Project description:Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometry complemented with two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. A total of 563 proteins were identified in this study. Altered expression of 65 proteins, including upregulation of the L. interrogans virulence factor Loa22 and 5 novel proteins with homology to virulence factors found in other pathogens, was observed between the comparative conditions. Immunoblot analyses confirmed upregulation of 5 of the known or putative virulence factors in L. interrogans exposed to the in vivo-like environmental conditions. Further, ELISA analyses using serum from patients with leptospirosis and immunofluorescence studies performed on liver sections derived from L. interrogans-infected hamsters verified expression of all but one of the identified proteins during infection. These studies, which represent the first documented comparative global proteome analysis of Leptospira, demonstrated proteome alterations under conditions that mimic in vivo infection and allowed for the identification of novel putative L. interrogans virulence factors.
Project description:Infectious diseases are the leading causes of death worldwide. Hence, there is a need to develop new antimicrobial agents. Traditional method of drug discovery is time consuming and yields a few drug targets with little intracellular information for guiding target selection. Thus, focus in drug development has been shifted to computational comparative genomics for identifying novel drug targets. Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. Availability of L. interrogans serovars and human genome sequences facilitated to search for novel drug targets using bioinformatics tools. The genome sequence of L. interrogans serovar Copenhageni has 5,124 genes while that of serovar Lai has 4,727 genes. Through subtractive genomic approach 218 genes in serovar Copenhageni and 158 genes in serovar Lai have been identified as putative drug targets. Comparative genomic approach had revealed that 88 drug targets were common to both the serovars. Pathway analysis using the Kyoto Encyclopaedia of Genes and Genomes revealed that 66 targets are enzymes and 22 are non-enzymes. Sixty two common drug targets were predicted to be localized in cytoplasm and 16 were surface proteins. The identified potential drug targets form a platform for further investigation in discovery of novel therapeutic compounds against Leptospira.
Project description:L. interrogans, a causative agent of leptospirosis, can survive in the environment for lengthy periods of time in between infection of mammalian hosts. In order to identify genes involved in survival in the early spirochetemic phase of infection, we performed a transcriptional analysis of L. interrogans serovar Copenhageni upon exposure to serum in comparison with EMJH medium. Analysis used RNA derived from serum- and EMJH-treated L. interrogans serovar Copenhageni as experimental and control samples, respectively. The samples were composed of 3 biological replicates with dye swap for each replicate, resulting in 6 arrays. Direct comparisons were made between arrays of experimental and control samples using raw data pulled from two different channels for data analysis.
Project description:L. interrogans, a causative agent of leptospirosis, can survive in the environment for lengthy periods of time in between infection of mammalian hosts. In order to identify genes involved in survival in the early spirochetemic phase of infection, we performed a transcriptional analysis of L. interrogans serovar Copenhageni upon exposure to serum in comparison with EMJH medium. Overall design: Analysis used RNA derived from serum- and EMJH-treated L. interrogans serovar Copenhageni as experimental and control samples, respectively. The samples were composed of 3 biological replicates with dye swap for each replicate, resulting in 6 arrays. Direct comparisons were made between arrays of experimental and control samples using raw data pulled from two different channels for data analysis.
Project description:Bacteria activate a regulatory network in response to the challenges imposed by DNA damage to genetic material, known as the SOS response. This system is regulated by the RecA recombinase and by the transcriptional repressor lexA. Leptospira interrogans is a pathogen capable of surviving in the environment for weeks, being exposed to a great variety of stress agents and yet retaining its ability to infect the host. This study aims to investigate the behavior of L. interrogans serovar Copenhageni after the stress induced by DNA damage. We show that L. interrogans serovar Copenhageni genome contains two genes encoding putative LexA proteins (lexA1 and lexA2) one of them being potentially acquired by lateral gene transfer. Both genes are induced after DNA damage, but the steady state levels of both LexA proteins drop, probably due to auto-proteolytic activity triggered in this condition. In addition, seven other genes were up-regulated following UV-C irradiation, recA, recN, dinP, and four genes encoding hypothetical proteins. This set of genes is potentially regulated by LexA1, as it showed binding to their promoter regions. All these regions contain degenerated sequences in relation to the previously described SOS box, TTTGN 5CAAA. On the other hand, LexA2 was able to bind to the palindrome TTGTAN10TACAA, found in its own promoter region, but not in the others. Therefore, the L. interrogans serovar Copenhageni SOS regulon may be even more complex, as a result of LexA1 and LexA2 binding to divergent motifs. New possibilities for DNA damage response in Leptospira are expected, with potential influence in other biological responses such as virulence.
Project description:A new repetitive DNA element was identified in an isolate of Leptospira interrogans serovar copenhageni from a patient in Salvador, Brazil. A Sau3A genomic library from this strain was constructed and screened for repetitive DNA elements. An insert of 438 bp (Rep1) from one library clone hybridized to multiple chromosomal DNA fragments resolved electrophoretically after digestion with BamHI, HindIII, and MfeI. A single oligonucleotide primer, designated iRepl, was designed to generate multiple PCR amplicons of various electrophoretic mobilities in a PCR typing method. The method distinguished strains belonging to the eight pathogenic and three saprophytic species of the genus Leptospira. Clinical isolates obtained during urban epidemics between 1996 and 1998 in Salvador, Brazil, were analyzed by this PCR method. Although the iRep1 primer was unable to discriminate strains among L. interrogans serovar copenhageni isolates, it was able to differentiate strains belonging to different species and serogroups of Leptospira identified in Salvador. This PCR-based method may provide a faster and less expensive alternative to serologic tests used in reference laboratories.
