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The COVID-19 pandemic has imposed several challenges and strains at all levels of the educational system, especially as a consequence of lockdown and social distance measures. After a period of exclusive use of the online educational environment, educators have adapted to the new circumstances and, by a combination of different strategies, have fought to overcome the limitations and deficiencies of virtual learning. Student motivation, productivity, and creativity continue to be the main pedagogical issues that have to be reached with the new didactic tools developed during the pandemic. At the same time, this pandemic has shown the importance of the inclusion of microbiology as a core element of the educational curriculum and the opportunity to raise public awareness of the importance of microbes to everyday life.

Gasdermin D (GSDMD) and mixed lineage kinase domain-like protein (MLKL) are the pore-forming effectors of pyroptosis and necroptosis, respectively, with the capacity to disturb plasma membrane selective permeability and induce regulated cell death. The budding yeast Saccharomyces cerevisiae has long been used as a simple eukaryotic model for the study of proteins associated with human diseases by heterologous expression. In this work, we expressed in yeast both GSDMD and its N-terminal domain (GSDMD(NT)) to characterize their cellular effects and compare them to those of MLKL. GSDMD(NT) and MLKL inhibited yeast growth, formed cytoplasmic aggregates and fragmented mitochondria. Loss-of-function point mutants of GSDMD(NT) showed affinity for this organelle. Besides, GSDMD(NT) and MLKL caused an irreversible cell cycle arrest through TORC1 inhibition and disrupted endosomal and autophagic vesicular traffic. Our results provide a basis for a humanized yeast platform to study GSDMD and MLKL, a useful tool for structure–function assays and drug discovery.

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.

Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human Caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of Caspase-8. Overexpression of both caspases in the heterologous model led to their activation, and caused mitochondrial depolarization, ROS production, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and Caspase-8 was more aggressive than Caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which Caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of Caspase-1 enhanced its activity, while the deletion of the death effector domain (DED) of Caspase-8 diminished it. We propose the yeast model as a useful and manageable tool to explore Caspase-1 structure-function relationships, the impact of mutations or the activity of putative inhibitors or regulators.

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 to autism. PTEN nonsense mutations generating premature termination codons (PTC) and producing non-functional truncated PTEN proteins are frequent in association with human disease. However, there are no studies addressing the restoring of full-length PTEN proteins from 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 of PTEN functional restoration in response to readthrough-inducing compounds. Comprehensive readthrough analyses of disease-associated PTComes will be valuable tools for implementation of readthrough-based precision interventions in specific groups of patients. This article is protected by copyright. All rights reserved.

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., Author summary Brucella is a genus of zoonotic bacteria that cause severe disease in a variety of mammals, ranging from farm animals (as bovines, swine and ovine) to marine mammals. Transmission to humans, often by ingestion of non-treated dairy products, leads to serious systemic infection. Brucella abortus invades host cells and replicates intracellularly. Such behavior relies on the injection of bacterial proteins into the host cytoplasm via specialized secretion systems. Our work focuses on the study of two of these factors, BtpA and BtpB, previously described to contain Toll/Interleukin-1 Receptor (TIR)-domains that modulate innate immunity. We use here two biological models: the yeast Saccharomyces cerevisiae and human cell lines. We found that the TIR domains of both Brucella proteins were necessary and sufficient to collapse energy metabolism in yeast by depleting ATP and NAD+. This result was translatable to higher cells and consistent with the recently described NADase activity of some TIR domains both in mammalian and bacterial proteins. Importantly, we demonstrate that Brucella down-regulates total NAD levels in host cells by using both BtpA and BtpB effectors. Our results show that NAD+ is targeted by Brucella during infection, which may constitute a novel mechanism for its pathogenicity.

