Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species

Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Clayton, VIC 3800, Australia.
Infection and immunity (Impact Factor: 3.73). 02/2009; 77(2):810-6. DOI: 10.1128/IAI.01293-08
Source: PubMed


Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of
these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon
insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon
mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot
spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed
few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated
using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus
validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding
of the biology of L. interrogans.

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Available from: Julio Croda
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    • "The assay is detailed in the flow chart in Fig. 1(b). Ninety-five mutants were selected for analysis from a library constructed previously (Murray et al., 2009a). Eighty-two mutants were detected in input or output pools by PCR (Table S1, available in JMM Online). "
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    ABSTRACT: The molecular basis for leptospirosis remains poorly understood, with no efficient methods available for screening libraries of mutants for attenuation. We have analysed the attenuation of leptospiral transposon mutants in vivo using a high-throughput method by infecting animals with pooled sets of transposon mutants. A total of 95 mutants was analysed by this method in the hamster model of acute infection, and one mutant was identified as attenuated (M1233, lb058 mutant). All virulence factors identified in Leptospira to date have been characterised in the acute model of infection, neglecting the carrier host. To address this, a BALB/c mouse colonisation model was established. The lb058 mutant and two mutants defective in LPS synthesis were colonisation-deficient in the mouse model. By applying the high-throughput screening method a further five colonisation-deficient mutants were identified for the mouse model; these included two mutants in genes encoding proteins with a predicted role in iron uptake (LB191/HbpA and LB194). Two attenuated mutants had transposon insertions in either la0589 or la2786 (encoding proteins of unknown function). The final attenuated mutant had an unexpected deletion of five genes (la0969-la0975) at the point of transposon insertion. This is the first description of defined, colonisation-deficient mutants in a carrier host for Leptospira. These mutants were either not attenuated, or only weakly attenuated, in the hamster model of acute leptospirosis, thus illustrating different factors that may be required in the carrier and acute models of leptospiral infection. High-throughput screening can reduce the number of animals used in virulence studies and increase the capacity to screen mutants for attenuation, thereby enhancing the likelihood of detecting unique virulence factors. A comparison of virulence factors required in the carrier and acute models of infection will help to unravel colonisation and dissemination mechanisms of leptospirosis.
    Full-text · Article · Jun 2013 · Journal of Medical Microbiology
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    • "The results of phage display showed LipL32 to interact with chloride channel accessory 2, glycoprotein VI, scavenger receptor expressed by endothelial cell isoform I, coronin 2A, laminin alpha 5, collagen XX and prostaglandin receptor EP1 of the host cells (Chaemchuen et al., 2011). Though large number of studies provided strong evidence for LipL32 in the role of pathogenesis, the unexpected results of Murray et al. (2009a, b) revealed by transposon mutagenesis studies in Leptospira interrogans denied the fact of LipL32 in pathogenesis. These concluding results arises several questions on the role of this abundant gene which is nearly 40 000 copies per cell. "
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    ABSTRACT: Abstract Pathogenic Leptospira species are important human and animal pathogen that causes leptospirosis, with more than half a million cases reported annually but little is known regarding the true incidence of leptospirosis due to the limitations in diagnosis. Proteins embedded in the outer membrane are found to be prime drug targets due to its key role as receptors for cellular communication and gatekeepers for iron and substrate transport across cell membranes. The major key issues to be addressed to overcome the disease burden of leptospirosis are: need to identify the genes that turn on in vivo; development of rapid diagnostic methods to facilitate the early diagnosis and to develop a universal vaccine. Recent whole genome sequencing of Leptospira species and development of in silico analysis tools have led to the identification of a large number of leptospiral virulence genes, metabolic pathways and surface protein secretion systems that represent potential new targets for the development of anti-leptospiral drug, vaccine and diagnostic strategies. This review surveys the different types of outer membrane proteins (OMPs) of Leptospira and combines all the novel features of OMPs reported till date and put forth some views for future research.
    Full-text · Article · May 2013 · Critical Reviews in Microbiology
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    • "In conclusion, by using L. biflexa as a surrogate host, we have shown that Lig proteins are factors involved in the attachment to fibronectin, fibrinogen, and laminin and to host cells and can act as microbial surface components recognizing host extracellular matrix proteins. Although important advances in the genetic system of the pathogen L. interrogans have been made in the last years [5,7], this bacterium remains poorly transformable and few mutants have been fully characterized [3]. We believe that L. biflexa can serve as a model bacterium for investigating the function of additional leptospiral pathogenesis mechanisms. "
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    ABSTRACT: In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.
    Full-text · Article · Jun 2011 · BMC Microbiology
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