Spirochetes Of Leptospira Research Articles

Leptospiral endostatin-like protein A is a bacterial cell surface receptor for human plasminogen.

The spirochete Leptospira interrogans is a highly invasive pathogen of worldwide public health importance. Studies from our laboratories and another have demonstrated that L. interrogans can acquire host plasminogen on its surface. Exogenous plasminogen activators can then convert bound plasminogen into the functionally active protease plasmin. In this study, we extend upon those observations and report that leptospiral endostatin-like protein A (LenA) binds human plasminogen in a dose-dependent manner. LenA-plasminogen interactions were significantly inhibited by the lysine analog xi-aminocaproic acid, suggesting that the lysine-binding sites on the amino-terminal kringle portion of the plasminogen molecule play a role in the binding. Previous studies have shown that LenA also binds complement regulator factor H and the extracellular matrix component laminin. Plasminogen competed with both factor H and laminin for binding to LenA, which suggests overlapping ligand-binding sites on the bacterial receptor. Finally, LenA-bound plasminogen could be converted to plasmin, which in turn degraded fibrinogen, suggesting that acquisition of host-derived plasmin by LenA may aid bacterial dissemination throughout host tissues.

High-coverage proteome analysis reveals the first insight of protein modification systems in the pathogenic spirochete Leptospira interrogans

Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira that infects humans and a wide range of animals. By combining computational prediction and high-accuracy tandem mass spectra, we revised the genome annotation of Leptospira interrogans serovar Lai, a free-living pathogenic spirochete responsible for leptospirosis, providing substantial peptide evidence for novel genes and new gene boundaries. Subsequently, we presented a high-coverage proteome analysis of protein expression and multiple posttranslational modifications (PTMs). Approximately 64.3% of the predicted L. interrogans proteins were cataloged by detecting 2 540 proteins. Meanwhile, a profile of multiple PTMs was concurrently established, containing in total 32 phosphorylated, 46 acetylated and 155 methylated proteins. The PTM systems in the serovar Lai show unique features. Unique eukaryotic-like features of L. interrogans protein modifications were demonstrated in both phosphorylation and arginine methylation. This systematic analysis provides not only comprehensive information of high-coverage protein expression and multiple modifications in prokaryotes but also a view suggesting that the evolutionarily primitive L. interrogans shares significant similarities in protein modification systems with eukaryotes.

Bilateral facial palsy associated with leptospirosis

Leptospirosis is a zoonosis of worldwide occurrence caused by the spirochete Leptospira interrogans. It is an acute feverish disease with a broad clinical spectrum and follows a characteristic biphasic course. Bilateral facial palsy is a rare clinical condition and the differential diagnosis of its causes is extensive. The objective of this exploratory study, presented as a case report, is to describe the occurrence of bilateral facial palsy as an unusual manifestation of leptospirosis. This suggestion should not be overlooked when analyzing the causes for bilateral facial palsy, and should be considered with other possible differential diagnoses, some of which are potentially fatal.

Acute Disseminated Encephalomyelitis After Leptospirosis

We report a case of acute disseminated encephalomyelitis after infection by the spirochete Leptospira interrogans and review the few cases of acute disseminated encephalomyelitis and central nervous system disorders described in literature. The high prevalence of leptospirosis in developing countries and the possibility of acute disseminated encephalomyelitis, per se, highlights the importance of complete investigation for diagnosis and early treatment, leading to a better prognosis with reduction of morbidity and mortality rates.

Leptospira interrogans Endostatin-Like Outer Membrane Proteins Bind Host Fibronectin, Laminin and Regulators of Complement

The pathogenic spirochete Leptospira interrogans disseminates throughout its hosts via the bloodstream, then invades and colonizes a variety of host tissues. Infectious leptospires are resistant to killing by their hosts' alternative pathway of complement-mediated killing, and interact with various host extracellular matrix (ECM) components. The LenA outer surface protein (formerly called LfhA and Lsa24) was previously shown to bind the host ECM component laminin and the complement regulators factor H and factor H-related protein-1. We now demonstrate that infectious L. interrogans contain five additional paralogs of lenA, which we designated lenB, lenC, lenD, lenE and lenF. All six genes encode domains predicted to bear structural and functional similarities with mammalian endostatins. Sequence analyses of genes from seven infectious L. interrogans serovars indicated development of sequence diversity through recombination and intragenic duplication. LenB was found to bind human factor H, and all of the newly-described Len proteins bound laminin. In addition, LenB, LenC, LenD, LenE and LenF all exhibited affinities for fibronectin, a distinct host extracellular matrix protein. These characteristics suggest that Len proteins together facilitate invasion and colonization of host tissues, and protect against host immune responses during mammalian infection.

