title: Aging and recovery of Listeria monocytogenes ScottA
Creators: name: Alharbi, MAS
Publisher: name: University of Tasmania
Year: 2018

description: The bacterial growth curve is considered to involve four phases: lag, exponential, stationary and death/decline phase. The lag phase is the time that bacteria use to recover and adapt to a new environment before shifting to exponential growth at a rate determined by the environmental conditions and available resources. Aging, on the other hand, is the process in which the inheritance of “new” and “old” cell poles by bacterial cells during cell division as well as putative accumulation of protein aggregates. Stress has been shown to be needed to make aging inevitable, and aging leads to cells that lose fitness thus providing a finite duration to a stressed population. Significant knowledge gaps still exist about the lag phase and bacterial aging, in particular, mechanistic details of the processes and whether broad scale principals apply. The thesis study aims to describe the microbiological, biochemical and regulatory-level mechanisms of the effect of cell aging and lag phase recovery in Listeria monocytogenes (strain Scott A). L. monocytogenes was utilised in the study due to its comparatively streamlined genome, high level of genetic and protein-level characterisation and the fact it is an important food pathogen that causes the disease listeriosis. The species is adept at switching its lifestyle from an environmental saprophyte to a human parasite and thus has a capacity to respond and adapt to relatively hostile environments. In the environment, it can survive due to this high degree of adaptability, and this has resulted in a ubiquitous distribution. How it adapts in the scenarios of enforced aging and subsequent recovery was approached by mainly using quantitative proteomics, allowing assessment of most essential cell functions.
To enable understanding of the effect of culture-based aging on L. monocytogenes ScottA growth and survival behaviour, morphological and metabolic changes, virulence potential, and ATP levels during aging processes were assessed. Aging was enforced by holding cells at 37°C deep into the stationary growth phase for up to 21 days. The temperature used represents the optimal temperature for growth rate (T\(_{opt}\)). In older cultures (aged =≥7 d) lag phase was proceeded by possible short, rapid recovery of a small segment (0.5-1.5 log units) of the population. The duration of the lag phase increased with the length of time the cells were initially incubated. The generation time of outgrowth increased with the duration of initial incubation suggesting more aged cells lost fitness. Extended incubation resulted in increasingly poor growth on agar. Aged cultures lose the ability to retain crystal violet, indicative of a thinner, less networked peptidoglycan layer. They also demonstrated cell elongation (2-10 fold increase), evidence of failed septation completion, and autolysis. After 7 d incubation, ATP levels in the cells collapsed to &lt;10\(^{-17}\) moles/cell from approximately 10\(^{-15}\) moles/cell. Catabolic enzymes associated with protein, lipid, phosphate, and carbohydrate metabolism though readily detected in young cultures become inactive in populations incubated ≥7 d. Haemolysis assays revealed no detectable listeriolysin activity was present in the bacterial supernatant fluid after ≥7 d, possibly suggesting reduced protein export. Overall, the results indicate that cell aging at the T\(_{opt}\) is associated with extensive loss of cell viability and results in lag phase extension and loss of fitness. Fitness loss may be attributable to depletion of ATP and reduced metabolism affecting protein secretion, cytokinetic and cell wall biogenic processes in the cell population.
