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The food-borne pathogen Listeria monocytogenes can live in extreme environmental conditions since it has numerous ways to respond to stress (van der Veen, Hain et al. 2007). The heat-shock response of the bacteria was done using its whole genome with the use of DNA microarrays. Differences in expression were observed at 37Â°C and at 48Â°C (the heat-shock). These results were obtained at different times (3, 10, 20 and 40 minutes). Virtually all the genes showed a differential expression pattern whether they were upregulated or downregulated. Furthermore, often genes belonging sharing a similar function would show comparable expression levels. The results were then confirmed by using quantitative PCR. A novel finding during the study was that certain heat-shock induced genes are activated during the SOS response in L. monocytogenes.
Listeria monocytogenes is a harmful food-borne pathogen. It is a Gram-positive bacillus-shaped microorganism. It also produces lysteriolysin O, a protein toxin that contributes to the virulence of this bacterium since this protein allows it to live intracellularly such that it can evade factors of the immune system like immunoglobulins and complement (Farber, J. M. and Peterkin, P. I., 1991). Furthermore, despite not being able to form spores, it can grow and survive at relatively high temperatures compared to other microorganism that do not form protective spores. It can withstand high environmental stress such as heat, high salt concentrations of up to 12% and low pH (as low as pH 5) (van der Veen, S., T. Hain, et al. 2007).
Temperature fluctuations, and heat stress in particular, is a concern because they are commonly encountered during food processing such as in the processes of pasteurization, canning, dehydration, etc. One of the main reasons why food gets processed is the need to develop its flavours, improve its texture, but most importantly to ensure public safety because microorganisms that may cause disease that may potentially be found in the food are destroyed in this process. Of course, not all of them can be eliminated because a few may be resistant to heat or other stresses encountered in food processing like high salt concentrations and highly basic or acidic environments (Scott Smith, J. and Hui, Y. H., 2004). A big problem encountered these days is that a new generation of people generally want less-processed foods (Alegre, Abadias et al. 2010). Hence, the conditions used to make sure our food is safe are becoming less harsh. Even though the untreated food will maintain more of its natural state, it is necessary to treat them to prevent the spread of disease. This signifies that more detrimental bacteria may end up in our food supplies. Among these bacteria is L. monocytogenes which is very potent at proliferating easily during food processing (van der Veen, Hain et al. 2007). Another worrisome fact is that it can also withstand low temperatures and can thus reside in refrigerators (Rhoades, Duffy et al. 2009). In fact, at a refrigeration temperature of 4Â°C, the quantity of ferric iron n some food stimulates the proliferation of L. monocytogenes (Dykes and Dworaczek 2002).
L. monocytogenes can be found in a wide variety of plants and vegetables since this bacterium can live in dirt or it can also be located in some water sources and thus can affect many crops growing in these infected environments (Rhoades, Duffy et al. 2009). In fact, cattle and other farm animal that give the impression that they are healthy may be harboring L. monocytgenes. As a result, contamination of beef, chicken, pork and even dairy products is sometimes inevitable. Listeria is usually linked to foods such as milk (raw or pasteurized), soft cheeses, ice cream, vegetables, sausages, poultry, smoked fish, and others (Destro, de Melo Serrano et al. 1991).
When L. monocytogenes enters the body, it soon breaks into the hosts' immune cells, notably monocytes, macrophages and neutrophils. Thus, it becomes blood-borne and can divide and circulate in the body. The body's defense against Listeria is due to cell-mediated immunity. Its pathogenicity relies on the fact that it is capable of on growing and dividing in these phagocytic cells. Consequently, people with a suppressed cell-mediated immunity are much more likely to be affected by L. monocytogenes (Yeung and Davies 2005).
Infection by Listeria monocytogenes leads to the disease listeriosis (Larsson, Lundqvist et al. 2009). This ailment may manifest itself as septicemia, meningitis, encephalitis (Alexandru, Gonzalez et al.), corneal ulcer (Holland, S., E. Alfonso et al. 1987), pneumonia, and even cervical infections in pregnant women (Yeung and Davies 2005), which may result in spontaneous abortion or stillbirth. Its occurrence in cells like macrophages and dendritic cells also permits its entry into the brain or it can even migrate through the placenta and affect the fetus in pregnant women. Pregnant women naturally have a decreased cell-mediated immune system so they are more predisposed to L. monocytogenes infections. Moreover, the immune system of fetuses are not fully developed, and thus rendering them very susceptible to this bacterium (Yeung and Davies 2005). Other adults, especially transplant recipients and patients with lymphomas, are given necessary therapies with the specific intent of depressing T cells, and these individuals become especially susceptible to Listeria as well. However, healthy adults and children can get infected with L. monocytogenes but can withstand it much better due to their better immune system (Alexandru, Gonzalez et al.)
