Elsevier

Microbial Pathogenesis

Volume 47, Issue 6, December 2009, Pages 289-298
Microbial Pathogenesis

Differential efficiency of induction of various lambdoid prophages responsible for production of Shiga toxins in response to different induction agents

https://doi.org/10.1016/j.micpath.2009.09.006Get rights and content

Abstract

Shiga toxin-producing Escherichia coli (STEC) is a group of pathogenic strains responsible for bloody diarrhea and hemorrhagic colitis, with often severe complications. Shiga toxins are the main factors causing the phathogenicity of STEC. Production of these toxins depends on the presence of stx1 and stx2 genes, which are located on lambdoid prophages, and their expression is stimulated upon prophage induction. Therefore, a transition of the phage genome from the prophage state to an extrachromosomal genetic element, and its further propagation, is crucial for the pathogenic effects. However, our knowledge on specific conditions for induction of these prophages in bacteria occurring in human intestine is very limited. In this report we present results of our studies on five different phages, originally occurring in STEC strains, in comparison to bacteriophage lambda. We found that efficiencies of induction of prophages and their further development vary considerably in response to different induction agents. Moreover, efficiency of progeny phage production might be modulated by other factors, like temperature or bacterial growth rate. Therefore, it is likely that pathogenicity of different STEC strains may be significantly different under specific conditions in their natural habitats.

Introduction

Pathogenicity of a number of bacteria depends on the presence of extrachromosomal genetic elements in their cells. For example, many pathogenicity factors are encoded in genomes of bacteriophages [1]. Shiga toxin-producing Escherichia coli (STEC) is a group of strains which are pathogenic for humans provided that they carry prophages bearing genes capable for producing specific toxins [2]. STEC strains are responsible for bloody diarrhea and hemorrhagic colitis, with often severe complications, and STEC infections are especially dangerous in children [3], [4].

Shiga toxins are the main factors causing the pathogenicity of STEC. Production of these toxins depends on the presence of stx1 and stx2 genes [3], [4], [5]. Although the STEC phenotype was initially correlated to the O157 serotype and to an inability to ferment sorbitol [6], subsequent studies indicated that some E. coli O157 strains are sorbitol-positive [7], [8]. Moreover, it was found that E. coli serotypes other than O157 can be responsible for the STEC phenotype [9]. Thus, it is not possible to determine STEC phenotype by microbiological and immunological assays and it was postulated that only genetic tests, based on detection of stx1 and stx2, can be adequate for identification of STEC [10], [11].

The stx1 and stx2 genes are located on lambdoid prophages, and at this stage their expression is repressed. Effective production of Shiga toxins occurs only upon prophage induction and its further lytic development, including replication of the phage genome as an extrachromosomal element [5], [12], [13], [14], [15], [16]. Therefore, determination of specific conditions causing induction of stx1- and stx2-bearing prophages in cells occurring in human intestine is crucial to understand pathogenicity of STEC and then to prevent symptoms of infections or to treat them.

The prophages bearing genes coding for Shiga toxins belong to the family of viruses called lambdoid phages, in which bacteriophage λ is the best investigated member. The mechanism of bacteriophage λ induction has been investigated in details, however, this appears to be true only for standard laboratory conditions and a few induction agents, like UV irradiation and mitomycin C [17], [18]. Generally, it is believed that any agent which can provoke the bacterial SOS response is a prophage induction agent, as the first step in this process is a RecA-dependent cleavage of the λ cI repressor and subsequent activation of lytic promoters [for reviews see [17], [18]]. On the other hand, it appears that under conditions which are not considered to be typical SOS inducers, efficient induction of some lambdoid prophages may occur. For example, Shkilnyj and Koudelka [19] demonstrated that an increased salt concentration caused induction of a prophage, whose lysogenic maintenance was dependent on the cI repressor of phage 434. Interestingly, this prophage induction was RecA-independent, suggesting that a high salt concentration may influence either the structure of cI or DNA topology (leading to impairment of cI binding to the operator sequences) rather than cause a RecA-stimulated auto-cleavage of the repressor protein due to induction of the SOS response [19].

Since UV irradiation, a classical procedure which is used for lambdoid prophage induction under laboratory conditions, is unlikely in intestine of humans infected with STEC, and treatment with mitomycin C (another commonly used induction agent) is relatively rare, other factors must be considered as main agents causing induction of prophages bearing stx genes. In this work, we aimed to compare efficiency of prophage induction and further lytic development of a series of five such phages, after treatment of bacterial cultures with various agents potentially affecting prophage maintenance.

Section snippets

Differential prophages' induction caused by various inducers

We found differential efficiency of prophage induction and production of progeny phages in response of various lysogenic strains to different induction agents (Fig. 1). When 3 mM H2O2 was used, the relative phage titer of 27Δtox was over 50-times higher than that of 933WΔtox, and other phages showed intermediate titers (Fig. 1A). Similar-order differences were observed after prophage induction with 1 μg/ml mitomycin C (Fig. 1B), where some differences in the length of the lag phase were evident.

Bacteriophages and bacterial strains

Five phages: φ24B (Δstx2::cat) [24], 933 WΔtox (Δstx2::cat), 22Δtox (Δstx2::cat), 27Δtox (Δstx2::cat), 32Δtox (Δstx2::cat) (derivatives of phages 933 W, 22, 27 and 32 were described by Gamage et al., [25]), devoid of the toxin genes (due to a safety reason) but bearing all other features of these phages, as well as bacteriophage λ papa (from our collection) were employed in this study. E. coli MG1655 strain [26] was lysogenized by these phages. E. coli C600 [27] was used as indicator strain.

Prophage induction agents

Four

Acknowledgments

This work was supported by the Ministry of Science and Higher Education (project grant no. N301 122 31/3747 to AW), and – in part – by the European Union, within European Regional Development Fund, through grant Innovative Economy (POIG.01.01.02-00-008/08). The research by JMŁ was also supported by the European Union within the European Social Fund in the framework of the project “InnoDoktorant - Scholarships for PhD students”, 1st edition. MŁ acknowledges supports from Foundation for Polish

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