The D-alanyl-d-alanine carboxypeptidase enzyme is essential for virulence in the Schu S4 strain of Francisella tularensis and a dacD mutant is able to provide protection against a pneumonic challenge

• Published in: Microbial pathogenesis 2019-12, Vol.137, p.103742-103742, Article 103742
• Main Authors: Kijek, Todd M., Mou, Sherry, Bachert, Beth A., Kuehl, Kathleen A., Williams, Janice A., Daye, Sharon P., Worsham, Patricia L., Bozue, Joel A.
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins - genetics; Bacterial Vaccines - immunology; Cell Line; D-alanyl-d-alanine carboxypeptidase; DacD; Disease Models, Animal; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Francisella tularensis; Francisella tularensis - enzymology; Francisella tularensis - genetics; Francisella tularensis - pathogenicity; Lung - microbiology; Macrophages - microbiology; Mice; Mice, Inbred BALB C; Mutation; Penicillin binding proteins; Peptidoglycan; Serine-Type D-Ala-D-Ala Carboxypeptidase - genetics; Serine-Type D-Ala-D-Ala Carboxypeptidase - metabolism; Tularemia; Tularemia - immunology; Tularemia - microbiology; Vaccine; Vaccines, Attenuated - immunology; Virulence; Virulence Factors - genetics
• ISSN: 0882-4010; 1096-1208
• DOI: 10.1016/j.micpath.2019.103742

Low molecular mass penicillin binding proteins (LMM PBP) are bacterial enzymes involved in the final steps of peptidoglycan biosynthesis. In Escherichia coli, most LMM PBP exhibit dd-carboxypeptidase activity, are not essential for growth in routine laboratory media, and contributions to virulent phenotypes remain largely unknown. The Francisella tularensis Schu S4 genome harbors the dacD gene (FTT_1029), which encodes a LMM PBP with homology to PBP6b of E. coli. Disruption of this locus in the fully virulent Schu S4 strain resulted in a mutant that could not grow in Chamberlain's Defined Medium and exhibited severe morphological defects. Further characterization studies demonstrated that the growth defects of the dacD mutant were pH-dependent, and could be partially restored by growth at neutral pH or fully restored by genetic complementation. Infection of murine macrophage-like cells showed that the Schu S4 dacD mutant is capable of intracellular replication. However, this mutant was attenuated in BALB/c mice following intranasal challenge (LD50 = 603 CFU) as compared to mice challenged with the parent (LD50 = 1 CFU) or complemented strain (LD50 = 1 CFU). Additionally, mice that survived infection with the dacD mutant showed significant protection against subsequent challenge with the parent strain. Collectively, these results indicate that the DacD protein of F. tularensis is essential for growth in low pH environments and virulence in vivo. These results also suggest that a PBP mutant could serve as the basis of a novel, live attenuated vaccine strain. •The DacD enzyme was necessary for F. tularensis virulence in a murine tularemia model.•Mice which received the dacD mutant were protected from the wild-type Schu S4 strain.•Growth of the dacD mutant in defined broth medium was found to be pH dependent.•The size of the dacD mutant was altered at lower pH conditions.