Various studies have taken a look at the heat-shock response in a range of bacteria (van der Veen, Hain et al. 2007). It is important to consider this because heat stress is often found in nature and in food processing. Upon heat-induced stress, the bacteria undergo various processes to protect themselves, including the heat-shock response that allows them to survive the elevated change in temperature. This response is important for normal growth of bacteria since it helps maintain the proteins from denaturing or misfolding (van der Veen, Hain et al. 2007). This can be done by activating gene transcription and translation for proteins called chaperones involved in folding proteins or by activating pathways such as the protein degradation pathway that sends proteins to be degraded through the proteases because they were initially denatured or misfolded (Lodish, H. et al. 2007).
L. monocytogenes has two main heat-shock mechanisms: class I and III heat-shock reponses and also a class II stress response. Class I genes are under the condrol of the HrcA repressor expression of the major heat shock genes, the groESL and dnaKJ operons, by binding to the CIRCE (controlling inverted repeat of chaperone expression) element. Class II genes encode for molecular chaperones and Clp proteases which are involved in degrading misfolded/denatured proteins. These genes are under the control of the CtsR repressor. Finally, the Class II genes encode for general stress proteins. These genes are regulated by SigB (van der Veen, Hain et al. 2007).
The whole heat-shock regulon of L. monocytogenes had not been studied before. Thus, the researchers tested to see how different genes from the microorganism will change when the temperature changes from 37Â°C to 48Â°C (a heat-shock). In fact, they did find a differential expression for a number of genes. Also, it was observed for the first time in L. monocytogenes that heat shock played a role in the SOS response, a DNA repair mechanism. Another finding was that there was a decrease in the amount of expressed genes implicated with cell wall synthesis (van der Veen, Hain et al. 2007).
Results and Discussion
The strains of bacteria used were L. monocytogenes EGD-e which were placed in a BHI (brain heart infusion) broth. Exponentially growing cells were placed in conical flasks and placed in a 37Â°C incubator until an optical density of 1.0 at 600 nm was observed. Then, 5 mL of this was used for RNA extraction and 10 mL flasks at 48Â°C (van der Veen, S., T. Hain, et al. 2007).
The researches then extracted RNA from the bacteria and converted it to cDNA so that they could use DNA microarrays to observe which genes were expressed at different times during the heat shock (3, 10, 20 and 40 minutes) and compared these results to time zero. The researchers decided to set their minimum false discovery rate for differentially expressed genes at less than or equal to 1% and at greater or equal to 2 for a fold-change for the microarray analysis they used (van der Veen, S., T. Hain, et al. 2007). Cy3 and Cy5 dyes were used to label the DNA and were then hybridized to microchips using an ASP base hybridizer at a temperature of 315 K for twelve hours (Chatterjee, S. S. et al. 2006).
A microscopy analysis was also performed where 1 mL of the samples were centrifuged and then placed in a dilute nigrosin solution to produce a negative staining. A program called ImageJ was then used to count the pixels that each cell occupied in order to determine the size of the cells quite accurately (van der Veen, S., T. Hain, et al. 2007).
Finally, quantitative PCR was also used to validate the microarray analysis using the Superscript III reverse transcriptase. Each of the primers used set a standard curve with both genomic DNA and cDNA. Negative controls used water as a solution. Forty cycles of PCR were done to amplify the DNA (van der Veen, S., T. Hain, et al. 2007).
From the DNA microarray, the researchers found that there was a differential expression of the cells expressed at the various times of heat-shock with the time zero expression. 714 genes showed a 2-fold change or greater in expression where 427 showed increased expression but 287 showed decreased expression. Also, many of the expression profiles were transient. They observed that the genes showing the highest upregulation involved genes used for carbohydrate transport and metabolism and genes for transcription and those least expressed were those involved in translation (van der Veen, S., T. Hain, et al. 2007).
Class I genes were found to have between a two and four-fold in increased expression, whereas the class III genes showed a transient expression pattern. One of the stress genes htrA, which encodes for HtRA (a serine protease) was highly expressed and this was also found in Bacillus subtilis, and homologues of it were found in other bacteria. Thus, this sugested more evidence that it is involved in heat-stress. It is important to know that SigB controls this stress genes. When L. monocytogenes is present within a host organism, quorum sensing causes further activation of several virulence (Vir) genes. Depending on the location of the bacteria within the host organism different activators up regulate the virulence genes. SigB, an alternative sigma factor, up regulates Vir genes in the intestines whereas another protein called PrfA up regulates gene expression when the bacteria is present in blood (Pallen and Wren 1997). The results showed though that it was only expressed at a level of 1.5 higher at any time point (van der Veen, S., T. Hain, et al. 2007).
