Project description:The overall goal of these experiments was to determine how human endothelial cells respond to pathogenic Leptospira interrogans. Leptospira interrogans causes leptospirosis, the most widespread zoonotic infection in the world. A hallmark of leptospirosis is widespread endothelial damage, which in severe cases leads to hemorrhage. In these experiments, we infected two endothelial cell lines with pathogenic Leptospira interrogans serovar Canicola strain Ca12-005, and as controls, with the non-pathogenic Leptospira biflexa serovar Patoc strain Pfra. As additional controls, uninfected cells were also included in the analyses.
Project description:The overall goal of these experiments was to determine how human endothelial cells respond to pathogenic Leptospira interrogans. Leptospira interrogans causes leptospirosis, the most widespread zoonotic infection in the world. A hallmark of leptospirosis is widespread endothelial damage, which in severe cases leads to hemorrhage. In these experiments, we infected two endothelial cell lines with pathogenic Leptospira interrogans serovar Canicola strain Ca12-005, and as controls, with the non-pathogenic Leptospira biflexa serovar Patoc strain Pfra. As additional controls, uninfected cells were also included in the analyses.
Project description:Leptospirosis is a global zoonotic, neglected tropical disease. Interestingly, a high level of species specificity (both bacteria and host) plays a major role in the severity of disease presentation which can vary from asymptomatic to multi-organ failure. Pathogenic Leptospira colonize the kidneys of infected individuals and are shed in urine into the environment where they can survive until they are contracted by another host. This study looks at two strains of L. borgpetersenii, HB203 and JB197 which are genetically very similar, and identical by serotyping as serovar Hardjo, yet HB203 causes a chronic infection in the hamster while JB197 causes organ failure and mortality. To better characterize bacterial factors causing different disease outcomes, we examined the gene expression profile of these strains in the context of temperatures that would reflect natural Leptospira life cycles (environmentally similar 29oC and 37oC which is more indicative of host environment). We found vast differences in gene expression both between the strains and within strains between temperatures. Characterization of the transcriptome of L. borgpetersenii serovar Hardjo strains JB197 and HB203 provides insights into factors that can determine acute versus chronic disease in the hamster model of infection. Additionally, these studies highlight strain to strain variability within the same species, and serovar, at different growth temperatures, which needs to be considered when serovars are selected and propagated for use as bacterin vaccines used to immunize domestic animal species.
Project description:Leptospirosis, caused by bacteria of the genus Leptospira, is a zoonotic disease affecting humans, companion animals, and all major livestock species. Typical propagation of the highly fastidious Leptospira borgepetesenii serovar Hardjo is limited to 29°C. However, newer culture media formulations now facilitate isolation and propagation at 37°C, a temperature that more closely emulates in vivo conditions and is hypothesized to regulate the expression of virulence factors during host infection. Since protein expression by leptospires is temperature dependent, and therefore the proteome of bacterin vaccines can differ whether grown at 37°C compared to 29°C, we compared the proteome of strains of Leptospira borgpetersenii serovar Hardjo at each temperature; two well-established strains that causes acute (strain JB197) or chronic asymptomatic disease (strain HB203) in the hamster challenge model of leptospirosis and two more recently isolated strains designated TC129 and TC273 (both of which cause chronic asymptomatic disease in the hamster). We found proteomic expression differences within strains propagated at the routine temperature of 29°C, and compared to the newly achieved culture temperature of 37°C. Results highlight significant differential protein expression, including virulence factors, amongst identical serovars of L. borgpetersenii when propagated at 29oC, the collective variation of which can be diminished when propagated at 37oC. Collectively, there is increasingly more evidence available to suggest bacterin vaccine design would benefit from consideration of strains employed, and potential effects of growth temperature related to specific behavior of pathogens in vaccine composition.
Project description:The overall goal of these experiments was to determine how human endothelial cells respond to pathogenic Leptospira interrogans. Leptospira interrogans causes leptospirosis, the most widespread zoonotic infection in the world. A hallmark of leptospirosis is widespread endothelial damage, which in severe cases leads to hemorrhage. In these experiments, we infected two endothelial cell lines with pathogenic Leptospira interrogans serovar Canicola strain Ca12-005, and as controls, with the non-pathogenic Leptospira biflexa serovar Patoc strain Pfra. As additional controls, uninfected cells were also included in the analyses. The cell line used fhere was a microvascular endothelial line, HMEC (Ades et al, 1992. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 99:683-690); due to loss of the original analysis files, only raw data files are provided. Infection times were performed at a multiplicity of infection (# bacteria/endothelial cell) of 10 for either 1 hour or 3 hours, after which RNA was harvested and reverse transcribed. Labeled cDNAs were used to probe HEEBO arrays purchased from Microarrays Inc. (Nashville, TN). In each of three biological replicate experiments, for each time point, three comparisons were made. First, the L. interrogans-infected cells were compared to the L. biflexa-infected cells. Second, the L. Interrogans-infected cells were compared to the uninfected cells. Third, the L. biflexa-infected cells were compared to the uninfected cells. A second endothelial cell line,
Project description:The overall goal of these experiments was to determine how human endothelial cells respond to pathogenic Leptospira interrogans. Leptospira interrogans causes leptospirosis, the most widespread zoonotic infection in the world. A hallmark of leptospirosis is widespread endothelial damage, which in severe cases leads to hemorrhage. In these experiments, we infected two endothelial cell lines with pathogenic Leptospira interrogans serovar Canicola strain Ca12-005, and as controls, with the non-pathogenic Leptospira biflexa serovar Patoc strain Pfra. As additional controls, uninfected cells were also included in the analyses. The cell line used was Ea.hy926, a macrovascular line (Edgell, C. J.,et al. 1990. In vitro Cell. & Dev. Biol. 26:1167-1172, and Edgell, C. J., et al. 1983. Proc. Natl. Acad. Sci. 80:3734-3737). Infection times were performed at a multiplicity of infection (# bacteria/endothelial cell) of 10 for either 1 hour or 3 hours, after which RNA was harvested and reverse transcribed. Labeled cDNAs were used to probe HEEBO arrays purchased from Microarrays Inc. (Nashville, TN). In each of three biological replicate experiments, for each time point, three comparisons were made. First, the L. interrogans-infected cells were compared to the L. biflexa-infected cells. Second, the L. Interrogans-infected cells were compared to the uninfected cells. Third, the L. biflexa-infected cells were compared to the uninfected cells. A second endothelial cell line, HMEC (Ades et al, 1992. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 99:683-690), which is of microvascular origin, was also used; raw data files are provided separately.
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.
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.