The effect of two ribonucleases on the production of Shiga toxin and stx-bearing bacteriophages in Enterohaemorrhagic Escherichia coli

Abstract

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.

Figure 1

Growth of EHEC TEA028 and its RNases E and G derivative strains in cultures treated or non-treated with mitomycin C (MMC). Cultures of TEA028 (parental strain) and its RNases E and G derivatives were grown in Luria Bertani medium to optical density at 600 nm (OD₆₀₀) of 0.30–0.35 at which point the cultures were split and an aliquot was treated with MMC (1 µg/mL) to induce the stx2-phage lytic cycle. Thereafter, samples were collected at various time points to measure OD₆₀₀. The strains TEA028-rne and TEA028-rne-Δrng underproduce RNase E when the medium is supplemented with low levels of isopropyl β-d-1-thiogalactopyranoside (IPTG), as indicated. For each time point, the OD₆₀₀ of MMC-treated cultures was subtracted from the OD₆₀₀ of non-treated aliquots (ΔOD₆₀₀). Means and standard errors or at least 4 biological replicates are graphed. Lack of error bars indicates that the standard error was smaller than the plot symbol.

Figure 2

Production of phages by EHEC containing normal levels of RNases E and G versus derivative strains deficient in either or both enzymes. Cultures of TEA028 (parental strain) and its RNases E and G derivatives were grown in LB medium to an optical density at 600 nm of 0.30–0.35 at which point the cultures were treated with MMC (1 µg/mL) to induce the phage lytic cycle. Supernatants were collected at 6 h, and phage titers were determined by the double agar assay as described in “Methods” section. The strains TEA028-rne, TEA028-rne-Δrng, and TEA028-rne-Δrng (prng) underproduce RNase E when the medium is supplemented with 0.1 µM IPTG. Means and standard errors of 3–4 biological replicates are shown. Abbreviations: rng: TEA028-Δrng; rne: TEA028-rne; rne-rng: TEA028-rne-Δrng; rne-rng (prng): TEA028-rne-Δrng (prng); p.f.u.: plaque forming units. * Adjusted P value versus TEA028 = 0.0095; ** adjusted P value versus TEA028 = 0.0001; # adjusted P value versus TEA028-rne = 0.0007; & adjusted P value versus TEA028-rne-Δrng = 0.0015.

Figure 3

Toxin (Stx2a subtype) production by EHEC containing normal levels of RNases E and G versus derivative strains deficient in either or both enzymes. Cultures of TEA028 (parental strain) and its RNase E/G derivatives were grown in LB medium to an optical density at 600 nm of 0.30–0.35 at which point the cultures were treated with MMC (1 µg/mL) to induce the phage lytic cycle and toxin production. Thereafter, samples were collected at the indicated time intervals, and toxin concentrations were determined in whole cell lysates. The strains TEA028-rne, TEA028-rne-Δrng, and TEA028-rne-Δrng (prng) underproduce RNase E when the medium is supplemented with 0.1 µM IPTG. Means and standard errors of at least 4 biological replicates are graphed. Abbreviations: rng: TEA028-Δrng; rne: TEA028-rne; rne rng: TEA028-rne-Δrng; rne rng (prng): TEA028-rne-Δrng (prng). * Adjusted P value versus TEA028 = 0.0001; ** adjusted P value versus TEA028 < 0.0001; # adjusted P value versus TEA028-rne at 10 h = 0.0001; & adjusted P value versus TEA028-rne at 10 h = 0.0024.

Figure 4

Kinetics of mRNA concentrations of stx2B, recA, and Q genes in EHEC TEA028 and its RNases E and G derivatives. Cultures of TEA028 (parental strain) and its RNase E/G derivatives were grown in Luria Bertani medium to an optical density at 600 nm of 0.30–0.35 (time 0) at which point the cultures were treated with MMC (1 µg/mL) to induce the phage lytic cycle and toxin production. Thereafter, samples were collected at the indicated time intervals for total RNA extraction and reverse transcription real-time PCR. The strains TEA028-rne and TEA028-rne-Δrng underproduce RNase E when the medium is supplemented with 0.1 µM IPTG. The concentration of cDNA molecules for each gene was normalized to the concentration of cDNA molecules of 16S rRNA. Fold change was calculated as the ratio of the normalized cDNA molecules at the indicated time points after MMC addition to the normalized cDNA molecules before the addition of MMC (time 0). Adjusted P values versus TEA028 (control) are indicated in each graph, ns not significant. Means and standard errors of 3–4 biological replicates are shown. (a) TEA028, stx2B gene. (b) TEA028, recA gene. (c) TEA028, Q gene. (d) TEA028-rne 0.1 µM IPTG, stx2B gene. (e) TEA028-rne 0.1 µM IPTG, recA gene (f) TEA028-rne 0.1 µM IPTG, Q gene. (g) TEA028-rne-Δrng 0.1 µM IPTG, stx2B gene. (h) TEA028-rne-Δrng 0.1 µM IPTG, recA gene. (i) TEA028-rne-Δrng 0.1 µM IPTG, Q gene.