espanolLa produccion bibliografica del siglo XVIII se sirvio de las Gacetas de Mexico para la promocion de autores y titulos de impresos que aparecian cotidianamente. Bajo los titulos Libros nuevos de Mexico, Libros nuevos de Espana o Libros nuevos, varios de los fasciculos de las Gacetas ofrecen a los lectores informacion sobre novedades bibliograficas. El presente texto se enfoca particularmente en la identificacion del conjunto de noticias sobre impresos espanoles que aparecen en las dos primeras Gacetas de Mexico, que en total representan 15 anos de publicacion y mas de 160 fasciculos. Se ofrecen datos sobre autores, titulos, temas y lugares de venta de los libros espanoles que circularon en Mexico durante la primera mitad del siglo XVIII. EnglishBibliographic production in the 18th century utilized the Gacetas de Mexico to promote the authors and printed titles that appearing daily under such headings as “New Mexican Books”, “New Spanish Books”, or “New Books” (Libros nuevos de Mexico, Libros nuevos de Espana, Libros nuevos). Several fascicles of the Gacetas offered to their readers information on recent publications. This paper focuses particularly on the identification of all the news on Spanish print appearing in the first two Gazettes of Mexico, which together represent 15 years of publication and over 160 fascicles. Data on authors, titles, subjects and places selling Spanish books that circulated in Mexico during the first half of the eighteenth century.

Small World Initiative and Tiny Earth are popular citizen science programs that are implemented worldwide in response to the global antibiotic resistance crisis. When starting up the program in Albacete (Spain), we noted that rates of isolated antibiotic-producing bacteria are generally low. To make the activity more stimulating for participating students, we modified the protocol to obtain more positive results by focusing on isolation of actinomycetes, the main producers of most clinically used antibiotics. Adaptations involved redesigning culture media, incubation times and temperatures, and modification of the ESKAPE antibiosis experiment by employing an agar-transplantation step. Of 390 bacterial isolates tested, almost 6% tested positive in antibiosis experiments and DNA sequence analysis confirmed that all positives are actinomycetes, demonstrating that our protocol is efficient toward isolating antibiotic-producing actinomycetes from soil. Evaluation forms filled by participating students indicated that the program was received very positively and that our modifications contribute to make this educational program more stimulating and efficient.

Brucellaspecies 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.BrucellaToll/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 yeastSaccharomyces cerevisiaeas 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 inBrucellapathogenesis.

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., Author summary Klebsiella pneumoniae has been singled out as an “urgent threat to human health” due to extremely drug resistant strains. Numerous studies investigate the molecular mechanisms underlying antibiotic resistance in K. pneumoniae, while others dissect the virulence strategies of this pathogen. However, there is still limited knowledge on the fitness of Klebsiella in the environment, and, particularly, the competition of Klebsiella with other species. Here, we demonstrate that Klebsiella exploits the type VI secretion system (T6SS) nanoweapon to kill bacterial competitors and fungi. K. pneumoniae perceives T6SS attacks from bacterial competitors, resulting in retaliation against the aggressive cell. The perception of the attack involved the sensor PhoPQ and led to the up-regulation of the T6SS. We identified one of the toxins deployed by the T6SS to antagonize other microbes and revealed how Klebsiella protects itself from this toxin. Our findings provide a better understanding of the T6SS role in microbial competition and uncover new aspects on how bacteria regulate T6SS-mediated microbial antagonism.

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 Small World Initiative (SWI) and Tiny Earth are a consolidated and successful education programs rooted in the USA that tackle the antibiotic crisis by a crowdsourcing strategy. Based on active learning, it challenges young students to discover novel bioactive-producing microorganisms from environmental soil samples. Besides its pedagogical efficiency to impart microbiology content in academic curricula, SWI promotes vocations in research and development in Experimental Sciences and, at the same time, disseminates the antibiotic awareness guidelines of the World Health Organization. We have adapted the SWI program to the Spanish academic environment by a pioneering hierarchic strategy based on service-learning that involves two education levels (higher education and high school) with different degrees of responsibility. Throughout the academic year, 23 SWI teams, each consisting of 3-7 undergraduate students led by one faculty member, coordinated off-campus programs in 22 local high schools, involving 597 high school students as researchers. Post-survey-based evaluation of the program reveals a satisfactory achievement of goals: acquiring scientific abilities and general or personal competences by university students, as well as promoting academic decisions to inspire vocations for science- and technology-oriented degrees in younger students, and successfully communicating scientific culture in antimicrobial resistance to a young stratum of society.

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.

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.

