Salmonella enterica: a surprisingly well-adapted intracellular lifestyle

The infectious intracellular lifestyle of Salmonella enterica relies on the adaptation to nutritional conditions within the Salmonella-containing vacuole (SCV) in host cells. We summarize latest results on metabolic requirements for Salmonella during infection. This includes intracellular phenotypes of mutant strains based on metabolic modeling and experimental tests, isotopolog profiling using (13)C-compounds in intracellular Salmonella, and complementation of metabolic defects for attenuated mutant strains towards a comprehensive understanding of the metabolic requirements of the intracellular lifestyle of Salmonella. Helpful for this are also genomic comparisons. We outline further recent studies and which analyses of intracellular phenotypes and improved metabolic simulations were done and comment on technical required steps as well as progress involved in the iterative refinement of metabolic flux models, analyses of mutant phenotypes, and isotopolog analyses. Salmonella lifestyle is well-adapted to the SCV and its specific metabolic requirements. Salmonella metabolism adapts rapidly to SCV conditions, the metabolic generalist Salmonella is quite successful in host infection

A suicide gene approach using the human pro-apoptotic protein tBid inhibits HIV-1 replication

<p>Abstract</p> <p>Background</p> <p>Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, however, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. These preconditions have not yet been completely fulfilled and, thus, success of suicide approaches has been limited so far. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection.</p> <p>Results</p> <p>When tBid was introduced into the HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector pLRed(INS)₂R, very efficient induction of apoptosis was observed within 24 hours, but only in the presence of both HIV-1 regulatory proteins Tat and Rev. Induction of apoptosis was not observed in their absence. Cells containing this vector rapidly died when transfected with plasmids containing full-length viral genomic DNA, completely eliminating the chance for HIV-1 replication. Viral replication was also strongly reduced when cells were infected with HIV-1 particles.</p> <p>Conclusions</p> <p>This suicide vector has the potential to establish a safe and effective gene therapy approach to exclusively eliminate HIV-1 infected cells before infectious virus particles are released.</p

De novo missense variants in FBXO11 alter its protein expression and subcellular localization.

Recently, we and others identified de novo FBXO11 variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs

De novo missense variants in FBXO11 alter its protein expression and subcellular localization.

Recently, we and others identified de novo FBXO11 variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs

PHIP-associated Chung-Jansen syndrome: Report of 23 new individuals

In 2016 and 2018, Chung, Jansen and others described a new syndrome caused by haploinsufficiency of PHIP (pleckstrin homology domain interacting protein, OMIM *612,870) and mainly characterized by developmental delay (DD), learning difficulties/intellectual disability (ID), behavioral abnormalities, facial dysmorphism and obesity (CHUJANS, OMIM #617991). So far, PHIP alterations appear to be a rare cause of DD/ID. “Omics” technologies such as exome sequencing or array analyses have led to the identification of distinct types of alterations of PHIP, including, truncating variants, missense substitutions, splice variants and large deletions encompassing portions of the gene or the entire gene as well as adjacent genomic regions. We collected clinical and genetic data of 23 individuals with PHIP-associated Chung-Jansen syndrome (CHUJANS) from all over Europe. Follow-up investigations (e.g. Sanger sequencing, qPCR or Fluorescence-in-situ-Hybridization) and segregation analysis showed either de novo occurrence or inheritance from an also (mildly) affected parent. In accordance with previously described patients, almost all individuals reported here show developmental delay (22/23), learning disability or ID (22/23), behavioral abnormalities (20/23), weight problems (13/23) and characteristic craniofacial features (i.e. large ears/earlobes, prominent eyebrows, anteverted nares and long philtrum (23/23)). To further investigate the facial gestalt of individuals with CHUJANS, we performed facial analysis using the GestaltMatcher approach. By this, we could establish that PHIP patients are indistinguishable based on the type of PHIP alteration (e.g. missense, loss-of-function, splice site) but show a significant difference to the average face of healthy individuals as well as to individuals with Prader-Willi syndrome (PWS, OMIM #176270) or with a CUL4B-alteration (Intellectual developmental disorder, X-linked, syndromic, Cabezas type, OMIM #300354). Our findings expand the mutational and clinical spectrum of CHUJANS. We discuss the molecular and clinical features in comparison to the published individuals. The fact that some variants were inherited from a mildly affected parent further illustrates the variability of the associated phenotype and outlines the importance of a thorough clinical evaluation combined with genetic analyses for accurate diagnosis and counselling

