Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.

• Keywords
• Francisella, glyceraldehyde-3-phosphate dehydrogenase, infection, interacting partners, multitasking, pleiotropy, secretion,
• MeSH
• Cytokines metabolism   MeSH
• Gene Expression MeSH
• Francisella tularensis * genetics   metabolism   MeSH
• Glyceraldehyde-3-Phosphate Dehydrogenases genetics   metabolism   MeSH
• Proteomics MeSH
• Virulence genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• Glyceraldehyde-3-Phosphate Dehydrogenases MeSH

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well known for its involvement in numerous non-metabolic processes inside mammalian cells. Alternative functions of prokaryotic GAPDH are mainly deduced from its extracellular localization ability to bind to selected host proteins. Data on its participation in intracellular bacterial processes are scarce as there has been to date only one study dealing with this issue. We previously have reported several points of evidence that the GAPDH homolog of Francisella tularensis GapA might also exert additional non-enzymatic functions. Following on from our earlier observations we decided to identify GapA's interacting partners within the bacterial proteome to explore its new roles at intracellular level. The quantitative proteomics approach based on stable isotope labeling of amino acids in cell culture (SILAC) in combination with affinity purification mass spectrometry enabled us to identify 18 proteins potentially interacting with GapA. Six of those interactions were further confirmed by alternative methods. Half of the identified proteins were involved in non-metabolic processes. Further analysis together with quantitative label-free comparative analysis of proteomes isolated from the wild-type strain strain with deleted gapA gene suggests that GapA is implicated in DNA repair processes. Absence of GapA promotes secretion of its most potent interaction partner the hypothetical protein with peptidase propeptide domain (PepSY) thereby indicating that it impacts on subcellular distribution of some proteins.

• Keywords
• Francisella tularensis, SILAC, glyceraldehyde-3-phosphate dehydrogenase, multifunctional enzyme, protein–protein interaction,
• Publication type
• Journal Article MeSH

Bacterial proteins exhibiting two or more unrelated functions, referred to as moonlighting proteins, are suggested to contribute to full virulence manifestation in pathogens. An expanding number of published studies have revealed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be a multitasking protein with virulence impact in a number of pathogenic bacteria. This protein can be detected on the bacterial surface or outside the bacterial cell, where it interacts with host proteins. In this way, GAPDH is able to modulate various pathogenic processes. Moreover, it has been shown to be involved in non-enzymatic processes inside the bacterial cell. In this mini review, we summarize main findings concerning the multiple localization and protein interactions of GAPDH derived from bacterial pathogens of humans. We also briefly discuss problems associated with using GAPDH as a vaccine antigen and endeavor to inspire further research to fill gaps in the existing knowledge.

• Keywords
• glyceraldehyde-3-phosphate dehydrogenase, localization, moonlighting proteins, pathogenic bacteria, protein-protein interaction (PPI),
• MeSH
• Bacteria enzymology   pathogenicity   MeSH
• Bacterial Infections microbiology   prevention & control   MeSH
• Bacterial Proteins metabolism   MeSH
• Bacterial Vaccines immunology   MeSH
• Glyceraldehyde-3-Phosphate Dehydrogenases immunology   metabolism   MeSH
• Humans MeSH
• Proteins metabolism   MeSH
• Protein Binding MeSH
• Virulence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Bacterial Vaccines MeSH
• Glyceraldehyde-3-Phosphate Dehydrogenases MeSH
• Proteins MeSH

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.

• Keywords
• DacD, Francisella, carboxypeptidase, membrane defects, penicillin binding proteins, phagosomal escape, virulence,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Cell Wall metabolism   MeSH
• Serine-Type D-Ala-D-Ala Carboxypeptidase genetics   metabolism   MeSH
• Francisella tularensis drug effects   growth & development   pathogenicity   MeSH
• Cells, Cultured MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Peptidoglycan biosynthesis   MeSH
• Protein Disulfide-Isomerases genetics   MeSH
• Penicillin-Binding Proteins genetics   metabolism   MeSH
• Microscopy, Electron, Transmission MeSH
• Tularemia microbiology   pathology   MeSH
• Virulence genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Serine-Type D-Ala-D-Ala Carboxypeptidase MeSH
• Peptidoglycan MeSH
• Protein Disulfide-Isomerases MeSH
• Penicillin-Binding Proteins MeSH