Phosphatidylinositol 3-kinase (PI3K) is a key regulator of phosphoinositide-dependent signaling in mammalian cells and its dysfunction is related to multiple syndromes, including cancer. By heterologous expression in Saccharomyces cerevisiae, we have developed a humanized yeast system as a tool for functional studies on higher eukaryotic PI3K. Here we restrict PI3K activity in yeast to specific plasma membrane (PM) microdomains by fusing the p110α PI3K catalytic subunit to either a septin or an eisosome component. We engineered a Dual Reporter for PI3K (DRAPIK), useful to monitor activity on cellular membranes in vivo at a single-cell level, by simultaneous PM staining of the enzyme substrate (PtdIns4,5P2) with GFP and its product (PtdIns3,4,5P3) with mCherry. We also developed a sensitive FLUorescence by PI3K Inhibition (FLUPI) assay based on a GFP transcriptional reporter that is turned off by PI3K activity. This reporter system proved useful to monitor PI3K inhibition in vivo by active compounds. Such novel tools were used to study the performance of yeast PM microdomain-directed PI3K. Our results show that tethering heterologous PI3K to discrete PM domains potentiates its activity on PtdIns4,5P2 but different locations display distinct effects on yeast growth and endocytosis.

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.

López-Goñi, Ignacio et al., Social networks are already being exploited for searching, storing, and sharing knowledge, demonstrating that they are an efficient vehicle for social learning. Consequently, they could be implemented as a competent tool for formal learning. Twitter is among the 10 most popular online social networks, integrating a community of over 500 million users around the world. Twitter has already been used in several educational programs and evaluated as a positive teaching experience with an outstanding potential in academic and educational environments (1–6). However, there are scarce examples available in the literature about its use in science teaching and communication. In this work, we present and analyze the application of Twitter to create an online space for communication and learning of basic microbiology. With this aim, a team of professionals in the field has imparted, to our knowledge, the first worldwide open access microbiology course via Twitter. Here we assess the results of our experience of using this social network as a tool for teaching, promoting, and communicating scientific knowledge to a wide audience.

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, β, 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.

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 C&alpha, 2 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.

Summary 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.

Saccharomyces cerevisiae has been widely used as a model eukaryotic organism to elucidate the molecular mechanisms that operate upon activation of signalling pathways. For over two decades, many clues to the regulation of mitogen-activated protein kinase (MAPK) pathways have derived from basic research in yeast. Here we review aspects of MAPK pathway fine-tuning, such as the functional implication of feedback loops or regulatory inputs from other pathways, mediated by post-transcriptional modifications on their components. The impact of recent phosphoproteomic approaches in this particular field is also discussed. Copyright © 2010 John Wiley & Sons, Ltd.

Model organisms have emerged as suitable and reliable biological tools to study the properties of proteins whose function is altered in human disease. In the case of the PI3K and PTEN human cancer-related proteins, several vertebrate and invertebrate models, including mouse, fly, worm and amoeba, have been exploited to obtain relevant functional information that has been conserved from these organisms to humans along evolution. The yeast Saccharomyces cerevisiae is an eukaryotic unicellular organism that lacks a canonical mammalian-like PI3K/PTEN pathway and PIP3 as a physiological second messenger, PIP2 being essential for its life. The mammalian PI3K/PTEN pathway can be reconstituted in S. cerevisiae, generating growth alteration phenotypes that can be easily monitored to perform in vivo functional analysis of the molecular constituents of this pathway. Here, we review the current nonmammalian model systems to study PTEN function, summarize our knowledge of PTEN orthologs in yeast species and propose the yeast S. cerevisiae as a sensitive biological sensor of PI3K oncogenicity and PTEN tumor suppressor activity.

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.

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.

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.

© 2015 Gil et al., 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., This work was supported in part by grants SAF2009-10226 from Ministerio Ciencia e Innovación (Spain and Fondo Europeo de Desarrollo Regional), SAF2013-48812-R from Ministerio de Economía y Competitividad (Spain), BIO2010-22369-C02-01 from Ministerio de Ciencia e Innovación (Spain), and S2011/BMD-2414 from Comunidad Autónoma de Madrid (Spain). A. Gil has been the recipient of CP04/00318 research contract from Instituto de Salud Carlos III (Spain).

