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.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics ASCR v v i Prague Czechia

Department of Microbiology and Parasitology Medical Faculty University of Rijeka Rijeka Croatia

Department of Molecular Pathology and Biology Faculty of Military Health Sciences University of Defence Hradec Kralove Czechia

Microscopy Center Institute of Molecular Genetics ASCR v v i Electron Microscopy Core Facility Prague Czechia

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Alkhuder K., Meibom K. L., Dubail I., Dupuis M., Charbit A. (2009). Glutathione provides a source of cysteine essential for intracellular multiplication of PubMed DOI PMC

Asare R., Abu Kwaik Y. (2010). Molecular complexity orchestrates modulation of phagosome biogenesis and escape to the cytosol of macrophages by PubMed DOI PMC

Asare R., Akimana C., Jones S., Abu Kwaik Y. (2010). Molecular bases of proliferation of PubMed DOI PMC

Baquero M. R., Bouzon M., Quintela J. C., Ayala J. A., Moreno F. (1996). dacD, an PubMed DOI PMC

Biswas S., Raoult D., Rolain J.-M. (2008). A bioinformatic approach to understanding antibiotic resistance in intracellular bacteria through whole genome analysis. Int. J. Antimicrob. Agents 32, 207–220. 10.1016/j.ijantimicag.2008.03.017 PubMed DOI

Bönquist L., Lindgren H., Golovliov I., Guina T., Sjöstedt A. (2008). MglA and Igl proteins contribute to the modulation of PubMed DOI PMC

Brambilla L., Morán-Barrio J., Viale A. M. (2014). Low-molecular-mass penicillin binding protein 6b (DacD) is required for efficient GOB-18 metallo-β-lactamase biogenesis in PubMed DOI PMC

Brotcke A., Weiss D. S., Kim C. C., Chain P., Malfatti S., Garcia E., et al. (2006). Identification of MglA-regulated genes reveals novel virulence factors in PubMed DOI PMC

Celli J., Zahrt T. C. (2013). Mechanisms of PubMed DOI PMC

Chamberlain R. E. (1965). Evaluation of live tularemia vaccine prepared in a chemically defined medium. Appl. Microbiol. 13, 232–235. PubMed PMC

Chandler C. E., Ernst R. K. (2017). Bacterial lipids: powerful modifiers of the innate immune response. F1000Res. 6:F1000. 10.12688/f1000research.11388.1 PubMed DOI PMC

Chong A., Wehrly T. D., Child R., Hansen B., Hwang S., Virgin H. W., et al. (2012). Cytosolic clearance of replication-deficient mutants reveals PubMed DOI PMC

Clemens D. L., Lee B.-Y., Horwitz M. A. (2009). PubMed DOI PMC

Dankova V., Balonova L., Link M., Straskova A., Sheshko V., Stulik J. (2016). Inactivation of PubMed DOI

Denome S. A., Elf P. K., Henderson T. A., Nelson D. E., Young K. D. (1999). PubMed PMC

Dieppedale J., Gesbert G., Ramond E., Chhuon C., Dubail I., Dupuis M., et al. (2013). Possible links between stress defense and the tricarboxylic acid (TCA) Cycle in PubMed DOI PMC

Egan A. J. F., Biboy J., van't Veer I., Breukink E., Vollmer W. (2015). Activities and regulation of peptidoglycan synthases. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 370:20150031. 10.1098/rstb.2015.0031 PubMed DOI PMC

Fuller J. R., Craven R. R., Hall J. D., Kijek T. M., Taft-Benz S., Kawula T. H. (2008). RipA, a cytoplasmic membrane protein conserved among PubMed DOI PMC

Ghosh A. S., Chowdhury C., Nelson D. E. (2008). Physiological functions of D-alanine carboxypeptidases in PubMed DOI

Glauner B., Höltje J. V. (1990). Growth pattern of the murein sacculus of PubMed

Henderson T. A., Dombrosky P. M., Young K. D. (1994). Artifactual processing of penicillin-binding proteins 7 and 1b by the OmpT protease of PubMed DOI PMC

Henderson T. A., Templin M., Young K. D. (1995). Identification and cloning of the gene encoding penicillin-binding protein 7 of PubMed DOI PMC

Henderson T. A., Young K. D., Denome S. A., Elf P. K. (1997). AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of PubMed DOI PMC

Matsuhashi M. (1994). Chapter 4: Utilization of lipid-linked precursors and the formation of peptidoglycan in the process of cell growth and division: membrane enzymes involved in the final steps of peptidoglycan synthesis and the mechanism of their regulation, in New Comprehensive Biochemistry, eds Ghuysen J.-M., Hakenbeck R. (Amsterodam: Elsevier; ), 55–71.

