Direct repeat‐mediated deletion of a type IV pilin gene results in major virulence attenuation of <i>Francisella tularensis</i>

Forslund, Anna-Lena; Kuoppa, Kerstin; Svensson, Kerstin; Salomonsson, Emelie; Johansson, Anders; Byström, Mona; Oyston, Petra C. F.; Michell, Stephen L.; Titball, Richard W.; Noppa, Laila; Frithz‐Lindsten, Elisabet; Forsman, Mats; Försberg, Åke

doi:10.1111/j.1365-2958.2006.05061.x

Cited by 91 publications

(147 citation statements)

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“…F. tularensis subsp. holarctica deleted for the pilE1 (pilA) gene is severely attenuated for virulence in mice when administered by the subcutaneous route, but only slightly defective for intramacrophage replication (Forslund et al, 2006). A deletion in pilE1 is also present in the LVS, and the authors of that study suggested that the LVS pilE1 mutation may be responsible, at least in part, for the attenuation of LVS.…”

Section: Introductionmentioning

confidence: 79%

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Characterization of the Francisella tularensis subsp. novicida type IV pilus

et al. 2008

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Francisella tularensis causes the disease tularaemia. Type IV pili (Tfp) genes are present in the genomes of all F. tularensis subspecies. We show that the wild-type F. tularensis subsp. novicida expresses pilus fibres on its surface, and mutations in the Tfp genes pilF and pilT disrupt pilus biogenesis. Mutations in other Tfp genes (pilQ and pilG) do not eliminate pilus expression. A mutation in pilE4 eliminates pilus expression, whereas mutations in the other pilin subunits pilE1-3 and pilE5 do not, suggesting that pilE4 is the major pilus structural subunit. The virulence regulator MglA is required for pilus expression, and it regulates the transcription of a putative Tfp glycosylation gene (FTN0431). However, MglA does not regulate transcription of pilF, pilT or pilE4, and a strain lacking FTN0431 still expresses pili; thus, it is unclear how MglA regulates pilus expression. Only pilF was also required for protein secretion, while pilE4 and pilT were not, indicating that there is very little overlap of the protein secretion/Tfp functions of the pil genes. The protein secretion component pilE1 was more important for in vitro intramacrophage growth and mouse virulence than the Tfp component pilE4. Our results provide the first genetic characterization of the novel Tfp system of F. tularensis. INTRODUCTIONFrancisella tularensis is a highly infectious bacterium that causes tularaemia. F. tularensis is found in many different animal hosts and can be transmitted by arthropod vectors (Ellis et al., 2002). Humans can acquire the bacteria by a number of different routes, but inhalation of low doses of the organism can lead to a serious pneumonic form of disease that has a high mortality rate, and this has led to the classification of this bacterium as a category A biothreat agent (Dennis et al., 2001). F. tularensis is further divided into different subspecies (Oyston et al., 2004). F. tularensis subsp. tularensis has historically been associated with bioweapons development, and is reported to have the highest virulence for humans (McLendon et al., 2006). F. tularensis subsp. holarctica is also infectious in humans but typically with a less severe outcome (Sjostedt, 2003). An attenuated 'live vaccine strain' (LVS) was derived by repeated passage of subsp. holarctica in the laboratory; despite the unclear nature of the attenuating mutation(s) in this strain, it is frequently used as a laboratory model for tularaemia (Anthony & Kongshavn, 1987;Eigelsbach et al., 1951). F. tularensis subsp. novicida has low virulence for humans, but maintains high virulence in mice (Kieffer et al., 2003;Lauriano et al., 2004). Whole-genome sequencing has revealed that all three subspecies are closely related, with the less virulent (for humans) subsp. novicida showing less genomic decay than the more virulent subsp. holarctica and subsp. tularensis (Larsson et al., 2005;Petrosino et al., 2006;Rohmer et al., 2006).The ability of F. tularensis to survive and replicate within macrophages has been linked to its virulence (Anthony et al., 19...

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Section: Introductionmentioning

confidence: 79%

“…These results are consistent with the reported attenuation in intramacrophage growth seen with an F. tularensis subsp. holarctica pilE1 mutant (Forslund et al, 2006). These results demonstrate that PilE1 and protein secretion, but not Tfp biogenesis, contribute to intramacrophage growth in vitro.…”

Section: Tfp/protein Secretion Functional Overlapmentioning

confidence: 99%

Characterization of the Francisella tularensis subsp. novicida type IV pilus

et al. 2008

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“…We have seen similar effects in other studies where we examined the role of a pilin gene, pilA, in virulence. In one study, where we used a moderately virulent strain isolated from a hare, we found that PilA mutants were severely attenuated compared to a strain expressing PilA (16). In contrast, when pilA mutants of virulent strains like FSC200 and the type A strain Schu S4 were used to infect mice, the level of attenuation was lower (unpublished results).…”

