Stanley Brul | University of Amsterdam (original) (raw)

Papers by Stanley Brul

Applied and Environmental Microbiology, 1998

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous mole... more The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type Saccharomyces cerevisiae . This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to ...

International Journal of Molecular Sciences

Bacillus cereus can survive in the form of spores for prolonged periods posing a serious problem ... more Bacillus cereus can survive in the form of spores for prolonged periods posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. Our study aims at increasing knowledge of B. cereus spores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of B. cereus spores are a core with the genetic material encased by multiple protective layers, an important one being the spores′ inner membrane (IM), the location of many important germination proteins. To study mechanisms involved in germination of B. cereus spores, we have examined the organization of germinant receptors (GRs) in spores′ IM. Previous studies have indicated that in spores of B.cereus ATCC 14579 the L-alanine responsive GR, GerR, plays a major role in the germination process. In our study, the location of the GerR GR subunit, GerRB, in spores was examined as a C-terminal SGFP2 fusion protein expressed under the control of the gerR op...

mSphere

Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoil... more Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore’s inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg,...

Sorbic acid is a commonly used food preservative against yeast and fungal food spoilage. Understa... more Sorbic acid is a commonly used food preservative against yeast and fungal food spoilage. Understanding its effect on the molecular physiology of yeast cells will allow the food industry to develop knowledge-based strategies to make more optimal use of its preservative action. Here we show that the yeast membrane protein Pdr12, previously shown to be prominently involved in sorbic acid resistance development in laboratory strains, was strongly induced by the presence of sorbic acid in the culture medium in Saccharomyces strains isolated from spoiled foods. Induction of Pdr12 expression was seen both under laboratory conditions and upon growth in a commercial soft drink. Induction was rapid and maintained for the duration of the stress. No Pdr12-like protein induction was seen in Zygosaccharomyces bailii or Zygosaccharomyces lentus, two well-known beverages spoilage organisms. Finally, unexpectedly, our studies showed for the first time that pre-inducing Pdr12p to maximal levels by su...

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore ge... more Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination, and then inactivating the less heat resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores, and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single spore levels. The heat treatments used were 40-80 degrees Celcius, and for 0-300 min. The results were as follows. 1) Heat activation at 40-70 degrees Celcius promoted L-valine and L-asparagine-glucose-fructose-potassium (AGFK) induced germination in a time dependent manner. 2) The optimal heat activation temperatures for AGFK and L-valine germination via the GerB ...

Journal of Proteome Research

The resistance properties of the bacterial spores are partially due to spore surface proteins, ∼3... more The resistance properties of the bacterial spores are partially due to spore surface proteins, ∼30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis of the proteinaceous component of the spore coat, we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods. Young (day 1) and matured (day 5) Bacillus subtilis spores of wild-type and transglutaminase mutant strains were digested with formic acid and trypsin, and cross-linked peptides were enriched using strong cation exchange chromatography. The enriched crosslinked peptide fractions were subjected to Fourier-transform ion cyclotron resonance tandem mass spectrometry, and the high-quality fragmentation data obtained were analyzed using two specialized software tools, pLink2 and XiSearch, to identify cross-links. This analysis identified specific disulfide bonds between coat proteins CotE−CotE and CotJA−CotJC, obtained evidence of disulfide bonds in the spore crust proteins CotX, CotY, and CotZ, and identified dityrosine and ε-(γ)-glutamyl-lysine cross-linked coat proteins. The findings in this Letter are the first direct biochemical data on protein cross-linking in the spore coat and the first direct evidence of the cross-linked building blocks of the highly ordered and resistant structure called the spore coat.

International Journal of Molecular Sciences

The occurrence of bacterial pathogens in the food chain has caused a severe impact on public heal... more The occurrence of bacterial pathogens in the food chain has caused a severe impact on public health and welfare in both developing and developed countries. Moreover, the existence of antimicrobial-tolerant persisting morphotypes of these pathogens including both persister-cells as well as bacterial spores contributes to difficulty in elimination and in recurrent infection. Therefore, comprehensive understanding of the behavior of these persisting bacterial forms in their environmental niche and upon infection of humans is necessary. Since traditional antimicrobials fail to kill persisters and spores due to their (extremely) low metabolic activities, antimicrobial peptides (AMPs) have been intensively investigated as one of the most promising strategies against these persisting bacterial forms, showing high efficacy of inactivation. In addition, AMP-based foodborne pathogen detection and prevention of infection has made significant progress. This review focuses on recent research on ...

