THE PHENOMENON OF PERSISTENCE IN ESCHERICHIA COLI IN RESPONSE TO THERMAL SHOCK
Lina Contreras1, Juan Manuel Pedraza2, Silvia Restrepo1
1Biological Sciences Department, Universidad de los Andes, Bogotá, Colombia. 2 PhysicsDepartment, Universidad de los Andes, Bogotá, Colombia.
ABSTRACT
When a population of bacteria is exposed to a stress such as an antibiotic, the majority of the population is killed. However, there is a small fraction of slow growing cells stochastically formed that can survive and become a new population susceptible to the antibiotic. This phenomenon is called bacterial persistence. Given the importance of this type of cells in the environment, there is a need to understand their nature and discover the different factors that trigger their formation. Here this phenomenon was studied in the wild type strain and the hipQ mutant of Escherichia coli. Both wild type and mutant stains were grown and exposed to thermal shock to study if the conditions of growth affect the fraction of persistent cells formed. The results of this experiment suggest that bacterial persistence is affected by the external conditions to which the population is exposed, implying that it is a phenomenon that is between stochastic and deterministic.
Keywords: Persistence, Thermal shock, E. coli, hipQ.
INTRODUCTION
In an isogenic bacterial population, there is a small fraction of slow growing cells highly tolerant to antibiotics (Kint, Verstraeten, Fauvart, & Michiels, 2012). When exposed to antibiotics, most of the population is killed. However, a small fraction of cells is able to survive and when the antibiotic is removed they can regrow a new population of susceptible cells (Balaban, 2004). This phenomenon is called bacterial persistence. Persisters are of great importance to chronic recalcitrant bacterial infections (Lewis, 2010) since it has been shown that they play an important role in survival of bacterial biofilms (Lewis, 2001). Bacterial biofilms are the responsible for nearly 65% of human infections (Keren, Shah, Spoering, Kaldalu, & Lewis, 2004) therefore the study and understanding of this phenomenon could lead to a change in the way these infections are treated.
It is important to clarify that persistence and resistance are two different phenomena. In order to act, antibiotics need an active target to bind to (Lewis, 2010). Unlike resistant cells, that have mutations that prevent the antibiotic from binding to its target, persisters are genetically identical to the wild type but their phenotype is different (Keren et al., 2004). They are known to be cells that stochastically enter into a “dormant” state (Balaban, 2004), that in some way allows them to be highly tolerant to antibiotics. However, it is known that
dormancy per se cannot entirely account for the persister phonotype, the action of different active mechanisms in antibiotic tolerance, like the SOS response might play an important role (Kint et al., 2012).
The SOS response is a DNA repair network, in which two proteins play important roles: the LexA repressor and the RecA inducer. In normal growing cells, LexA binds to the SOS box prsenten in the promoter of the SOS genes preventing their expression, once the cell senses an increased level of DNA damage, the RecA is activated and it induces the cleavage of LexA. Allowing the SOS genes to be expressed.(Sassanfar & Roberts, 1990)
The toxin-antitoxin (TA) module has been recognized as the first gene in persister formation. TA elements were found to be increased in E. coli persisters. There are different types of TA modules; bacterial persistence is considered to be related with type II (Kint et al., 2012), in which two molecules are generated: a stable toxin, capable of reducing the cellular growth and an antitoxin which binds to the toxin to inhibit its action. So high levels of toxin would induce persister generation. (Keren et al., 2004)
Bacterial persistence was discovered by Joseph Bigger in 1944 while working with penicillin and Staphylococcus, but it was poorly understood and difficult to study, which is why it was forgotten for approximately 40 years. Later, while working with non hereditable variation, Harris Moyed discovered the hipA7 mutant (Lewis, 2010) , and by the end of the 90s the hipQ mutant was described by Wolfson et al (1990). This phenomenon has been mostly studied in the hipA7 mutant, and because of their differences it is important to study and understand both mutants.
As it was just mentioned, there are two types of persistent cells; type I persisters (hipA7)
are non-growing cells that are generated at stationary phase, as they completely stop their growth, they have a longer lag phase than usual. Type II persisters (hipQ) on the other hand, are slow-growing cells continuously generated at exponential phase via a phenotype switching mechanism, they have a normal lag phase. (Balaban, 2004; Kussell, Kishony, Balaban, & Leibler, 2005). This phenomenon in which a small fraction of the population is phenotypically different to the rest might constitute an “insurance policy” to reduce the risk of death in changing environments (Kussell et al., 2005).
The experimental study of the phenomenon of persistence has been difficult because of the continual changes in the persistent subpopulation and mostly because of the very small fraction they represent (Kussell et al., 2005). Current methods to study bacterial persistence rely on the activity of antibiotics and their killing kinetics for prolonged periods of time, this may activate an SOS response in the cells and induce the generation of persisters (Cañas-Duarte, Restrepo, & Pedraza, 2014). Studies with the SOS response in E. coli,
showed that mutants unable to establish an SOS response had reduced persister fraction (Dörr, Lewis, & Vulić, 2009).
