Francisella tularensis D-Ala D-Ala Carboxypeptidase DacD Is Involved in Intracellular Replication and It Is Necessary for Bacterial Cell Wall Integrity

Abstract

D-alanyl-D-alanine carboxypeptidase, product of dacD gene in Francisella, belongs to penicillin binding proteins (PBPs) and is involved in remodeling of newly synthetized peptidoglycan. In E. coli, PBPs are synthetized in various growth phases and they are able to substitute each other to a certain extent. The DacD protein was found to be accumulated in fraction enriched in membrane proteins from severely attenuated dsbA deletion mutant strain. It has been presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are the primary virulence factors. Here we demonstrate that Francisella DacD is required for intracellular replication and virulence in mice. The dacD insertion mutant strain showed higher sensitivity to acidic pH, high temperature and high osmolarity when compared to the wild-type. Eventually, transmission electron microscopy revealed differences in mutant bacteria in both the size and defects in outer membrane underlying its SDS and serum sensitivity. Taken together these results suggest DacD plays an important role in Francisella pathogenicity.

A Francisella novicida Mutant, Lacking the Soluble Lytic Transglycosylase Slt, Exhibits Defects in Both Growth and Virulence

“…The Fn slt mutant cells generally appeared to be larger than WT but not to this degree as observed for the F. holarctica dacD mutant. The authors also demonstrate that while intracellular replication of the dacD mutant was affected during infection of murine macrophages, phagosomal escape was increased compared to WT, possibly due to the increased sensitivity to acidic environment of the phagosome (Spidlova et al, 2018). While our studies did not address phagosomal escape, we show that the slt mutant indeed has a partial replicative defect within the macrophages as compared to parent, which may reflect its pH sensitivity.…”

“…Interestingly, FTT_0924, encoding an unknown protein highly conserved among Francisella spp., was shown to be necessary to maintaining peptidoglycan stability and for intracellular replication in macrophages (Brunton et al, 2015). Similarly, the D -alanyl- D -alanine carboxypeptidase DacD was found to be necessary for maintaining the integrity of the cell wall as well as being involved in intracellular replication and virulence in F. holarctica (Spidlova et al, 2018). While knowledge of the specific peptidoglycan remodeling proteins in Francisella is currently limited, we demonstrate that the Slt enzyme described in this study has a critical role in maintaining cell growth and morphology in acidic conditions.…”

“…The pH-dependent phenotype of the slt mutant is a unique finding. Recent studies on the D -alanyl- D -alanine carboxy-peptidase DacD, another peptidoglycan-remodeling enzyme, showed a significant pH sensitivity of a F. holarctica dacD mutant, as well as morphological defects including increased cell size up to ten times larger than WT, and discontinuous membranes that left cells more susceptible to osmotic stress (Spidlova et al, 2018). The Fn slt mutant cells generally appeared to be larger than WT but not to this degree as observed for the F. holarctica dacD mutant.…”

A Francisella tularensis L,D-carboxypeptidase plays important roles in cell morphology, envelope integrity, and virulence

“…In fact, Gram-negative bacteria recycle up to 60% of their PG with every generation, suggesting that both PG synthesis and PG recycling are dynamic (Dhar, 2018, Typas, 2011, Park, 2008). A number of proteins are involved in these processes and, while they are well-characterized in E. coli , very little is known about these pathways in intracellular pathogens such as Burkholderia pseudomallei , Legionella pneumophila , or F. tularensis (van Heijenoort, 2011, Jenkins, 2019, Spidlova, 2018, Kijek, 2019).…”

“…It should be noted that although N. gonorrhoeae LdcA also has been reported to have L,D-endopeptidase activity, this activity was shown to cleave tetra-tri and tri-tri dimers, but not tetra-tetra dimers, suggesting a specificity for 3-3 cross linked dimers (Lenz, 2017). Interestingly, only two previous studies have examined putative F. tularensis PG modifying enzymes and both studies primarily focused on the role of an F. tularensis DacD ortholog in virulence, with no PG activity assays to confirm function (Spidlova, 2018, Kijek, 2019). In our PG cleavage analysis, F. tularensis LdcA demonstrated the highest specific activity on disaccharide-tetrapeptide PG substrates (GlcNAc-anhydroMurNAc-L-Ala-γ-D-Glu- meso -A 2 pm-D-Ala [TCT] and GlcNAc-MurNAc-L-Ala-γ-D-Glu- meso -A 2 pm-D-Ala [reducing PG monomer]), followed by cleavage of pentapeptide PG substrates (MurNAc-L-Ala-γ-D-Glu- meso -A 2 pm-D-Ala-D-Ala and UDP-MurNAc-L-Ala-γ-D-Glu- meso -A 2 pm-D-Ala-D-Ala).…”