Cancer cell metabolism is dependent on cell-intrinsic factors like genetics, and cell-extrinsic f... more Cancer cell metabolism is dependent on cell-intrinsic factors like genetics, and cell-extrinsic factors like nutrient availability. In this context, understanding how these two aspects interact and how diet influences cellular metabolism is important for developing personalized treatment. In order to achieve this goal, genome-scale metabolic models (GEMs) are used, however, genetics and nutrient availability are rarely considered together. Here, we propose an integrated metabolic profiling, a framework that allows to enrich GEMs with metabolic gene expression data and information about nutrients. First, the RNA-seq is converted into Reaction Activity Score (RAS) to further scale reaction bounds. Second, nutrient availability is converted to Maximal Uptake Rate (MUR) to modify exchange reactions in a GEM. We applied our framework to the human osteosarcoma cell line (U2OS). Osteosarcoma is a common and primary malignant form of bone cancer with poor prognosis, and, as indicated in our...

mSphere

Bacillus subtilis spores can reactivate their metabolism through germination upon contact with ge... more Bacillus subtilis spores can reactivate their metabolism through germination upon contact with germinants and can develop into vegetative cells upon outgrowth. However, the mechanisms at the basis of the molecular machinery that triggers the spore germination and outgrowth processes are still largely unclear. To gain further insights into these processes, the transcriptome and proteome changes occurring during the conversion of spores to vegetative cells were analyzed in the present study. For each time point sampled, the changes in the spore proteome were quantitatively monitored relative to the proteome of metabolically 15N-labeled vegetative cells. Of the quantified proteins, 60% are shared by vegetative cells and spores, indicating that the spores have a minimal protein set, sufficient to resume metabolism upon completion of germination. These shared proteins thus represent the most basic “survival kit” for spore-based life. We observed no significant change in the proteome or t...

Clostridioides difficile-associated infection (CDI) is a health-care-associated infection mainly ... more Clostridioides difficile-associated infection (CDI) is a health-care-associated infection mainly transmitted via highly resistant endospores from one person to the other. In vivo, the spores need to germinate in to cells prior to establishing an infection. Bile acids and glycine, both available in sufficient amounts inside the human host intestinal tract, serve as efficient germinants for the spores. It is therefore, for better understanding of Clostridioides difficile virulence, crucial to study both the cell and spore states with respect to their genetic, metabolic and proteomic composition. In the present study, mass spectrometric relative protein quantification, based on the 14N/15N peptide isotopic ratios, has led to quantification of over 700 proteins from combined spore and cell samples. The analysis has revealed that the proteome turnover between a vegetative cell and a spore for this organism is moderate. Additionally, specific cell and spore surface proteins, vegetative ce...

Bacillus subtilis forms highly resistant, metabolically inactive dormant spores upon nutrient lim... more Bacillus subtilis forms highly resistant, metabolically inactive dormant spores upon nutrient limitation. These endospores pose challenges to the food and medical sectors. Spores reactivate their metabolism upon contact with germinants and develop into vegetative cells. The activation of the molecular machinery that triggers the progress of germination and spore outgrowth is still unsettled. To gain further insight in spore germination and outgrowth processes, the transcriptome and proteome changeover during spore germination and outgrowth to vegetative cells, was analysed. B. subtilis transcriptome analysis allow us to trace the different functional groups of genes expressed. For each time-point sample, the change in the spore proteome was quantitatively monitored relative to the reference proteome of 15N metabolically labelled vegetative cells. We observed until the phase transition, i.e. completion of germination, no significant change in the proteome. We have identified 36 trans...

Aging Cell

Calorie restriction is the only physiological intervention that extends lifespan throughout all k... more Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pH c) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pH c and the central nutrient signalling cAMP-Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pH c correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pH c showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.

