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Deletion of TolC orthologs in Francisella tularensis identifies roles in multidrug resistance and virulence.

ABSTRACT: The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia. Interest in this zoonotic pathogen has increased due to its classification as a category A agent of bioterrorism, but little is known about the molecular mechanisms underlying its virulence, and especially what secretion systems and virulence factors are present. In this study, we characterized two genes in the F. tularensis genome, tolC and a gene we term ftlC, whose products have high homology with the Escherichia coli TolC protein. TolC functions as the outer membrane channel component for both type I secretion and multidrug efflux systems. We constructed deletion mutations of these genes in the F. tularensis live vaccine strain by allelic replacement. Deletion of either tolC or ftlC caused increased sensitivity to various antibiotics, detergents, and dyes, indicating both genes are involved in the multidrug resistance machinery of F. tularensis. Complementation of the deletion mutations in trans restored drug resistance. Neither tolC nor ftlC was required for replication of the live vaccine strain in murine bone marrow-derived macrophages. However, deletion of tolC, but not ftlC, caused a significant attenuation of virulence in a mouse model of tularemia that could be complemented by addition of tolC in trans. Thus, tolC is a critical virulence factor of F. tularensis in addition to its role in multidrug resistance, which suggests the presence of a functional type I secretion system.

SUBMITTER: Gil H

PROVIDER: S-EPMC1568944 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Deletion of TolC orthologs in Francisella tularensis identifies roles in multidrug resistance and virulence.

Gil Horacio H Platz Gabrielle J GJ Forestal Colin A CA Monfett Michael M Bakshi Chandra Shekhar CS Sellati Timothy J TJ Furie Martha B MB Benach Jorge L JL Thanassi David G DG

Proceedings of the National Academy of Sciences of the United States of America 20060814 34

The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia. Interest in this zoonotic pathogen has increased due to its classification as a category A agent of bioterrorism, but little is known about the molecular mechanisms underlying its virulence, and especially what secretion systems and virulence factors are present. In this study, we characterized two genes in the F. tularensis genome, tolC and a gene we term ftlC, whose products have high homology with the Esch ...[more]

PMID: 16908853

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Project description:The alarmone ppGpp is a critical regulator of virulence gene expression in Francisella tularensis In this intracellular pathogen, ppGpp is thought to work in concert with the putative DNA-binding protein PigR and the SspA protein family members MglA and SspA to control a common set of genes. MglA and SspA form a complex that interacts with RNA polymerase (RNAP), and PigR functions by interacting with the RNAP-associated MglA-SspA complex. Prior work suggested that ppGpp indirectly exerts its regulatory effects in F. tularensis by promoting the accumulation of polyphosphate in the cell, which in turn was required for formation of the MglA-SspA complex. Here we show that in Escherichia coli, neither polyphosphate nor ppGpp is required for formation of the MglA-SspA complex but that ppGpp promotes the interaction between PigR and the MglA-SspA complex. Moreover, we show that polyphosphate kinase, the enzyme responsible for the synthesis of polyphosphate, antagonizes virulence gene expression in F. tularensis, a finding that is inconsistent with the notion that polyphosphate accumulation promotes virulence gene expression in this organism. Our findings identify polyphosphate kinase as a novel negative regulator of virulence gene expression in F. tularensis and support a model in which ppGpp exerts its positive regulatory effects by promoting the interaction between PigR and the MglA-SspA complex.IMPORTANCE In Francisella tularensis, MglA and SspA form a complex that associates with RNA polymerase to positively control the expression of key virulence genes. The MglA-SspA complex works together with the putative DNA-binding protein PigR and the alarmone ppGpp. PigR functions by interacting directly with the MglA-SspA complex, but how ppGpp exerts its effects was unclear. Prior work indicated that ppGpp acts by promoting the accumulation of polyphosphate, which is required for MglA and SspA to interact. Here we show that formation of the MglA-SspA complex does not require polyphosphate. Furthermore, we find that polyphosphate antagonizes the expression of virulence genes in F. tularensis Thus, ppGpp does not promote virulence gene expression in this organism through an effect on polyphosphate.

Dataset Information

Deletion of TolC orthologs in Francisella tularensis identifies roles in multidrug resistance and virulence.

Publications

Deletion of TolC orthologs in Francisella tularensis identifies roles in multidrug resistance and virulence.

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