Pseudomonas aeruginosa is an opportunistic pathogen that is a serious problem for immuno-compromised patients. Toxins such as exoenzyme (Exo) S, ExoT, ExoY and ExoU are secreted and translocated from the bacteria into the eukaryotic cell via the bacterial encoded type III secretion system. Our research focuses on ExoS, a bifunctional toxin comprising a Rho-GTPase-activating protein domain (RhoGAP) and a 14-3-3 dependent ADP-ribosyltransferase domain. In addition, ExoS contains a membrane localization domain termed MLD. In this study, cell lines expressing activated forms of various components of the Ras signaling pathway have been used to understand the functional and mechanical activation of ExoS-ADP-ribosyltransferase activity and to reveal its cellular targets in the cell. Our observations suggested that Ras GTPase is the dominant target by which ExoS mediates cell death and activated Ras is able to protect cells against cell death, regardless of whether it has been ADP-ribosylated by ExoS. It has been reported that the 14-3-3 cofactor protein is required for ADP-ribosyltransferase activity of ExoS and a phosphorylation-independent interaction occurs between 14-3-3 and the C-terminal part of ExoS. We have undertaken a deeper analysis including structural and biological investigation of this interaction. Our results suggested that leucine-428 of ExoS is the most critical residue for ExoS enzymatic activity. Structural analysis showed that ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. Our structure was supported by biochemical and cytotoxicity analyses, which revealed that the substitution of important residues of ExoS significantly weakens the ability of ExoS to modify endogenous targets such as RAS/RAP1 and to induce cell death. Further, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. Leucine residues-422, 423, 426, and 428 of ExoS are important for the interaction with the ″roof″ of the amphiphatic groove of 14-3-3. In conclusion, we show the mechanism of cell signal transduction pathways affected upon ExoS infection and also demonstrate that the hydrophobic residues of ExoS in 14-3-3 interaction motif have a significant role for ExoS enzymatic activity.

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system.

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system.

Doxorubicin (DOX) is an incessantly used chemotherapeutic drug that can cause detrimental dose-dependent effects such as cardiotoxicity and congestive heart failure. Studies have focused on therapeutic strategies such as exosomes derived from embryonic stem cell (ES-Exos) and antioxidants for example resveratrol; however, the function of mesenchymal stem cell-derived exosomes (MSC-Exos) have never been examined in DOX-induced pyroptosis. MSC-Exos maintains the therapeutic potential of exosome therapy without the ethical concerns. Hence, the current study focuses on determining whether MSC-Exos has the potential to ameliorate inflammation-induced cell death pyroptosis in our established in vitro DOX-induced cardiotoxicity (DIC) model. Rat embryonic cardiomyocytes (H9c2) were first exposed to DOX to stimulate pyroptosis, followed by subsequent treatment with MSC-Exos, with further analysis performed through immunocytochemistry, western blotting, and RT-PCR. We evaluated the therapeutic potential of MSC-Exos by investigating the pyroptotic initiator HMGB1 which binds to TLR4 resulting in the formation of the NLRP3 inflammasome that initiates pyroptosis by activating the pyroptotic markers, caspase-1, IL-1β and IL-18, and the pyroptotic executioner GSDMD. Our data depicted that treatment with MSC-Exos significantly (p

<p><i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP-ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity <i>in vitro</i>. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of <i>Yersinia pseudotuberculosis</i>. We found that Ras was ADP-ribosylated <i>in vivo</i> and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily <i>in vivo</i>, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by <i>P. aeruginosa</i>. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 <i>in vivo</i>, and that ExoT shows GAP activity towards RhoA <i>in vitro</i>. </p><p>The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids ⁴²⁴DALDL⁴²⁸ on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS <i>in vivo</i>.</p>

Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP-ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity in vitro. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of Yersinia pseudotuberculosis. We found that Ras was ADP-ribosylated in vivo and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily in vivo, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by P. aeruginosa. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 in vivo, and that ExoT shows GAP activity towards RhoA in vitro. The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids 424DALDL428 on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS in vivo.

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