PloS one, 2017

Use of some HIV-1 nucleoside reverse transcriptase inhibitors (NRTI) is associated with severe ad... more Use of some HIV-1 nucleoside reverse transcriptase inhibitors (NRTI) is associated with severe adverse events. However, the exact mechanisms behind their toxicity has not been fully understood. Mitochondrial dysfunction after chronic exposure to specific NRTIs has predominantly been assigned to mitochondrial polymerase-γ inhibition by NRTIs. However, an increasing amount of data suggests that this is not the sole mechanism. Many NRTI induced adverse events have been linked to the incurrence of oxidative stress, although the causality of events leading to reactive oxygen species (ROS) production and their role in toxicity is unclear. In this study we show that short-term effects of first generation NRTIs, which are rarely discussed in the literature, include inhibition of oxygen consumption, decreased ATP levels and increased ROS production. Collectively these events affect fitness and longevity of C. elegans through mitohormetic signalling events. Furthermore, we demonstrate that th...

Biochimica et biophysica acta, Jan 9, 2018

Three amphipathic cationic antimicrobial peptides (AMPs) were characterized by determining their ... more Three amphipathic cationic antimicrobial peptides (AMPs) were characterized by determining their effect on Gram-positive bacteria using Bacillus subtilis strain 168 as a model organism. These peptides were TC19 and TC84, derivatives of thrombocidin-1 (TC-1), the major AMPs of human blood platelets, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide based on human bactericidal permeability increasing protein (BPI). To elucidate the possible mode of action of the AMPs we performed a transcriptomic analysis using microarrays. Physiological analyses were performed using transmission electron microscopy (TEM), fluorescence microscopy and various B. subtilis mutants that produce essential membrane bound proteins fused to green fluorescent protein (GFP). The transcriptome analysis showed that the AMPs induced a cell envelope stress response (cell membrane and cell wall). The cell membrane stress response was confirmed with the physiological observations that TC19, TC84 and BP2 ...

FEMS Microbiology Letters

Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in... more Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in patients admitted to intensive care units. Resistance rapidly develops against two drugs of choice: ceftazidime and meropenem. Several therapeutic protocols were compared for reduction in viable cells and limiting development of resistance. Chemostat cultures were exposed to antibiotic concentrations measured in the blood of patients at low (5th percentile), medium (50th percentile) or high (95th percentile) levels in several therapy protocols to simulate therapy. Cultures exposed to ceftazidime recovered after 1 day at low, 2 days at medium and 3 days at high concentrations and developed corresponding levels of resistance. Patterns were very similar for meropenem except that recovery was delayed. Fluctuating levels and intermittent treatment achieved similar reduction of cell numbers at lower resistance costs. Treatment alternating ceftazidime and meropenem reduced cell numbers more th...

Applied and environmental microbiology, 2017

FEMS Microbiology Letters, 2016

The contribution of antibiotic resistance originally selected for in the agricultural sector to r... more The contribution of antibiotic resistance originally selected for in the agricultural sector to resistance in human pathogens is not known exactly, but is unlikely to be negligible. It is estimated that 50% to 80% of all antibiotics used are applied in agriculture and the remainder for treating infections in humans. Since dosing regimens are less controlled in agriculture than in human health care, veterinary and environmental microbes are often exposed to sublethal levels of antibiotics. Exposure to sublethal drug concentrations must be considered a risk factor for de novo resistance, transfer of antimicrobial resistant (AMR) genes, and selection for already existing resistance. Resistant zoonotic agents and commensal strains carrying AMR genes reach the human population by a variety of routes, foodstuffs being only one of these. Based on the present knowledge, short treatments with the highest dose that does not cause unacceptable side-effects may be optimal for achieving therapeutic goals while minimizing development of resistance. Novel approaches such as combination or alternating therapy are promising, but need to be explored further before they can be implemented in daily practice.