The mammalian signalling pathway involving class I PI3K (phosphoinositide 3-kinase), PTEN (phosphatidylinositol 3-phosphatase) and PKB (protein kinase B)/c-Akt has roles in multiple processes, including cell proliferation and apoptosis. To facilitate novel approaches for genetic, molecular and pharmacological analyses of these proteins, we have reconstituted this signalling pathway by heterologous expression in the unicellular eukaryote, Saccharomyces cerevisiae (yeast). High-level expression of the p110 catalytic subunit of mammalian PI3K dramatically inhibits yeast cell growth. This effect depends on PI3K kinase activity and is reversed partially by a PI3K inhibitor (LY294002) and reversed fully by co-expression of catalytically active PTEN (but not its purported yeast orthologue, Tep1). Growth arrest by PI3K correlates with loss of PIP2 (phosphatidylinositol 4,5-bisphosphate) and its conversion into PIP3 (phosphatidylinositol 3,4,5-trisphosphate). PIP2 depletion causes severe rearrangements of actin and septin architecture, defects in secretion and endocytosis, and activation of the mitogen-activated protein kinase, Slt2. In yeast producing PIP3, PKB/c-Akt localizes to the plasma membrane and its phosphorylation is enhanced. Phospho-specific antibodies show that both active and kinase-dead PKB/c-Akt are phosphorylated at Thr308 and Ser473. Thr308 phosphorylation, but not Ser473 phosphorylation, requires the yeast orthologues of mammalian PDK1 (3-phosphoinositide-dependent protein kinase-1): Pkh1 and Pkh2. Elimination of yeast Tor1 and Tor2 function, or of the related kinases (Tel1, Mec1 and Tra1), did not block Ser473 phosphorylation, implicating another kinase(s). Reconstruction of the PI3K/PTEN/Akt pathway in yeast permits incisive study of these enzymes and analysis of their functional interactions in a simplified context, establishes a new tool to screen for novel agonists and antagonists and provides a method to deplete PIP2 uniquely in the yeast cell.

Summary The internalization of Salmonella into epithelial cells relies on the function of bacterial proteins which are injected into the cell by a specialized type III secretion system. Such bacterial effectors interfere with host cell signalling and induce local cytoskeletal rearrangements. One of such effectors is SigD/SopB, which shares homology with mammalian inositol phosphatases. We made use of the Saccharomyces cerevisiae model for elucidating new aspects of SigD function. Endogenous expression of SigD in yeast caused severe growth inhibition. Surprisingly, sigD alleles mutated in the catalytic site or even deleted for the whole C-terminal phosphatase domain still inhibited yeast growth by inducing loss of actin polarization and precluding the budding process. Accordingly, when expressed in HeLa cells, the same sigD alleles lost the ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, but still caused disappearance of actin fibres and loss of adherence. We delineate a region of 25 amino acids (residues 118–142) that is necessary for the effect of SigD on actin in HeLa cells. Our data indicate that SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity.

EspG is a conserved protein encoded by the locus of enterocyte effacement (LEE) of attaching and effacing (A/E) pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli and Citrobacter rodentium . EspG is delivered into infected host cells by a type III secretion system. The role of EspG in virulence has not yet been defined. Here we describe experiments that probe the virulence characteristics and biological activities of EspG in vitro and in vivo. A C. rodentium espG mutant displayed a significantly reduced ability to colonize C57BL/6 mice and to cause colonic hyperplasia. Epitope-tagged EspG was detected in the apical regions of infected colonic epithelial cells in infected mice, partially localizing with another LEE-encoded effector protein, Tir. EspG was found to interact with mammalian tubulin in both genetic screens and gel overlay assays. Binding to tubulin by EspG caused localized microtubule depolymerization, resulting in actin stress fiber formation through an undefined mechanism. Heterologous expression of EspG in yeast resulted in loss of cytoplasmic microtubule structure and function, preventing coordination between bud development and nuclear division. Yeast expressing EspG were also unable to control cortical actin polarity. We suggest that EspG contributes to the ability of A/E pathogens to establish infection through a modulation of the host cytoskeleton involving transient microtubule destruction and actin polymerization in a manner akin to the Shigella flexneri VirA protein.

