Structural and Proteomic Changes in Viable but Non-culturable Vibrio cholerae.

Susanne Brenzinger,Jean-Marie Lacroix,Ariane Briegel,Gilles P Van Wezel,Lizah T Van Der Aart,Timo Glatter

doi:10.3389/fmicb.2019.00793

Abstract

Aquatic environments are reservoirs of the human pathogen Vibrio cholerae O1, which causes the acute diarrheal disease cholera. Upon low temperature or limited nutrient availability, the cells enter a viable but non-culturable (VBNC) state. Characteristic of this state are an altered morphology, low metabolic activity, and lack of growth under standard laboratory conditions. Here, for the first time, the cellular ultrastructure of V. cholerae VBNC cells raised in natural waters was investigated using electron cryo-tomography. This was complemented by a comparison of the proteomes and the peptidoglycan composition of V. cholerae from LB overnight cultures and VBNC cells. The extensive remodeling of the VBNC cells was most obvious in the passive dehiscence of the cell envelope, resulting in improper embedment of flagella and pili. Only minor changes of the peptidoglycan and osmoregulated periplasmic glucans were observed. Active changes in VBNC cells included the production of cluster I chemosensory arrays and change of abundance of cluster II array proteins. Components involved in iron acquisition and storage, peptide import and arginine biosynthesis were overrepresented in VBNC cells, while enzymes of the central carbon metabolism were found at lower levels. Finally, several pathogenicity factors of V. cholerae were less abundant in the VBNC state, potentially limiting their infectious potential. This study gives unprecedented insight into the physiology of VBNC cells and the drastically altered presence of their metabolic and structural proteins.

Highlights

Changes in the physical and chemical properties of their environment, such as heat, cold and salt stress, oxygen and nutrient deprivation, desiccation and changes in osmolarity, threaten the survival of bacteria, and they have evolved various strategies to evade detrimental effects (Merchant and Helmann, 2012; Alvarez-Ordóñez et al, 2015; Guan et al, 2017)
In order to simulate the aquatic habitats as closely as possible in our laboratory setting, we used water samples collected from environmental sources, including fresh water (NFW), brackish water (NBW) and salt water (NSW)
Cells introduced into the 50 ml microcosms of natural fresh water (NFW), natural brackish water (NBW) and natural salt water (NSW) had all lost their ability to form colonies on lysogeny broth (LB) plates over the course of 200 days (Figure 1)

Summary

Introduction

Changes in the physical and chemical properties of their environment, such as heat, cold and salt stress, oxygen and nutrient deprivation, desiccation and changes in osmolarity, threaten the survival of bacteria, and they have evolved various strategies to evade detrimental effects (Merchant and Helmann, 2012; Alvarez-Ordóñez et al, 2015; Guan et al, 2017). A characteristic morphological feature of V. cholerae VBNC cells is a smaller size and a round, coccoid shape with an increased gap between the cytoplasmic and outer membrane (OM), which was previously observed in transmission electron microscopy (TEM) studies (Chaiyanan et al, 2007; Krebs and Taylor, 2011; Kim et al, 2018). The toxin co-regulated pilus (TCP) and the flagellum were detected in cultures that are transitioning into, or have already entered, the VBNC state using transcription based methods or immunolabeling (Asakura et al, 2007; Krebs and Taylor, 2011; Xu et al, 2018) These methods can only detect the presence of macromolecular complex components, but whether those machineries are still properly assembled and embedded in the envelope remained unclear. The transcriptional studies are difficult to compare with each other as gene targets were not featured in all data sets or the results were contradicting each other (González-Escalona et al, 2006; Asakura et al, 2007; Xu et al, 2018)

Methods

Results

Conclusion