Project description:BACKGROUND: Leptospirosis is a world-widely distributed zoonosis. Humans become infected via exposure to pathogenic Leptospira spp. from contaminated water or soil. The availability of genomic sequences of Leptospira interrogans serovar Lai and serovar Copenhageni opened up opportunities to identify genetic diversity among different pathogenic strains of L. interrogans representing various kinds of serotypes (serogroups and serovars). RESULTS: Comparative genomic hybridization (CGH) analysis was used to compare the gene content of L. interrogans serovar Lai strain Lai with that of other 10 L. interrogans strains prevailed in China and one identified from Brazil using a microarray spotted with 3,528 protein coding sequences (CDSs) of strain Lai. The cutoff ratio of sample/reference (S/R) hybridization for detecting the absence of genes from one tested strain was set by comparing the ratio of S/R hybridization and the in silico sequence similarities of strain Lai and serovar Copenhageni strain Fiocruz L1-130. Among the 11 strains tested, 275 CDSs were found absent from at least one strain. The common backbone of the L. interrogans genome was estimated to contain about 2,917 CDSs. The genes encoding fundamental cellular functions such as translation, energy production and conversion were conserved. While strain-specific genes include those that encode proteins related to either cell surface structures or carbohydrate transport and metabolism. We also found two genomic islands (GIs) in strain Lai containing genes divergently absent in other strains. Because genes encoding proteins with potential pathogenic functions are located within GIs, these elements might contribute to the variations in disease manifestation. Differences in genes involved in O-antigen biosynthesis were also identified for strains belonging to different serogroups, which offers an opportunity for future development of genomic typing tools for serological classification. CONCLUSION: CGH analyses for pathogenic leptospiral strains prevailed in China against the L. interrogans serovar Lai strain Lai CDS-spotted microarrays revealed 2,917 common backbone CDSs and strain specific genes encoding proteins mainly related to cell surface structures and carbohydrated transport/metabolism. Of the 275 CDSs considered absent from at least one of the L. interrogans strains tested, most of them were clustered in the rfb gene cluster and two putative genomic islands (GI A and B) in strain Lai. The strain-specific genes detected via this work will provide a knowledge base for further investigating the pathogenesis of L interrogans and/or for the development of effective vaccines and/or diagnostic tools.
Project description:Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.
Project description:Over 230 serovars of Leptospira interrogans have been identified; however few have been completely characterised. The aim of this study was to characterise the proteome of serovar Canicola and to compare this against the serovars of Copenhageni and Pomona. 2D-LC/MS analysis identified 1653 Leptospira proteins in serovar Canicola; 60 of these proteins were common to Copenhageni and Pomona, 16 of which are known to be immunogenic. This study provides the first reported proteome for serovar Canicola and suggests that proteomic comparison of different serovars could be used as a tool for identification of novel target molecules for vaccine development.
Project description:Leptospira interrogans is the etiological agent of leptospirosis, a globally distributed zoonotic disease. Human infection usually occurs through skin exposure with water and soil contaminated with the urine of chronically infected animals. In this study, we aimed to quantitatively characterize the survival of Leptospira interrogans serovar Copenhageni in environmental matrices. We constructed laboratory microcosms to simulate natural conditions and determined the persistence of DNA markers in soil, mud, spring water and sewage using a quantitative PCR (qPCR) and a propidium monoazide (PMA)-qPCR assay. We found that L. interrogans does not survive at high concentrations in the tested matrices. No net growth was detected in any of the experimental conditions and in all cases the concentration of the DNA markers targeted decreased from the beginning of the experiment following an exponential decay with a decreasing decay rate over time. After 12 and 21 days of incubation the spiked concentration of 106L. interrogans cells/ml or g decreased to approximately 100 cells/ml or g in soil and spring water microcosms, respectively. Furthermore, culturable L. interrogans persisted at concentrations under the limit of detection by PMA-qPCR or qPCR for at least 16 days in soil and 28 days in spring water. Altogether, our findings suggest that the environment is not a multiplication reservoir but a temporary carrier of L. interrogans Copenhageni, although the observed prolonged persistence at low concentrations may still enable the transmission of the disease.IMPORTANCE Leptospirosis is a zoonotic disease caused by spirochetes of the genus Leptospira that primarily affects impoverished populations worldwide. Although leptospirosis is transmitted by contact with water and soil, little is known about the ability of the pathogen to survive in the environment. In this study, we quantitatively characterized the survival of L. interrogans in environmental microcosms and found that although it cannot multiply in water, soil or sewage, it survives for extended time periods (days to weeks depending on the matrix). The survival parameters obtained here may help to better understand the distribution of pathogenic Leptospira in the environment and improve the predictions of human infection risks in areas where such infections are endemic.