(27o) Effect of Oxidative Stress on Pertactin Productivity in Bordetella Pertussis Fermentations: A Study on Glutamate-Induced ROS Inhibition

1. Motivation
Whooping cough, also referred to as pertussis, is a highly contagious
respiratory tract disease. It is caused by the bacteria Bordetella pertussis,
which is a gram-negative, aerobic, pathogenic, encapsulated coccobacillus of
the genus Bordetella [1]. Pertactin is a key virulence factor of B. Pertus-
sis, the bacterium that causes whooping cough [2]. The acellular vaccine for
whooping cough comprises a mixture of pertussis toxin, fimbriae, filamentous
hemagglutinin, and pertactin [3]. Currently, the isolation of pertactin is the
bottleneck of the vaccine manufacturing process due to its low abundance in
fermentation broth and variability in the final yield. Oxidative stress plays
an important role in the growth and productivity of B. Pertussis fermen-
tations and can be a main cause of variable productivity [4, 5]. A higher
concentration of the carbon source in the media can induce higher oxidative
stress, which can lead to inhibition of growth and productivity of pertactin
[6, 7].
In this work, the impact of glutamate-induced oxidative stress on growth
and productivity of pertactin antigen was investigated. Surface expression of
pertactin, and its concentration in the culture broth was observed. Oxidative
stress among the cells and the secretion of NADPH, a key reactant in anti-
oxidative reactions, in the culture supernatant was monitored throughout
the culture run. Clear correlations were established between NADPH in the
broth with oxidative stress and productivity for different levels of oxidative
stress induced by high concentrations of glutamate. The results are highly
relevant for optimizing nutrient media, and also for identifying and addressing
possible sources of process variability.

2. Methods
The effect of oxidative stress due to high glutamate (carbon source)
concentration on pertactin productivity was investigated. The study fo-
cuses on glutamate since it is the most abundant nutrient in the growth
media and changes in glutamate may occur in the industrial case process
due to measurement error and other operating procedures. Flow cytome-
try was used to observe the oxidative stress and surface expression of per-
tactin at different growth phases. Oxidative stress was assessed using 2’,7’-
dichlorodihydrofluorescein diacetate (H2DCFDA) dye (Thermo Fisher). Anti-
pertactin monoclonal antibodies (Sanofi) conjugated with Cy5 dye (abcam,
ab188288) were used for monitoring surface PRN expression. An in-line 2-D
fluorometer probe (developed in-house) with 365 and 405 nm excitation wave-
length was used to monitor the extracellular NADPH (anti-oxidant) which
was secreted by the bacterial cells in 3.5-liter fermentors. Affinity chro-
matography was used to purify and quantify pertactin secreted in fermenta-
tion broth. Correlation between surface-bound pertactin and the pertactin
secreted in the broth was established. The weighted average of fluorescence
intensities measured by cytometry for surface-bound pertactin showed a clear
inverse correlation between ROS and pertactin productivity.

3. Results
The findings of this work highlight the importance of monitoring both
oxidative stress and PRN expression levels on bacterial cultures to better
understand their growth dynamics and potential virulence. Figure (1) com-
pares the growth of bacterial cultures with two different concentrations of
glutamate and a positive control for oxidative stress. The growth of cultures
with high glutamate concentration is inhibited, likely due to inhibition of
TCA cycle enzymes [7, 8]. Figure 2 shows the weighted average of ROS
and surface PRN expression for the cell population for different glutamate
concentration with a positive control for oxidative stress.

4. References

[1] D. J. Nieves and U. Heininger, Emerging Infections 10 pp. 311–339 (2016).
[2] J. T. Poolman and H. O. Hallander, Expert review of vaccines 6, 47
(2007).
[3] N. Guiso, Clinical infectious diseases 49, 1565 (2009).
[4] V. Zavatti, H. Budman, R. L. Legge, and M. Tamer, Biochemical Engi-
neering Journal 151, 107359 (2019).
[5] V. Zavatti, H. Budman, R. L. Legge, and M. Tamer, Biotechnology
progress 36, e2899 (2020).
[6] J. D. Wang and P. A. Levin, Nature Reviews Microbiology 7, 822 (2009).
[7] K. E. Wellen and C. B. Thompson, Molecular cell 40, 323 (2010).
[8] R. Singh, J. Lemire, R. J. Mailloux, and V. D. Appanna, PLoS One 3,
e2682 (2008).