Milton D. L., O'Toole R., Horstedt P., Wolf-Watz H. (1996). Flagellin A is essential for the virulence of PubMed PMC

Nelson D. E., Young K. D. (2001). Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in PubMed DOI PMC

Pavkova I., Kopeckova M., Klimentova J., Schmidt M., Sheshko V., Sobol M., et al. (2017). The multiple localized glyceraldehyde-3-phosphate dehydrogenase contributes to the attenuation of the PubMed DOI PMC

Pechous R. D., McCarthy T. R., Mohapatra N. P., Soni S., Penoske R. M., Salzman N. H., et al. (2008). A PubMed DOI PMC

Peters K., Kannan S., Rao V. A., Biboy J., Vollmer D., Erickson S. W., et al. (2016). The redundancy of peptidoglycan carboxypeptidases ensures robust cell shape maintenance in PubMed DOI PMC

Qin A., Zhang Y., Clark M. E., Moore E. A., Rabideau M. M., Moreau G. B., et al. (2016). Components of the type six secretion system are substrates of PubMed DOI PMC

Ren G., Champion M. M., Huntley J. F. (2014). Identification of disulfide bond isomerase substrates reveals bacterial virulence factors. Mol. Microbiol. 94, 926–944. 10.1111/mmi.12808 PubMed DOI PMC

Rioseras B., Yagüe P., López-García M. T., Gonzalez-Quiñonez N., Binda E., Marinelli F., et al. (2016). Characterization of SCO4439, a D-alanyl-D-alanine carboxypeptidase involved in spore cell wall maturation, resistance, and germination in PubMed DOI PMC

Rodriguez S. A., Yu J.-J., Davis G., Arulanandam B. P., Klose K. E. (2008). Targeted inactivation of PubMed DOI PMC

Rowe H. M., Huntley J. F. (2015). From the outside-in: the PubMed DOI PMC

Santic M., Molmeret M., Barker J. R., Klose K. E., Dekanic A., Doric M., et al. (2007). A PubMed DOI

Sarkar S. K., Dutta M., Chowdhury C., Kumar A., Ghosh A. S. (2011). PBP5, PBP6 and DacD play different roles in intrinsic β-lactam resistance of PubMed DOI

Schiffer G., Höltje J. V. (1999). Cloning and characterization of PBP 1C, a third member of the multimodular class A penicillin-binding proteins of PubMed DOI

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682. 10.1038/nmeth.2019 PubMed DOI PMC

Simon R., Priefer U., Pühler A. (1983). A broad host range mobilization system for DOI

Sommer C., Strähle C., Köthe U., Hamprecht F. A. (2011). ilastik: interactive learning and segmentation toolkit, in Proceedings of Eighth IEEE International Symposium on Biomedical Imaging (ISBI) (Chigaco, IL: ), 230–233.

Sorokin V. M., Pavlovich N. V., Prozorova L. A. (1996). PubMed DOI

Spidlova P., Senitkova I., Link M., Stulik J. (2017). Identification of two substrates of FTS_1067 protein - An essential virulence factor of PubMed DOI

Spratt B. G. (1975). Distinct penicillin binding proteins involved in the division, elongation, and shape of PubMed DOI PMC

Szybalski W., Bryson V. (1952). Genetic studies on microbial cross resistance to toxic agents. I. Cross resistance of PubMed PMC

Vollmer W., Blanot D., de Pedro M. A. (2008a). Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167. 10.1111/j.1574-6976.2007.00094.x PubMed DOI

Vollmer W., Joris B., Charlier P., Foster S. (2008b). Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol. Rev. 32, 259–286. 10.1111/j.1574-6976.2007.00099.x PubMed DOI

Wallqvist A., Memišević V., Zavaljevski N., Pieper R., Rajagopala S. V., Kwon K., et al. (2015). Using host-pathogen protein interactions to identify and characterize PubMed DOI PMC

Wehrly T. D., Chong A., Virtaneva K., Sturdevant D. E., Child R., Edwards J. A., et al. (2009). Intracellular biology and virulence determinants of PubMed DOI PMC

Breaking the cellular defense: the role of autophagy evasion in Francisella virulence

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Arginine 58 is indispensable for proper function of the Francisella tularensis subsp. holarctica FSC200 HU protein, and its substitution alters virulence and mediates immunity against wild-type strain