Leptospirosis vaccines

Leptospirosis is a serious infection disease caused by pathogenic strains of the Leptospira spirochetes, which affects not only humans but also animals. It has long been expected to find an effective vaccine to prevent leptospirosis through immunization of high risk humans or animals. Although some leptospirosis vaccines have been obtained, the vaccination is relatively unsuccessful in clinical application despite decades of research and millions of dollars spent. In this review, the recent advancements of recombinant outer membrane protein (OMP) vaccines, lipopolysaccharide (LPS) vaccines, inactivated vaccines, attenuated vaccines and DNA vaccines against leptospirosis are reviewed. A comparison of these vaccines may lead to development of new potential methods to combat leptospirosis and facilitate the leptospirosis vaccine research. Moreover, a vaccine ontology database was built for the scientists working on the leptospirosis vaccines as a starting tool.

Acute renal failure after a holiday in the tropics

A 20-year-old, previously healthy woman, presented with high fever, headache and myalgia 3 days after her return from a holiday in Southeast Asia. Laboratory data on admission demonstrated a pronounced increase in plasma creatinine, marked thrombocytopenia and moderately elevated liver aminotransferases. After having ruled out malaria, dengue fever was primarily suspected and supportive intravenous fluid therapy was initiated. Still, 1 day after admission, platelet counts dropped even further and she became anuric although she did not appear hypovolemic. On day 2 after admission, urine production commenced spontaneously and the patient slowly recovered. All laboratory test results had returned to normal approximately 2 months later. Serological analysis for dengue fever was negative. It turned out that the patient had been trekking in the jungle while in Thailand and we, therefore, analyzed serology for Leptospira spirochetes which was clearly positive. The patient was diagnosed with leptospirosis which is a serious condition associated with a high mortality when complicated by acute renal failure. Differential diagnoses in patients with acute renal failure and tropical infections are reviewed. The importance of early recognition of leptospirosis, and prompt treatment with antibiotics in suspected cases, is emphasized.

Improvement in Motion Efficiency of the Spirochete Brachyspira pilosicoli in Viscous Environments

Spirochetes are unique among swimming bacteria in terms of their lack of external flagella. They actively move in viscous environments, and, surprisingly, the swimming speed of the spirochete Leptospira interrogans has been reported to increase with viscosity in methylcellulose solutions. Many researchers consider that the presence of a loose, quasi-rigid network formed by linear polymer molecules is related to this strange phenomenon. One of the authors has proposed a theory that expresses this idea mathematically and successfully explains the speed properties of an externally flagellated bacterium in viscous environments. This theory predicts that the ratio of swimming speed to wave frequency ( v/ f ratio, motion efficiency in a sense) increases with viscosity. In this study, we demonstrated a new method of measuring the swimming speed and wave frequency of spirochetes and the motion characteristics of a swine intestinal spirochete, Brachyspira pilosicoli strain NK1f, measured in viscous environments. Several sets of swimming speed and wave frequency data were simultaneously derived from an animation obtained by our method. The v/ f ratio of NK1f displayed a tendency to increase with increasing viscosity, suggesting the validity of the above-mentioned theory. Improvement of motion efficiency is at least one of the factors that maintain spirochete motility in viscous environments.

Regulation of complement activation at the C3-level by serum resistant leptospires

Leptospires are spirochetes that are transmitted to humans through contacts with wild or domestic animals or via an exposure to contaminated soil or water. In this study we have compared the serum-sensitivity of five pathogenic strains of leptospires ( L. interrogans) to an environmental isolate (strain Patoc). Different levels of sensitivities to human serum were seen. Interestingly, the most sensitive strain was the non-pathogenic Patoc strain. The fully and intermediately resistant strains have been isolated from human patients. Testing was performed in the absence of specific antibodies, and killing was found to be dependent on the complement system. The serum sensitive Patoc strain was killed in human serum within minutes, whereas the most resistant strains tolerated serum up to 4 h. We also tested the deposition of the complement components C3, C5, C6, C8 and C5b-9 to the surfaces of the sensitive and resistant strains of Leptospira by immunofluorescence microscopy and ELISA. C3 was deposited on both the sensitive and resistant strains, but the terminal complement components were detected only on the surface of the complement-sensitive strain. The complement resistant and intermediate strains were found to bind more factor H from human serum than the complement sensitive strain. Thus, binding of this major alternative complement pathway inhibitor is related to serum resistance in Leptospira spirochetes.