Integrated global proteomic responses of L. monocytogenes ScottA incubated for 1 to 21 days at 37°C was performed to understand the metabolic mechanisms behind the loss of fitness and culturability in populations after extended incubation. The continued lack of availability of carbohydrates leads to an overall decrease in cellular metabolism, loss of culturability on agar, elongated multi-septated cells, autolysis, and non-detectable haemolysis. Protein samples were analysed by nano-LC-MS/MS using an LTQ-Orbitrap XL instrument with 686 proteins assessed. Metabolism reduction observed for aged cells is linked to empty ATP and precursor pools; there was no evidence of a harsh response. The process is demonstrated by a decline in phosphotransferase system protein abundances and associated compensatory adjustments to enzyme abundances within the glycolysis pathway, amongst mixed fermentation enzymes, and a possible shift in F\(_1\)F\(_0\) ATPase complex to a synthesis mode. The observed greatly enhanced abundances of glutamate dehydrogenase RocG in aged cells could suggest the use of oxidative deamination for resupply of pyruvate and NADH. Many central processes of the cell linked to growth such as the translational apparatus, ribosomal proteins and DNA methods showed a significant reduction. There is a greater emphasis on proteins involved in cytoplasmic homoeostasis, proteostasis, substrate scavenging, and amino acid/nucleic acid salvage processes in aged cells. Internalin A and B abundances were elevated in aged cells but were accompanied by reduced levels of wall teichoic acid synthesis enzymes. The abundances of several autolysins (including Spl, Iap, Auto, Ami) were substantially elevated, explaining aged induced autolysis. The increase in autolysins is potentially linked to cytokinetic dysfunction associated with the septal ring. The increase would likely explain the reason why the bulk of the cell population loses culturability though apparently, a subpopulation survives. The proteome data provides a perspective of L. monocytogenes in a state of decline deep in its stationary growth phase. 
The proteome dynamics of L. monocytogenes ScottA recovering during the lag phase was investigated. A quantitative analysis of ScottA proteome was based on cells first being grown at 37°C over an extended stationary growth phases (1, 7, 14 and 21 days) and then allowed to recover in new TSYE media over 0.5 to 4 hours. A total 459 proteins were estimated for abundance for all treatments. During lag phase L. monocytogenes ScottA rapidly altered its proteome (within 0.5 h) partially rebounding from the aged state but not completely reattaining the original exponential phase state; this was evident at all time points. The length of initial incubation had no substantial effect on lag phase responses at the protein level. Proteins associated with carbohydrate catabolism became relatively abundant in the lag phase. Recovery of the intermediary metabolism based on protein abundance data revealed a shift to mixed acid fermentation occurs. The data also points to activation of initial stages of biosynthetic pathways for cell components including peptidoglycan, fatty acids, and menaquinone, but not broad-scale increased abundance of whole pathways. The molecular chaperone Hsp70 complex, detoxification-associated proteins, compatible solute uptake transporters, and metal ion homoeostasis transporters were detected at higher abundances relative to aged cells, potentially indicating processes are involved in remediation of the cytoplasmic contents. Overall, lag phase cells adjustments cover a wide range number of anabolic and remedial processes; however, the activation responses collectively do not represent a “full steam ahead” approach but is constrained possibly to ensure precursor and energy resources can be accumulated before the exponential growth phase.
Utilising proteomic data an original insight into the global regulation of protein production by L. monocytogenes ScottA during aging and lag phase processes at 37°C was performed. The data suggest that in lag phase SigB, SigH, CtsR, HrcA and AgrA regulons are more activated relative to old and exponential cells while the PrfA and SigC regulons were instead strongly repressed. CodY levels were highly elevated in lag phase resulting in intense repression and derepression of its previously defined regulon. The activation responses collectively link to increased fermentation and anabolic processes, protein folding and rescue, remediation of the cytoplasm from toxic compounds, and buffering of cytosolic proteins. At the same time the combined increased abundance of CodY and catabolite control protein CcpA seems to lead to constraints on the levels of enzymes that rely on expenditure ATP possibly allowing build-up of the precursor, ATP and reducing equivalent pools prior to exponential growth. The data support the observation that lag phase responses are rapid and that the proteomes do not rebound back to the previous exponential phase state. The data also suggest that the extent of aged incubation results in largely homogenous responses throughout the lag phase though subtle differences were observed in the degree of activation of regulons. This incomplete rebound effect creates the possibility that inputted cell recovery in lag phase manifests various outcomes for outgrowth in the exponential phase depending on the physiological nature of the coded cells.
Collectively, the results of this research provide an insight into the mechanisms engaged by L. monocytogenes ScottA during aging and recovery. The mechanism includes overviews if the morphological, metabolic and cell energetic changes during aging and the associated underlying mechanisms as revealed by proteomic assessments. Renewal of cells in lag phase showed the critical role of CodY and SigB in these processes as master regulators as well as providing some initial information of possible protein responses that could be linked to adaptation.