Furthermore, it was shown that the opuC operon expression was increased and this codes for the betaine/carnitine/choline ABC transporter which has been shown to be involved in an increase of osmolytes upon salt and acid stress, and provide protection during heat stress in B. subtilis as well as Escherichia coli. One study showed that osmolytes like glycine betaine and carnitine, as they accumulate in the cell, are critical for the survival of L. monocytogenes at concentrations of salt as high as 0.7 molar and at temperatures of 4oC. OpuC is an important protein for the regulation of these osmolytes. Thus, as shown in infected mice, OpuC promotes the survival of L. monocytogenes (Sleator, R. D. et al 2001).
The researchers also found that there were genes involved in the SOS repair mechanism, which repairs DNA that was damaged or mutated, that were highly and less expressed. RecA (an SOS activator) were expressed at higher levels than at 37Â°C. Their results also showed that there was an increase in transcription and translation of various virulence genes such as prfA whose role is to activate virulence genes. RecA's expression augmented significantly from 1.4-fold to 5.2-fold from 3 min to 40 min. Furthermore, polymerases involved in repairing DNA such as DinB and UmuDC were upregulated. Other proteins in DNA repair like UvrA and UvrB also showed an increase in expression. Another important protein called YneA is the cause for cells elongating instead of dividing during a heat response. These results were shown during the microscopy analysis which showed bigger and bigger cells appearing at the different times of heat-shock. They also determined this since the optical density increased whereas the concentration remained the same by performing a colony-forming-unit (c.f.u.) count (van der Veen, Hain et al. 2007).
The SOS response is a DNA repair system that is critical for DNA replication to occur when there are slight mutations in the DNA. RecA is a protein that becomes activated when there is single-stranded DNA present as a result of DNA damage. It works to inactivate another protein called LexA. The latter is a transcriptional repressor. If LexA is not inactivated, its function is to prevent the SOS repair system to become engaged. Despite the SOS repair system's role to repair damage, it is an error-prone repair system. Under normal circumstances, the SOS genes are inhibited by the action of LexA since the latter binds to a sequence in the SOS operator. However, there is still some leaky scanning occurring, such that some of the genes in the operator are transcribed at low levels (Michel 2005).
When an accumulation of ssDNA is present, RecA becomes active and interacts with LexA which helps cleave LexA to become inactivated. As a result, more SOS genes can be transcribed. Operators that bind LexA weakly are the first to be fully expressed. In this way LexA can sequentially activate different mechanisms of repair (Michel 2005).
On one hand, some genes (such as lexA, recA, uvrA, uvrB, and uvrD) are fully induced in response to even weak SOS signal. Thus, because these genes are involved in nucleotide excision repair (NER), this is generally the first mechanism to occur before a full-blown SOS repair mechanism is involved. On the other hand, if NER fails to repair the DNA sufficiently, then concentrations of LexA are reduced even further and genes such as sulA, umuD and umuC, which happen to be expressed late, become transcribed. SulA functions to halt cellular division. Then, UmuC and UmuD precisely hinder the shift from stationary phase to exponential growth and that this is associated with a quick inactivation of DNA production (Murli, S. et al 2000). Due to these properties, some genes may be activated partially when these is a lot of DNA damage. But, some genes instead are only activated when persistent levels of DNA mutations and damage are located in the cell (Murli, S. et al 2000).
Other results in van der Veen et al's research showed consistent results with the gene expression profile related to the aforementioned SOS genes: ynzC and yneA, other SOS genes, were shown to have increased expression. They saw elongated cells in the microscopic analysis, but did not observe a significant different in the number of cells, which confirms why YneA would be activated. (van der Veen, Hain et al. 2007). YneA is a protein that causes cell elongation, and thus indirectly causing the downregulation of cell division during the SOS response. Cell division first occurs by synthesizing a protein ring called FtsZ. Then, 6 other proteins join to help form the septum. It has been suggested though, through a study, that YneA is involved in downregulating the synthesis of FtsZ, and thus contributing to cell elongation (Kawai, Y.S. Moriya, et al. 2003). Consistent with these results is that two genes encoding for proteins that are used specifically for cell division (such as ftsX and ftsE) were for the most part showed lower expression levels. In addition, all cell wall hydrolases (proteins that also play a role in cell division) were found to be downregulated and included genes like ami, murA and aut showing a particularly constant 3-fold repression pattern (van der Veen, Hain et al. 2007).