International Journal of Food Microbiology, 2016

Frontiers in Cell and Developmental Biology, 2016

Antimicrobial peptides (AMPs) have been proposed as a novel class of antimicrobials that could ai... more Antimicrobial peptides (AMPs) have been proposed as a novel class of antimicrobials that could aid the fight against antibiotic resistant bacteria. The mode of action of AMPs as acting on the bacterial cytoplasmic membrane has often been presented as an enigma and there are doubts whether the membrane is the sole target of AMPs. Progress has been made in clarifying the possible targets of these peptides, which is reported in this review with as focus gram-positive vegetative cells and spores. Numerical estimates are discussed to evaluate the possibility that targets, other than the membrane, could play a role in susceptibility to AMPs. Concerns about possible resistance that bacteria might develop to AMPs are addressed. Proteomics, transcriptomics, and other molecular techniques are reviewed in the context of explaining the response of bacteria to the presence of AMPs and to predict what resistance strategies might be. Emergent mechanisms are cell envelope stress responses as well as enzymes able to degrade and/or specifically bind (and thus inactivate) AMPs. Further studies are needed to address the broadness of the AMP resistance and stress responses observed.

Frontiers in Microbiology, 2016

Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage orga... more Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene were found to be hypersensitive to the lipophilic weak organic acid. RodZ is involved in determining the cell's rod-shape and believed to interact with the bacterial actin-like MreB cytoskeleton. Since rodZ lies upstream in the genome of the essential gene pgsA (phosphatidylglycerol phosphate synthase) we hypothesized that expression of the latter might also be affected in rodZ mutants and hence contribute to the phenotype observed. We show that both genes are co-transcribed and that both the rodZ::mini-Tn10 mutant and a conditional pgsA mutant, under conditions of minimal pgsA expression, were sensitive to sorbic and acetic acid. Both strains displayed a severely altered membrane composition. Compared to the wild-type strain, phosphatidylglycerol and cardiolipin levels were lowered and the average acyl chain length was elongated. Induction of rodZ expression from a plasmid in our transposon mutant led to no recovery of weak acid susceptibility comparable to wild-type levels. However, pgsA overexpression in the same mutant partly restored sorbic acid susceptibility and fully restored acetic acid sensitivity. A construct containing both rodZ and pgsA as on the genome led to some restored growth as well. We propose that RodZ and PgsA play intertwined roles in membrane homeostasis and tolerance to weak organic acid stress.

Applied and Environmental Microbiology, 1998

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous mole... more The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type Saccharomyces cerevisiae . This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to ...

International Journal of Molecular Sciences

Bacillus cereus can survive in the form of spores for prolonged periods posing a serious problem ... more Bacillus cereus can survive in the form of spores for prolonged periods posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. Our study aims at increasing knowledge of B. cereus spores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of B. cereus spores are a core with the genetic material encased by multiple protective layers, an important one being the spores′ inner membrane (IM), the location of many important germination proteins. To study mechanisms involved in germination of B. cereus spores, we have examined the organization of germinant receptors (GRs) in spores′ IM. Previous studies have indicated that in spores of B.cereus ATCC 14579 the L-alanine responsive GR, GerR, plays a major role in the germination process. In our study, the location of the GerR GR subunit, GerRB, in spores was examined as a C-terminal SGFP2 fusion protein expressed under the control of the gerR op...

mSphere

Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoil... more Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore’s inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg,...

Sorbic acid is a commonly used food preservative against yeast and fungal food spoilage. Understa... more Sorbic acid is a commonly used food preservative against yeast and fungal food spoilage. Understanding its effect on the molecular physiology of yeast cells will allow the food industry to develop knowledge-based strategies to make more optimal use of its preservative action. Here we show that the yeast membrane protein Pdr12, previously shown to be prominently involved in sorbic acid resistance development in laboratory strains, was strongly induced by the presence of sorbic acid in the culture medium in Saccharomyces strains isolated from spoiled foods. Induction of Pdr12 expression was seen both under laboratory conditions and upon growth in a commercial soft drink. Induction was rapid and maintained for the duration of the stress. No Pdr12-like protein induction was seen in Zygosaccharomyces bailii or Zygosaccharomyces lentus, two well-known beverages spoilage organisms. Finally, unexpectedly, our studies showed for the first time that pre-inducing Pdr12p to maximal levels by su...

