A Putative Regulatory Genetic Locus Modulates Virulence in the Pathogen Leptospira interrogans

Eshghi, Azad; Bécam, Jérôme; Lambert, Ambroise; Sismeiro, Odile; Dillies, Marie‐Agnès; Jagla, Bernd; Wunder, Elsio A.; Ko, Albert I.; Coppée, Jean‐Yves; Goarant, Cyrille; Picardeau, Mathieu

doi:10.1128/iai.01803-14

Cited by 33 publications

(36 citation statements)

References 47 publications

(56 reference statements)

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“…DNA fragments containing the transcriptional fusions were released by KpnI and XhoI digestions and cloned into the corresponding sites of pAL614 (a generous gift from Gerald Murray, Monash University). The lpxD1 complementation construct was introduced by conjugation in the Manilae lpxD1 mutant strain as previously described (35), and complementation of the lpxD1 mutant strain was confirmed by using primers that PCR amplified a region of the spectinomycin resistance cassette and primers lpxD1F and lpxD1R, using genomic DNA as the template.…”

Section: Methodsmentioning

confidence: 99%

“…Leptospira strains were cultured in triplicate at 30°C in EMJH medium to a density of 1 ϫ 10 8 bacteria/ml, and a total of 10 10 bacteria of each strain from each replicate were used for RNA extraction and reverse transcription-quantitative real-time PCR (RT-qPCR), as previously described (35)(36)(37)(38). The following modifications were implemented for RT-qPCR: the primers used to quantify the genes were lpxD1f (5=-ATCCGAACGTTGTCATTGAA) and lpxD1r (5=-GATCACCGTATTCGCATGAA) to quantify lpxD1 mutant transcripts and lpxD2f (5=-TCATCCTTCTGCAAAGTTGG) and lpxD2r (5=-AACGCCGTCTTCCAAATAAG) to quantify lpxD2 mutant transcripts.…”

Section: Methodsmentioning

confidence: 99%

“…Leptospira burdens in kidneys and liver were determined by qPCR, as previously described (35,44), with the following modifications. The Leptospira Manilae wild-type, lpxD1 mutant, and lpxD1 complemented mutant strains were injected intraperitoneally into groups of 4 gerbils (10 4 bacteria per animal).…”

Section: Figmentioning

confidence: 99%

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Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

et al. 2015

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Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model. Members of the genus Leptospira are spirochete bacteria encompassing saprophytic and pathogenic species and are considered to be the most widespread zoonotic bacteria worldwide (1). Leptospira is the etiological agent of the disease leptospirosis, which in severe manifestations leads to hemorrhage in the lungs, meningitis, and liver and/or kidney failure (1). Leptospirosis is an emerging disease, and the worldwide annual occurrence is estimated to be over 1 million human cases, with a 5 to 20% mortality rate (2, 3). Leptospira cannot breach the host epidermal lining, and transmission requires direct contact of the bacteria with cuts or abrasions in the skin (4, 5). Rats and other rodent species serve as reservoir hosts for Leptospira, which colonizes the urinary systems of these animals (6). Leptospira is shed back into the environment through the urine of reservoir hosts and can persist in freshwater and soil until direct contact with an animal recommences an infection cycle (7,8).A prominent Leptospira feature is the ability to proliferate under significantly different environmental conditions. Other Gram-negative bacterial pathogens with this ability include species of the genera Escherichia (9), Salmonella (10), Yersinia (11), Vibrio (12), and Pseudomonas (13). The capacity of bacteria to adapt to disparate environments is likely imparted by numerous evolved strategies that probably include modi...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

et al. 2015

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“…Another TALE construct, named the TALE lig gene, was designed to anneal to the sequence 5=-TCCAATAAATCTTAAGAGA-3=, which is located in homologous promoter regions of ligA and ligB in L. interrogans 170 bp upstream of position ϩ1 (15). The TALE lig gene was inserted downstream of the B. burgdorferi flgB promoter (as described above), and the DNA fragment containing the fusion was released by ApaI and KpnI digestions (Thermo Scientific, Waltham, MA), gel purified (Qiagen, Venlo, Netherlands), and subcloned into the corresponding sites of pAL614 (16). Plasmid constructs (ptale lig ) were introduced into Leptospira strains by conjugation with E. coli, as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

