A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection.

Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFß-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria.

Functional analysis of PTEN variants of unknown significance from PHTS patients unveils complex patterns of PTEN biological activity in disease.

Heterozygous germline mutations in PTEN gene predispose to hamartomas and tumors in different tissues, as well as to neurodevelopmental disorders, and define at genetic level the PTEN Hamartoma Tumor Syndrome (PHTS). The major physiologic role of PTEN protein is the dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), counteracting the pro-oncogenic function of phosphatidylinositol 3-kinase (PI3K), and PTEN mutations in PHTS patients frequently abrogate PTEN PIP3 catalytic activity. PTEN also displays non-canonical PIP3-independent functions, but their involvement in PHTS pathogeny is less understood. We have previously identified and described, at clinical and genetic level, novel PTEN variants of unknown functional significance in PHTS patients. Here, we have performed an extensive functional characterization of these PTEN variants (c.77 C > T, p.(Thr26Ile), T26I; c.284 C > G, p.(Pro95Arg), P95R; c.529 T > A, p.(Tyr177Asn), Y177N; c.781 C > G, p.(Gln261Glu), Q261E; c.829 A > G, p.(Thr277Ala), T277A; and c.929 A > G, p.(Asp310Gly), D310G), including cell expression levels and protein stability, PIP3-phosphatase activity, and subcellular localization. In addition, caspase-3 cleavage analysis in cells has been assessed using a C2-domain caspase-3 cleavage-specific anti-PTEN antibody. We have found complex patterns of functional activity on PTEN variants, ranging from loss of PIP3-phosphatase activity, diminished protein expression and stability, and altered nuclear/cytoplasmic localization, to intact functional properties, when compared with PTEN wild type. Furthermore, we have found that PTEN cleavage at the C2-domain by the pro-apoptotic protease caspase-3 is diminished in specific PTEN PHTS variants. Our findings illustrate the multifaceted molecular features of pathogenic PTEN protein variants, which could account for the complexity in the genotype/phenotype manifestations of PHTS patients.

A global analysis of the reconstitution of PTEN function by translational readthrough of PTEN pathogenic premature termination codons.

The PTEN tumor suppressor gene is mutated with high incidence in tumors and in the germline of patients with cancer predisposition or with macrocephaly associated with autism. PTEN nonsense mutations generating premature termination codons (PTC) and producing nonfunctional truncated PTEN proteins are frequent in association with human disease. However, there are no studies addressing the restoration of full-length PTEN proteins from the PTC-mutated PTEN gene by translational readthrough. Here, we have performed a global translational and functional readthrough analysis of the complete collection of PTEN PTC somatic or hereditary mutations found in tumors or in the germline of patients (disease-associated PTEN PTCome), and we set standards for the analysis of the potential of readthrough functional reconstitution in disease-relevant genes. Our analysis indicates that prevalent pathogenic PTEN PTC mutations are susceptible to PTEN functional restoration in response to readthrough-inducing compounds. Comprehensive readthrough analyses of disease-associated PTComes will be valuable tools for the implementation of readthrough-based precision interventions in specific groups of patients.

The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism.

Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent.

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.

Modeling human disease in yeast: recreating the PI3K-PTEN-Akt signaling pathway in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a model organism that has been thoroughly exploited to understand the universal mechanisms that govern signaling pathways. Due to its ease of manipulation, humanized yeast models that successfully reproduce the function of human genes permit the development of highly efficient genetic approaches for molecular studies. Of special interest are those pathways related to human disease that are conserved from yeast to mammals. However, it is also possible to engineer yeast cells to implement functions that are naturally absent in fungi. Along the years, we have reconstructed several aspects of the mammalian phosphatidylinositol 3-kinase (PI3K) pathway in S. cerevisiae. Here, we briefly review the use of S. cerevisiae as a tool to study human oncogenes and tumor suppressors, and we present an overview of the models applied to the study of the PI3K oncoproteins, the tumor suppressor PTEN, and the Akt protein kinase. We discuss the application of these models to study the basic functional properties of these signaling proteins, the functional assessment of their clinically relevant variants, and the design of feasible platforms for drug discovery.

Expression of Human PTEN-L in a Yeast Heterologous Model Unveils Specific N-Terminal Motifs Controlling PTEN-L Subcellular Localization and Function.