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore ge... more Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination, and then inactivating the less heat resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores, and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single spore levels. The heat treatments used were 40-80 degrees Celcius, and for 0-300 min. The results were as follows. 1) Heat activation at 40-70 degrees Celcius promoted L-valine and L-asparagine-glucose-fructose-potassium (AGFK) induced germination in a time dependent manner. 2) The optimal heat activation temperatures for AGFK and L-valine germination via the GerB ...

Journal of Proteome Research

The resistance properties of the bacterial spores are partially due to spore surface proteins, ∼3... more The resistance properties of the bacterial spores are partially due to spore surface proteins, ∼30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis of the proteinaceous component of the spore coat, we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods. Young (day 1) and matured (day 5) Bacillus subtilis spores of wild-type and transglutaminase mutant strains were digested with formic acid and trypsin, and cross-linked peptides were enriched using strong cation exchange chromatography. The enriched crosslinked peptide fractions were subjected to Fourier-transform ion cyclotron resonance tandem mass spectrometry, and the high-quality fragmentation data obtained were analyzed using two specialized software tools, pLink2 and XiSearch, to identify cross-links. This analysis identified specific disulfide bonds between coat proteins CotE−CotE and CotJA−CotJC, obtained evidence of disulfide bonds in the spore crust proteins CotX, CotY, and CotZ, and identified dityrosine and ε-(γ)-glutamyl-lysine cross-linked coat proteins. The findings in this Letter are the first direct biochemical data on protein cross-linking in the spore coat and the first direct evidence of the cross-linked building blocks of the highly ordered and resistant structure called the spore coat.

International Journal of Molecular Sciences

The occurrence of bacterial pathogens in the food chain has caused a severe impact on public heal... more The occurrence of bacterial pathogens in the food chain has caused a severe impact on public health and welfare in both developing and developed countries. Moreover, the existence of antimicrobial-tolerant persisting morphotypes of these pathogens including both persister-cells as well as bacterial spores contributes to difficulty in elimination and in recurrent infection. Therefore, comprehensive understanding of the behavior of these persisting bacterial forms in their environmental niche and upon infection of humans is necessary. Since traditional antimicrobials fail to kill persisters and spores due to their (extremely) low metabolic activities, antimicrobial peptides (AMPs) have been intensively investigated as one of the most promising strategies against these persisting bacterial forms, showing high efficacy of inactivation. In addition, AMP-based foodborne pathogen detection and prevention of infection has made significant progress. This review focuses on recent research on ...

Cancer cell metabolism is dependent on cell-intrinsic factors like genetics, and cell-extrinsic f... more Cancer cell metabolism is dependent on cell-intrinsic factors like genetics, and cell-extrinsic factors like nutrient availability. In this context, understanding how these two aspects interact and how diet influences cellular metabolism is important for developing personalized treatment. In order to achieve this goal, genome-scale metabolic models (GEMs) are used, however, genetics and nutrient availability are rarely considered together. Here, we propose an integrated metabolic profiling, a framework that allows to enrich GEMs with metabolic gene expression data and information about nutrients. First, the RNA-seq is converted into Reaction Activity Score (RAS) to further scale reaction bounds. Second, nutrient availability is converted to Maximal Uptake Rate (MUR) to modify exchange reactions in a GEM. We applied our framework to the human osteosarcoma cell line (U2OS). Osteosarcoma is a common and primary malignant form of bone cancer with poor prognosis, and, as indicated in our...

mSphere

Bacillus subtilis spores can reactivate their metabolism through germination upon contact with ge... more Bacillus subtilis spores can reactivate their metabolism through germination upon contact with germinants and can develop into vegetative cells upon outgrowth. However, the mechanisms at the basis of the molecular machinery that triggers the spore germination and outgrowth processes are still largely unclear. To gain further insights into these processes, the transcriptome and proteome changes occurring during the conversion of spores to vegetative cells were analyzed in the present study. For each time point sampled, the changes in the spore proteome were quantitatively monitored relative to the proteome of metabolically 15N-labeled vegetative cells. Of the quantified proteins, 60% are shared by vegetative cells and spores, indicating that the spores have a minimal protein set, sufficient to resume metabolism upon completion of germination. These shared proteins thus represent the most basic “survival kit” for spore-based life. We observed no significant change in the proteome or t...

