Stanley Brul

Future challenges to microbial food safety

Despite significant efforts by all parties involved, there is still a considerable burden of foodborne illness, in which micro-organisms play a prominent role. Microbes can enter the food chain at different steps, are highly versatile and can adapt to the environment allowing survival, growth and production of toxic compounds. This sets them apart from chemical agents and thus their study from food toxicology. We summarize the discussions of a conference organized by the Dutch Food and Consumer Products Safety Authority and the European Food Safety Authority. The goal of the conference was to discuss new challenges to food safety that are caused by micro-organisms as well as strategies and methodologies to counter these. Management of food safety is based on generally accepted principles of Hazard Analysis Critical Control Points and of Good Manufacturing Practices. However, a more pro-active, science-based approach is required, starting with the ability to predict where problems might arise by applying the risk analysis framework. Developments that may influence food safety in the future occur on different scales (from global to molecular) and in different time frames (from decades to less than a minute). This necessitates development of new risk assessment approaches, taking the impact of different drivers of change into account. We provide an overview of drivers that may affect food safety and their potential impact on foodborne pathogens and human disease risks. We conclude that many drivers may result in increased food safety risks, requiring active governmental policy setting and anticipation by food industries whereas other drivers may decrease food safety risks. Monitoring of contamination in the food chain, combined with surveillance of human illness and epidemiological investigations of outbreaks and sporadic cases continue to be important sources of information. New approaches in human illness surveillance include the use of molecular markers for improved outbreak detection and source attribution, sero-epidemiology and disease burden estimation. Current developments in molecular techniques make it possible to rapidly assemble information on the genome of various isolates of microbial species of concern. Such information can be used to develop new tracking and tracing methods, and to investigate the behavior of micro-organisms under environmentally relevant stress conditions. These novel tools and insight need to be applied to objectives for food safety strategies, as well as to models that predict microbial behavior. In addition, the increasing complexity of the global food systems necessitates improved communication between all parties involved: scientists, risk assessors and risk managers, as well as consumers.

Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis.

We have used complementation analysis after somatic cell fusion to investigate the genetic relationships among various genetic diseases in humans in which there is a simultaneous impairment of several peroxisomal functions. The activity of acyl-coenzyme A:dihydroxyacetonephosphate acyltransferase, which is deficient in these diseases, was used as an index of complementation. In some of these diseases peroxisomes are deficient and catalase is present in the cytosol, so that the appearance of particle-bound catalase could be used as an index of complementation. The cell lines studied can be divided into at least five complementation groups. Group 1 is represented by a cell line from a patient with the rhizomelic form of chondrodysplasia punctata. Group 2 consists of cell lines from four patients with the Zellweger syndrome, a patient with the infantile form of Refsum disease and a patient with hyperpipecolic acidemia. Group 3 comprises one cel line from a patient with the Zellweger syndrome, group 4 one cell line from a patient with the neonatal form of adrenoleukodystrophy, and group 5 one cell line from a patient with the Zellweger syndrome. We conclude that at least five genes are required for the assembly of a functional peroxisome.

Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae.

The cell wall perturbants Calcofluor white and Zymolyase activate the Pkc1–Rho1‐controlled Slt2p MAP kinase pathway in Saccharomyces cerevisiae. A downstream transcription factor of this pathway, Rlm1p, is known to control expression of about 20 cell wall‐related genes. Global transcript analysis of Calcofluor white and Zymolyase treatment was performed to determine whether cell wall stress affects transcription of these and other genes. Transcript profiles were analysed using two recently developed algorithms, viz. REDUCE, which correlates upstream regulatory motifs with expression, and Quontology, which compares expression of genes from functional groups with overall gene expression. Both methods indicated upregulation of Rlm1p‐controlled cell wall genes and STRE‐controlled genes, and downregulation of ribosomal genes and rRNA genes. Comparison of these expression profiles with the published profiles of two constitutively active upstream activators of the Slt2p–MAP kinase pathway, viz. Pkc1‐R398A and Rho1‐Q68A, revealed significant similarity. In addition, a new putative regulatory motif, CCC(N)10GGC, was found. In Zymolyase ‐treated cells a regulatory site was identified, ATGACGT, which resembles the AFT/CRE binding site. Interestingly, Sko1p, a downstream regulator of the high osmolarity pathway is known to bind to the AFT/CRE binding site, suggesting a possible role for the Hog1 pathway in the response to cell wall stress. Finally, using REDUCE, an improved version of the Rlm1 binding motif, viz. TA(W)4TAGM, was discovered. We propose that this version can be used in combination with REDUCE as a sensitive indicator of cell wall stress. Taken together, our data indicate that cell wall stress results in activation of various signalling pathways including the cell wall integrity pathway. Copyright

Measuring enzyme activities under standardized in vivo-like conditions for systems biology

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme–kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme–kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113‐7D grown in aerobic glucose‐limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h−1. The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo‐like medium containing 300 mm potassium, 50 mm phosphate, 245 mm glutamate, 20 mm sodium, 2 mm free magnesium and 0.5 mm calcium, at a pH of 6.8. The Vmax values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme‐specific, optimal conditions.

Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulence.