The tumour suppressor PTEN is frequently downregulated, mutated or lost in several types of tumours and congenital disorders including PHTS (PTEN Hamartoma Tumour Syndrome) and ASD (Autism Spectrum Disorder). PTEN is a lipid phosphatase whose activity over the lipid messenger PIP3 counteracts the stimulation of the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway. Recently, several extended versions of PTEN produced in the cell by alternative translation initiation have been described, among which, PTEN-L and PTEN-M represent the longest isoforms. We previously developed a humanized yeast model in which the expression of PI3K in Saccharomyces cerevisiae led to growth inhibition that could be suppressed by co-expression of PTEN. Here, we show that the expression of PTEN-L and PTEN-M in yeast results in robust counteracting of PI3K-dependent growth inhibition. N-terminally tagged GFP-PTEN-L was sharply localized at the yeast plasma membrane. Point mutations of a putative membrane-binding helix located at the PTEN-L extension or its deletion shifted localization to nuclear. Also, a shift from plasma membrane to nucleus was observed in mutants at basic amino acid clusters at the PIP2-binding motif, and at the Cα2 and CBR3 loops at the C2 domain. In contrast, C-terminally tagged PTEN-L-GFP displayed mitochondrial localization in yeast, which was shifted to plasma membrane by removing the first 22 PTEN-L residues. Our results suggest an important role of the N-terminal extension of alternative PTEN isoforms on their spatial and functional regulation.

Heterologous mammalian Akt disrupts plasma membrane homeostasis by taking over TORC2 signaling in Saccharomyces cerevisiae.

The Akt protein kinase is the main transducer of phosphatidylinositol-3,4,5-trisphosphate (PtdIns3,4,5P3) signaling in higher eukaryotes, controlling cell growth, motility, proliferation and survival. By co-expression of mammalian class I phosphatidylinositol 3-kinase (PI3K) and Akt in the Saccharomyces cerevisiae heterologous model, we previously described an inhibitory effect on yeast growth that relied on Akt kinase activity. Here we report that PI3K-Akt expression in yeast triggers the formation of large plasma membrane (PM) invaginations that were marked by actin patches, enriched in PtdIns4,5P2 and associated to abnormal intracellular cell wall deposits. These effects of Akt were mimicked by overproduction of the PtdIns4,5P2 effector Slm1, an adaptor of the Ypk1 and Ypk2 kinases in the TORC2 pathway. Although Slm1 was phosphorylated in vivo by Akt, TORC2-dependent Ypk1 activation did not occur. However, PI3K-activated Akt suppressed the lethality derived from inactivation of either TORC2 or Ypk protein kinases. Thus, heterologous co-expression of PI3K and Akt in yeast short-circuits PtdIns4,5P2- and TORC2-signaling at the level of the Slm-Ypk complex, overriding some of its functions. Our results underscore the importance of phosphoinositide-dependent kinases as key actors in the homeostasis and dynamics of the PM.

A pathogenic role for germline PTEN variants which accumulate into the nucleus.

The PTEN gene encodes a master regulator protein that exerts essential functions both in the cytoplasm and in the nucleus. PTEN is mutated in the germline of both patients with heterogeneous tumor syndromic diseases, categorized as PTEN hamartoma tumor syndrome (PHTS), and a group affected with autism spectrum disorders (ASD). Previous studies have unveiled the functional heterogeneity of PTEN variants found in both patient cohorts, making functional studies necessary to provide mechanistic insights related to their pathogenicity. Here, we have functionally characterized a PTEN missense variant [c.49C>G; p.(Gln17Glu); Q17E] associated to both PHTS and ASD patients. The PTEN Q17E variant displayed partially reduced PIP3-catalytic activity and normal stability in cells, as shown using S. cerevisiae and mammalian cell experimental models. Remarkably, PTEN Q17E accumulated in the nucleus, in a process involving the PTEN N-terminal nuclear localization sequence. The analysis of additional germline-associated PTEN N-terminal variants illustrated the existence of a PTEN N-terminal region whose targeting in disease causes PTEN nuclear accumulation, in parallel with defects in PIP3-catalytic activity in cells. Our findings highlight the frequent occurrence of PTEN gene mutations targeting PTEN N-terminus whose pathogenicity may be related, at least in part, with the retention of PTEN in the nucleus. This could be important for the implementation of precision therapies for patients with alterations in the PTEN pathway.

