A Model System for Studying the Transcriptomic and Physiological Changes Associated with Mammalian Host-Adaptation by Leptospira interrogans Serovar Copenhageni

Abstract

Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, 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. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of “core” housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants.

Cyclic di-GMP Modulates Gene Expression in Lyme Disease Spirochetes at the Tick-Mammal Interface To Promote Spirochete Survival during the Blood Meal and Tick-to-Mammal Transmission

“…Prior to library construction, rRNA was removed from each pooled sample using a Ribo-Zero rRNA removal core kit (Epicentre, Madison, WI). Paired-end Illumina TruSeq libraries were generated as previously described (50). Postrun data analyses were performed by the use of the RNA-Rocket program (http: //rnaseq.pathogenportal.org/) (51).…”

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

“…No large-scale microarray data or extensive proteomic analyses have been reported for the saprophyte, although Thongboonkerd and colleagues compared total protein lysate of L. biflexa to multiple pathogenic strains to identify potential virulence factors (10). In contrast, extensive microarray and proteomic studies have been performed on various host-associated species (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). These limitations in our basic knowledge of L. biflexa reduce its utility as a model organism, since there are no known protein markers for the outer membrane and no abundantly synthesized proteins have been reported.…”

Screening of a Leptospira biflexa Mutant Library To Identify Genes Involved in Ethidium Bromide Tolerance

“…Gene LEPBIa2665 (ybiT) was originally annotated as an ABC transporter, but it lacks a necessary membrane-spanning domain (36). The YbiT protein is conserved among all sequenced pathogenic Leptospira strains, and it is among the 100 most highly expressed L. interrogans genes during growth within dialysis membrane chambers implanted in rats (19,48), suggesting its importance in Leptospira biology. Gene LEPBIa2665 has close orthologues in the ART ABC2 family, which is involved in cell regulatory processes and antibiotic resistance (34).…”

“…Protein YbiT (LEPBI2665) is involved in tolerance to three antibiotics with different mechanisms of action, not only in L. biflexa, but also in pathogenic L. interrogans. Determination of its precise function would be of a great interest, since its homologue, LIC10538, is highly expressed in animal models of infection (48).…”