Clostridioides difficile-associated infection (CDI) is a health-care-associated infection mainly ... more Clostridioides difficile-associated infection (CDI) is a health-care-associated infection mainly transmitted via highly resistant endospores from one person to the other. In vivo, the spores need to germinate in to cells prior to establishing an infection. Bile acids and glycine, both available in sufficient amounts inside the human host intestinal tract, serve as efficient germinants for the spores. It is therefore, for better understanding of Clostridioides difficile virulence, crucial to study both the cell and spore states with respect to their genetic, metabolic and proteomic composition. In the present study, mass spectrometric relative protein quantification, based on the 14N/15N peptide isotopic ratios, has led to quantification of over 700 proteins from combined spore and cell samples. The analysis has revealed that the proteome turnover between a vegetative cell and a spore for this organism is moderate. Additionally, specific cell and spore surface proteins, vegetative ce...

Bacillus subtilis forms highly resistant, metabolically inactive dormant spores upon nutrient lim... more Bacillus subtilis forms highly resistant, metabolically inactive dormant spores upon nutrient limitation. These endospores pose challenges to the food and medical sectors. Spores reactivate their metabolism upon contact with germinants and develop into vegetative cells. The activation of the molecular machinery that triggers the progress of germination and spore outgrowth is still unsettled. To gain further insight in spore germination and outgrowth processes, the transcriptome and proteome changeover during spore germination and outgrowth to vegetative cells, was analysed. B. subtilis transcriptome analysis allow us to trace the different functional groups of genes expressed. For each time-point sample, the change in the spore proteome was quantitatively monitored relative to the reference proteome of 15N metabolically labelled vegetative cells. We observed until the phase transition, i.e. completion of germination, no significant change in the proteome. We have identified 36 trans...

Aging Cell

Calorie restriction is the only physiological intervention that extends lifespan throughout all k... more Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pH c) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pH c and the central nutrient signalling cAMP-Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pH c correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pH c showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.

PloS one, 2017

Use of some HIV-1 nucleoside reverse transcriptase inhibitors (NRTI) is associated with severe ad... more Use of some HIV-1 nucleoside reverse transcriptase inhibitors (NRTI) is associated with severe adverse events. However, the exact mechanisms behind their toxicity has not been fully understood. Mitochondrial dysfunction after chronic exposure to specific NRTIs has predominantly been assigned to mitochondrial polymerase-γ inhibition by NRTIs. However, an increasing amount of data suggests that this is not the sole mechanism. Many NRTI induced adverse events have been linked to the incurrence of oxidative stress, although the causality of events leading to reactive oxygen species (ROS) production and their role in toxicity is unclear. In this study we show that short-term effects of first generation NRTIs, which are rarely discussed in the literature, include inhibition of oxygen consumption, decreased ATP levels and increased ROS production. Collectively these events affect fitness and longevity of C. elegans through mitohormetic signalling events. Furthermore, we demonstrate that th...

Biochimica et biophysica acta, Jan 9, 2018

Three amphipathic cationic antimicrobial peptides (AMPs) were characterized by determining their ... more Three amphipathic cationic antimicrobial peptides (AMPs) were characterized by determining their effect on Gram-positive bacteria using Bacillus subtilis strain 168 as a model organism. These peptides were TC19 and TC84, derivatives of thrombocidin-1 (TC-1), the major AMPs of human blood platelets, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide based on human bactericidal permeability increasing protein (BPI). To elucidate the possible mode of action of the AMPs we performed a transcriptomic analysis using microarrays. Physiological analyses were performed using transmission electron microscopy (TEM), fluorescence microscopy and various B. subtilis mutants that produce essential membrane bound proteins fused to green fluorescent protein (GFP). The transcriptome analysis showed that the AMPs induced a cell envelope stress response (cell membrane and cell wall). The cell membrane stress response was confirmed with the physiological observations that TC19, TC84 and BP2 ...