The spirochetal chpK-chromosomal toxin–antitoxin locus induces growth inhibition of yeast and mycobacteria

Toxin–antitoxin systems encoded by bacterial plasmids and chromosomes typically consist of a toxin that inhibits growth of the host cell and a specific antitoxin. In this report, the chpK gene from the chromosomal toxin–antitoxin locus of the spirochete Leptospira interrogans was studied in both prokaryotic and eukaryotic systems. Cloning of the the spirochetal chpK gene into a mycobacterial expressing vector led to dramatic reductions of transformation efficiency in both Mycobacterium smegmatis and Mycobacterium bovis BCG. However, few mycobacterial transformants were obtained. This result could be due to plasmid structural modifications leading to disruption of chpK expression, suggesting that L. interrogans ChpK is highly toxic for mycobacteria. Presence of the L. interrogans chpK gene was also found to inhibit cell growth of the yeast Saccharomyces cerevisiae. These results show that ChpK possesses a broad activity against both prokaryotes and eukaryotes, suggesting that the cellular target of the toxin is conserved in these organisms.

Genetic evidence for the existence of two pathways for the biosynthesis of methionine in the Leptospira spp

There are two major pathways for methionine biosynthesis: the enterobacterial type transsulfuration pathway and the sulfhydrylation pathway as previously identified in the spirochete Leptospira meyeri. Sequence analysis of the L. meyeri metYX locus allows the identification of a third gene, called metW, which encodes a protein exhibiting similarities with homologs in many organisms belonging to the α-, β-, and γ-subdivisions of proteobacteria. The metW, metX and metY genes of L. meyeri were disrupted by a resistance cassette by homologous recombination. While the L. meyeri metX mutant shows methionine auxotrophy, the metY mutant (as well as the metW and metYmetW mutants) conserves methionine prototrophy, suggesting the presence of additional route(s) which may bypass the direct sulfhydrylation pathway. In addition, a L. interrogans gene, called metZ, was found to complement an Escherichia coli metB mutant, further suggesting that the transsulfuration pathway is also present in Leptospira spp.

Leptospirosis in India and the rest of the world.

Leptospirosis is an acute anthropo-zoonotic infection of worldwide significance caused by spirochaete Leptospira interrogans which has 23 serogroups and >200 serovars. Various factors influencing the animal activity, suitability of the environment for the survival of the organism and behavorial and occupational habits of human beings can be the determinants of incidence and prevalence of the disease. The disease was considered inconsequential till recently, but it is emerging as an important public health problem during the last decade or so due to sudden upsurge in the number of reported cases and outbreaks. Since isolation rate of the microorganism from clinical specimens is low due to prior indiscriminate use of antibiotics, serological techniques remain the cornerstone of diagnosis.

Construction and complementation of the first auxotrophic mutant in the spirochaete Leptospira meyeri.

In bacteria, the first reaction of the tryptophan biosynthetic pathway involves the conversion of chorismate and glutamine to anthranilate by the action of anthranilate synthase, which is composed of the alpha (trpE gene product) and beta (trpG gene product) subunits. In this study, the tryptophan biosynthetic gene trpE of the spirochaete Leptospira meyeri was interrupted by a kanamycin-resistance cassette by homologous recombination. The trpE double cross-over mutant was not able to grow on solid or in liquid EMJH medium. In contrast, the trpE mutant showed a wild-type phenotype when tryptophan or anthranilate was added to the media, therefore showing that disruption of the L. meyeri trpE gene resulted in tryptophan auxotrophy. The authors have also characterized a second selectable marker that allows the construction of a spectinomycin-resistant L. meyeri-E. coli shuttle vector and the functional complementation of the L. meyeri trpE mutant.

Ocular leptospirosis.

Uveitis is a well-known late complication of systemic leptospirosis, a zoonotic disease caused by the water-borne spirochete Leptospira. Although it is one of the world's most widespread febrile diseases, it remains underdiagnosed, mainly because of protean manifestations, lack of awareness, and nonavailability of laboratory support. Systematic collection of published literature was conducted using Medline, the Cochrane library, and bibliographies of retrieved reports. Articles directly applicable to ocular leptospirosis and current reports on the epidemiology, basic research, clinical presentations, and management of leptospirosis were reviewed. Changing trends in risk factors and an expanding spectrum of ocular and systemic findings have been reported. Molecular research on leptospirosis has shown remarkable progress; several rapid diagnostic modalities are currently under study. Awareness of this entity is absolutely essential to arrive at an accurate diagnosis and to prevent its potential reversible and irreversible ocular complications.

Characterization of the Leptospira interrogans S10-spc-alpha operon.

