Bioengineered Lactobacillus casei expressing internalin AB genes for control of Listeria monocytogenes infection

Abstract

Listeria monocytogenes is one of the common food pathogens implicated in different outbreaks. It has recently (2017-2018) been implicated in the South African listeriosis outbreak, ever reported, where 1060 people were infected resulting in 214 deaths. The ability of listeriosis to cause high case fatality rate (20 to 30%) when compared to most foodborne pathogens makes it an important pathogen and a substantial public health concern. Listeria is an intracellular pathogen that employs different virulence factors to cross the three significant barriers, namely, the intestinal epithelial, the blood-brain endothelial, and the feto-placental endothelial cell barrier, thereby causing listeriosis. As it is the case for most pathogenic infections, antibiotics have been the first line of defence against listeriosis, however, these undesirable effects in the gastrointestinal (GI) infections keep increasing and thus posing major clinical problems. Coupled with that is the increase in the number of bacteria referred to as “superbugs”, those bacteria which have developed resistance against most of the commonly used antibiotics. The rise in these clinical problems, the increase in foodborne infections and the development of antibiotic resistance have led to a need for an alternative solution for these infections. There has been a growing interest in exploring probiotics as an alternative to antibiotics. Probiotics offer beneficial effects to the host and are able to inhibit pathogens through the use of different mechanisms including among others, competing for food and space with foodborne pathogens. They grow rapidly and colonize the gastrointestinal tract (GIT) either permanently or temporarily, consequently alleviate and prevent foodborne infections through mechanisms such as competitive exclusion. However, these probiotics are generic in their action, that is, they are non discriminatory in their action. Furthermore, they are not equally effective in all hosts nor against all pathogens. These limitations inspired the development or design of probiotics strains that will be targeted against specific pathogens. This can be achieved through a systematic understanding of the infection cycle of the pathogens, their virulence factors and disease mechanisms. Virulence genes from food-borne pathogens are cloned and expressed into probiotics through bioengineering in an effort to offer them direct competition for the same receptor sites to which pathogens attach, or for enhanced production of antimicrobial peptides and ultimately inhibition of the specific pathogen. Listeria monocytogenes in its disease progression uses virulence factors such as Listeria adhesion protein (LAP), autolysin amidase (AmiA) for adhesion, while the bacterial surface proteins internalin A (InlA) and internalin B (InlB) are responsible for invasion through the host cells. Cloning and expression of these virulence factors into probiotics will potentially offer the recombinant probiotics an enhanced ability to compete and ultimately inhibit L. monocytogenes. Taking this into consideration, the current study intended to determine whether cloning and expressing the invasion proteins internalins A and B of L. monocytogenes into Lactobacillus casei using the expression vector pLP401-T would alleviate or prevent the Listeria associated damages in vitro. The current study and its findings are organized into the five chapters of this thesis as follows. The first chapter of this thesis (Chapter 1- Literature Review) gives an overview of L. monocytogenes characteristics and pathogenesis, highlighting the virulence genes important for its infection. The various control measures used in clinical environment and food industry, their advantages and disadvantages are discussed. The limitations of these control measures and the need for an alternative measure are justified. Then probiotics as an alternative control for L. monocytogenes are described, taking into consideration their different modes of action. This gives a comprehensive explanation of the limitations of the wild type probiotics, including that they at times fail to inhibit pathogens, which emphasizes the demand for a robust strategy for their improvement. This is followed by discussion of the concept of probiotic engineering as an alternative strategy for improving the efficiency of probiotics for enhanced and targeted control of specific pathogens, explaining some applications where such recombinant strains have been explored. Recombinant probiotics are genetically modified organisms, therefore, due to the ethical reasons surrounding genetically modified organisms, safety concerns regarding recombinant probiotics were briefly addressed. This chapter ends by giving future perspectives regarding the use of recombinant probiotics. In the first experimental chapter (Chapter 2- Construction of recombinant Lactobacillus casei strain expressing the invasion proteins internalins A and B of Listeria monocytogenes), the research followed a stepwise procedure to clone and express the proteins. Firstly, the genomic DNA from L. monocytogenes F4244 (serotype 4b) was extracted and using the specific InlAB primers, the genes was amplified using PCR. The amplification of the InlAB genes was successful, and the genes was subsequently purified for cloning. Using the specific restriction digestion enzymes, the genes and expression vector pLP401- T were digested and ligated using T4 DNA Ligase. Ligation of the two was successful and this was visualized by a band larger than that of the vector alone. The ligated pLP401- InlAB was transformed into L. casei through electroporation. A total of twenty-five transformants were obtained, which were subsequently tested for the presence of InlA, InlB and InlAB genes with their specific primers using PCR. The full length InlA, InlB and the genes InlAB were all amplified confirming their presence in the transformants (recombinant L. casei). The SDS-PAGE and Western blot were used to determine whether the internalins were expressed in the recombinants. The results showed that both InlA and InlB were expressed by the recombinant L. casei but not in its wild- type counterpart. The growth patterns of the wild-type L. casei strains (L. casei WT(LbcWT)), L. casei with the vector without InlAB (LbcV) and L. casei with InlAB (LbcInlAB)) were compared. Interestingly, there was no difference in the growth patterns of all the L. casei strains. The results from this chapter demonstrates that the cloning and expression of the proteins InlAB into the probiotic was successful and that expression of the foreign genes did not have observable negative effects on L. casei growth characteristics as growth curves of all the L. casei strains were comparable. The successful cloning and expression of the invasion proteins InlAB allowed an opportunity to test if there were any differences in the effects that the recombinant L. casei would have on the inhibition of L. monocytogenes in vitro. In the second experimental chapter (Chapter 3- Prevention of Listeria monocytogenes adhesion, invasion and translocation in vitro by the recombinant Lactobacillus casei expressing the internalin AB), the study investigated the ability of L. casei expressing the invasion genes internalin AB (InlAB) (LbcInlAB) to affect L. monocytogenes progression in vitro using the Caco-2 cells grown and maintained in the cell culture medium, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS). This construct was compared with a previously developed L. casei expressing Listeria adhesion protein (LAP) (LbcLAP). To achieve this aim, the ability of the L. casei strains to adhere to, invade and translocate through the Caco-2 cells were first investigated. The results showed a difference in all stages, with the recombinant L. casei showing enhanced activity then the wild- type counterpart. For microorganisms to be deemed a probiotic, they have to be able to competitively exclude pathogens. Taking that into consideration, the ability of all L. casei strains to inhibit L. monocytogenes adhesion using three different mechanisms, namely, Compe