FEMS Microbiology Letters

Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in... more Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in patients admitted to intensive care units. Resistance rapidly develops against two drugs of choice: ceftazidime and meropenem. Several therapeutic protocols were compared for reduction in viable cells and limiting development of resistance. Chemostat cultures were exposed to antibiotic concentrations measured in the blood of patients at low (5th percentile), medium (50th percentile) or high (95th percentile) levels in several therapy protocols to simulate therapy. Cultures exposed to ceftazidime recovered after 1 day at low, 2 days at medium and 3 days at high concentrations and developed corresponding levels of resistance. Patterns were very similar for meropenem except that recovery was delayed. Fluctuating levels and intermittent treatment achieved similar reduction of cell numbers at lower resistance costs. Treatment alternating ceftazidime and meropenem reduced cell numbers more th...

Applied and environmental microbiology, 2017

FEMS Microbiology Letters, 2016

The contribution of antibiotic resistance originally selected for in the agricultural sector to r... more The contribution of antibiotic resistance originally selected for in the agricultural sector to resistance in human pathogens is not known exactly, but is unlikely to be negligible. It is estimated that 50% to 80% of all antibiotics used are applied in agriculture and the remainder for treating infections in humans. Since dosing regimens are less controlled in agriculture than in human health care, veterinary and environmental microbes are often exposed to sublethal levels of antibiotics. Exposure to sublethal drug concentrations must be considered a risk factor for de novo resistance, transfer of antimicrobial resistant (AMR) genes, and selection for already existing resistance. Resistant zoonotic agents and commensal strains carrying AMR genes reach the human population by a variety of routes, foodstuffs being only one of these. Based on the present knowledge, short treatments with the highest dose that does not cause unacceptable side-effects may be optimal for achieving therapeutic goals while minimizing development of resistance. Novel approaches such as combination or alternating therapy are promising, but need to be explored further before they can be implemented in daily practice.

International Journal of Food Microbiology, 2016

Frontiers in Cell and Developmental Biology, 2016

Antimicrobial peptides (AMPs) have been proposed as a novel class of antimicrobials that could ai... more Antimicrobial peptides (AMPs) have been proposed as a novel class of antimicrobials that could aid the fight against antibiotic resistant bacteria. The mode of action of AMPs as acting on the bacterial cytoplasmic membrane has often been presented as an enigma and there are doubts whether the membrane is the sole target of AMPs. Progress has been made in clarifying the possible targets of these peptides, which is reported in this review with as focus gram-positive vegetative cells and spores. Numerical estimates are discussed to evaluate the possibility that targets, other than the membrane, could play a role in susceptibility to AMPs. Concerns about possible resistance that bacteria might develop to AMPs are addressed. Proteomics, transcriptomics, and other molecular techniques are reviewed in the context of explaining the response of bacteria to the presence of AMPs and to predict what resistance strategies might be. Emergent mechanisms are cell envelope stress responses as well as enzymes able to degrade and/or specifically bind (and thus inactivate) AMPs. Further studies are needed to address the broadness of the AMP resistance and stress responses observed.

Frontiers in Microbiology, 2016

Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage orga... more Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene were found to be hypersensitive to the lipophilic weak organic acid. RodZ is involved in determining the cell's rod-shape and believed to interact with the bacterial actin-like MreB cytoskeleton. Since rodZ lies upstream in the genome of the essential gene pgsA (phosphatidylglycerol phosphate synthase) we hypothesized that expression of the latter might also be affected in rodZ mutants and hence contribute to the phenotype observed. We show that both genes are co-transcribed and that both the rodZ::mini-Tn10 mutant and a conditional pgsA mutant, under conditions of minimal pgsA expression, were sensitive to sorbic and acetic acid. Both strains displayed a severely altered membrane composition. Compared to the wild-type strain, phosphatidylglycerol and cardiolipin levels were lowered and the average acyl chain length was elongated. Induction of rodZ expression from a plasmid in our transposon mutant led to no recovery of weak acid susceptibility comparable to wild-type levels. However, pgsA overexpression in the same mutant partly restored sorbic acid susceptibility and fully restored acetic acid sensitivity. A construct containing both rodZ and pgsA as on the genome led to some restored growth as well. We propose that RodZ and PgsA play intertwined roles in membrane homeostasis and tolerance to weak organic acid stress.