Analyses of IgE-epitope profiles from legume allergens as approach towards the development of novel diagnostic and therapeutic reagents in food allergy

Legumes are an important source of high-value proteins and edible oils in the human diet. However, they are also a known and frequent source of severe allergic reactions to foods. Immediate-type allergic reactions are elicited by allergenic proteins (allergens), mediated by their interaction with the immune system via allergen-specific antibodies of the isotype immunoglobulin E (IgE). These specific IgE antibodies to total protein of the allergenic food, e.g. peanut, can be measured in vitro to diagnose an allergic sensitization. While specific IgE to total protein extracts indicates a sensitization, it is not necessarily a proof for clinical reactivity. In contrast, the measurement of specific IgE to selected allergen components, such as the peanut 2S albumin Ara h 2, has been proposed to improve the in vitro diagnostic specificity for a clinical reactivity. In this context, specific recognition patterns of IgE binding at the protein level, and further at the peptide level, may allow the development of advanced diagnostic approaches, and innovative therapeutic reagents. However, knowledge about the relevance of allergenic storage proteins, such as 2S albumins and 7S globulins, of the legumes peanut, pea and soybean and their IgE-binding peptides is in part controversial or limited. Therefore, this study aimed to determine the serum IgE binding of legume-allergic versus sensitized but clinically tolerant children to homologous 2S and 7S legume allergens from peanut, pea and soybean at the protein and peptide level. In this study, sera from legume-allergic as well as sensitized but clinically tolerant children with legume extract-specific IgE ≥ 0.35 kUA/L were included. Total protein extracts and recombinant 7S globulins and 2S albumins of peanut, pea, and soybean, respectively, were prepared. Afterwards, serum IgE binding to legume extracts and recombinant legume allergens was individually determined by means of densitometric immunoblot analysis. Overlapping peptides (15 AA; offset 4 AA) representing the full-length allergens were synthesized and analyzed for their ability to bind serum IgE on CelluSpotTM multipeptide microarrays. The influence of post-translational hydroxylation of proline residues in peanut Ara h 2 on the capacity to bind serum IgE and thus on the diagnostic value was additionally investigated on the peptide level. Potential candidate diagnostic peptides specific for peanut and pea allergy were identified, according to predefined selection criteria that were established in this study. Finally, the diagnostic value of the investigated recombinant legume allergens and of the potential candidate diagnostic peptides was determined as area under curve (AUC) by receiver operating characteristic (ROC) curve analysis. By doing so, it should be determined whether peptides may serve as additional or alternative reagents in the in vitro diagnosis of legume allergy. However, due to the very limited number of available and included soybean-allergic patients, soybean was excluded from a detailed analysis of candidate diagnostic peptides and ROC curve analysis. According to immunoblot analysis and the specific limit of detection, serum IgE of 48%, 79% and 50% of peanut-, pea- and soybean-allergic children bound to the 7S globulins rAra h 1, rPis s 1 and rGly m 5.03, respectively. Of the peanut-, pea- and soybean-sensitized but tolerant children 8%, 20% and 20% showed a serum IgE binding to the respective 7S globulins. The most striking difference between the three legumes could be observed regarding the relevance of the 2S albumins. In immunoblot analysis, 65% and 78% of peanut-allergic children showed a serum IgE binding to rAra h 2.01 and rAra h 2.02, respectively. In contrast, serum IgE of peanut-sensitized but tolerant patients did not bind to any of both Ara h 2 isoforms. Hence, the relevance and the diagnostic value of both 2S albumin isoforms, especially of rAra h 2.02, in peanut allergy were further highlighted. Based on immunoblot analyses, such clinical relevance of 2S albumins could not be found in pea and soybean. Here, negligible or no serum IgE binding to 2S albumins was detectable in pea- as well as soybean-allergic children. Based on the ROC curve analysis of the investigated recombinant proteins analyzed in immunoblot, rAra h 2.02 (AUC 0.86) and rPis s 1 (AUC 0.86) had the highest diagnostic value in peanut and pea allergy, respectively, also compared to the respective total legume protein extract. With regard to potential diagnostic peptides, two Ara h 2-derived peptide pairs (AUC 0.87-0.90) with a diagnostic value comparable to that of full-length rAra h 2.02 (AUC 0.86) could be identified for peanut allergy. Each of the two peptide pairs contains at least one peptide with hydroxylated proline residues. In addition, it could be observed that hydroxylation of proline residues in Ara h 2-derived peptides increased the sensitivity while maintaining the specificity which makes this post-translational modification an interesting target for future diagnostic approaches. Moreover, for pea allergy eleven candidate diagnostic peptides of Pis s 1 could be identified. Compared to pea extract and rPis s 1, the candidate peptides had, with an AUC of 0.99, the highest diagnostic value in this pea study population. In this pediatric study population, general differences in allergenic potency of the 2S and 7S storage proteins of the three investigated legumes were apparent. In peanut, Ara h 1 and Ara h 2 could be identified as relevant allergens. However, based on immunoblot analysis, the 2S albumin Ara h 2, in particular the isoform Ara h 2.02, was the most relevant allergen with the highest diagnostic value in this peanut study population. In contrast, in pea and soybean 2S albumins were excluded as relevant allergens in this study. In pea, the 7S globulin Pis s 1 was identified as a major and immunodominant allergen. A similar trend seemed to be observed for Gly m 5.03 and soybean, but due to the limited number of soybean-allergic patients included in this study, no further conclusions can be drawn. In addition, the investigation of IgE binding at the linear peptide level revealed that peptides with a diagnostic value that is comparable or even better than that of the respective full-length recombinant proteins could be identified. These peptides may potentially serve as additional or alternative reagents in the in vitro diagnosis of legume allergy; however, this requires verification in a prospective study. Moreover, based on this study, additional knowledge about relevant allergens and their IgE-binding sites was obtained. This knowledge can support the development of novel therapeutic reagents with improved characteristics. In this context, IgE binding to short peptides of allergens was investigated to localize IgE-binding sites in approximation to linear (continuous) IgE epitopes. It could be shown that linear IgE-binding epitopes of natural Ara h 2, as represented by a 27-mer peptide, still induced relevant mast cell degranulation. This finding underpins the potential relevance of linear IgE-binding sites with regard to total allergenic potency, and should thus be considered in the development of novel reagents for the immunotherapeutic treatment of peanut allergy as otherwise patients might be at high risk of unintended side effects. In addition, identified immunodominant IgE-binding sites of Pis s 1 may allow the development of hypoallergenic substitution variants for allergen-specific immunotherapy of pea allergy. The relevance of these findings with regard to efficacy and safety can be further addressed in preclinical models of peanut and pea allergy, respectively