Section: Discussionmentioning

confidence: 99%

Hfq, a Novel Pleiotropic Regulator of Virulence-Associated Genes in Francisella tularensis

et al. 2009

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Francisella tularensis is a highly infectious pathogen that infects animals and humans, causing tularemia. The ability to replicate within macrophages is central for virulence and relies on expression of genes located in the Francisella pathogenicity island (FPI), as well as expression of other genes. Regulation of FPI-encoded virulence gene expression in F. tularensis involves at least four regulatory proteins and is not fully understood. Here we studied the RNA-binding protein Hfq in F. tularensis and particularly the role that it plays as a global regulator of gene expression in stress tolerance and pathogenesis. We demonstrate that Hfq promotes resistance to several cellular stresses (including osmotic and membrane stresses). Furthermore, we show that Hfq is important for the ability of the F. tularensis vaccine strain LVS to induce disease and persist in organs of infected mice. We also demonstrate that Hfq is important for stress tolerance and full virulence in a virulent clinical isolate of F. tularensis, FSC200. Finally, microarray analyses revealed that Hfq regulates expression of numerous genes, including genes located in the FPI. Strikingly, Hfq negatively regulates only one of two divergently expressed putative operons in the FPI, in contrast to the other known regulators, which regulate the entire FPI. Hfq thus appears to be a new pleiotropic regulator of virulence in F. tularensis, acting mostly as a repressor, in contrast to the other regulators identified so far. Moreover, the results obtained suggest a novel regulatory mechanism for a subset of FPI genes.Regulation of gene expression by noncoding or small RNAs (sRNAs) is prevalent in both prokaryotes and eukaryotes, and recent studies have identified numerous sRNAs in different organisms. Most sRNAs encoded by bacterial chromosomes act by base pairing with mRNA targets that are encoded in trans, and these sRNAs commonly require Hfq in order to function (47). Hfq is a bacterial RNA-binding protein that was initially recognized as a host factor for replication of the Q␤ RNA phage in Escherichia coli (17). The Hfq protein is very abundant, and it belongs to the eukaryotic and archaeal families of Sm and Sm-like proteins, respectively, that form homohexameric structures (for reviews, see references 5 and 53). The importance of Hfq became clear when an E. coli hfq null mutant was created. This mutant had pleiotropic phenotypes, such as a decreased growth rate, increased sensitivity to cellular stresses, and increased cell length (51). Hfq is a posttranscriptional regulator that binds sRNAs and mRNA and facilitates RNA-RNA interaction (1,18,23,32,35,55). For the most part, sRNA-mRNA interactions result in mRNA degradation and/or inhibition of translation, but they can also increase translation (for reviews, see references 1, 20, and 48). As might be expected from the pleiotropic phenotypes of an hfq mutant, deletion of hfq has been correlated with consider-

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“…The genes required for the production of type IV pili exist in Francisella, and ultrastructural analysis of the LVS revealed the presence of long protrusions with the appearance of type IV pili [12]. Deletion of one of the putative pilin genes in a type B strain of F. tularensis results in attenuation of virulence [13], further implicating type IV pili in the pathogenesis of tularemia. Finally, deletion of the tolC or ftlC genes in the LVS decreases the resistance of the organism to anti-bacterial agents, and, in the case of the former, causes attenuation of virulence in mice [14].…”

Section: Introductionmentioning

confidence: 99%

A conserved and immunodominant lipoprotein of Francisella tularensis is proinflammatory but not essential for virulence

Forestal

Gil

Monfett

et al. 2008

Microbial Pathogenesis

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Francisella tularensis is a highly virulent bacterium that causes tularemia, a disease that is often fatal if untreated. A live vaccine strain (LVS) of this bacterium is attenuated for virulence in humans but produces lethal disease in mice. F. tularensis has been classified as a Category A agent of bioterrorism. Despite this categorization, little is known about the components of the organism that are responsible for causing disease in its hosts. Here, we report the deletion of a well-characterized lipoprotein of F. tularensis, designated LpnA (also known as Tul4), in the LVS. An LpnA deletion mutant was comparable to the wild-type strain in its ability to grow intracellularly and cause lethal disease in mice. Additionally, mice inoculated with a sublethal dose of the mutant strain were afforded the same protection against a subsequent lethal challenge with the LVS as were mice initially administered a sublethal dose of the wild-type bacterium. The LpnA-deficient strain showed an equivalent ability to promote secretion of chemokines by human monocyte-derived macrophages as its wild-type counterpart. However, recombinant LpnA potently stimulated primary cultures of human macrophages in a Toll-like receptor 2-dependent manner. Although human endothelial cells also were activated by recombinant LpnA, their response was relatively modest. LpnA is clearly unnecessary for multiple functions of the LVS, but its inflammatory capacity implicates it and other Francisella lipoproteins as potentially important to the pathogenesis of tularemia.

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Direct repeat‐mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis

Cited by 91 publications

References 57 publications

Characterization of the Francisella tularensis subsp. novicida type IV pilus

Characterization of the Francisella tularensis subsp. novicida type IV pilus

Hfq, a Novel Pleiotropic Regulator of Virulence-Associated Genes in Francisella tularensis

A conserved and immunodominant lipoprotein of Francisella tularensis is proinflammatory but not essential for virulence

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