Distribution of the leptospiral immunoglobulin-like (lig) genes in pathogenic Leptospira species and application of ligB to typing leptospiral isolates

Cerqueira, Gustavo M.; McBride, Alan J. A.; Picardeau, Mathieu; Ribeiro, Samuel G.; Moreira, Ângela Nunes; Morel, Viviane; Reis, Mitermayer Galvão dos; Ko, Albert I.; Dellagostin, Odir A.

doi:10.1099/jmm.0.009175-0

Cited by 50 publications

(45 citation statements)

References 51 publications

(76 reference statements)

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“…Furthermore, single-gene mutants of either ligA in L. interrogans serovar Manilae strain L495 or ligB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 did not reduce virulence in standard animal models and remained recoverable from animal tissue (25,26). The distribution and sequences of lig genes, including ligA, ligB, and ligC (27,28) Previous analyses demonstrated homology in the N-terminal repeat domains of LigA and LigB (encompassing 266 bp upstream of their start codon to 1,890 bp into ligA and ligB), with variation at the C-terminal region of LigB (22). As was previously suggested, LigA and LigB may work as a redundant system to ensure spirochete adhesion and survival within a mammalian host (25,29).…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…12 , 20 Several groups have reported using shorter sequences (£ 500 bp) in other genes to improve the discriminatory power over the 16S rRNA gene, including secY , 19 gyrB , 21 and ligB . 17 Alternative techniques such as…”

Section: Resultsmentioning

confidence: 99%

Monitoring Leptospira Strain Collections: The Need for Quality Control

Cerqueira

McBride²,

Queiroz

et al. 2010

The American Society of Tropical Medicine and Hygiene

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The purpose of this study was to perform a 16S sequence-based quality control of two Leptospira strain collections. 16S rRNA gene sequencing was used to verify two Leptospira reference collections provided by the World Health Organization and maintained at a reference laboratory for leptospirosis in Brazil. Among the 89 serovars evaluated, four conflicting strains were identified in one of the collections. Although 16S rRNA gene sequencing cannot identify Leptospira beyond the species level, it is suitable for the identification of contamination and quality control of leptospiral reference collections. This study highlights the importance of the availability of high-quality 16S rRNA sequences in public databases. In addition, it emphasizes the need for periodical verifications and quality control of Leptospira reference collections.

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“…The latter codes for a high-molecular-weight leptospiral immunoglobulin-like repeat (Lig) protein that was previously identified as a putative virulence factor in pathogenic Leptospira 9 . Furthermore, the ligB gene has been found in all pathogenic Leptospira species identified to date 10 . Our results show that the duplex endpoint PCR assay is rapid, as well as highly specific and sensitive for the simultaneous detection and differentiation of Leptospira strains.…”

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“…This gene has also been used in previous studies on the genus Leptospira 3,13 . However, to determine the pathogenicity of Leptospira strains, we designed primers that targeted the ligB gene, a decision that was based on previous typing of pathogenic Leptospira strains at the species level 9,10 , though only a few studies have used this gene as a marker for differentiating pathogenic and non-pathogenic Leptospira species 14,15 . Lig proteins, including LigA, LigB, and LigC, belong to a superfamily of bacterial immunoglobulin-like proteins 9 .…”

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A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains

Benacer

Zain

Lewis

et al. 2017

Rev. Soc. Bras. Med. Trop.

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Introduction: This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains. Methods: Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively. Results: The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 10 3 leptospires/ml in both spiked urine and water. Conclusions: Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

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Distribution of the leptospiral immunoglobulin-like (lig) genes in pathogenic Leptospira species and application of ligB to typing leptospiral isolates

Cited by 50 publications

References 51 publications

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

Monitoring Leptospira Strain Collections: The Need for Quality Control

A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains

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