Salmonella—how a metabolic generalist adopts an intracellular lifestyle during infection

The human-pathogenic bacterium Salmonella enterica adjusts and adapts to different environments while attempting colonization. In the course of infection nutrient availabilities change drastically. New techniques, “-omics” data and subsequent integration by systems biology improve our understanding of these changes. We review changes in metabolism focusing on amino acid and carbohydrate metabolism. Furthermore, the adaptation process is associated with the activation of genes of the Salmonella pathogenicity islands (SPIs). Anti-infective strategies have to take these insights into account and include metabolic and other strategies. Salmonella infections will remain a challenge for infection biology

Blocks in Tricarboxylic Acid Cycle of Salmonella enterica Cause Global Perturbation of Carbon Storage, Motility, and Host-Pathogen Interaction.

Noster J, Hansmeier N, Persicke M, et al. Blocks in Tricarboxylic Acid Cycle of Salmonella enterica Cause Global Perturbation of Carbon Storage, Motility, and Host-Pathogen Interaction. mSphere. 2019;4(6): e00796-19.The tricarboxylic acid (TCA) cycle is a central metabolic hub in most cells. Virulence functions of bacterial pathogens such as facultative intracellular Salmonella enterica serovar Typhimurium (S Typhimurium) are closely connected to cellular metabolism. During systematic analyses of mutant strains with defects in the TCA cycle, a strain deficient in all fumarase isoforms (DeltafumABC) elicited a unique metabolic profile. Alongside fumarate, S Typhimurium DeltafumABC accumulates intermediates of the glycolysis and pentose phosphate pathway. Analyses by metabolomics and proteomics revealed that fumarate accumulation redirects carbon fluxes toward glycogen synthesis due to high (p)ppGpp levels. In addition, we observed reduced abundance of CheY, leading to altered motility and increased phagocytosis of S Typhimurium by macrophages. Deletion of glycogen synthase restored normal carbon fluxes and phagocytosis and partially restored levels of CheY. We propose that utilization of accumulated fumarate as carbon source induces a status similar to exponential- to stationary-growth-phase transition by switching from preferred carbon sources to fumarate, which increases (p)ppGpp levels and thereby glycogen synthesis. Thus, we observed a new form of interplay between metabolism of S Typhimurium and cellular functions and virulence.IMPORTANCE We performed perturbation analyses of the tricarboxylic acid cycle of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium. The defect of fumarase activity led to accumulation of fumarate but also resulted in a global alteration of carbon fluxes, leading to increased storage of glycogen. Gross alterations were observed in proteome and metabolome compositions of fumarase-deficient Salmonella In turn, these changes were linked to aberrant motility patterns of the mutant strain and resulted in highly increased phagocytic uptake by macrophages. Our findings indicate that basic cellular functions and specific virulence functions in Salmonella critically depend on the proper function of the primary metabolism. Copyright © 2019 Noster et al