As for virulence genes, many of the genes regulated under the control of PrfA showed more expression including plcA (a phospholipase), inlA and inlB which code for internalins specific to L. monocytogenes, lmo2067 which is a conjugated bile acid hydrolase and lmo0596 which codes for an unknown protein (van der Veen, Hain et al. 2007). Hence, we can understand how not only is L. monocytogenes harmful because of its stress-resistant properties, but some virulence factors are also increased and thus putting us at more risk for infection.
The results of the microarray analysis were confirmed by using a quantitative-PCR approach. However, it was observed that the microarray analysis underestimated the differential expression by usually a 5-fold difference. However, other papers such as those by Gao, H. et al. and Helmann, J. D. et al. found similar results. Furthermore, in the microarray analysis, there was no difference in expression see for LexA (which represses the SOS response), but there was a slight increase in the Q-PCR analysis. This fact may be explained by the fact that the microarray used cDNA which has its introns removed, whereas in Q-PCR, the full DNA with its introns is used. Thus, perhaps some minor reverse transcription error occurred during the conversion from RNA to cDNA cause this differential expression, leading to perhaps a frameshift mutation or some point mutations.
From this research article, it appears that the authors have appeared to have searched the topic fairly well enough as they incorporate in their study a few times that they might found conflicting results with other papers or found the same kinds of results. This aspect shows at least that some research about the topic has been done already and includes some recent sources (at the time of writing). However, there are not really that many papers that observe the whole transcription of L. monocytogenes although there are some providing information on a few genes or at different temperatures of heat stress.
As a result, from their findings, we see that there is definitely a change in gene expression in L. monocytogenes at 37Â°C and 48Â°C. It uses different strategies to counteract the stress or to help relieve it by upregulating or downregulating genes to the advantage of the cell.
The development of the researchers ideas are fairly clear for the most part and they progress through the article in an organized fashion. However, they do not really have a hypothesis, instead they are mainly just looking at the expression patterns of the genes. Thus, the research report does not really describe an explicit theoretical or conceptual framework for the study. But, the absence of such a framework does not detract from the usefulness of the research because their research results are very open-ended in the sense that the results obtained can lead the way to a number of different experiments. The results can provide other researchers with a stepping stone as to how they might want to look at certain gene profiles in much greater depth. But overall, it may be that the study was a bit too general to make significant inferences in the results especially since there were differences in the expression patterns found in Q-PCR and the microarray analysis. In addition, some of the steps in their methods lack a bit of clarity as they do not always quantitatively describe how much of a solution was used for example. Nevertheless, the results were organized and clear. Furthermore, the microscopy analysis which could have been just a qualitative assessment was actually quantitative with the use of the ImageJ program. And, these results were used in complementary fashion with the other experiments (at least for the cell division proteins). But, they still provide controls as need, such as using the tpi, rpoB and 16S rRNA genes in Q-PCR which normalized the conditions as they do not change expression upon varying temperatures.
The authors nicely tie up most of the different proteins (even those not belonging to the same class) and how they relate to each other in terms of their effects on heat stress. In other words, the researchers tie the findings of the study back to the framework at the end of the report. They go on saying how a certain protein's expression result is consistent with a different protein that might share a similar role but not necessarily be directly involved with the protein.
Sometimes the authors use terms like "proven" or "conclude" when they reference results from author researchers. However, it is not quite right to use such terms since they have performed an experiment and their results may not be conclusive but just support their hypothesis.
Furthermore, it would have probably been more useful to have done a Q-PCR validation for all the genes they used for expression or simply more of them. Of course, this would probably have been very long and expensive, it would probably have offered a better representation of all the genes in the organism since 13 genes is a rather small sample compared to the number of genes compared in the whole study.
Conclusions and Future Research Questions
The results from the researchers' findings of showing which genes in L. Monocytogenes were more activated or inhibited by an increase in temperature could be one of the reasons why this bacterium can be so problematic and pathogenic as it can cope with stress quite well. Furthermore, the results suggest how the SOS mechanism is a significant way to protect the organism under heat-shock. Of course, by doing additional research, researchers could better understand the genetic mechanism occurring in these stress-induced genes so that there could be specific targets that could render L. Monocytogenes less virulent. This factor would be of particular for the food-processing industry. Thus, perhaps in the future, health government agencies can become more stringent again in regards to how they deal with food processing to help diminish listeriosis. Additionally, other researchers could use the results from the experiment as a tool to further describe certain genes much more specifically to understand their mechanisms of action better.