The prevalent use of antimicrobials in humans for therapeutic purposes and in animals for promoting growth has led to the rise of resistant bacteria. Moreover, the use of antibiotic in therapies results in eliminating the natural microflora and carries the risk of developing the pathogens to multi drug resistant strains. As an alternative approach, recently, use of probiotics has gained interest for fighting infectious diseases. World Health Organization (WHO) defines probiotic as ”live microorganisms which when administered in adequate amounts confer a health benefit on the host” (http://www.who.int). The advantages of probiotics compared to antibiotics are their specificity of action and lesser pressure of selection on bacteria. Probiotics may use different mechanisms to inhibit the growth of pathogenic bacteria. The mechanism to inhibit pathogens may include production of antimicrobial agents, depletion of nutrients, preventing adhesion to the gut mucosa and inhibition of bacterial toxins (reviewed by (Sikorska & Smoragiewicz, 2013). The probiotic bacteria may show effectiveness by interacting with the host, either by reinforcing the function of the epithelium barrier or by modifying the immune system response. However, the mode of actions, especially those involved in adhesion, are specific to species and even strains. The majority of probiotics are bacterial genera including Lactobacillus, Bifidobacterium, Escherichia, Enterococcus, Bacillus and Streptococcus as well as some fungal strains belonging to Saccharomyces (Alvarez-Olmos & Oberhelman, 2001; Gibson et al., 2004; Gupta & Garg, 2009).
Lactobacilli and Bifidobacterium are generally recognized as safe due to its long history of safe consumption supported together with epidemiological data. Lactobacillus plantarum is one of the most important and widespread members of the genus Lactobacillus, and is also used as a probiotic because of its beneficial effects on human and animal health. L. plantarum has shown therapeutic potential for wound infections caused by Pseudomonas Aeruginosa and Staphylococcus aureus (Jabbar et al., 2008; Valdéz et al., 2005). Till date, 246 genome sequences of L. plantarum are publically available, with 48 completely sequenced genomes (NCBI genome database accessed on 28.0.2018). L. plantarum WCFSI, isolated from human saliva, was fully sequenced and first to be published among any Lactobacillus species. The size of this genome which is approximately 3.4 Mbp, with a GC content of 44.4%, is one of the largest in the Lactobacillus group (Siezen et al., 2010).
My intended study group has isolated 120 strains of L. plantarum from the vaginal bacterial flora. Of the isolated strains, ten strains were found to be resistant to gastrointestinal stress conditions and also highly adhesive to human tissue cultures. One strain, L. plantarum 2025, which revealed symbiotic and synergistic effects upon application with other probiotic strains, identified to highly efficient probiotic strain. This strain, which is of considerable interest for the development of antibacterials, has been sequenced using Ion Torrent sequencing technology and draft genome sequence has been published.
Putative specific adhesins and its host cell receptors
The NCBI GenBank genome annotation pipeline allowed the identification of 2,819 protein-coding genes, 47 of which encode putative cell surface/adhesion proteins (up to 1,135 amino acids in size). As adhesion is crucial for colonization of host, biofilm formation and preventing adhesion of pathogens to the gut, presence of putative cell surface proteins indicate their role in crosstalk between our strain of interest and their host. A study evaluating the ability of 31 different L. plantarum strains suggested lectin-like adhesins and other proteinacious cell surface structures to be involved in adhesion (Tallon et al., 2007). Another study showed multifunctional proteins on the cell surface of L. plantarum 423 to play a role in adherence to Caco-2 cells (Ramiah et al., 2008). The surface expressed ?-enolase of L. plantarum LM3 involved in binding with fibronectin, plasminogen and collagen was also shown (Castaldo et al., 2009; Salzillo et al., 2015; Vastano et al., 2013). The ?-enolase was shown to modulate immune response in Caco-2 cells and in development of biofilms (Vastano et al., 2015). The detection of a gene-encoding fibronectin-binding adhesin in our strain could explain the property of high adhesion to human tissues. The studies showed that difference in adherence to Caco-2 cells for different L. plantarum strains are strain specific and speculates the reason could be due to interaction between strain specific bacterial adhesins and host cell receptors. Thus, our interest is to identify putative adhesins specific to our strain and its host cell receptors.
Extra polysaccharide, biofilm and adherence to the host cell:
The differences in adherence levels for the strains were also attributed to factors other than surface-bound proteins. Interestingly, like other strains of the species, L. plantarum 2025 contains the genes responsible for capsular polysaccharide (CPS) biosynthesis. The extra polysaccharide (EPS) encoding CPS gene clusters in L. plantarum have shown to be contributing independently to cell surface architecture of the bacterium (Remus et al., 2012). The presence of unique polysaccharide gene clusters in L. plantarum was also shown to contribute to strain specific attributes like adhesion, biofilm development and immunomodulation. EPS together with strain-specific adhesins and other surface molecules have been postulated to influence each other on the host interaction to varying levels in different strains (Lee et al., 2016). Thus, we would like to study how strain specific adhesin influence interaction of the strain with the host, production of EPS and formation of biofilms.
Bacteriocin biosynthesis genes:
Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria. Most of the gram positive bacteria producing bacteriocins are 20 – 70 aminoacids in size. The small peptides are released in to the extracellular medium and offer advantage to colonization and competition in the gastro intestinal tract. The peptides activity against pathogenic bacteria makes them a potential alternative to antibiotics in veterinary and medical applications. The plantaricin (pln) locus is responsible for bacteriocin biosynthesis in L.
plantarum. The pln gene cluster of L. plantarum contains about 25 genes and encodes various class II bacteriocins. The pln loci of L. plantarum strains were highly variable (Diep et al., 2009; Nissen-Meyer et al., 2010). The genes encoding bacteriocin can be located at the chromosome or at the plasmid. The presence of genes responsible for bacteriocin production and immunity in our strain implies its superior probiotic potential. Thereby, we are interested in heterologous expression of bacteriocin encoding genes specific to the strain in E. coli, to study the antimicrobial activity of the purified bacteriocin and understand its genetic characterization.
