Identification of Mechanisms for Attenuation of the FSC043 Mutant of Francisella tularensis SCHU S4

Abstract

bPreviously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 of Francisella tularensis subsp. tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that the pdpE gene and most of the pdpC gene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fused fupA and fupB genes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the ⌬fupA mutant were <10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the ⌬pdpC and ⌬pdpC ⌬pdpE mutants. Complementation of FSC043 with the intact fupA and fupB genes did not affect the phenotype, whereas complementation with the pdpC and pdpE genes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in the pdpC gene makes an essential contribution to the phenotype.

Evaluation of Francisella tularensis ΔpdpC as a candidate live attenuated vaccine against respiratory challenge by a virulent SCHU P9 strain of Francisella tularensis in a C57BL/6J mouse model

“…showed that Δ pdpC confers protective immunity against intradermal challenge with Schu S4, but does not protect against i.n. challenge . In the present study, our Δ pdpC conferred protective immunity against i.n.…”

“…Pathogenicity determinant protein C ( pdpC ) is one of the genes within the Francisella pathogenicity island. Δ pdpC , in which pdpC has been disrupted by TargeTron mutagenesis, exhibits a different phenotype . In our previous study, we reported that PdpC is crucial in determining the virulence of F. tularensis SCHU P9 because Δ pdpC displays significantly decreased intracellular growth in J774.1 cells and does not induce severe disease in experimentally infected mice .…”

Identification of an Attenuated Substrain of Francisella tularensis SCHU S4 by Phenotypic and Genotypic Analyses

“…Two such spontaneously derived mutants of SCHU S4 have been identified and are referred to as SCHU S4 P0 and FSC043 [ 26 , 27 ]. Both substrains were shown to be significantly attenuated in murine challenge models, and genomic sequencing showed that both variants contained multiple mutations [ 26 , 28 , 29 ]. In the current study, the characterization of four available SCHU S4 stocks has now revealed the existence of another highly attenuated substrain of SCHU S4 containing multiple genetic differences compared to the SCHU S4 reference strain.…”

“…However, it is unclear at this time which one, or combination of the nine identified genetic mutations, was responsible for the attenuated virulence observed in the NR-643/FTS-635 substrain. Unlike the FSC043 and SCHU S4 P0 SCHU S4 mutant strains, which both lacked functional PdpC and were essentially avirulent in mice [ 26 , 27 , 28 ], no mutations were found within the critical virulence genes of the Francisella pathogenicity island (FPI) in NR-643/FTS-635. Although the virulence of NR-643/FTS-635 was reduced, it was not completely abolished.…”

Deletion of the Major Facilitator Superfamily Transporter fptB Alters Host Cell Interactions and Attenuates Virulence of Type A Francisella tularensis

“…In addition to an intracellular replication defect, these data support an escape delay for SchuS4Δ fptB . Multiple studies have demonstrated that F. tularensis does not replicate until it escapes from the phagosome ( 47 – 49 ). The positive intracellular replication rate of SchuS4Δ fptB , measured as early as 3 to 9 hpi, demonstrates that the mutant strain has escaped from the phagosome, similarly to the WT, and is replicating in the cytosol albeit at a reduced rate.…”