As shown by Cañas (2014), the fraction of persister cells formed in a population varies in response to the changes in the environment. When tested with different antibiotics, the fractions of survival cells changed drastically (Fig 1). The same study demonstrated that an antibiotic had differences in the stabilization time when used in the same strain in different phases of the curve, and when used in two strains in the same phase of the curve (Fig 2). This implies that although persistence is a stochastic phenomenon, it is also a response to changes in the environment, meaning that it is also in some ways a deterministic phenomenon.
In order to observe if the phenomenon of bacterial persistence was affected or not in changing environments, wild type E. coli and mutant hipQ were studied under two conditions of stress, heat and cold shock. This was achieved by the use of the novel protocol described by Cañas et al and comparing the formed fraction of persisters under both stressors and optimal conditions.
MATERIALS AND METHODS
Bacterial Strains and Growth Conditions
The strains used in this work were the K12 Wt and DS1 (hipQ) mutant of E. coli. The experiments were conducted in Luria-Bertani (LB) minimum medium at 37°C and 200rpm.
Novel Protocol for Persister Cells Isolation by Cañas et al (2014)
Bacterial strains were grown under the conditions described above for approximately six hours until OD reached 0,45. An aliquot of one milliliter was taken from the culture and placed into a 15 mL falcon, 200 µL of lysis solution (Sigma, Miniprep Kit) was added. The mixture was homogenized with vortex for 10 seconds and cultured for ten minutes at room temperature. After ten minutes, 200 µL of enzymatic lysis solution was added. The mixture was homogenized by inverting the capped falcon and cultured for 15 minutes at initial conditions.
Stress conditions
For all the experiments, 300 ml of LB minimum medium were inoculated with 10 ul of ON culture of each strain until it reached an OD of about 0.45 (approximately 6 hours of incubation at previously mentioned conditions). At that point the cells were exposed for ten minutes to low temperatures (10°C), and 15 minutes to high temperature (45°C). After the respective time was complete, the protocol for persister cells isolation proposed by Cañas et al (2014) and described above was applied, then an aliquot was serially diluted and plated on LB plates for determination of persister cell frequencies.
Statistical analysis
All obtained data were tested for normality and a non-parametric approach was used to analyze the data with a Kruskal-Wallis test for the analysis of the variance.
RESULTS
In order to investigate the generation of persisters cells during temperature shifts from 37˚ to 10˚C and from 37˚ to 42˚C, the wild type and hipQ mutant strains of E.coli were growth at normal conditions until OD of approximately 0.45 was reached. At this point cells were exposed for ten and fifteen minutes to low and high temperature respectively and then the protocol for persister isolation was applied. The obtained data for both treatments and for the control group of each strain was tested for normality using a Shapiro test, and the data obtained did not show a normal distribution. The Kruskal-Wallis test showed that there wasn’t a significant difference between the groups in the Wt strain (Fig 3), which means that in this case the persister generation is not affected in a negative or positive way under the thermal shock. On the contrary, the results for the hipQ mutant show a significant difference between groups (Fig 4).
DISCUSSION
As mentioned before, in bacteria, control of cell growth is an important insurance policy to reduce the risk of death and various mechanisms are involved; one of the most important mechanisms in this process are TA module. Persister formation has been related with TA modules, in which the generation of persisters depends on the concentrations of toxins in the cells. The levels of active toxin must be high to generate a persister cell, for this to occur, the levels of antitoxin must be low. Lon protease is known to degrades all antitoxin of type II TA modules.
Previous studies have demonstrated that persistence in E. coli depends on the activity of Lon protease and toxin-antitoxin loci. The proposed model established that Lon protease activated the mRNases encoded by TA loci by degrading the antitoxin and that the activation of the mRNases induces dormancy and persistence. It was also proposed that Lon protease was stochastically turned ON at a low frequency (Maisonneuve, Shakespeare, Jørgensen, & Gerdes, 2011), however further investigation showed this wasn’t the case and that the activation on Lon depended on the inorganic polyphosphate (PolyP), whose concentration is controlled by (p)ppGpp levels. A new model was proposed (fig. 5) in which it was established that the formation of persisters depend hierarchically on the signaling nucleotide (p)ppGpp, Lon protease, inorganic polyphosphate, and toxin-antitoxins (Maisonneuve, Castro-Camargo, & Gerdes, 2013).