Insights into the pathological mechanisms of p85α mutations using a yeast-based phosphatidylinositol 3-kinase model.

In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdIns(4,5)P2 into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, ß, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85-p110 interactions typical of cancer.

Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.

Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.

The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.

The yeast cell wall integrity pathway signals from recycling endosomes upon elimination of phosphatidylinositol (4,5)-bisphosphate by mammalian phosphatidylinositol 3-kinase.

Phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] is essential for recognition of the plasma membrane inner leaf by protein complexes. We expressed mammalian class I phosphoinositide 3-kinase (PI3K) in Saccharomyces cerevisiae to eliminate PtdIns(4,5)P(2) by its conversion into PtdIns(3,4,5)P(3), a lipid naturally missing in this yeast. This led to loss of actin function and endocytosis defects, causing a blockage in polarized secretion. Also, the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway was activated, triggering a typical transcriptional response. In the absence of PtdIns(4,5)P(2) at the plasma membrane, the Pkc1 protein kinase upstream the CWI MAPK module localized to post-Golgi endosomes marked by SNARE Snc1 and Rab GTPases Ypt31 and Ypt32. Other components at the head of the pathway, like the mechanosensor Wsc1, the GTPase Rho1 and its activator the GDP/GTP exchange factor Rom2, co-localized with Pkc1 in these compartments. Chemical inhibition of PI3K proved that both CWI activation and Pkc1 relocation to endosomes are reversible. These results suggest that the CWI pathway is able to respond to loss of plasma membrane identity from recycling endosomes.

A functional dissection of PTEN N-terminus: implications in PTEN subcellular targeting and tumor suppressor activity.

Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.

Yeast-based methods to assess PTEN phosphoinositide phosphatase activity in vivo.

The PTEN phosphoinositide 3-phosphatase is a tumor suppressor commonly targeted by pathologic missense mutations. Subject to multiple complex layers of regulation, its capital role in cancer relies on its counteracting function of class I phosphoinositide 3-kinase (PI3K), a key feature in oncogenic signaling pathways. Precise assessment of the involvement of PTEN mutations described in the clinics in loss of catalytic activity requires either tedious in vitro phosphatase assays or in vivo experiments involving transfection into mammalian cell lines. Taking advantage of the versatility of the model organism Saccharomyces cerevisiae, we have developed different functional assays by reconstitution of the mammalian PI3K-PTEN switch in this lower eukaryote. This methodology is based on the fact that regulated PI3K expression in yeast cells causes conversion of PtdIns(4,5)P2 in PtdIns(3,4,5)P3 and co-expression of PTEN counteracts this effect. This can be traced by monitoring growth, given that PtdIns(4,5)P2 pools are essential for the yeast cell, or by using fluorescent reporters amenable for microscopy or flow cytometry. Here we describe the methodology and review its application to evaluate the functionality of PTEN mutations. We show that the technique is amenable to both directed and systematic structure-function relationship studies, and present an example of its use for the study of the recently discovered PTEN-L variant.

A yeast-based in vivo bioassay to screen for class I phosphatidylinositol 3-kinase specific inhibitors.

The phosphatidylinositol 3-kinase (PI3K) pathway couples receptor-mediated signaling to essential cellular functions by generating the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate. This pathway is implicated in multiple aspects of oncogenesis. A low-cost bioassay that readily measures PI3K inhibition in vivo would serve as a valuable tool for research in this field. Using heterologous expression, we have previously reconstituted the PI3K pathway in the model organism Saccharomyces cerevisiae. On the basis of the fact that the overproduction of PI3K is toxic in yeast, we tested the ability of commercial PI3K inhibitors to rescue cell growth. All compounds tested counteracted the PI3K-induced toxicity. Among them, 15e and PI-103 were the most active. Strategies to raise the intracellular drug concentration, specifically the use of 0.003% sodium dodecyl sulfate and the elimination of the Snq2 detoxification pump, optimized the bioassay by enhancing its sensitivity. The humanized yeast-based assay was then tested on a pilot scale for high-throughput screening (HTS) purposes using a collection of natural products of microbial origin. From 9600 extracts tested, 0.6% led to a recovery of yeast growth reproducibly, selectively, and in a dose-dependent manner. Cumulatively, we show that the developed PI3K inhibition bioassay is robust and applicable to large-scale HTS.

A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes.

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.