Pappas

Picardeau

2015

Appl Environ Microbiol

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bLeptospirosis is a zoonotic disease that affects ϳ1 million people annually, with a mortality rate of >10%. Currently, there is an absence of effective genetic manipulation tools for targeted mutagenesis in pathogenic leptospires. Transcription activator-like effectors (TALEs) are a recently described group of repressors that modify transcriptional activity in prokaryotic and eukaryotic cells by directly binding to a targeted sequence within the host genome. To determine the applicability of TALEs within Leptospira spp., two TALE constructs were designed. First, a constitutively expressed TALE gene specific for the lacO-like region upstream of bgaL was trans inserted in the saprophyte Leptospira biflexa (the TALE ␤gal strain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALE ␤gal strain. Second, to study the role of LigA and LigB in pathogenesis, a constitutively expressed TALE gene with specificity for the homologous promoter regions of ligA and ligB was cis inserted into the pathogen Leptospira interrogans (TALE lig ). LigA and LigB expression was studied by using three independent clones: TALE lig1 , TALE lig2 , and TALE lig3 . Immunoblot analysis of osmotically induced TALE lig clones demonstrated 2-to 9-fold reductions in the expression levels of LigA and LigB, with the highest reductions being noted for TALE lig1 and TALE lig2 , which were avirulent in vivo and nonrecoverable from animal tissues. This study reconfirms galactosidase activity in the saprophyte and suggests a role for LigA and LigB in pathogenesis. Collectively, this study demonstrates that TALEs are effective at reducing the expression of targeted genes within saprophytic and pathogenic strains of Leptospira spp., providing an additional genetic manipulation tool for this genus. L eptospirosis, a bacterial infection transmitted by animal reservoirs, has emerged to become a major public health concern in much of the developing world. There are Ͼ1 million cases of severe leptospirosis reported each year, for which the mortality rate is Ͼ10% (1). As spirochetes, Leptospira spp., which include the causative agent of leptospirosis, differ considerably from other Gram-positive and Gram-negative bacteria. Progress in our understanding of the general biology and virulence mechanisms of pathogenic Leptospira strains has been slow and difficult. This is mainly due to the lack of adequate and efficient genetic tools (2). Genetic modifications of the pathogen are limited primarily to random transposon mutagenesis, and there are only a few examples of mutants obtained by targeted mutagenesis (3). Thus, there is a clear need for additional tools to develop genetic studies of Leptospira spp.The transcription activator-like effector (TALE) family forms a subset of proteins made by Xanthomonas bacterial species that are injected into plants to modulate host gene expression, with each effector directly binding a specific DNA target (4, 5). TALEs are composed of three domains: (i) a central ...

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“…The findings in our study reflect the total potential transporters available to respective Leptospira species. Other investigators have studied the transcriptional profiles of specific pathovars under various environmental conditions [97–99]. Future efforts will attempt to integrate transporter proteomic studies with transcriptomic studies and the bioinformatic analyses explored here.…”

Section: Discussionmentioning

confidence: 99%

Comparative genomic analyses of transport proteins encoded within the genomes of Leptospira species

Buyuktimkin

Saier

2015

Microbial Pathogenesis

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Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, L. interrogans serovar Lai str. 56601 and L. borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, L. biflexa serovar Patoc str. ‘Patoc 1 (Ames)’. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity was accompanied by progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles.

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A Putative Regulatory Genetic Locus Modulates Virulence in the Pathogen Leptospira interrogans

Cited by 33 publications

References 47 publications

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

Comparative genomic analyses of transport proteins encoded within the genomes of Leptospira species

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