The Saccharomyces cerevisiae CDC15, DBF2, TEM1 and CDC14 genes encode regulatory proteins that play a crucial role in the latest stages of the M phase of the cell cycle. By complementation of a S. cerevisiae cdc15-lyt1 mutant with a Candida albicans centromeric-based genomic library, we have isolated a homologue of the protein phosphatase-encoding gene CDC14. The sequence analysis of the C. albicans CDC14 gene reveals a putative open reading frame of 1626 base pairs interrupted by an intron located close to the 5′ region. Analysis of C. albicans cDNA proved that the intron is processed in vivo. The CaCDC14 gene shares 49% of amino acid sequence identity with the S. cerevisiae CDC14 gene, 46% with Schizosaccharomyces pombe homologue, 35% with Caenorhabditis elegans and 37% and 38% with human CDC14A and CDC14B genes, respectively. As expected, the C. albicans CDC14 gene complemented a S. cerevisiae cdc14-1 mutant. We found that this gene was able to efficiently suppress not only a S.cerevisiae cdc15-lyt1 mutant but also a dbf2-2 mutant in a low number of copies and allowed growth, although very slightly, of a tem1 deletant. Overexpression of the human CDC14A and CDC14B genes complemented, although very poorly, S. cerevisiae cdc15-lyt1 and dbf2-2 mutants, suggesting a conserved function of these genes throughout phylogeny. The sequence of CaCDC14 was deposited in the EMBL database under Accession No. AJ243449. Copyright © 2001 John Wiley & Sons, Ltd.

The complete yeast genome contains a large number of genes of unknown biological function. Simple, rapid and reliable specific screens are valuable tools in exploring gene function via systematic phenotypic analysis of large mutant collections. This report provides a new approach for monitoring changes in cell wall strength, based on the deleterious effects caused by ultrasound on the yeast cell surface. Sonication can thus be used for the screening of mutants affected in the architecture or stability of the cell wall, since such mutants are expected to have an altered sensitivity to this treatment compared to that of a wild-type. The experimental procedure, consisting in the quantification of damaged cells after a mild sonication treatment, by means of flow cytometry, can be applied on a large scale. The usefulness of the sonication assay as a primary screen for cell wall-related mutants is evaluated on the collection of calcofluor white-hypersensitive and -resistant mutants obtained by Lussier et al. (1997). A further phenotypic characterization of the sonication-hypersensitive mutants within the calcofluor white collection is also presented. Copyright © 1999 John Wiley & Sons, Ltd.

We have deleted six different ORFs of unknown function located on the right arm of Saccharomyces cerevisiae chromosome VII; namely, YGR187c/HGH1, YGR189c, YGR194c, YGR195w, YGR196c and YGR198w. No basic phenotypes could be attributed to the strains deleted in any of genes YGR187c/HGH1, YGR189c, YGR194c and YGR196c. These deletants did not show mating, sporulation or growth defects under any of the conditions tested. However, spores bearing deletions in either the YGR195w or YGR198w genes were unable to develop into macroscopical colonies. The YGR195w gene product shows significant homology with bacterial ribonuclease PH, an enzyme hitherto undescribed in yeasts, and its deletion causes a loss of viability after one to three rounds of cell division. Overexpression of this gene, using a tetracycline-regulatable promoter system, did not cause any effect on the cells. Contrary to what has been reported for prokaryotic homologs, this enzyme could play an essential role in yeast cell biology. The product encoded by the other essential ORF, YGR198w, shows no significant homology with any protein of known function in the databases. Spores bearing the deletion usually germinate and give rise to microcolonies of 50–100 non-viable cells. © 1998 John Wiley & Sons, Ltd.

We report the finding of a Saccharomyces cerevisiae gene necessary for growth in culture media with D-xylulose as the sole carbon source. This gene corresponds to the YGR194c open reading frame that we have previously described, and it is renamed now XKS1. Data bank comparisons of the protein encoded by the XKS1 gene showed significant homology with different xylulokinases, indicating a possible role in xylulose phosphorylation. The wild-type gene in a centromeric plasmid complemented defective growth of xks1 S. cerevisiae mutant strains in xylulose. By contrast, overexpression negatively influenced cell growth in this carbon source.

