The Impact of Protease during Recovery from Viable but Non-Culturable (VBNC) State in Vibrio cholerae

Abstract

Vibrio cholerae can survive cold stress by entering into a viable but non-culturable (VBNC) state, and resuscitation can be induced either by temperature upshift only or the addition of an anti-dormancy stimulant such as resuscitation-promoting factors (Rpfs) at suitable temperature. In this study, the role of proteinase K was analyzed as an Rpf in V. cholerae. A VBNC state was induced in V. cholerae AN59 in artificial seawater (ASW) media at 4 °C, and recovery could be achieved in filtered VBNC microcosm, called spent ASW media, merely by a temperature upshift to 37 °C. The resuscitation ability of spent ASW was further enhanced by the addition of proteinase K. The mode of action of proteinase K was investigated by comparing its effect on the growth of the VBNC and culturable state of V. cholerae in ASW and spent ASW media. The presence of proteinase K allowed culturable cells to grow faster in ASW by reducing the generation time. However, this effect of proteinase K was more pronounced in stressed VBNC cells. Moreover, proteinase K-supplemented spent ASW could also accelerate the transition of VBNC into recovered cells followed by rapid growth. Additionally, we found that dead bacterial cells were the substrate on which proteinase K acts to support high growth in spent ASW. So, the conclusion is that the proteinase K could efficiently promote the recovery and growth of dormant VBNC cells at higher temperatures by decreasing the duration of the initial lag phase required for transitioning from the VBNC to recovery state and increasing the growth rate of these recovered cells.

Keywords: VBNC; growth; protease; proteinase K; recovery.

Figure 1

Progress curve of V. cholerae culturability and viability during the course of VBNC induction.(a) Culturable cell count was determined for V. cholerae AN59 during the induction of VBNC state at 4 °C (●) in ASW. Recovery was induced on days 56, 63, 70 and 77 after entry into VBNC state by temperature upshift from 4 °C to 37 °C (●). Each bar represents the mean ± SE of three independent experiments. (b) The relative mRNA expression of transcripts of molecular chaperones (■) dnaK and (●) GroEL were analyzed during the course of VBNC induction as an indicator of cell viability.

Figure 2

Effect of proteinase K on VBNC and culturable cells in ASW and spent ASW media. (a) Growth of culturable cells (CI) and recovery of VBNC cells (VI) in ASW media (FM) without or with 100 μg mL⁻¹ of proteinase K (PK); cell count was determined at 0 h and 32 h. # represents non-significant difference in growth of CI between FM and PK-supplemented FM. Asterisk (*) represents statistically significant difference between the growth of CI and VI in FM + PK (p < 0.05). (b) Growth of CI cells and recovery of VI cells in SM (spent ASW media) and SM+PK. Each bar represents the mean ± SE of three independent experiments. Asterisk (**) represents statistically significant difference in growth of CI between SM and SM+PK. (p < 0.001).Asterisk (***) represents statistically significant difference in growth of VI between SM and SM+PK. (p < 0.02). Asterisk (*) represents statistically significant difference in growth between CI and VI in SM+PK (p < 0.05).

Figure 3

Time-dependent growth curve of culturable cells and resuscitation curve of VBNC cells in ASW (FM), ASW with proteinase K ( FM + PK), spent ASW (SM) and spent ASW with proteinase K (SM + PK). (a) Growth curve of culturable cells (CI cells) in FM (■) and FM + PK (●) for 32 h at 37 °C. The viable cell count was determined by the plate count method at an interval of 2 h until the end of incubation time. (b) Resuscitation curve of VBNC cells (VI cells) in FM (■) and FM + PK (●) for 32 h at 37 °C. (c) Growth curve of CI cells in SM (■) and SM+PK (●) for 32 h at 37 °C. (d) Resuscitation curve of VI cells in SM (■) and SM+PK (●) for 32 h at 37 °C. Each data point represents the mean ± SE of three independent experiments.

Figure 4

Effect of protease on recovery of V. cholerae from the VBNC state is a general trait. Recovery was induced in VBNC cells at 37 °C in spent ASW media (SM) and heat-treated spent ASW media (∆SM) without or with proteinase K, subtilisin, trypsin and BSA. The recovered cell count was determined after 32 h. Asterisk (*) represents statistically significant difference in growth of VBNC cells with and without proteases in spent ASW media (p < 0.05). # represents non-significant difference in growth of VBNC cells with and without proteases in heat-treated spent ASW media.

Figure 5

Growth-promoting components of spent ASW media. The growth of culturable cells was checked in ASW media (FM), ASW media with 10⁷ cells of dead bacteria (+dead cells), ASW media with 10 µg mL⁻¹ genomic DNA (+gDNA), ASW media with 100 of µg mL⁻¹ BSA (+BSA) and spent ASW media (SM). The cell count was determined after 32 h of incubation and represented as (−) PK. The samples in the presence of proteinase K are represented as (+) PK.

Figure 6

Protease expression at mRNA and protein level during recovery. (a) We checked the mRNA expression of serine proteases such as vc0099, vc1200, vca0803 and vc1989 (yaeZ) in a time-dependent manner (4 h, 8 h, 16 h) using dnaK as reference gene. (b) Skim milk plate assay for the detection of protease secretion during recovery from VBNC state. Protease activity on 1% skim milk plate.