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The LetA/S two-component system is essential for the survival of Legionella pneumophila in water

ABSTRACT: Surviving the nutrient-poor aquatic environment for extended periods of time is important for transmission of various water-borne pathogen to the host, including Legionella pneumophila (Lp). Lp is a leading cause of community-acquired and nosocomial pneumonia called Legionnaires’ disease. The remarkable ability of the bacterium to survive in water for periods ranging from several months to years under starvation conditions alludes to regulatory pathways that mediate adaptation to the water environment. In the present study, we investigated a potential role for the LetA/LetS signal transduction system in the successful survival of Lp in water. During infection of host cells, the LetA/LetS two-component system controls the transition from the replicative phase to the transmissive phase in response to nutrient deprivation. In accordance with previous work, the letS mutant used in the present study is defective for pigment production and contributed to cell size reduction in the post-exponential phase. LetS also contributed to cell size reduction when Lp was exposed to water. Importantly, absence of the sensor kinase resulted in a significantly lower survival rate in water at various temperatures, as well as an increase sensitivity to heat shock. Transcriptomic analysis indicated that a general transcriptomic downshift of major pathways is orchestrated by LetA/LetS upon water exposure leading to better survival, suggesting a potential link with the stringent response. However, the expression of the LetA/S regulated small regulatory RNAs RsmY and RsmZ was not changed in a relAspoT mutant, which indicates that the stringent response and the LetA/S response are two distinct regulatory systems important for the survival of Lp in water.

ORGANISM(S): Legionella pneumophila subsp. pneumophila str. Philadelphia 1

PROVIDER: GSE98743 | GEO | 2018/05/09

REPOSITORIES: GEO

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Project description:Legionella pneumophila (Lp) is an opportunistic pathogen and its survival in water is critical for human infection. Therefore, identifying the genes of Lp that are required for survival in water may help devise strategies to prevent Legionella outbreaks. In this study, we exposed Lp in rich medium and in an artificial freshwater medium (Fraquil) for 2, 6 and 24 hours to uncover the global transcriptomic changes of Lp in water. The repression of major metabolic pathways, such as division, transcription and translation, suggests that Lp enters a dormant state in water. The induction of the flagellar associated genes (flg, fli and mot), enhance entry genes (enh) and some Icm/Dot effectors suggests that Lp may be waiting to establish intracellular replication in suitable host. Moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that Lp may be more tolerant to environmental stresses in water. Indeed, Lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than those cultured in rich medium. Apart from this, the gene bdhA involved in the degradation of the intracellular energy storage compound poly-hydroxybutyrate is highly expressed in water. Further characterization shows that bdhA is positively regulated by RpoS during short-term exposure to water. The deletion mutant of bdhA had a survival defect in water at 37°C, demonstrating that this gene is important for maintaining the long-term survivorship of Lp in water. Other identified genes highly induced upon exposure to water could also be necessary for Lp to survive in water. Legionella pneumophila Philadelphia-1 strain JR32 was grown in AYE broth at 25°C shaking to OD600 of 1 in triplicate. Samples were taken for analysis; this is the control. Then the cultures were washed three times in Fraquil and resuspended in Fraquil to an OD600 of 1 and transfered to vessels of bioreactior (Biostat Q-plus). Samples were taken after 2h, 6h and 24h.

Project description:In the intracellular pathogen Brucella spp., the activation of the stringent response, a global regulatory network providing rapid adaptation to a variety of growth-affecting stress conditions such as nutrient deficiency, is essential for replication in the host. A single, bi-functional enzyme Rsh catalyzes synthesis and hydrolysis of the alarmone (p)ppGpp, responsible for differential gene expression under stringent conditions. cDNA microarray analysis allowed characterization of the transcriptional profiles of the B. suis 1330 wild-type and rsh mutant in a synthetic minimal medium, partially mimicking the nutrient-poor environment of the intramacrophagic vacuole. A total of 379 genes (11.6% of the genome) were differentially expressed in a rsh-dependent manner, of which 52% were up-regulated and 48% were down-regulated. The pleiotropic character of the response was confirmed, as the genes encoded factors belonging to various functional groups, comprising an important number of transcriptional regulators, cell envelope proteins, stress factors, transport systems, and energy metabolism proteins. Several virulence genes were under the positive control of (p)ppGpp. Methionine was the only amino acid whose biosynthesis was absolutely dependent on stringent response in B. suis. The study illustrated the complexity of the processes involved in adaptation to nutrient starvation, and contributed to a better understanding of the correlation between stringent response and Brucella virulence. Most interestingly, it clearly indicated (p)ppGpp-dependent cross-talk between at least three stress responses playing a central role in Brucella adaptation to the host: nutrient, oxidative, and low-oxygen stress. Two-condition experiment, wild-type against rsh mutant. Biological replicates: 4 wild-type, 4 rsh mutant, independently grown in minimal medium (under nutrient starvation condition) and harvested. One replicate per array. Please note that Channel 1 (Cy3) on each hybridization was a universal reference (Uref) used as a reference point to merge two (2 channel arrays) into a virtual 2 channel array with wild-type versus rsh mutant.

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The LetA/S two-component system is essential for the survival of Legionella pneumophila in water

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