Dataset Information

Isolation and propagation of leptospires at 37?°C directly from the mammalian host.

ABSTRACT: The causative agent of leptospirosis includes multiple serovars and species of pathogenic leptospires that are excreted via urine from reservoir hosts of infection. Primary isolation takes weeks to months, and is limited to semi-solid media at 28-30?°C. Here we present an alternative media formulation, HAN, compared to commercially available EMJH and the more specialized T80/40/LH media formulations, in semi-solid and liquid compositions, for the primary isolation of two diverse species and serovars of pathogenic leptospires directly from host kidney tissue. All three media types supported the isolation and propagation of L. interrogans serovar Copenhageni strain IC:20:001 in semi-solid media at 29?°C. However, only HAN and T80/40/LH supported the growth of L. borgpetersenii serovar Hardjo strain HB15B203 at 29?°C. In addition, HAN supported primary isolation at 37?°C. Both T80/40/LH and HAN supported primary isolation of strain IC:20:001 in liquid media at 29?°C but only HAN supported growth of strain HB15B203 in liquid media, at both 29 and 37?°C. HAN media supports the primary isolation of fastidious pathogenic leptospires directly from infected host tissue at either 29 or 37?°C: this formulation represents a more defined media for the continued optimization of growth factors required to support the primary isolation of the large and diverse range of species and serovars within the genus Leptospira circulating within domestic and wild animal populations.

SUBMITTER: Hornsby RL

PROVIDER: S-EPMC7296004 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Isolation and propagation of leptospires at 37 °C directly from the mammalian host.

Hornsby Richard L RL Alt David P DP Nally Jarlath E JE

Scientific reports 20200615 1

The causative agent of leptospirosis includes multiple serovars and species of pathogenic leptospires that are excreted via urine from reservoir hosts of infection. Primary isolation takes weeks to months, and is limited to semi-solid media at 28-30 °C. Here we present an alternative media formulation, HAN, compared to commercially available EMJH and the more specialized T80/40/LH media formulations, in semi-solid and liquid compositions, for the primary isolation of two diverse species and sero ...[more]

PMID: 32541841

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Project description:Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed (p < 0.05, fold change >1.25 or < -1.25) across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with trimethyllysine and acetyllysine occurs to a different degree in response to mammalian host signals encountered during persistent renal colonization. These results provide novel insights into differential protein and PTMs present in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.

Project description:A large body of work has demonstrated that leptospires regulate and modify gene expression in response to environmental cues, as encountered during disease transmission, including changes in temperature, osmolarity, concentration of iron, the presence of serum, and interaction with macrophages. However, since leptospires are not readily amenable to genetic manipulation, the functional and biological significance of these differentially expressed genes often remains unclear. More recently, it is been shown that proteins, in response to changing environmental conditions, can be modified further by specific post-translational modifications. Indeed, both saprophytic and pathogenic leptospires have comprehensive systems to modify proteins. The paucibacillary nature of spirochetal infections, combined with the challenges associated with acquiring pathogens free from contaminating host proteins makes the study of these bacteria in a mammalian host-adapted state inherently difficult. As an alternative approach, we developed a model in which leptospires are cultivated in a dialysis membrane chamber (DMC) implanted within the peritoneal cavity of rats, where they are exposed to the environmental cues encountered during host infection. This strategy has been successfully applied to compare the transcriptome of L. interrogans cultivated within DMC with that of leptospires grown under standard in vitro conditions. In addition to determining the relative expression levels of ‘‘core’’ housekeeping genes under both growth conditions, we identified 166 genes that were differentially-expressed by L. interrogans in response to mammalian host signals. The proteome of DMC (dialysis membrane chamber) cultivated leptospires was compared to that of in vivo derived leptospires and with leptospires cultivated in vitro at 30°C or 37°C by 2-dimensional difference in gel electrophoresis (2-D DIGE). Our analysis indicates that the abundance of leptospire-proteins is modulated the expression of a range of proteins which are differentially expressed in response to mammalian host signals, and not temperature alone . In addition, we confirm that in several proteins there is a change in the presence of modify the expression of the post-translational modifications trimethyllysine and acetyllysine in response to environmental cues encountered during persistent renal colonization in a reservoir host of infection. These results provide novel insights in the proteome level changes, including to differential protein and post-translational modifications, expressed in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.

Dataset Information

Isolation and propagation of leptospires at 37?°C directly from the mammalian host.

Publications

Isolation and propagation of leptospires at 37 °C directly from the mammalian host.

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