Food-Related Gram-Positive Bacteria: Enterotoxin A Expression in Staphylococcus aureus and a New Regulation Mechanism in Lactococcus lactis

Abstract

Staphylococcal enterotoxin A (SEA) is the most common enterotoxin found in outbreaks of staphylococcal food poisoning (SFP). Based on the amount of SEA produced, S. aureus strains were divided into two main groups, high- and low-SEA-producing strains. The differences in the amounts of SEA formed were found to be associated with the levels of expression of the sea gene and the bacteriophage version of sea, i.e. sea1 or sea2. Furthermore, differences in the nucleotide sequence of the Siphoviridae phage region showed two clonal lineages of the high-SEA-producing strains. One of these lines was associated with the capacity for a massive release of SEA through prophage induction, as demonstrating using mitomycin C (MC). This was also established by the detection of additional sea mRNA transcripts presumed to be initiated by a latent phage promoter located upstream of the endogenous sea promoter. The low-SEA-producing group and the high-SEA-producing subgroup lacking prophage-activated transcript of sea showed no increase in the production of SEA after the addition of MC. Thus, sea gene expression was found to be associated with the clonal lineage of the sea-encoding Siphoviridae phages.

During the processing and preservation of food, organic acids such as acetic acid and lactic acid are widely used. The effects of acetic acid on the expression and formation of SEA in S. aureus were investigated in pH-controlled batch cultures carried out with two strains belonging to two clonal lineages of the high-SEA-producing strains. The sea expression profiles of both strains were comparable, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase at all pH values tested. The elevated sea expression in S. aureus was also found to be influenced by acetic acid, which induced the sea-encoding prophage and increased the intracellular copy number of the sea gene, linking SEA formation to the life cycle of the phage.

The expression and formation of SEA was also studied in situ in pork sausage at low temperature (15°C). Three strains with different sea genetic backbones, the two clonal lineages of the high-SEA-producing strains (sea1) and the low-producing-strains (sea2), respectively, were used. Prolonged periods of expression and formation of SEA—of at least fourteen days—were found relative to the short growth-associated periods of SEA production observed in planktonic batch cultivations. The growth patterns were similar in all three strains; however, a clear difference in SEA levels between the two high-SEA-producing strains and the low-SEA-producing strain was seen. More importantly, the SEA formation of the high-SEA-producing strain harbouring the prophage-inducible sea1 gene was about three times higher than in the high-SEA-producing strain lacking phage-activated sea transcription. These results were in agreement with the previous prophage-activation results using MC. Without MC, this difference was not seen, indicating on-going prophage induction in situ. The complex food matrix of the frankfurter sausage and low temperature most likely influenced the formation of SEA in a way similar to that in MC treatment. In food, the high-SEA-producing group, and in particular the prophage-inducible sea1 group, may be more relevant in SFP than the low-SEA-producing group, which mainly harbours sea2. Environmental factors, including many preservatives and unfavourable temperature, can stimulate the expression and formation of SEA.

Lactococcus lactis is very commonly used in the manufacture of many food products and as a biopreservative in food. The main function of L. lactis in food is to lower the pH by producing acids, e.g. formate, acetate, and lactate. A regulation mechanism that contributes to readjustment of the flux of ATP production in L. lactis was investigated. To varying degrees, ATP and ADP inhibit several dehydrogenases of the central carbon metabolism of Lactococcus lactis ATCC 19435 in vitro, i.e. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH). Inhibition types and parameters by single and multiple inhibitors were determined; a mathematical model using Hill-type kinetics was introduced and developed, and showed greater flexibility than the usual parabolic inhibition equation. Model discrimination suggested that the weak allosteric inhibition of GAPDH had no relevance when multiple inhibitors were present. Interestingly, with ADH and GAPDH, the combination of ATP and ADP showed lower dissociation constants than with either inhibitor alone. Moreover, the concerted inhibition of ADH and GAPDH, but not of LDH, showed synergy between the two nucleotides. Similar kinetics, but without synergies, were found for ADHs from horse liver and yeast, indicating that dehydrogenases can be modulated by these nucleotides in a non-linear manner in many organisms. The action of an elevated pool of ATP and ADP may effectively inactivate lactococcal ADH, but not GAPDH and LDH, providing leverage for the observed metabolic shift to homolactic acid formation in resting lactococcal cells supplied with maltose.

Abstract (Swedish)

Popular Abstract in English

Foods, microorganisms, and humans have been tightly associated from the start of human history. Foods are not only the source of nutrients for humans, but also good media for microorganisms. Although we humans discovered the existence of microorganisms relatively recently, we have been dealing with them for hundreds and thousands of years. We have used some microorganisms to transfer raw food materials to ready-to-eat products, e.g. beer, wine, soy sauce, cheese, and so on, for preservation purposes and/or good flavour. On the other hand, microorganisms have spoiled foods, even causing food poisoning diseases in humans and cattle.

Control of pathogens is an important and on-going task, which continues to influence human health. Many effective measures, such as heat processing (e.g. pasteurization of milk), irradiation (e.g. UV lamps for surface sterilization), high-pressure processing, low-temperature storage, chemical preservation (e.g. organic acids), packaging (e.g. modification of atmosphere), water activity (e.g. dryness) and so on, are studied and performed in order to prevent the growth of pathogens. Most of these physical and chemical treatments affect (to a greater or lesser extent) the nutritional value and flavour. Nowadays, people prefer foods that are fresher, less processed, and nutrient-rich. This leads to opportunities and challenges for food researchers and industry. More information is needed to understand microorganisms and their mechanisms of virulence. With the development of natural science and technology, studies of microbial pathogens are not only limited to the growth behaviour of microbes. Nowadays, we can study the molecular mechanisms of virulence and their genetics by using tools such as PCR and high-throughput DNA sequencing.

