Chromosomal Type II Toxin-Antitoxin Systems May Enhance Bacterial Fitness of a Hybrid Pathogenic Escherichia coli Strain Under Stress Conditions

Abstract

The functions of bacterial plasmid-encoded toxin-antitoxin (TA) systems are unambiguous in the sense of controlling cells that fail to inherit a plasmid copy. However, its role in chromosomal copies is contradictory, including stress-response-promoting fitness and antibiotic treatment survival. A hybrid pathogenic Escherichia coli strain may have the ability to colonize distinct host niches, facing contrasting stress environments. Herein, we determined the influence of multiple environmental stress factors on the bacterial growth dynamic and expression profile of previously described TA systems present in the chromosome of a hybrid atypical enteropathogenic and extraintestinal E. coli strain. Genomic analysis revealed 26 TA loci and the presence of five type II TA systems in the chromosome. Among the tested stress conditions, osmotic and acid stress significantly altered the growth dynamics of the hybrid strain, enhancing the necessary time to reach the stationary phase. Using qPCR analyses, 80% of the studied TA systems were differentially expressed in at least one of the tested conditions, either in the log or in the stationary phase. These data indicate that type II TA systems may contribute to the physiology of pathogenic hybrid strains, enabling their adaptation to different milieus.

Keywords: gene transcription; hybrid strain; stress conditions; toxin–antitoxin type II.

Figure 1

(A) Experimental design for bacterial growth under stress. The illustration depicts how the experiments were designed for cultivation under stress conditions, the media employed for bacterial growth, and the bacterial cultivation phase in which samples were collected for qPCR analyses. Created with BioRender.com. Count of BA1250 Colony Forming Units (CFUs/mL) under different culture conditions in the (B) logarithmic and (C) stationary phase. Statistical analysis was performed using the non-parametric t-test, compared to bacteria growth in the LB medium. * p-value < 0.02; ** p-value < 0.002.

Figure 2

Relative expression of toxin–antitoxins in a log growth phase in duplicates of three independent experiments. E. coli BA1250 toxin–antitoxin gene pairs (ccdB/ccdA, yhaV/prlF, mazE/mazF, yoeB/yefM, and pasT/pasI) were evaluated in the log growth phase under nutritional scarcity, oxidative stress, acid shock, osmotic stress, and LB medium condition. Genes were considered up/downregulated when relative average expression was −1 > Log2Fc > 1 in comparison to the LB group. Statistical significance was considered when the p value < 0.05 in 2-way ANOVA test comparing toxin and antitoxin at the same condition. (*) Represent statistical significance with p < 0.05; (**) represent statistical significance with p < 0.01.

Figure 3

Relative expression of toxin–antitoxins in the stationary growth phase in duplicates of three independent experiments. E. coli BA1250 toxin–antitoxin gene pairs (ccdB/ccdA, yhaV/prlF, mazE/mazF, yoeB/yefM, and pasT/pasI) were evaluated in the stationary growth phase under nutritional scarcity, oxidative stress, acid shock, osmotic stress, and stress-free LB medium condition. Genes were considered up/downregulated when relative average expression was −1 > Log2Fc > 1 in comparison to the LB group. Statistical significance was considered when the p value < 0.05 in a 2-way ANOVA test comparing toxin and antitoxin at the same condition. (*) Represent statistical significance with p < 0.05; (****) represent statistical significance with p < 0.0001.

Figure 4

(A) Barplot demonstrating the predicted presence and absence of the 39 gene components of TA systems detected into aEPEC, ExPEC, Hybrid, and tEPEC strains chromosomes and plasmids. Bars indicate the percentage of predicted gene presence among the bacterial strains within each pathotype. Gene predicted rate of type I, II, IV, and V TA system components present in aEPEC, ExPEC, Hybrid, and tEPEC strains in the (B) chromosome and in the (C) plasmid. Bars indicate the percentage of predicted gene presence among the bacterial strains within each TA system type. * p < 0.05, as determined by a non-parametric one-way ANOVA test.