Random Insertional Mutagenesis of<i>Leptospira interrogans</i>, the Agent of Leptospirosis, Using a<i>mariner</i>Transposon

Bourhy, Pascale; Louvel, Hélène; Girons, Isabelle Saint; Picardeau, Mathieu

doi:10.1128/jb.187.9.3255-3258.2005

Cited by 96 publications

(117 citation statements)

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“…Leptospira interrogans serovar Manilae strain L495 was culture maintained in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (25,26) at 30°C. L. interrogans transposon mutagenesis has been described previously (27)(28)(29), and the L495 transposon mutants used in this study were obtained from an in-house-maintained library of mutants. The mutant strains were culture maintained as described above for the parent strain.…”

Section: Methodsmentioning

confidence: 99%

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

et al. 2015

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bLeptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.

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Section: Methodsmentioning

confidence: 99%

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

et al. 2015

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“…Transposon mutagenesis was conducted as described previously (1,13). In brief, the leptospires were grown to late log phase and made electrocompetent by repeated washing in ultrapure water.…”

Section: Methodsmentioning

confidence: 99%

“…-like promoter from a flagellar biogenesis cluster of B. burgdorferi and is active in a number of different bacteria, including L. interrogans (1,5).…”

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confidence: 99%

“…Recently, an L. interrogans mutagenesis method was developed with a mariner transposon, TnSC189 (1,12). This is currently the only efficient means for constructing defined leptospiral mutants.…”

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confidence: 99%

“…A very slow rate of growth (Leptospira spp. take up to 4 weeks to form colonies on plates [1]) makes growth-dependent outputs such as CFU counts and minimal dilution slow and prone to overgrowth by contaminants. The unusual morphology of leptospires makes identification by light microscopy difficult, while a low maximum culture density makes estimation of the concentration by measurement of the optical density (OD) of limited use.…”

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Use of Luminescent Leptospira interrogans for Enumeration in Biological Assays

Murray¹,

King

Srikram³

et al. 2010

J Clin Microbiol

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Rapid and reliable in vitro methods for the detection of pathogenic leptospires, such as Leptospira interrogans, are lacking. The present study investigated the use of luminescence to replace the existing enumeration techniques. Transposon TnSC189 was modified to incorporate the luxCDABE cassette from Photorhabdus luminescens and was used to construct luminescent Leptospira spp. There was a linear relationship between luminescence and cell number, with the theoretical detection limit being less than 10 4 leptospires. A comparison of enumeration by a standard method (counting by dark-field microscopy) and enumeration by luminescence was conducted with luminescent L. interrogans. There was a good correlation between the two methods of enumeration (R 2 ‫؍‬ 0.766), although variation in the luminescence early and late in growth phase reduced the degree of correlation. To demonstrate the utility of luminescence as a viability and cell number reporter, in vitro assays, including MIC determination, an extracellular matrix binding experiment, and a complement killing experiment, were conducted. In each case, the results obtained by luminescence matched those obtained by traditional means with high correlations (binding assay R 2 ‫؍‬ 0.916, complement killing assay R 2 ‫؍‬ 0.988). A strain expressing the luxCDABE transposon retained virulence in the hamster model of infection. Despite some variation in luminescence as a result of the growth phase or the particular assay conditions, enumeration by luminescence was found to be a quick, reliable, and highly sensitive method for the in vitro detection of leptospires that has the potential to replace more time-consuming methods of enumeration.

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Leptospira

Adler¹,

Moctezuma²

2010

Pathogenesis of Bacterial Infections in Animals

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Random Insertional Mutagenesis ofLeptospira interrogans, the Agent of Leptospirosis, Using amarinerTransposon

Cited by 96 publications

References 20 publications

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

Use of Luminescent Leptospira interrogans for Enumeration in Biological Assays

Leptospira

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