Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexaconfers enhanced adhesion to cultured cells and fibronectin

Figueira, Cláudio Pereira; Croda, Júlio; Choy, Henry A.; Haake, David A.; Reis, Mitermayer Galvão dos; Ko, Albert I.; Picardeau, Mathieu

doi:10.1186/1471-2180-11-129

Cited by 56 publications

(76 citation statements)

References 51 publications

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“…We used for further characterization nine mutants in which we inactivated genes encoding exoproteins that previous studies suggested are involved in the infection process (LigA [54] and LruB [55][56][57]) or that were detected in high abundance in supernatants in the present study. In vitro growth rates identified two genes, lruB (LIC10713) and the gene for O-acetylhomoserine (thiol) lyase (LIC11852), that, when inactivated, resulted in significantly reduced in vitro growth of L. interrogans (Table 5).…”

Section: Validation Of Methodology and Overview Of Wcps And Exoproteinsmentioning

confidence: 99%

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

et al. 2015

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bLeptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.

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Section: Validation Of Methodology and Overview Of Wcps And Exoproteinsmentioning

confidence: 99%

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

et al. 2015

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“…Interestingly, previous studies have also demonstrated LigB-and Sph2-mediated binding of host homeostatic proteins (38,39), suggesting a role for these proteins in leptospiral attachment to host cells. In addition to…”

Section: Fig 2 the Lb139mentioning

confidence: 99%

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

et al. 2014

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Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139 ؊ mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

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“…The TALE ␤gal gene was digested with NdeI and XbaI, purified, and inserted between the NdeI and SpeI restriction sites of pCRPromFlgB (13) to generate a transcriptional fusion between the TALE gene and the Borrelia burgdorferi flgB promoter. The DNA fragment containing the transcriptional fusions was released by PvuII digestion and cloned into the SmaI site of the E. coli-L. biflexa shuttle vector pMAT (14) to generate plasmid ptale ␤gal , which replicates in L. biflexa.…”

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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Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexaconfers enhanced adhesion to cultured cells and fibronectin

Cited by 56 publications

References 51 publications

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

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

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

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