RESUMO
Francisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensis subspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pil genes. We show here that deletion of the pilF putative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilT putative disassembly ATPase also caused a complete loss of pili, indicating that pilT functions differently in F. tularensis than in model Tfp systems such as those found in Pseudomonas aeruginosa and Neisseria spp. The LVS pilF and pilT mutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilF and pilT mutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pil genes and provides evidence that the Francisella pili contribute to host cell adhesion and virulence.
Assuntos
Adenosina Trifosfatases/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Francisella tularensis/patogenicidade , Proteínas Motores Moleculares/metabolismo , Tularemia/fisiopatologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Vacinas Bacterianas , Células Cultivadas , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Francisella tularensis/genética , Francisella tularensis/metabolismo , Hepatócitos/microbiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/genética , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Tularemia/microbiologia , Vacinas Atenuadas , VirulênciaRESUMO
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 were also 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.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Francisella tularensis/patogenicidade , Mediadores da Inflamação/imunologia , Lipoproteínas/imunologia , Tularemia/imunologia , Animais , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Quimiocinas/imunologia , Conjugação Genética , Células Endoteliais/imunologia , Células Endoteliais/microbiologia , Feminino , Francisella tularensis/genética , Francisella tularensis/metabolismo , Humanos , Lipoproteínas/genética , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Deleção de Sequência , Tularemia/microbiologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , VirulênciaRESUMO
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.