Evaluation of Blood-Brain-Barrier Permeability, Neurotoxicity, and Potential Cognitive Impairment by Pseudomonas aeruginosa's Virulence Factor Pyocyanin

Abstract

Pyocyanin (PCN) is a redox-active secondary metabolite produced by Pseudomonas aeruginosa as its primary virulence factor. Several studies have reported the cytotoxic potential of PCN and its role during infection establishment and progression. Considering its ability to diffuse through biological membranes, it is hypothesized that PCN can gain entry into the brain and induce oxidative stress across the blood-brain barrier (BBB), ultimately contributing towards reactive oxygen species (ROS) mediated neurodegeneration. Potential roles of PCN in the central nervous system (CNS) have never been evaluated, hence the study aimed to evaluate PCN's probable penetration into CNS through blood-brain barrier (BBB) using both in silico and in vivo (Balb/c mice) approaches and the impact of ROS generation via commonly used tests: Morris water maze test, novel object recognition, elevated plus maze test, and tail suspension test. Furthermore, evidence for ROS generation in the brain was assessed using glutathione S-transferase assay. PCN demonstrated BBB permeability albeit in minute quantities. A significant hike was observed in ROS generation (P < 0.0001) along with changes in behavior indicating PCN permeability across BBB and potentially affecting cognitive functions. This is the first study exploring the potential role of PCN in influencing the cognitive functions of test animals.

Figure 1

(a) Pyocyanin molecular structure. (b) Tricyclic antidepressants (TCAs) share great deal of structural similarity with PCN. TCAs are blood-brain barrier permeable and regulate neurotransmitters in the synapse.

Figure 2

Schematic depiction of entry routes into and across the blood-brain barrier.

Figure 3

Schematic representations of behavioral tests performed. Morris water maze (a and b), novel object recognition (c and d), elevated plus maze (e and f), and tail suspension (g).

Figure 4

Pyocyanin BBB permeability simulations. Most simulations predicted PCN to be BBB permeable/active.

Figure 5

HPLC confirmation of pyocyanin BBB permeability. (a) HPLC analysis of murine brain homogenate with intraperitonial PCN injection and (b) control murine brain homogenate without PCN injection.

Figure 6

PCN redox activity assessment across BBB. Experimental mice with PCN IP injection exhibited about~2.54 μM/cm/min enzyme activity in comparison to ~1.20 μM/cm/min enzyme activity for control mice.

Figure 7

(a) Murine behavioral tests for assessment of potential PCN-induced impairment of cognitive functions. Morris water maze test was used to assess memory development in Balb/C mice. PCN-C and PCN-C ip (n = 8) were control with single and 5 day saline injections, respectively, PCN-I and II were test groups (n = 8 each). Mice were trained for 5 days and tested on the 6th. The time required to reach the destination was recorded and analyzed. At day 4, the mice took (PCN-C) 29.65 sec, (PCN-C ip) 12.9 sec, (PCN-I) 39.1 sec, and (PCN-II) 42.9 sec. While on the fifth day, the time recorded was 21.92, 10.9, 36.9, and 43.97 seconds, respectively. At day 4, PCN-I and PCN-II took significantly more time for reaching the destination in comparison to PCN-C ip (^##P value < 0.005), while on day 5, both the test groups took greater time to complete the task. Hence, indicating hampered memory formation in comparison to control group. Data is presented as mean ± SEM and analyzed with two way ANOVA followed by Bonferroni's post hoc test (^∗P value < 0.005 compared to PCN-C, ##P value < 0.005, and ###P value < 0.001 compared to PCN-C ip). (b) Murine behavioral tests for assessment of potential PCN-induced impairment of cognitive functions. Balb/C mice were subjected to the elevated plus maze with two open arms and two closed ones for assessing anxiety patterns. The time spent in each group of arms was recorded and analyzed. Group PCN-II spent slightly more time in open arms in comparison to PCN-C and PCN-I (P value > 0.5). The impact of extended PCN exposure was estimated using y = 13.982ln(x) + 253.37 (the calculated R² = 0.9999 is representing good fit of data in the prediction of extended exposure of PCN). (c) Murine behavioral tests for assessment of potential PCN-induced impairment of cognitive functions. Tail suspension test studies depression patterns in stranded situations. Mice were suspended inverted via their tails. The time spent struggling was noted and analyzed. Group PCN-II spent slightly less time struggling to correct their inverted position in comparison to PCN-C and PCN-I (P value > 0.5). The impact of extended PCN exposure was estimated using y = −33.7ln(x) + 122.13 (the calculated R² = 0.9955 is representing good fit of data in the prediction of extended exposure of PCN). (d) Murine behavioral tests for assessment of potential PCN-induced impairment of cognitive functions. Group PCN-II were relatively unable to discriminate between the two objects presented during the trials in comparison to PCN-C and PCN-I. The significant difference was observed between PCN-C and PCN-II (P value < 0.5).