Summary: Saccharomyces cerevisiae strain V918 was previously isolated in a search for thermosensitive autolytic mutants and found to bear a recessive mutation that caused the development of multinucleate swollen cells undergoing cell lysis. The BEM2 gene has been isolated by complementation of the phenotype of a V918 segregant. BEM2 encodes a Rho-GTPase-activating protein (GAP) which is thought to act as a modulator of the Rho1 small GTPase. It is shown that the mutation causing the morphogenetic and autolytic phenotype in strain V918 and its segregants lies in the BEM2 gene, defining a new mutant allele, bem2-21. Mutants in the BEM2 gene have been reported to display loss of cell polarity and depolarization of the actin cytoskeleton, causing a bud-emergence defect. Low resistance to sonication and to hydrolytic enzymes proved that the cell wall is less protective in bem2-21 mutants than in wild-type strains. Moreover, bem2-21 mutants are more sensitive than the wild-type to several antifungal drugs. Transmission electron microscopy revealed the development of abnormally thick and wide septa and the existence of thin areas in the cell wall which probably account for cell lysis. The depolarization of actin in bem2-21 mutants did not preclude morphogenetic events such as cell elongation in homozygous diploid strains during nitrogen starvation in solid media, hyperpolarization of growth in a background bearing a mutated septin, or sporulation. Multinucleate cells from bem2-21 homozygous diploids underwent sporulation giving rise to multispored asci (‘polyads’), containing up to 36 spores. This phenomenon occurred only under osmotically stabilized conditions, suggesting that the integrity of the ascus wall is impaired in cells expressing the bem2-21 mutation. It is concluded that the function of the BEM2 gene product is essential for the maintenance of a functional cell wall.

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

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.

Yeast exo-1,3-beta-glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(beta-D-glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of the EXG1 gene product of Saccharomyces cerevisiae and its homologous gene in Candida albicans to function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions in S. cerevisiae. This technique has the advantage over other reporter gene systems--such as beta-galactosidase fusions--that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells--by sorting--after flow cytometry analysis.

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.

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.

Infection of cells and tissues by pathogenic microorganisms often involves severe reprogramming of host cell signaling. Typically, invasive microorganisms manipulate host cellular pathways seeking advantage for replication and survival within the host, or to evade the immune response. Understanding such subversion of the host cell by intracellular pathogens at a molecular level is the key to possible preventive and therapeutic interventions on infectious diseases. Reverse Protein Arrays (RPAs) have been exploited in other fields, especially in molecular oncology. However, this technology has not been applied yet to the study of infectious diseases. Coupling classic in vitro infection techniques used by cellular microbiologists to proteomic approaches such as RPA analysis should provide a wealth of information about how host cell pathways are manipulated by pathogens. The increasing availability of antibodies specific for phosphorylated epitopes in signaling proteins allows monitoring global changes in phosphorylation through the infection process by utilizing RPA analyses. In our lab, we have shown the potential of RPA technology in this field by devising a microarray consisting of lysates from cell cultures infected by Salmonella typhimurium. The protocols used are described here.

Infection of cells and tissues by pathogenic microorganisms often involves severe reprogramming of host cell signaling. Typically, invasive microorganisms manipulate host cellular pathways seeking advantage for replication and survival within the host, or to evade the immune response. Understanding such subversion of the host cell by intracellular pathogens at a molecular level is the key to possible preventive and therapeutic interventions on infectious diseases. Reverse Protein Arrays (RPAs) have been exploited in other fields, especially in molecular oncology. However, this technology has not been applied yet to the study of infectious diseases. Coupling classic in vitro infection techniques used by cellular microbiologists to proteomic approaches such as RPA analysis should provide a wealth of information about how host cell pathways are manipulated by pathogens. The increasing availability of antibodies specific for phosphorylated epitopes in signaling proteins allows monitoring global changes in phosphorylation through the infection process by utilizing RPA analyses. In our lab, we have shown the potential of RPA technology in this field by devising a microarray consisting of lysates from cell cultures infected by Salmonella typhimurium. The protocols used are described here.

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.