Staphylococcus aureus is an important pathogen of humans and animals. It is associated with both infection and intoxication Overall, staphylococcal food poisoning (SFP) is said to be the fourth most common causative agent of food-borne illness in the EU, but this depends on the region. Due to the generally unsevere symptoms, people may not report it or go to hospital; this leads to underestimation of the total number of cases. S. aureus is carried in humans and other warm-blooded animals. About 25% of healthy humans carry S. aureus in their nose and skin all the time, and about 50% of people have it intermittently. Due to this, one of the most important sources of S. aureus contamination in food is people. Other sources of contamination can be mainly raw materials and food-processing equipment. The challenges in controlling SFP are not only to prevent bacterial growth in food, but also to prevent the formation of enterotoxins. Staphylococcal enterotoxins are extremely thermo-tolerant. Normal cooking in the kitchen is good enough to kill the bacteria in food, but is not sufficient to deactivate the enterotoxins entirely. S. aureus produces many types of enterotoxins, and enterotoxin A (SEA) is the one most commonly associated with cases of food poisoning.

In this thesis, I have studied the expression and formation of SEA in S. aureus. The growth behaviour was also studied. The results show that there is no great difference regarding the growth rate and final optical density among the various strains tested, using either controlled laboratory conditions or food matrices. But a very important phenomenon was confirmed, namely the huge variation in amounts of SEA produced by the S. aureus strains investigated. It was also found that this variation was linked to the genetic backbone of the SEA gene. From a number of genome sequences of S. aureus analysed, there were found to be two versions of the sea gene. One version of the gene (sea1) is carried by strains that produce high amounts of SEA. The strains that produce minor amounts of SEA have the other version of the sea gene (sea2). Both versions of the gene have a common, endogenous promoter, but the expression of sea2 was found to be less pronounced than that of sea1. The sea gene is encoded by a specific family of bacteriophages (bacterial viruses, also called phages). Based on genome analysis of sea-encoding phages, three major branches were described. One branch contained sea2 and the two other branches harboured sea1 with different genetic surroundings. By using a classic phage-inducing reagent, it was found that one of these branches produced high amounts of SEA after prophage induction. These strains were also genetically mapped to verify phage relationships. By monitoring gene expression, it was possible to compare the levels of sea mRNA with the amount of SEA released from the bacteria. Interestingly, a new transcript associated with the phage life cycle was observed in the prophage-induced branch, producing a boost of SEA. Thus, there appears to be a close connection between the biology of the phage and the amount of SEA formed—as also observed in situ in sausages. However, in the food-processing, storage, and consumption environments, many factors such as temperature, preservatives, and starvation can trigger phage induction. For instance, food preservatives are used widely in food products, e.g. organic acids, which have been used for centuries. It was found that expression of the sea gene was 9 times higher at pH 6.0 and 4 times higher at pH 5.5 (adjusted with acetic acid) than at natural pH. SEA formation peaks at pH 6.0, which is a very common pH in food products. Strains with different prophage background behave similarly, but with differences in the effect of acetic acid on the virulence and degree of expression of SEA.

One study was performed to test S. aureus strains with different prophage background in a real food matrix, i.e. frankfurter sausage. The results confirmed that some S. aureus strains are more virulent than others, and that counting the number of living S. aureus bacteria in the product is not a reliable way of judging whether or not the food is safe to eat.

In the context of this thesis, I have also investigated the metabolic regulation of weak acids in Lactococcus lactis. Weak acids and lactic acid bacteria (LAB) are frequently used to preserve food products and prevent the growth of S. aureus. LAB are widespread in our environments and an important part of our lives. Many food products are produced with the help of LAB, such as cheese, yoghurt, sauerkraut, and wine. They are also added to food as biopreservatives, mainly because they produce lactic acids together with mixed acids, which lower the pH and therefore inhibit other microorganisms. In this work, I studied three important enzymes of the glycolytic pathway, all dehydrogenases. The aim was to understand the metabolic switch between homolactic fermentation and mixed-acid fermentation in LAB. I also studied the inhibitory effects of natural metabolites such as ADP and ATP, the energy currency molecules of the cell. It was found that the pool of ADP and ATP can effectively inactivate some of the dehydrogenases, e.g. ADH, but it has less influence on GAPDH and LDH. This regulatory mechanism contributes to readjustment of the flux of ATP production in L. lactis, and consequently the production of weak acids.

• author: Cao, Rong
• supervisor: Peter Rådström
• opponent: Dr. Frees, Dorte, Section of Microbiology, Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Denmark
• organization: Applied Microbiology
• publishing date: 2012
• type: Thesis
• publication status: published
• subject: Industrial Biotechnology
• keywords: Lactococcus lactis, multiple inhibition kinetics, dehydrogenase, ATP, ADP, food factors, biopreservatives, staphylococcal food poisoning, SEA, prophage induction, enterotoxin A, Staphylococcus aureus
• pages: 156 pages
• defense location: Lecture hall C, Kemicentrum, Getingevägen 60, Lund University Faculty of Engineering
• defense date: 2012-05-25 10:15:00
• ISBN: 978-91-7422-297-5
• language: English
• LU publication?: yes
• id: 63979e6b-bdd7-40d2-a10e-44ab56a46251 (old id 2517895)
• date added to LUP: 2016-04-04 13:07:19
• date last changed: 2018-11-21 21:12:17