The cell wall of Candida albicans consists of an internal skeletal layer and an external protein coat. This coat has a mosaic-like nature, containing c. 20 different protein species covalently linked to the skeletal layer. Most of them are GPI proteins. Coat proteins vary widely in function. Many of them are involved in the primary interactions between C. albicans and the host and mediate adhesive steps or invasion of host cells. Others are involved in biofilm formation and cell-cell aggregation. They further include iron acquisition proteins, superoxide dismutases, and yapsin-like aspartic proteases. In addition, several covalently linked carbohydrate-active enzymes are present, whose precise functions remain hitherto largely elusive. The expression levels of the genes that encode covalently linked cell wall proteins (CWPs) can vary enormously. They depend on the mode of growth and the combined inputs of several signaling pathways that sense environmental conditions. This is reflected in the unusually long intergenic regions of most of these genes. Finally, the precise location of several covalently linked CWPs is temporally and spatially regulated. We conclude that covalently linked CWPs of C. albicans play a crucial role in fitness and virulence and that their expression is tightly controlled.

Parallel and comparative analysis of the proteome and transcriptome of sorbic acid-stressed Saccharomyces cerevisiae.

Exposure of Saccharomyces cerevisiae to 0.9 mM sorbic acid at pH 4.5 resulted in the upregulation of 10 proteins; Hsp42, Atp2, Hsp26, Ssa1 or Ssa2, Ssb1 or Ssb2, Ssc1, Ssa4, Ach1, Zwf1 and Tdh1; and the downregulation of three proteins; Ade16, Adh3 and Eno2. In parallel, of 6144 ORFs, 94 (1.53%) showed greater than a 1.4‐fold increase in transcript level after exposure to sorbic acid and five of these were increased greater than two‐fold; MFA1, AGA2, HSP26, SIP18 and YDR533C. Similarly, of 6144 ORFs, 72 (1.17%) showed greater than a 1.4‐fold decrease in transcript level and only one of these, PCK1, was decreased greater than two‐fold Functional categories of genes that were induced by sorbic acid stress included cell stress (particularly oxidative stress), transposon function, mating response and energy generation. We found that proteomic analysis yielded distinct information from transcript analysis. Only the upregulation of Hsp26 was detected by both methods. Subsequently, we demonstrated that a deletion mutant of Hsp26 was sensitive to sorbic acid. Thus, the induction of Hsp26, which occurs during adaptation to sorbic acid, confers resistance to the inhibitory effects of this compound. Copyright

Intracellular pH is a tightly controlled signal in yeast

BACKGROUND Nearly all processes in living cells are pH dependent, which is why intracellular pH (pH(i)) is a tightly regulated physiological parameter in all cellular systems. However, in microbes such as yeast, pH(i) responds to extracellular conditions such as the availability of nutrients. This raises the question of how pH(i) dynamics affect cellular function. SCOPE OF REVIEW We discuss the control of pH(i,) and the regulation of processes by pH(i), focusing on the model organism Saccharomyces cerevisiae. We aim to dissect the effects of pH(i) on various aspects of cell physiology, which are often intertwined. Our goal is to provide a broad overview of how pH(i) is controlled in yeast, and how pH(i) in turn controls physiology, in the context of both general cellular functioning as well as of cellular decision making upon changes in the cells environment. MAJOR CONCLUSIONS Besides a better understanding of the regulation of pH(i), evidence for a signaling role of pH(i) is accumulating. We conclude that pH(i) responds to nutritional cues and relays this information to alter cellular make-up and physiology. The physicochemical properties of pH allow the signal to be fast, and affect multiple regulatory levels simultaneously. GENERAL SIGNIFICANCE The mechanisms for regulation of processes by pH(i) are tightly linked to the molecules that are part of all living cells, and the biophysical properties of the signal are universal amongst all living organisms, and similar types of regulation are suggested in mammals. Therefore, dynamic control of cellular decision making by pH(i) is therefore likely a general trait. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.

Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

ABSTRACT This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105°C, 120°C, and 131°C, respectively. The estimated Z values were 6.3°C, 6.1°C, and 9.7°C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (Tc), the temperature at which half the DPA content has been released within a fixed incubation time. We found Tc values for spores from Bacillus strains 168, A163, and IC4 of 108°C, 121°C, and 131°C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.

Analysis of temporal gene expression during Bacillus subtilis spore germination and outgrowth.

Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of the B. subtilis genome.

Mass spectrometric analysis of the secretome of Candida albicans

The pathogenic fungus Candida albicans secretes a considerable number of hydrolases and other proteins. In‐depth studies of the C. albicans secretome could thus provide new candidates for diagnostic markers and vaccine development. We compared various growth conditions differing in pH, temperature and the presence of the hyphal inducer N‐acetylglucosamine. The polypeptide content of the growth media was ca. 0.1–0.2% of the total biomass. Using LC–tandem mass spectrometry, we identified 44 secretory proteins, the transmembrane protein Msb2, six secretory pathway‐associated proteins and 28 predicted cytosolic proteins. Many secretory proteins are wall‐related, suggesting that their presence in the growth medium is at least partially due to accidental release from the walls. Als3, Csa2, Rbt4, Sap4 and Sap6 were enriched in the medium of hyphal cultures; Bgl2, Cht3, Dag7, Eng1, Pir1, Rbe1, Scw11, Sim1/Sun42, Xog1 and Ywp1 in the medium of yeast cells; and Plb4.5 in pH 4 medium. Seven proteins (Cht3, Mp65, Orf19.5063/Coi1, Scw11, Sim1, Sun41 and Tos1) were consistently present under all conditions tested. These observations indicate that C. albicans tightly regulates its secretome. Mp65, Sun41, and Tos1 were each predicted to contain at least one highly immunogenic peptide. In total, we identified 29 highly immunogenic peptides originating from 18 proteins, including two members of the family of secreted aspartyl proteases. Fifty‐six peptides were identified as proteotypic and will be useful for quantification purposes. In summary, the number of identified secretory proteins in the growth medium has been substantially extended, and growth conditions strongly affect the composition of the secretome. Copyright