Indian Journal of Animal Research

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Research Article

​Optimization of Cultural Conditions for Maximum Production of Outer Membrane Vesicles from Salmonella Typhimurium

Menguzotunuo Solo1,*, Shantanu Tamuly1, Luit Moni Barkalita3, Girin Kumar Saikia2, Dhruba Jyoti Kalita3
1Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.
2Department of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.
3Department of Veterinary Microbiology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.

ABSTRACT

Background: The non-typhoidal Salmonella causes gastroenteritis in humans that makes its way to the food chain mainly through the animal products. The multiple drug resistance imposes one of the major hurdle in the treatment of the disease. The vaccination appears to be the most important method for prevention of the disease. Unfortunately, there is no liscenced vaccine available against non-typhoidal Salmonellae. The use of outer membrane vesicles (OMVs) of Salmonella as a vaccine candidate has attained significant centre-stage in the recent years given to its protective immunogenicity. However, the large scale production of OMVs is difficult owing to low yield per liter of culture.

Methods: In the present study, we have optimized the culture conditions viz. pH, phase of growth and presence of oxidative stress for maximum production of OMVs from Salmonella Typhimurium. The OMVs were characterized based on yield based on protein concentration, lipopolysaccharide concentration and zeta size.

Result: In the present study, it was found that incubation of Salmonella Typhimurium up to peak of the growth phase at pH 7 in presence of oxidative stress was found to be the most suitable condition for maximum production of OMVs.

INTRODUCTION

Salmonellosis is one of the most common food borne diseases with reportedly 5.35 million burden of non-typhoidal Salmonella worldwide (Stanaway et al., 2019). The Salmonella serovars have been reported by various workers in food and animal products posing a potential public health issues (Badhe et al., 2013; Hassan et al., 2020; Singh et al., 2018). The antibiotic resistant Salmonella are increasingly being observed (Chakraborty et al., 2019; Chiu et al., 2002; Eng et al., 2015) and so far, only two vaccines, Ty21a and Vi capsular polysaccharide, are available (MacLennan et al., 2014; Xiong et al., 2017). Gram negative bacteria, including Salmonella Typhimurium, secrete vesicles from their outer membrane which can generate immune and inflammatory responses inside the host cells (Alaniz et al., 2007). These outer membrane vesicles possess range of intact outer-membrane proteins that are similar to the bacterium that has released it. The immunogenicity of these OMVs are similar to the parent bacterium in terms of uptake by macrophages. In addition, the vaccine based on OMVs do not require adjuvant as they have the potential of self-adjuvantation (van der Pol et al., 2015). A vaccine based on outer membrane vesicles, Bexsero (Novartis), has already been approved and licensed against meningococcal serogroup B (McCarthy et al., 2018). The Salmonella Typhimurium OMVs can produce cross-immunity among the various strains of Salmonella (Liu et al., 2016). Presently, the production of OMVs poses an important challenge in terms of yield. The yield of OMVs is generally less making it commercially expansive. The yield of OMVs is generally influenced by great majority of factors such as pH of the medium, type of media, presence of oxidative stress, presence of antibiotics etc. (Balhuizen et al., 2021; Klimentova and Stulík, 2015). Hence, it is important to standardize the cultural condition of growing Salmonella Typhimurium for obtaining maximum yield of OMVs. This study was conducted to optimize the conditions for Salmonella Typhimurium outer membrane vesicle production by altering pH of the medium, period of incubation of bacterial culture and induction of H2O2 mediated oxidative stress.

MATERIALS AND METHODS

Place and time of work
 
The work was carried out in the Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati in the year 2018.
 
Bacterial strains
 
For this study, the MTCC-98 strain of Salmonella Typhimurium was obtained from Department of Animal Biotechnology, College of Veterinary Science, AAU, Khanapara, Guwahati.
 
Design of experiment
 
For optimization, the full factorial design was utilized. The layout of the design is depicted in Table 1.

Table 1: Experimental layout for determination of maximum OMV yield.


 
Culture and isolation of outer membrane vesicles
 
The 30 ml BHI (brain heart infusion broth) broth was inoculated with 0.6 ml overnight grown culture and incubated at 37°C up to either mid-log phase or peak of growth phase or mid-stationary phase. The culture was then centrifuged at 5000 ×g for 5 minutes at room temperature. The supernatant was discarded and the pellet was re-suspended in 30 ml of fresh BHI medium having pH 6 or 7 with or without oxidative stress. The oxidative stress was generated by adding hydrogen peroxide to a final concentration of 250 µM. Incubation was done for 1 hour and the culture was centrifuged at 10,000 ×g for 10 minutes at room temperature. The OMVs were purified using the method described by Macdonald and Kuehn (2013). The protein concentration of OMVs was estimated using modified Lowry’s method (Lowry et al., 1951). The yield of OMV was expressed in terms of protein concentration. The three conditions were selected that showed the highest yield of OMVs. The OMVs from these three conditions were selected for further characterization in terms of protein profiling by SDS-PAGE (Sambrook and Russel, 2001) as recommended by Klimentova and Stulik, (2015) and lipopolysaccharide concentration and zeta size. The estimation of the lipopolysaccharide (LPS) content of OMVs was done by Kdo method (Sunayana and Reddy, 2015). The zeta size of the outer membrane vesicles were measured using the dyanamic light scattering in Institute of Advanced Study in Science and Technology, Guwahati, Assam.
 
Statistical analysis
 
The three factor ANOVA was carried out to determine the effect of different factors on yield of OMVs. The multiple comparison of means was carried out by Tukey’s HSD test at 5% significance level. Linear regression analysis was carried to assess the influence of different factors on LPS concentration and size of OMVs. All analysis was carried out using statistical software R version 4.0.3 (R Core Team, 2020).