Since it have been said that persister cells are affected by the environment and that TA modules act in response to external conditions, it was expected to obtain a higher fraction of persister cells under any kind of stressor. The results obtained here demonstrate that in fact the environment affects the fraction of persister cells, yet we have also shown that the different stress conditions might not produce a higher fractions of persisters as expected, validating the importance of these studies. Here thermal shock was used as stress, however other types of stress, such as nitrogen starvation, might have a different effect over the molecular mechanisms that are responsible for bacterial persistence.
The concentrations of (p)ppGpp in each cells varies with temperature shifts; therefore the persister generation is expected to be affected by changes in the temperature. Mackow and Chang (1983) demonstrated that the levels of (p)ppGpp decreased after prolonged periods of exposure to cold or heat shock. Decreased levels of (p)ppGpp means low persisters generation, however as it can be seen in the figure (Fig 6), when exposed to heat shock there is an initial increase for the first 15-20 minutes of the exposure (Mackow & Chang, 1983). Our results for the hipQ mutant are consistent with these data because our heat shock only lasted 15 minutes, time at which the levels of (p)ppGpp are still high and therefore a high level of persisters was expected.
As mentioned before, a higher fraction of persisters was also expected for the cold shock. Unlike the heat case, the (p)ppGpp levels rapidly decreased when cells were exposed to low temperatures (Fig 7), an so a smaller fraction of persisters is also consistent with Mackow and Chang results.
Since these results were also expected with the wild type strain, it might be necessary to increase the number of experiments in order to increase the statistical power. It is also important to repeat the same experiments using the clean puuP mutation, which was demonstrated by Cañas et al (unpublished data) to be the responsible for the hipQ mutant. The strain used in this study had numerous extra mutations; these mutations of unknown effect may be the responsible for the results obtained for the hipQ strain.
CONCLUSIONS
As partial result, it can be said that the environment affects the generation of cells formed in a population, in this case temperature shifts were used, however more studies using other stressors and all strains (hipA7, Wt and puuP) are needed to reach a more global conclusion.
Also, the use of puuP would help to understand more clearly the effect that the temperature has in the hipQ phenotype. Being a clean mutation the use of puuP can also elucidate the real role of the mutation in the generation of persisters.
The molecular mechanism controlling persister cells formation is being affected in different ways by the external conditions, therefore more studies that help elucidate how these molecular mechanisms are being affected are needed to understand more globally the phenomenon of bacterial persistence.
The data obtained here and the results obtained by Cañas et al (2014) previously explained, can be used as examples to illustrate the fact that this intracellular process is somewhere in between the stochastic and the deterministic.
FIGURES
Figures 1 and 2 were taken from Cañas et al (2014)
Figure 1 Comparison between Ampicillin and Ofloxacin based persister cells isolation methods in 3 E. coli strains. Using different antibiotics and methodologies for persister isolation generated markedly different survival fractions. Ampicillin and Ofloxacin were tested using exponentially growing cultures without dilution (1:1) at an OD of 0.5. As a reference in this figure, we used the results of ampicillin treatment with an initial dilution of the culture (1:100) reported by Balaban et al. in 2004 [5]. Error bars indicate the standard deviation (n = 3), except for the Ampicillin (1:100) treatment in which the error bars indicate the range reported by Balaban for exponentially growing cultures. doi:10.1371/journal.pone.0088660.g001
Figure 2 Persister cells isolation in E. coli strains using Ofloxacin. The killing kinetics of Ofloxacin showed differences in stabilization time when comparing its activity in stationary and exponential growth phase, requiring almost double time when acting in stationary phase cells, even in the same E. coli strain (A). Survival fractions from stationary phase cultures of two different E. coli strains also exhibited markedly different stabilization times, even in the same physiological state (B). Error bars indicate the standard deviation (n = 3). doi:10.1371/journal.pone.0088660.g002
Figure 3 Survival fraction Wild type strain. The wild type strain was exposed to both cold and heat shock, whit a p value of 0.166 the difference between groups are non significant.
Figure 4 Survival fraction DS1 (hipQ) strain. The hipQ strain was exposed to both cold and heat shock, whit a p value of 0.00029 the difference between groups is significant, showing a higher survival fraction with heat shock than with cold shock.
Figure 4 Molecular Model Explaining Bacterial Persistence RelA or SpoT synthesize (p)ppGpp that inhibits exopolyphosphatase (PPX), the cellular enzyme that degrades PolyP. PolyP is constitutively synthesized by polyphosphate kinase (PPK). PolyP combines with and stimulates Lon to degrade the 11 antitoxins of E. coli K-12, thereby activating the toxins that inhibit translation, and inhibits cell growth. The arrow from the toxin to the TA promoter (indicated by an arrow pointing right) indicates that excess toxin ([toxin] > [antitoxin]) derepresses the TA promoter that, in turn, may lead to rapid quenching of toxin activity as a prerequisite for resuscitation of persister cells (Cataudella et al., 2012) (see the Discussion for further details).
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