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Francisella tularensis virulence is dependent on a conserved putative catalytic triad within the Type VI secretion system component PdpC

ABSTRACT: Many Gram-negative bacteria utilize the ubiquitous and versatile type VI secretion systems (T6SS) for diverse purposes, such as outcompeting other microbes or to successfully colonize hosts. T6SS of common pathogens are classified as T6SSi, but an outlier is the T6SSii and it is present only in the genus Francisella. The highly virulent human pathogen Francisella tularensis harbors a variant of the T6SSii distinct from that of many other species of the genus, e.g., aquatic pathogens and tick endosymbionts. At the gene level, the most distinctive difference between the two variants is the presence in F. tularensis of a putative T6SS effector denoted PdpC. We here provide bioinformatic evidence that PdpC contains a conserved amino acid triad homologous to motifs present in toxins of the family Make Caterpillars Floppy. By site-directed mutagenesis, each of the three amino acids was substituted in cis in the live vaccine strain, LVS, of F. tularensis. Since the T6SS of F. tularensis is essential for the intracellular life cycle and virulence of the pathogen, the effects of the mutations were tested in mammalian cells and in a mouse model. It was observed that the resulting mutants demonstrated distinct phenotypes, evidenced as delayed, or no intramacrophage phagosomal escape, variable degrees of intramacrophage replication, and marked attenuation in mice. The corresponding mutants were also generated in the related species F. novicida, a commonly used model for F. tularensis, which conveniently allows studies of T6SS-mediated secretion. A mass spectrometry analysis of secreted proteins demonstrated that PdpC is one of several T6SS-dependent substrates, and that the lack of PdpC, or the presence of each of the amino acid-substituted mutant PdpC proteins, did not affect the secretion pattern of F. novicida. Collectively, the data demonstrate that the markedly attenuated phenotypes of the mutants were due to essential roles of the amino acid triad of PdpC.

INSTRUMENT(S):

ORGANISM(S): Francisella Tularensis Subsp. Novicida (strain U112)

TISSUE(S): Cell Culture

SUBMITTER: Shaochun Zhu

LAB HEAD: Anders Sjöstedt

PROVIDER: PXD059583 | Pride | 2026-03-09

REPOSITORIES: Pride

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			Action	DRS
	S00129_Exploris01_SZ_Igor_proteome_Ig1_1_20231115173017.raw	Raw
	S00129_Exploris01_SZ_Igor_proteome_Ig1_2_20231115190558.raw	Raw
	S00129_Exploris01_SZ_Igor_proteome_Ig1_3_20231115204126.raw	Raw
	S00129_Exploris01_SZ_Igor_proteome_Ig2_1_20231115224716.raw	Raw
	S00129_Exploris01_SZ_Igor_proteome_Ig2_2_20231116002244.raw	Raw

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Project description:Francisella tularensis is a Gram-negative bacterium that is highly virulent in humans, causing the disease tularemia. F. novicida is closely related to F. tularensis and exhibits high virulence in mice, but is avirulent in healthy humans. A F. novicida-specific gene cluster (FTN0451-FTN0456) encodes two proteins with diguanylate cyclase (DGC) and phosphodiesterase (PDE) domains that modulate synthesis and degradation of cyclic di-GMP (cdGMP). No DGC or PDE containing proteins are present in the F. tularensis genome. F. novicida strains lacking either the two DGC/PDE genes cdgA and cdgB or the entire gene cluster (KKF457) are defective for biofilm formation. In addition, expression of CdgB or a heterologous DGC in KKF457 stimulated F. novicida biofilms, even in a strain lacking the biofilm regulator QseB. Genetic evidence suggests that CdgA is predominantly a PDE, while CdgB is predominantly a DGC. The F. novicida qseB strain has reduced cdgA and cdgB transcript levels, demonstrating a F. novicida biofilm signaling cascade that controls cdGMP levels. Interestingly, KKF457 with elevated cdGMP levels exhibited a decrease in intramacrophage replication and virulence in mice, as well as increased growth yield and biofilm formation in vitro. Microarray analyses revealed that cdGMP stimulated the transcription of a chitinase (ChiB) known to contribute to biofilm formation. Our results indicate that elevated cdGMP in F. novicida stimulates biofilm formation and inhibits virulence. We suggest that differences in human virulence between F. novicida and F. tularensis may be due in part to the absence of cdGMP signaling in F. tularensis. A whole genome NibleGen microarray was developed for multiple Francisella subspecies, using shared probes for common genes as well as unique probes for subspecies-specific genes. Included were probes representing the F. novicida U112 genome, with a maximum of 6 probes/gene. RNA was isolated from bacteria and purified as described above. The double-strand cDNA was generated and labeled with Cy3 according to the NimbleGen Gene Expression Array protocol (www.nimblegen.com), using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) and the DNA Labeling Kit (NimbleGen). Microarray results were analyzed using the ArrayStar software (DNASTAR).

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Francisella tularensis virulence is dependent on a conserved putative catalytic triad within the Type VI secretion system component PdpC

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