RESULTS AND DISCUSSION

The conditions where the protein yield was found to be the highest are pH 7, peak of growth phase with oxidative stress; pH 7, peak of growth phase without oxidative stress and pH 7, mid-stationary phase with oxidative stress (Fig 1). The 3-factor ANOVA has indicated that all the factors that were taken has significant influence on the yield of OMVs in terms of protein concentration (Table 2). The interaction of factors were found to exist only between the pH of the medium and the phase of the growth. The pH was found to have the highest contribution in the yield of OMVs followed by phase of the growth and presence of oxidative stress. The protein profiling of the OMVs of the three conditions did not yield any significant difference (Fig 2).

Fig 1: Mean (± SEM) yield of outer membrane vesicles per liter of BHI broth in different cultural conditions (as depicted in Table 1). The different letters designation over the bars indicates the statistically significant difference (p<0.05).



Table 2: Three factor ANOVA depicting the influence of the three factors on the yield of outer membrane vesicles.



​

Fig 2: Protein profile of OMV OF Salmonella Typhimurium by SDS-PAGE. Lane M – protein marker, L1, L2, L3 – OMV (pH 7, peak, oxidative stress), L4, L5, L6 – OMV (pH7, peak, without oxidative stress) L7, L8, L9 – OMV (pH7, mid-stationary, oxidative stress).



The LPS was found to be lowest in the condition “G” followed by conditions “J” and “I”. The condition “G” where the media was grown up to peak of growth phase in presence of H2O2 mediated oxidative stress at pH 7, was found to yield minimum LPS concentration and maximum OMV yield (Fig 3). In addition, this condition appears to have yielded the OMVs of significantly lowest mean zeta size of 858.96 ± 83.29 nm (Fig 4). We observed that the oxidative stress and peak of growth phase has significantly negative influence on the LPS concentration and zeta size of OMVs (Table 3 and Table 4).

​

Fig 3: Graphical representation of mean (± SEM) LPS yield from OMV of Salmonella Typhimurium (grown in pH 7.00). The different letters designation over the bars indicates the statistically significant difference (p<0.05).



​

Fig 4: Graphical representation of mean (± SEM) zeta size of OMVs of Salmonella Typhimurium (grown in pH 7.00). The different letters designation over the bars indicates the statistically significant difference (p<0.05).



Table 3: Linear regression model showing the effect of oxidative stress and peak of growth phase on LPS concentration of OMVs (at pH 7.0).



Table 4: Linear regression model showing the effect of oxidative stress and peak of growth phase on zeta size of OMVs (at pH 7.0).



The H2O2 mediated oxidative stress mimics the reactive oxygen burst in intracellular environment of neutrophils in response to bacterial infection that eventually stimulates the bacteria to overproduce the OMVs through quorum sensing (Gerritzen et al., 2018; Macdonald and Kuehn, 2013).

The OMV production occurs in all stages of growth of bacteria but the maximum production of OMV occurs in later part of log phase that is at the peak of growth phase (Chatterjee and Das, 1967; Gamazo and Moriyon, 1987; Hoekstra et al., 1976; Mccaig et al., 2013). The higher yield of OMV may facilitate the cell to cell communication in the cells in the later stage of growth. The large amount of cellular materials are deposited during the log phase in the OMVs that continues till the peak of growth phase thereafter the cell death ensues leading to protease mediated degradation of OMVs  (Gamazo and Moriyon, 1987). The low pH causes damage to the membrane due to alteration in membrane fluidity, integrity and lipid composition (Guan and Liu, 2020), which could be the possible reason of lower yield of OMVs in acidic pH.

The genetic control of LPS synthesis occurs though the environmental stress (Klein and Raina, 2019) leading to reduction in LPS synthesis as observed in our study. The LPS is responsible for febrile and allergic response (Steiner et al., 2006; Williams et al., 2005). It may be assumed that lower concentration of LPS in the OMV-based vaccines may be beneficial in terms of febrile and possible allergic response.

The oxidative stress has significant influence on the size of OMVs produced by the Gram negative bacteria (Mozaheb and Mingeot-Leclercq, 2020). Though the exact mechanism of the influence on stress is not understood however as it is evident in the present study, the size of OMVs are significantly negatively influenced by the presence of oxidative stress which could probably be due to the fact that reduction in size of OMVs invariably increase surface to volume ratio, hence greater surface area of OMVs would probably be helpful to bacteria to capture the larger amount of free radicals. Similar findings were also reported by Baumgarten et al., (2012) in case of Pseudomonas putida. The small size of antigens are known to increase their immunogeniticy that could be due to ease in uptake by macrophages (Jia et al., 2018; Jia et al., 2012) and would be a useful candidate for vaccine development. The three best conditions recognized in the present study did not show any difference in protein profile in SDS-PAGE indicating the change in conditions within pH 7 did not have significant change in the protein contents. Based on the protein profile of the SDS-PAGE, we may conclude that the optimized conditions may not be having any significant alteration in the immune-potential.

CONCLUSION

The present study has indicated that the optimal conditions for OMV production from Salmonella Typhimurium is neutral pH of growth media, presence of oxidative stress mediated by H2O2 and growth of bacteria up to peak of growth phase. The further studies are needed to assess the immune response of the OMV produced by the optimized condition in a laboratory animal model.

ACKNOWLEDGEMENT

The authors are grateful to the DBT, New Delhi for providing financial support to carry out the work. The authors are also grateful to the Dean, Faculty of Veterinary Science, Assam Agricultural University, Khanapara for providing the laboratory facilities, Guwahati Biotech Park for helping in ultracentrifugation and CIF of IASST for the works related to dynamic light scattering.

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