The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment

Hu, Wei Lin; Pappas, Christopher J.; Zhang, Jun Jie; Yang, You Yun; Yan, Jie; Picardeau, Mathieu; Yang, Xianlin

doi:10.1128/aem.02377-16

Cited by 15 publications

(31 citation statements)

References 46 publications

(34 reference statements)

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“…A typical σ54-binding site was identified in the promoter regions of three genes encoding for putative lipoproteins (LMANv2_200027/LIC12503 and LMANv2_290065/LIC11935) and the ammonium transporter AmtB (LMANv2_310003/LIC10441) at both 30 and 37°C (Supplementary Table 8). Our previous EMSA results show that both recombinant σ54 and EbpA proteins are able to bind a 50-bp oligonucleotide encoding the predicted −24/−12 promoter regions of these three genes, indicating that the σ54-binding motif of L. interrogans , [TA][TG[CG][TAC]A <6 bp>T[GT][GC]CA, closely resembles the E. coli motif (Hu et al, 2017). The alternative sigma factor σ28 (sigma F) is known to regulate flagellar genes in most bacteria and predicted σ28-binding sites at position −35 and −10 from the TSS in L. interrogans promoter sequences comprise at least four genes coding for components of the endoflagellum (LMANv2_260046/FlaA1, LMANv2_290016/FlaB1, LMANv2_590023/FlaB4) and the flagellin-specific chaperone FliS (LMANv2_10030).…”

Section: Resultsmentioning

confidence: 92%

“…To date, few experimentally proven TFBS have been described (Cuñé et al, 2005; Morero et al, 2014; Hu et al, 2017) in the literature and promoter prediction algorithms and E. coli consensus sequences of DNA motifs are not applicable to the Leptospira genome. Here, we annotated 2865 and 2866 pTSSs in L. interrogans at 30 and 37°C, respectively.…”

Section: Discussionmentioning

confidence: 99%

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Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans

Zhukova

Fernandes

Hugon

et al. 2017

Front. Cell. Infect. Microbiol.

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Leptospira are emerging zoonotic pathogens transmitted from animals to humans typically through contaminated environmental sources of water and soil. Regulatory pathways of pathogenic Leptospira spp. underlying the adaptive response to different hosts and environmental conditions remains elusive. In this study, we provide the first global Transcriptional Start Site (TSS) map of a Leptospira species. RNA was obtained from the pathogen Leptospira interrogans grown at 30°C (optimal in vitro temperature) and 37°C (host temperature) and selectively enriched for 5′ ends of native transcripts. A total of 2865 and 2866 primary TSS (pTSS) were predicted in the genome of L. interrogans at 30 and 37°C, respectively. The majority of the pTSSs were located between 0 and 10 nucleotides from the translational start site, suggesting that leaderless transcripts are a common feature of the leptospiral translational landscape. Comparative differential RNA-sequencing (dRNA-seq) analysis revealed conservation of most pTSS at 30 and 37°C. Promoter prediction algorithms allow the identification of the binding sites of the alternative sigma factor sigma 54. However, other motifs were not identified indicating that Leptospira consensus promoter sequences are inherently different from the Escherichia coli model. RNA sequencing also identified 277 and 226 putative small regulatory RNAs (sRNAs) at 30 and 37°C, respectively, including eight validated sRNAs by Northern blots. These results provide the first global view of TSS and the repertoire of sRNAs in L. interrogans. These data will establish a foundation for future experimental work on gene regulation under various environmental conditions including those in the host.

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Section: Resultsmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans

Zhukova

Fernandes

Hugon

et al. 2017

Front. Cell. Infect. Microbiol.

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“…Little is known on survival strategies and physiological mechanisms used by leptospires [119]. However, strategies of positive interactions with environmental microbiota and biofilm formation are now well-known for other bacteria.…”

