacin were studied by transmission electron microscopy after staining with uranyl acetate. Irregularities in coiling, loss of hooks, spherical swellings, membrane vesicle formation, disruption of outer membrane, exposure and detachment of flagella, penetration of dye, and lysis of cells were observed. These changes appeared in a time-dependent manner and appeared to be associated with the sites of action of antimicrobial agents. This is the first report delineating the action of aminoglycoside and new quinolone on the morphology of Leptospira cells.

Biofilm formation by saprophytic and pathogenic leptospires.

Leptospires exist as saprophytic organisms that are aquatic or as pathogens that are able to survive in water. Leptospirosis is transmitted to humans through environmental surface waters contaminated by the urine of mammals, usually rodents, which are chronically infected by pathogenic strains. The ecology of Leptospira spp. prompted us to evaluate if these spirochaetes were able to form biofilms. This study investigated the characteristics of biofilm development by both saprophytic and pathogenic Leptospira species using microscopic examinations and a polystyrene plate model. Biofilms were formed preferentially on glass and polystyrene surfaces. Electron microscopic images showed cells embedded in an extracellular matrix. The formation of such a biofilm is consistent with the life of saprophytic strains in water and may help pathogenic strains to survive in environmental habitats and to colonize the host.

Comparison of invasion of fibroblasts and macrophages by high- and low-virulence Leptospira strains: colonization of the host-cell nucleus and induction of necrosis by the virulent strain.

The infection cycle of low- and high-virulence strains of Leptospira interrogans was compared in fibroblasts and macrophages. L. interrogans serovar Lai strain Lai was used as a representative high-virulence strain, while L. interrogans serovars Pomona strain Luo was used as a low-virulence strain. L. biflexa serovar Patoc strain Patoc I, a nonparasitic strain of Leptospira, was used as a control. Both the high- and low-virulence strains could adhere to fibroblasts and macrophages using one or both ends of the spirochete, which was followed by phagocytosis of both strains. Both strains adhered more strongly to macrophages than fibroblasts. However, the high-virulence strain could invade the host-cell nucleus, while the low-virulence strain remained in phagosomes. The L. biflexa strain neither adhered to nor invaded either cell type. Both of the L. interrogans strains also induced cell death (mostly necrosis) of macrophages, whether or not the spirochetes were viable, suggesting that leptospiral virulence is unrelated to macrophage death. However, the high-virulence strain induced mainly necrosis in fibroblasts, while the low-virulence strain induced more apoptosis. Thus, the main feature distinguishing the two L. interrogans strains is the ability of the high-virulence strain to invade the host-cell nucleus and induce pro-inflammatory necrosis in fibroblasts.

Recurrent uveitis in horses: vitreal examinations with ultrastructural detection of leptospires.

This study documents the examination of 17 horses (both sexes, 3-18 years old) suffering from spontaneous equine recurrent uveitis (ERU). Vitreal samples obtained by pars plana vitrectomy were examined macroscopically and ultrastructurally, and in most cases also by cultural examination, by microscopic agglutination test (MAT) and by polymerase chain reaction. In 24% (4/17) of the animals, ultrastructural examination by electron microscopy revealed intact leptospiral bacteria in the vitreous. The leptospires were detected freely in the vitreous and also incorporated by a phagocyte. They were surrounded by a rim of proteinaceous material which was reduced around a phagocytosed leptospira. Ninety-four per cent (16/17) of the vitreal samples presented significant antibody levels in the MAT, mostly against leptospiral serovar Grippotyphosa. Seventy-five per cent (9/12) of bacterial culture examinations were positive for leptospira. Polymerase chain reaction was positive in all (16/16) examinations performed. Our findings support previous reports suggesting that leptospires play an important role in the pathogenesis of ERU. Interestingly, this study found leptospires after secondary and later acute episodes. A persistent leptospiral infection is therefore suggested as the cause of ERU.

[Adaptation of an immunohistochemistry protocol for the detection of Leptospira spp. in samples of formaldehyde-fixed tissue]. / Adaptación de un protocolo de inmunohistoquímica para la detección de Leptospira spp. en muestras de tejido fijado en formaldehído.

The immunohistochemistry technique was evaluated in tissue samples fixed in formaldehyde saline solution 10 % and included in paraffin to be used as a lab method allowing to identify leptospires in tissues. Samples obtained from the experimental inoculation of 8 guinea pigs carriers of L. interrogans Pomona isolated from a clinical case were used. The disease was reproduced in a lab model. The histologic sections of the kidneys of the animals inoculated were subjected to histopathological studies, immunofluorescence, Warthin-Starry stain, and immunohistochemistry technique using formaldehyde-fixed samples. This technique proved to be an efficient tool for the diagnosis of leptospirosis.

[Detection of leptospira in the vitreous body of horses without ocular diseases and of horses with equine recurrent uveitis (ERU) using transmission-electron microscopy]. / Darstellung von Leptospiren im Glaskörper augengesunder und an ERU erkrankter Pferde mittels Transmissions-Elektronenmikroskopie.

Equine recurrent uveitis (ERU) is caused by persistent intraocular leptospira, which appear to use the vitreous body as a refuge. The detection of leptospira in the vitreous body of horses with spontaneous ERU by histological methods has not yet been described. Thirty eight vitreous body samples from 36 horses with ERU (collected during vitrectomy), and 10 vitreous body samples obtained from 5 horses without ocular disease (control group) were examined by transmission electron microscopy. Prior to sample collection, 2 ml of a leptospira culture suspension were injected into the vitreous body of 2 eyes enucleated from horses of the control group. The detection of leptospira in samples, experimentally inoculated with these bacteria was uncomplicated; in vitreous body samples from horses with spontaneous ERU the detection was successful in only a few cases (3/38). The morphologically varying envelope of leptospira in vitreous body samples of horses which developed ERU spontaneously suggests the existence of a bacterial masquerade in vivo.

The scc spirochetal coiled-coil protein forms helix-like filaments and binds to nucleic acids generating nucleoprotein structures.

The analysis of the genome of Leptospira spp., a group of bacteria of the phylum of spirochetes with several unique evolutionary and morphological features, has allowed the identification of a gene encoding a coiled-coil protein, called Scc, which is completely unrelated to any other eukaryotic or prokaryotic protein. Since coiled-coil proteins are often key elements of the cytoskeleton, we analyzed the protein Scc, which is a 24-kDa protein composed of a N-terminal coiled-coil domain, a proline-rich intermediate domain, and an acidic tail. The gene scc is located in an operon which also contains the genes encoding the initiation factor IF3 and the two ribosomal proteins L20 and L35. In this study, we showed that the presence of the coiled-coil domain was responsible for the polymerization of Scc in helix-like structures, in an ATP-independent manner, in both Escherichia coli living cells and in vitro. Analysis of the Scc polymers by electron microscopy showed filaments with a width of 6 to 10 nm, similar to that of eukaryotic intermediate filaments. Scc was also found to bind both RNA and double-stranded DNA without detectable sequence specificity. By electron microscopy, we showed that Scc polymer assembly was affected by the presence of nucleic acids, giving rise to rod-shaped structures with a width ranging from 45 to 155 nm. Finally, Leptospira biflexa cells depleted in Scc form small colonies, but the morphology of their helicoidal cell body was not affected. These results provide the first insight into a unique DNA binding filament-forming coiled-coil protein that could play an important role in the subcellular architecture of the spirochetal microorganism.