Pea (Pisum sativum) allergy in children: Pis s 1 is an immunodominant major pea allergen and presents IgE binding sites with potential diagnostic value

BACKGROUND: Food allergy to pea (Pisum sativum) has been rarely studied in children at the clinical and molecular levels. OBJECTIVE: To elucidate the allergenic relevance and diagnostic value of pea 7S globulin Pis s 1, nsLTP, and 2S albumins PA1 and PA2 in children. METHODS: Children with pea-specific IgE ≥ 0.35 kUA/L and clinical evidence of pea allergy or tolerance were included in the study. IgE binding against pea total protein extract, recombinant (r) rPis s 1, rPA1, rPA2, and natural nsLTP was analysed using IgE immunoblot/inhibition. Mediator release potency was investigated in passively sensitized rat basophil leukaemia (RBL) 2H3-cells. IgE binding to synthetic overlapping peptides of Pis s 1 was detected on multipeptide microarrays. RESULTS: 19 pea-sensitized children were included, 14 with doctors’ diagnosed allergy and 5 with tolerance to pea (median age 3.5 and 4.5 years, respectively). 11/14 (78%) pea-allergic and 1/5 (20%) tolerant children were sensitized to Pis s 1. Under the reducing conditions of immunoblot analysis, IgE binding to rPA1 was negligible, sensitization to rPA2 and nsLTP undetectable. Compared to pea total protein extract, rPis s 1 displayed on average 58% IgE binding capacity and a 20-fold higher mediator release potency. Selected Pis s 1-related peptides displayed IgE binding in pea-allergic but not in pea-tolerant children. CONCLUSIONS AND CLINICAL RELEVANCE: In this study group, Pis s 1 is a major immunodominant allergen in pea-allergic children. Evidence for sensitization to nsLTP and 2S albumins was low but requires further verification with regard to conformational epitopes. Recombinant Pis s 1 and related peptides which were exclusively recognized by pea-allergic children may improve in vitro diagnosis of pea allergy once verified in prospective studies with larger study groups

Pea ( Pisum sativum

BACKGROUND: Food allergy to pea (Pisum sativum) has been rarely studied in children at the clinical and molecular levels. OBJECTIVE: To elucidate the allergenic relevance and diagnostic value of pea 7S globulin Pis s 1, nsLTP, and 2S albumins PA1 and PA2 in children. METHODS: Children with pea-specific IgE ≥ 0.35 kUA/L and clinical evidence of pea allergy or tolerance were included in the study. IgE binding against pea total protein extract, recombinant (r) rPis s 1, rPA1, rPA2, and natural nsLTP was analysed using IgE immunoblot/inhibition. Mediator release potency was investigated in passively sensitized rat basophil leukaemia (RBL) 2H3-cells. IgE binding to synthetic overlapping peptides of Pis s 1 was detected on multipeptide microarrays. RESULTS: 19 pea-sensitized children were included, 14 with doctors’ diagnosed allergy and 5 with tolerance to pea (median age 3.5 and 4.5 years, respectively). 11/14 (78%) pea-allergic and 1/5 (20%) tolerant children were sensitized to Pis s 1. Under the reducing conditions of immunoblot analysis, IgE binding to rPA1 was negligible, sensitization to rPA2 and nsLTP undetectable. Compared to pea total protein extract, rPis s 1 displayed on average 58% IgE binding capacity and a 20-fold higher mediator release potency. Selected Pis s 1-related peptides displayed IgE binding in pea-allergic but not in pea-tolerant children. CONCLUSIONS AND CLINICAL RELEVANCE: In this study group, Pis s 1 is a major immunodominant allergen in pea-allergic children. Evidence for sensitization to nsLTP and 2S albumins was low but requires further verification with regard to conformational epitopes. Recombinant Pis s 1 and related peptides which were exclusively recognized by pea-allergic children may improve in vitro diagnosis of pea allergy once verified in prospective studies with larger study groups