A ribosomal protein gene cluster from the spirochaete Leptospira interrogans was characterized. This locus is homologous to the Escherichia coli S10, spc, and alpha operons. Analysis of L. interrogans RNA showed that the ribosomal protein genes within this cluster are co-transcribed, thus forming an operon. Two transcription initiation sites were mapped by primer extension, upstream of fus, the first gene in this cluster, and sequences from this region provided promoter activity in E. coli. Transcription terminates near a predicted stem-loop structure following rplQ, the last gene in the cluster. These data suggest that two promoters upstream of fus direct transcription of this 17.5-kb ribosomal protein gene cluster. Comparison of the L. interrogans S10-spc-alpha cluster to homologous loci from Borrelia burgdorferi and Treponema pallidum provided evidence that this region of the genome underwent several rearrangements during spirochaete evolution.

Characterization of the Leptospira interrogans S10- spc-α operon

A ribosomal protein gene cluster from the spirochaete Leptospira interrogans was characterized. This locus is homologous to the Escherichia coli S10, spc, and α operons. Analysis of L. interrogans RNA showed that the ribosomal protein genes within this cluster are co-transcribed, thus forming an operon. Two transcription initiation sites were mapped by primer extension, upstream of fus, the first gene in this cluster, and sequences from this region provided promoter activity in E. coli. Transcription terminates near a predicted stem-loop structure following rplQ, the last gene in the cluster. These data suggest that two promoters upstream of fus direct transcription of this 17.5-kb ribosomal protein gene cluster. Comparison of the L. interrogans S10- spc-α cluster to homologous loci from Borrelia burgdorferi and Treponema pallidum provided evidence that this region of the genome underwent several rearrangements during spirochaete evolution.

Low-stringency PCR with diagnostically useful primers for identification of Leptospira serovars

Primers proposed for the diagnosis of the pathogenic spirochete Leptospira spp. (C. Gravekamp, H. V. D. Kemp, M. Franzen, D. Carrington, G.J. Schoone, G.J.J.M. Van Eys, C. O. R. Everard, R.A. Hartskeel, and W.J. Terpstra, J. Gen. Microbiol. 139:1691-1700, 1993) have been found to produce complex serovar-specific patterns under low-stringency PCR conditions. Such patterns obtained by low-stringency PCR, which maintain the specific band as an internal control, offer, an approach to the standardized identification of Leptospira serovars in clinical laboratories.

Nucleotide Sequence Analysis of IS1533 from Leptospira borgpetersenii: Identification and Expression of Two IS-Encoded Proteins

The nucleotide sequence of IS1533, an insertion sequence-like element cloned from the spirochete Leptospira borgpetersenii, was determined. IS1533 contains imperfect terminal inverted repeats (IVR) of 31 bp flanking a 1402-bp internal sequence. A putative target sequence was identified, and insertion may result in duplication of 2 bp. The internal sequence has a single open reading frame (ORF). IS1533 encodes two proteins (43.5 and 41 kDa) initiating alternatively at either the first or the second AUG codons of the ORF. These proteins are related to a recently recognized family of IS-encoded transposases and bacterial recombinases, all which share a region of homology with the active site of the HIV reverse transcriptase. The IS1533-encoded proteins were expressed in Escherichia coli . Both the 43.5- and 41-kDa proteins bound IS1533 DNA probes in a Southwestern blot assay. These data suggest that one or both proteins function during transposition of IS1533.

Leptospira genomes are modified at 5'-GTAC

Genomic DNAs of 14 strains from seven species of the spirochete Leptospira were resistant to cleavage by the restriction endonuclease RsaI (5'-GTAC). A modified base comigrating with m4C was detected by chromatography. Genomic DNAs from other spirochetes, Borrelia group VS461, and Serpulina strains were not resistant to RsaI digestion. Modification at 5'-GTAm4C may occur in most or all strains of all species of Leptospira but not in all genera of spirochetes. Genus-wide DNA modification has rarely been observed in bacteria.

Cloning of Genes for a Hemolytic Factor of Leptospira interrogans Serovar Autumnalis Strain Congo 21‐543

A DNA fragment encoding a hemolytic factor was cloned from the parasitic spirochete Leptospira interrogans serovar autumnalis strain Congo 21-543. Initial clones were isolated by screening a genomic library in pBR322 in Escherichia coli for hemolytic activity. Hemolytic activity was coded by a 4.5 kilobase BamHI-HindIII fragment. Southern hybridization with DNAs from other strains of Leptospira using this gene as a probe showed that DNAs from non-parasitic strains failed to hybridize with the probe, whereas those from all parasitic strains tested had the sequence which hybridize to the probe.