Whole geneome sequencing and Comparative genome analysis:
Draft genome sequences represent the complete nucleotide base sequences of our species of interest with lower accuracy than finished sequences. The information assembled into contigs needs further sequence information to close the gaps between the contigs requiring more coverage along with additional analysis. Complete genome sequences are essential for detailed molecular and transcriptomic studies. The data about the organisms DNA must be in large, contiguous segments, and fully sequenced for performing comparative genomic analysis. Whole-genome comparisons are a powerful approach for revealing fascinating differences and similarities between genomes and a prerequisite for profiling rapidly evolving sequences (Cooper et al., 2005; Garber et al., 2009). Thereby, the approach provides a mean to classify species and to describe horizontal gene transfer events (Darling et al., 2004). Hence, Comparative genomic analysis from multiple species or strains can provide insights into the functional and evolutionary processes of the sequenced genomes. Therefore, our main objective will be to identify unique genetic features of L. plantarum 2025 and understand its role in the superior probiotic activity of the strain.
Aim and Objectives:
From the above short overview, Our aim and objectives of this project are as follows:
Our aim is to identify and investigate the factors associated with probiotic activity of a of the isolated strain Lactobacillus plantarum 2025
Following are the list of objectives intended to study in this project. They are:
Objective I: To complete genome sequence of strain Lactobacillus plantarum 2025 using IonTorrent next generation sequencing technology.
Objective II: To do comparative genome analysis for identification of unique genetic features of the strain
Objective III: Identify specific adhesins of this strain, as well as specific host cell receptors, using in vitro adhesion assays. Investigate the role of strain specific adhesin interaction with the host cell receptor, production of EPS and in formation of biofilms.
Objective IV: Heterologous expression of bacteriocin in E. coli to study the antimicrobial activity and understand the genetic characterization.
I intend to do this research in Prof. Andrey Karlyshev’s group. The intended research methods described below have been applied by Karlyshev’s group for studying Lactobacillus fermentum 3872 stain and in other projects. The objectives of this project will further exploit the following methods:
Genome sequencing and assembly:
For whole genome sequencing we will use IonTorrent personal genome machine. The genome sequences obtained can be annotated using tools like PROKKA, BASys, RAST and NCBI GenBank annotation pipeline. Further regularities can be corrected using Geneious software.
Bioinformatic analysis for comparative studies:
Till this date, 48 completely sequenced genomes of Lactobacillus plantarum are publicly available in the database and other draft genomes of the same species are available for our comparative genomic analysis. BLASTN and Mauve alignments are available for whole genome comparison. MUMmer program with default parameters can be used to compare whole genome at nucleotide levels and can be viewed using the Artemis comparison tool (ACT).
Mutant strain construction:
Using PCR reactions, forward and reverse primers are constructed. Strain specific adhesion gene is replaced with antibiotic gene flanked homologous sequences and cloned into a plasmid followed by transformation in to L. plantarum 2025. The adhesion level will be compared between mutant and wild type strain.
SDS page, Immunoblot overlay assay and peptide mass finger printing:
To identify putative bacterial adhesion, surface proteins have to separated and tested for binding of host protein. The putative adhesin are digested and analysed using mass spectrometry. Adhesin molecules are identified by searching against the protein database.
In vitro Caco-2 adhesion assay:
To test adhesion, Caco-2 cells are seeded in 96 well tissue culture plates at a respective concentration and cultured for 12 – 15 days. The obtained monolayers of differentiated cells were overlaid with stationary phase cells of our wild type and mutant strain. After incubation under anaerobic condition, unbound bacteria are removed.Caco-2 cells and adherent strainare removed. Serial dilutions of samples are plated on MRS agar and numbers of cell-bound bacteria are determined. Measurement of total bacterial load obtained from unwashed control wells are used for correcting the number of cell-bound bacteria.
Microtitre plate assay of L. plantarum biofilm formation:
The assay measures the level of cell adhering to the surface of microtitre plate wells. Overnight grown cultures of wild type strain and adhesin mutant strains were inoculated into the wells of a 96-well cell culture plate. Wells containing uninoculated growth medium were used as negative control. Unattached cells are removed and adherent bacteria are stained. After a period of time, the bound dye is extracted from the stained cells. Biofilm formation quantification for each well is measured by taking OD at 570 nm.
Quantification of EPS and determination of sugar composition:
To determine adhesin role in EPS production, the difference in amount of EPS production between the wild type strain and adhesion mutants will be analyzed using High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
Construction of recombinant expression vectors:
Genes encoding bacteriocin are synthesized from the available genome sequence and amplified in PCR using forward and reverse primers. After amplification, PCR products are resolved on agarose gel and purified DNA is extracted. Recombinant plasmids are constructed with the extracted DNA after restriction and ligation. Recombinant plasmids are transformed into competent E. coli cells. After transformation, the recombinant plasmids carrying the gene are made to express by inducing with IPTG and purified for checking anti microbial activity.
Agar well diffusion test:
This test is used to determine the anti microbial activity of our purified, recombinant bacteriocin. The agar plate surface is inoculated by spreading microbial inoculums over the entire agar surface. Small wells of size 6-8mm are made on the agar plates. Purified bacteriocin (20 – 100µl) is added to the wells and the plates are kept for incubation. The bacteriocin diffuses into the agar and inhibits the growth of the test microorganism. The diameters of inhibition growth zones are measured.