New Insights into the Formation of Viable but Nonculturable Escherichia coli O157:H7 Induced by High-Pressure CO2

Abstract

The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l-serine and l-threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation.

Importance: Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment.

FIG 1

The number of differentially expressed genes in VBNC cells assigned to the 14 biological processes. Genes were categorized according to the function of their encoded proteins from the KEGG pathways for Escherichia coli O157:H7 EDL933. The pie chart indicates the percentage of genes coding for membrane proteins compared to the total differentially expressed genes.

FIG 2

The number of differentially expressed proteins in VBNC cells assigned to the 13 biological processes. The pie chart indicates the percentage of membrane proteins compared to the total differentially expressed proteins.

FIG 3

VBNC state formation for recombinant Escherichia coli O157:H7 strains upon HPCD treatment. (A and B) Analysis of homologous expression of z2046 and ompF upon 1 mM IPTG induction using Tricine–SDS-PAGE and SDS-PAGE, respectively. Lanes 1, soluble fraction of E. coli pTrcHisA; lanes 2, soluble fraction of E. coli pTrc-z2046 (A) and E. coli pTrc-ompF (B); lanes 3, insoluble fraction of E. coli pTrcHisA; lanes 4, insoluble fraction of E. coli pTrc-z2046 (A) and E. coli pTrc-ompF (B); lanes M, marker. (C) The percentage of VBNC cells for the control, E. coli pTrc-z2046 and E. coli pTrc-ompF treated by HPCD at 5 MPa and 25°C for 15 min. Error bars represent standard deviation (SD).

FIG 4

Tolerance of oxidative stress in Escherichia coli O157:H7 cells. Error bars represent standard deviation (SD).

FIG 5

SEM image showing Escherichia coli O157:H7 adhering to HeLa cells. (A and B) HeLa cells adhered by the exponential-phase cells. (C and D) HeLa cells adhered by the VBNC cells. Left images, magnification of ×3,000; right images, magnification of ×10,000.

FIG 6

Effect of Escherichia coli O157:H7 on F-actin in HeLa cells. (A and B) HeLa cells infected by the exponential-phase cells. (C and D) HeLa cells infected by the VBNC cells. Left images, fluorescence micrographs; right images, phase-contrast micrographs.

FIG 7

TEM image showing Escherichia coli O157:H7 adhering to HeLa cells. (A) HeLa cells adhered by the exponential-phase cells. Dashed lines indicate dense concentrations of microfilaments composed of F-actin. (B) HeLa cells adhered by the VBNC cells. Magnification, ×30,000.