Good things come in small packages : delivery of vitamin K2 to human cells by extracellular vesicles from Lactococcus lactis

Vitamin K2 is essential for maintaining human health. It is required for blood coagulation and contributes to cardiovascular and bone health. Therefore, vitamin K2 enrichment in the diet is of high interest for human health. Bacteria are the natural producers of vitamin K2, and known producers such as Lactococcus lactis are key players in the production of fermented foods. This fact offers opportunities to enhance vitamin K2 levels in food. Knowledge on vitamin K2 production in L. lactis is important for vitamin K2 enriched in fermented foods (a background introduction is provided in chapter 1).

In this thesis study, initially, L. lactis strains were screened for vitamin K2 content, and the impact of various cultivation conditions was examined (chapter 3). It was observed that significant strain diversity existed in terms of specific concentrations and titers of vitamin K2. In L. lactis ssp. cremoris MG1363, aerated cultivation conditions and carbon sources like fructose or trehalose, were found to increase the vitamin K2 content as compared to static cultivation and glucose as carbon source. In quark fermentation, it was consistently observed that altered carbon source (fructose) and aerobic cultivation of the L. lactis MG1363 pre-culture resulted in elevated vitamin K2 concentrations in the quark product.

Next, an adaptive laboratory evolution (ALE) strategy was applied to obtain natural vitamin K2 overproducing L. lactis strains (chapter 4). By propagating strain MG1363 in aerated conditions, Three evolved strains were selected that showed improved stationary phase survival in oxygenated conditions. In comparison to the original strain MG1363, the evolved strains showed increased vitamin K2 content and exhibited high resistance against hydrogen peroxide-induced oxidative stress. Genome sequencing and proteomic analysis provided explanations for the enhanced oxidative stress resistance, but the mechanisms underlying elevated vitamin K2 content in the evolved strains remain to be elucidated.

Besides the long-chain forms of vitamin K2, mainly MK-8 and MK-9, L. lactis also produces a detectable amount of short-chain forms, mainly represented by MK-3. To examine the physiological significance of the various MK forms in L. lactis, mutants with different MK profiles were constructed: a non-MK producer, a presumed MK-1 producer and a MK-3 producer were obtained (Chapter 5). By examining the phenotypes of the MG1363 wildtype strain and respective mutants under aerobic, anaerobic and respiration-permissive conditions, we could infer that short-chain MKs like MK-1 and MK-3 are preferred to mediate extracellular electron transfer, while the long-chain MKs like MK-9 and MK-8 are more efficient in the aerobic respiratory electron transfer. The different electron transfer routes mediated by short-chain and long-chain MKs likely support growth and survival of L. lactis in a range of (transiently) anaerobic and aerobic niches including food fermentations.

While it is of interest to improve the content of vitamin K2 in L. lactis, the actual efficiency of delivering this vitamin to the human body is important as well. Opportunities for bacterial membrane-bound vitamin K2 delivery are expected with extracellular membrane vesicles (EVs). Bacterial EVs are derived from the cell membrane, forming membrane-enclosed spheres carrying various types of cargos in the lumen or the membrane, supporting exchange between bacterial cells and conceivably also cells from other organisms including humans. The impact of bacterial EVs on health and disease are being revealed gradually, and the potential of EVs in various applications has been highlighted (reviewed in chapter 2). Bacterial EVs are potentially ideal vehicles for efficient delivery of vitamin K2 to the human host with a similar principle as liposomes, improving the solubility and absorption of hydrophobic compounds. Given this hypothesis, the EV-producing potential of L. lactis as the vitamin K2 producer is of high interest.

The findings started with intriguing observations on L. lactis strains residing in an artisanal cheese fermentation starter, where bacteriophages are highly abundant. The L. lactis strains are hosts to prophages belonging to the family Siphoviridae. Many prophage genomes contain disruptions in the tail genes, resulting in tailless phenotype of released phage particles (chapter 6). The host-phage interaction was examined using L. lactis ssp. cremoris TIFN1 (harboring prophage proPhi1) as a representative (chapter 7). It was demonstrated that during the massive phage release, all bacterial cells remained viable. Further, by monitoring phage replication in vivo, it was confirmed that the majority of the bacterial population was actively producing phage particles when induced with mitomycin C. The released tailless phage particles were found to be engulfed in lipid membranes, which is proposed to be a chronic phage release mechanism, leaving the host intact.

To further understand the mechanism of the interesting behavior of L. lactis, an artisanal cheese isolate similar to TIFN1, namely FM-YL11, was used. EVs were observed in the culture supernatant of strain FM-YL11, whereas the prophage-inducing condition led to an over 10-fold increase in EV production in comparison to the non-inducing condition (chapter 8). In contrast, the prophage-encoded holin-lysin knock-out mutant and the prophage-cured mutant produced constantly low levels of EVs. Under the prophage-inducing condition, strain FM-YL11 did not show massive cell lysis. Defective phage particles were found to be released in and associated with holin-lysin induced EVs from FM-YL11. Taken together, evidence was provided that L. lactis produces EVs, and EV production is stimulated by the prophage-encoded holin-lysin system.

Finally, the possibility of using L. lactis EVs to deliver vitamin K2 to human cells was examined (chapter 9). It was demonstrated that EVs produced by L. lactis carry mainly long-chain vitamin K2 (MK-8 and MK-9). When these EVs were applied to in vitro grown osteosarcoma cells, the carboxylation status of osteocalcin improved, indicating functional delivery of bioactive vitamin K2 by bacterial EVs. The efficiency of vitamin K2 delivery by EVs was higher than adding solvent-dissolved pure compounds at similar concentrations. Therefore, this study provides proof of principle that bacterial EVs are ideal vehicles to deliver lipophilic compounds like vitamin K2 to the human host.

In the final chapter 10, discussion of the whole thesis work is presented, and the conclusion is highlighted that this thesis study not only examined the fundamental mechanisms of vitamin K2 and EV production in L. lactis, but also demonstrated the possibilities for vitamin K2 enrichment of fermented food as well as efficient delivery of vitamin K2 to the human host.