2. CHAPTER 2: GENOME SIZE AND PHENOTYPIC DIFFERENCES
2.5 DISCUSSION
The results of the TEER assays confirmed the previously noted differential effects of the two strains AGR1485 and AGR1487 on Caco-2 cell barrier integrity. AGR1485 maintained Caco-2 cell TEER similar to that of control and had a positive effect on TEER but only after 24 hours of exposure. AGR1487, on the other hand, reduced Caco-2 cell TEER as compared to control as early as 10 hours, thus compromising the barrier integrity of the Caco-2 cells.
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Figure 2.5 Agar spot test of (A) AGR1485 and (B) AGR1487 Both strains grown at pH 6 (growth in MRS broth used as control) compared to cultures exposed to pH 2 and pH 4 (MRS broth acidified with 6M HCl) for 0, 2 and 4 hours followed by spot test on MRS agar plates. The values plotted are the means of three replicates and the error bars show SEM. *P<0.001 compared to bacteria in control medium, the probability-values adjusted by Benjamini-Hochberg (BH) method for controlling the false discovery rate.
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These results are similar to previously noted effects of AGR1485 and AGR1487 on undifferentiated Caco-2 cells [163]. In addition, the strains showed differences in their genome size, sugar utilisation capabilities and in their response to stress tolerance assays.
The identification and characterisation of L. fermentum and other members of this
genus reported in studies were usually based on colony morphology, Gram stain reactions, 16 S rRNA sequencing [163] and sugar fermentation profiles in addition to studying enzymatic activities [5, 277]. For this study, PFGE of genomic DNA, after digestion with restriction enzymes AscI and I-CeuI, was used to differentiate the two bacterial strains from one another. Comparison of fragment sizes of the two strains resulting from the restriction digests with published data [278] indicate that the genome size of AGR1485 was approximately 2,225 kb and that of AGR1487 was 1,930 kb. This difference in genome size suggests that there was an insertion in the genome AGR1485 and a deletion in the genome AGR1487. The effect that AGR1487 has on the intestinal barrier integrity could be a result of the absence of genes and/or gene products that may play a role in the microbe-host interactions.
The compromising effect on barrier integrity may also be the result of morphological alteration caused mutations of genes that could be involved in structural biosynthesis and/or pathways that influence bacterial morphological characteristics. The phenotypic differences exhibited by these two strains could therefore be due to the presence, absence, or mutation of genes, resulting in strain-specific characteristics.
An observation made during genomic DNA extraction from both the strains, showed that AGR1485 was more resistant to lysozyme activity as compared to
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AGR1487 and had to be treated with 0.85% NaCl (to remove EPS) in order to facilitate cell lysis, suggesting a difference in cell surface structure between the two strains. Many bacteria modify their PG in a variety of ways as a level of control over endogenous autolysins and other lytic enzymes [279]. One such modification is the O-
acetylation of PG, that provides bacteria with resistance to endogenous autolysins as well as protection from lysozymes [280]. The resistance that AGR1485 exhibits towards lysozymes may be caused by a modification to the PG that contribute to its cell surface structure making this bacterium more resistant to lysozymes and autolysins.
The API50CH test highlighted the differences in sugar utilising capabilities between AGR1485 and AGR1487. Studies with Lactobacillus delbrueckii subsp. lactis
313 suggest that utilisation of different sugars stimulate the production of different cell surface proteins which has an effect on cell membrane associated proteinase activity [281]. Proteinase production is relative to CW biosynthesis and differences in CW biosynthesis may result in variation in its structural components. Variation in the peptidoglycan composition has been shown to impart strain-specific characteristics in lactobacilli and their interactions with host cells in activating metabolic pathways and eliciting immune responses [88, 222]. Therefore, variations in sugar utilisation capabilities of AGR1485 and AGR1487 may be indicative of differences in cell surface proteins and proteinase activity between the two strains, which in turn may influence host-microbe interactions and cause them to have differential effects on the host.
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The possible genotypic differences of these strains may also be related to the difference in tolerance these bacteria show towards environmental stressors, such as bile and acidic conditions. Lactobacilli encounter various harsh conditions (e.g. bile, low pH, oxidative and osmotic stress) as they move through the GIT. To survive these conditions, they need to display many resistance mechanisms. These stress responses are dependent on the co-ordinated expression of genes that control different cellular processes [137, 282]. Many resistance mechanisms have been found to be common for bile and acid stress and result in alteration of lactobacilli cell surface structures [142]. For instance, bile salts and cholesterol have been shown to induce changes in the lipid cell membrane of L. reuteri [11] while low pH causes alterations in the fatty acid
composition of an oral strain of L. casei [12]. Studies attribute differences in stress
phenotypes to variation in stability or binding specificities of stress response regulatory proteins [283]. Screenings of acid and bile salt responses in lactobacilli have identified genes involved in PG biosynthesis and cell envelope functions. Gene expression analysis of L. acidophilus identified a high number of genes involved in PG and cell
surface protein (e.g., SrtA) biosynthesis that are differentially expressed after bile exposure [143]. In L. reuteri, response to acidic conditions involves the ClpL
chaperone, an ATPase with chaperone activity and a putative CW-altering esterase, which are also reported to be induced by bile exposure [144, 145]. AGR1485 was less tolerant to both bile and low pH as compared to AGR1487. This may be due to mutation in genes that make this strain less resistant to bile exposure as has been noted in studies with L. reuteri ATCC55730 [145]. The differences that AGR1485 showed in
comparison to AGR1487 in bile and acid tolerance may be related to their cell surface proteins and/or secretory abilities. Structures of teichoic acids have been suggested to
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affect proper functioning of cell integrity in acidic conditions and in the presence of bile [144, 145]. The cell surface structure of the strains can be an important indicator for the different stress responses that these strains exhibit [9, 136, 145]. AGR1487 may have a surface structure displaying proteins and/or secreting molecules that make it more tolerant to bile and low pH than AGR1485. AGR1485 may either lack or poorly express the proteins and/or components necessary to survive acidic conditions or to tolerate bile stress. There could also be alteration of expression of genes due to point mutations that influence the bacterial structure, thereby affecting its stress handling capabilities.
Many Lactobacillus species, including L. fermentum, are considered GRAS
microorganisms. However, they have been implicated as opportunistic pathogens, under certain conditions, especially in immunocompromised individuals [284, 285]. The tolerance that AGR1487 exhibits towards high bile concentrations and low pH suggests that it may be better adapted to survive the passage through the GIT and that the GIT conditions of such immunosuppressed individuals may provide the ideal niche for AGR1487 to flourish and colonise. The detrimental effect that AGR1487 has on the intestinal barrier may play a role in the intestinal health of the genetically-susceptible individual and other such individuals, but based on current data such conclusions cannot be drawn [258]. AGR1485 however, has no such detrimental effects on intestinal barrier integrity thus stressing the strain-specific characteristics of lactobacilli.
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