Section: Discussionmentioning

confidence: 99%

A systematic review of Leptospira in water and soil environments

Bierque¹,

Thibeaux²,

Girault³

et al. 2020

PLoS ONE

140

151

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Background Leptospirosis, caused by pathogenic Leptospira, is a zoonosis of global distribution. This infectious disease is mainly transmitted by indirect exposure to urine of asymptomatic animals via the environment. As human cases generally occur after heavy rain, an emerging hypothesis suggests that rainfall re-suspend leptospires together with soil particles. Bacteria are then carried to surface water, where humans get exposed. It is currently assumed that pathogenic leptospires can survive in the environment but do not multiply. However, little is known on their capacity to survive in a soil and freshwater environment. Methods We conducted a systematic review on Leptospira and leptospirosis in the environment in order to collect current knowledge on the lifestyle of Leptospira in soil and water. In total, 86 scientific articles retrieved from online databases or institutional libraries were included in this study. Principals findings/significance This work identified evidence of survival of Leptospira in the environment but major gaps remain about the survival of virulent species associated with human and animal diseases. Studies providing quantitative data on Leptospira in soil and water are a very recent trend, but must be interpreted with caution because of the uncertainty in the species identification. Several studies mentioned the presence of Leptospira in soils more frequently than in waters, supporting the hypothesis of the soil habitat and dispersion of Leptospira with resuspended soil particles during heavy rain. In a near future, the growing use of high throughput sequencing will offer new opportunities to improve our understanding of the habitat of Leptospira in the environment. This better insight into the risk of leptospirosis will allow implementing efficient control measures and prevention for the human and animal populations exposed.

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“…In another study, a high-affinity binding site for the phosphorylated form of the L. biflexa HemR response regulator, which regulates heme metabolism, was enriched from a highly randomized oligonucleotide library by several rounds of affinity selection with the phosphorylated protein (35). In a recent study, the L. interrogans enhancer-binding protein EBP and the alternative sigma factor RpoN were shown to bind to various promoters by gel shift analysis (36). For L. interrogans genes such as sph2 for which the trans-acting regulatory factor remains to be identified, the pRAT724 gfp reporter plasmid will facilitate the identification of additional cis-acting sequences affecting gene expression.…”

Section: Discussionmentioning

confidence: 99%

cis -Acting Determinant Limiting Expression of Sphingomyelinase Gene sph2 in Leptospira interrogans, Identified with a gfp Reporter Plasmid

Matsunaga

Haake

2018

Appl Environ Microbiol

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Many strains of the spirochete Leptospira interrogans serovar Pomona express the osmotically inducible sphingomyelinase gene sph2 at much higher levels than strains from other serovars. We developed a new green fluorescent protein (GFP) reporter plasmid to examine sph2 gene expression determinants. The vector enables the fusion of the test promoter to the ribosome-binding site and coding region of gfp. We fused the sph2 promoters from the L. interrogans serovar Lai strain 56601 and from the L. interrogans serovar Pomona strain LC82-25 to gfp to examine the molecular determinants of differential sph2 expression between the two strains. Similar to what was observed with the native sph2 genes, the introduction of the plasmids into the Lai 56601 strain resulted in near background levels of gfp expression from the Lai sph2 promoter, while the expression from the Pomona sph2 promoter was high. The expression of both fusions increased at physiologic levels of osmolarity achieved by adding sodium chloride to the culture medium. We examined the role of a 17-bp upstream element found in all L. interrogans strains expressing low basal levels of sph2 and missing from Pomona strains that express sph2 at high levels. When the 17-bp sequence present upstream of the Lai sph2 promoter was deleted or scrambled, the fusion expression increased substantially. Conversely, the insertion of the 17-bp sequence upstream of the Pomona sph2 promoter diminished fusion expression. In contrast, the removal of an insertion sequence-like element that is found only in the Pomona sph2 upstream sequence had no effect on the expression from the Pomona sph2 fusion in the Lai strain. These findings demonstrate the utility of the gfp reporter plasmid in analyzing gene expression in L. interrogans. IMPORTANCE Genetic tools are needed to examine gene expression in the pathogen Leptospira interrogans. We developed a reporter plasmid that replicates in L. interrogans with green fluorescent protein (GFP) as the readout of promoter activity. We demonstrated an application of the new reporter plasmid by identifying an upstream element responsible for the poor basal expression of the sph2 sphingomyelinase gene in an L. interrogans serovar Lai strain. This new tool is useful for the discovery of the molecular determinants of L. interrogans gene expression.

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The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment

Cited by 15 publications

References 46 publications

Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans

Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans

A systematic review of Leptospira in water and soil environments

cis -Acting Determinant Limiting Expression of Sphingomyelinase Gene sph2 in Leptospira interrogans, Identified with a gfp Reporter Plasmid

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