Interaction of the Salmonella Typhimurium effector protein SopB with host cell Cdc42 is involved in intracellular replication.

The phosphoinositide phosphatase SopB/SigD is a type III secretion system effector that plays multiple roles in Salmonella internalization and intracellular survival. We previously reported that SopB complexed with and inhibited the small GTPase Cdc42 when expressed in a yeast model system, independently of its phosphatase activity. Here we show that human Cdc42, but not Rac1, interacts with catalytically inactive SopB when coexpressed in Saccharomyces cerevisiae. This interaction occurs with both constitutively active and non-activatable Cdc42, suggesting that SopB binds Cdc42 independently of its activation state. By mutational analysis we have narrowed the Cdc42-interacting region of SopB to the first 142 amino acids, and isolated a collection of point mutations in this region, mainly affecting leucine residues conserved in the related Shigella IpgD protein. Such mutations yielded SopB unable to interact with Cdc42 but maintained phosphatase activity. SopB mutant proteins defective for binding Cdc42 were ubiquitinated upon translocation in mammalian cells, but their localization to the Salmonella-containing vacuole was reduced compared with wild-type SopB. Whereas invasion of mammalian cells by Salmonella bearing these sopB mutations was not affected, intracellular replication was less efficient, suggesting that SopB-Cdc42 interaction contributes to the adaptation of Salmonella to the intracellular environment.

Addressing the effects of Salmonella internalization in host cell signaling on a reverse-phase protein array.

Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellularly into specialized vacuolae. This involves changes in host cell signaling elicited by bacterial proteins delivered via type III secretion systems (TTSS). One of the two TTSSs of Salmonella enterica serovar Typhimurium encoded by the Salmonella pathogenicity island-1, triggers bacterial internalization. Among the effector proteins translocated by this TTSS, the GTPase modulator SopE/E2 and the phosphoinositide phosphatase SigD are known to play key roles in these processes. To better understand their contribution to re-programming host cell pathways, we used ZeptoMARK reverse-phase protein array technology, which allows printing 32-sample lysate arrays that can be analyzed with phospho-specific antibodies to evaluate the phosphorylation of signaling proteins. Lysates were obtained at different times after infection of HeLa cells with WT, TTSS-deficient, sopE/E2 and sigD single and double deletants, as well as different sigD Salmonella mutants. Our analysis detected activation of p38, JNK and ERK mitogen-activated protein kinases, mainly dependent on SopE/E2, as well as SigD-dependent phosphorylation of PKB/Akt and its targets GSK-3beta and FKHR/FoxO. This is the first time that reverse-phase protein array technology is used in the cellular microbiology field, demonstrating its value to screen for host signaling events through bacterial infection.

Phosphatidylinositol 3-kinase-dependent activation of mammalian protein kinase B/Akt in Saccharomyces cerevisiae, an in vivo model for the functional study of Akt mutations.

In animal cells, Akt (also called protein kinase B) is activated by stimuli that elevate the level of phosphatidylinositol 3,4,5-trisphosphate and is a major effector for eliciting responses that support cell growth and survival. We have shown previously that co-expression of Akt1 in budding yeast (Saccharomyces cerevisiae) along with hyperactive p110alpha, the catalytic subunit of mammalian phosphatidylinositol 3-kinase, results in Akt1 relocalization to cellular membranes and activation. In the present study, we show that activation of all three mammalian Akt isoforms by wild-type p110alpha causes deleterious effects on yeast cell growth. Toxicity of Akt in S. cerevisiae required its catalytic activity, its pleckstrin homology domain, and phosphorylation of its activation loop, but not phosphorylation of its hydrophobic motif. We demonstrate that expression in yeast of the only purported oncogenic allele, Akt1(E17K), leads to enhanced phenotypes. Ala-scanning mutagenesis of the VL1 region within the phosphatidylinositol 3,4,5-trisphosphate-interacting pocket of the Akt1 pleckstrin homology domain revealed that most residues in this region are essential for Akt1 activity. We found that active Akt leads to enhanced signaling through the yeast cell wall integrity pathway. This effect requires the upstream Rho1 activator Rom2 and involves both phosphorylation of the MAPK Slt2 and expression of its transcriptional targets, thus providing a quantitative reporter system for heterologous Akt activity in vivo. Collectively, our results disclose a heterologous yeast system that allows the functional assessment in vivo of both loss-of-function and tumorigenic Akt alleles.