Optimisation of External Factors for the Growth of Francisella novicida within Dictyostelium discoideum

The amoeba Dictyostelium discoideum has been used as a model organism to study host-pathogen interaction in many intracellular bacteria. Francisella tularensis is a Gram-negative, highly infectious bacterium that causes the zoonotic disease tularemia. The bacterium is able to replicate in different phagocytic and nonphagocytic cells including mammalian, amoebae, and arthropod cells. The aim of this study was to determine the optimal temperature and infection dose in the interaction of Francisella novicida with D. discoideum in order to establish a model of Francisella infection in the social amoeba. The amoeba cells were infected with a different multiplicity of infection (5, 10, and 100) and incubated at different temperatures (22, 25, 27, 30, and 37°C). The number of intracellular bacteria within D. discoideum, as well as cytotoxicity, was determined at 2, 4, 24, 48, and 72 hours after infection. Our results showed that the optimal temperature for Francisella intracellular replication within amoeba is 30°C with the MOI of 10. We can conclude that this MOI and temperature induced the optimal growth of bacteria in Dictyostelium with low cytotoxicity.

Francisella is known as a fastidious organism with many growth requirements in vitro. e bacterium is difficult to culture and grows slowly at 37°C, requiring enriched growth medium [8]. Each species of the genus Francisella require different growth and medium conditions. Francisella requires medium supplemented with cysteine for cultivation. e bacterium grows on Buffered Charcoal-Yeast Extract Agar (BCYE), Chocolate Agar, ayer-Martin Agar, and on the media containing hemoglobin, such as Cysteine Heart Agar Base (CHAB). Bacterial growth on blood agar is very slow with a narrow zone of alpha-hemolysis. Francisella can also be cultivated in a liquid medium; however it shows a lower replication rate. For example, the most used is Mueller-Hinton medium (MHM) in which bacteria efficiently replicates after incubation of 7 to 10 days. Francisella species can be incubated with 5% CO 2 , but it only enhances the growth of F. tularensis subsp. holarctica LVS (Live Vaccine Strain). F. novicida, F. noatunensis, and F. philomiragia require less nutritive supplements in the medium for in vitro growth, possibly due to their adaptation to environmental conditions [8,9]. Further, the optimal temperature for the growth of F. tularensis is 37°C. F. tularensis grows slowly at room temperature, in contrast to F. novicida and F. philomiragia, which survive at 25°C. Because of the complex growth requirement of Francisella, most of the human tularemia cases are misdiagnosed by serology or clinical features [10,11]. Importantly, previous studies demonstrated the impact of temperature and growth medium on the virulence of bacteria and vaccine design [12]. e exact reservoir of Francisella spp. in the environment has not been definitely determined. However, bacteria have been strongly associated with water environments [13]. It is assumed that the bacterial existence in the aquatic environment is connected with the ability of Francisella to survive and replicate within amoeba cells [14]. Our and other previous in vitro studies showed the survival and replication of F. novicida in H. vermiformis and A. castellanii, showing the possible importance of protozoa in Francisella ecology [15][16][17].
However, due to limited tools and antigens to study intracellular lifestyle in free-living amoeba, such as A. castellanii and H. vermiformis, D. discoideum has been used as a model organism to study phagocytosis, cell motility, and virulence factors for many bacterial pathogens, such as Pseudomonas, Legionella, Mycobacterium, Salmonella, and Klebsiella [18][19][20][21][22][23]. Dictyostelium has been established as a model organism for studying the life cycle of the fish pathogen, F. noatunensis, but it has not been established for studying the strains of Francisella that cause the disease in humans [24][25][26]. It has been shown that Salmonella requires O-antigen and the type VI secretion system (T6SS) for the survival within amoebae [22]. Francisella uses the T6SS to avoid lysosomal fusion within the macrophages [27]. After establishing this model of F. novicida infection in Dictyostelium it would be interesting to investigate the role of T6SS in this social amoeba for prolonged survival of Francisella in nature.
Although the previous studies demonstrated the survival and replication of Francisella within macrophages and various cell types, little is known about the adaptation of Francisella to protozoa cells [28,29]. Our study was focused on establishing the optimal external factors required for survival and replication of F. novicida within D. discoideum. We examined the role of various incubation temperatures and the dose of infection on F. novicida capability to survive and replicate within D. discoideum.

Cultivation of the Bacteria at Different Temperatures.
To determine the survival of F. novicida in vitro at different growing temperatures, the bacterial suspensions (10 3 CFU·mL − 1 ) were inoculated in tubes with 50 mL of the buffered yeast extract (BYE) broth (Sigma, USA). e suspensions were incubated at 22, 25, 27, 30, 37°C, and 42°C for 5 days. Every 24 hours, the number of F. novicida was determined by plating the serial dilutions on BCYE agar at 37°C.

Infection of Cells.
e number of D. discoideum (10 5 amoebae·mL − 1 ) was counted using a Neubauer chamber ( ermo Fisher Scientific, USA). e cells were seeded in 96well plates, incubated overnight and infected with F. novicida at a multiplicity of infection (MOI) 5, 10, or 100. For infection, the HL5 medium was diluted with a phosphate buffer (1 : 1). In order to achieve synchronized infection, the cells were centrifuged immediately after infection at 240 g for 3 minutes at room temperature. e cells were then incubated at 27°C for 1 hour and washed 3 times with PBS to remove extracellular bacteria. is was considered as a time point zero. At each time point after infection (2,4,24,48, and 72 h), the cells were treated with 0.9% Triton X-100 (Sigma, USA) for 10 minutes to lyse the cells. e number of intracellular bacteria was determined by plating the serial dilutions on BCYE agar.

Growth of Bacteria within Amoeba at Different
Temperatures. To determine the influence of different incubation temperatures on the growth of bacteria within amoeba cells, the cells were infected with MOI 10 as described previously. e infected cells were incubated at 22, 25, 27, 30, or 37°C and the number of intracellular bacteria was determined by plating the serial dilutions on BCYE agar.

Prolonged Survival of Bacteria In Vitro Is Temperature Dependent.
e effect of different incubation temperatures on the survival of F. novicida in vitro was examined. e bacterial suspensions were incubated at temperatures of 22°C, 25°C, 27°C, 30°C, 37°C, or 42°C for 5 days. By every 24 hours, the number of F. novicida was determined by plating the serial dilutions on BCYE agar at 37°C.
Our results show that F. novicida replicates with prolonged time of incubation at 22, 25, 27, 30, and 37°C. By day 5, the number of bacteria increased up to 8.0 × 10 5 CFU·mL − 1 after incubation at 25°C. At 37°C the number of bacteria increased up to 1.5 × 10 5 CFU·mL − 1 , and at 22°C up to 8.0 × 10 6 CFU·mL − 1 , while the incubation temperatures of 27 and 30°C resulted in a higher replication of bacteria, 2.0 × 10 9 CFU·mL − 1 and 5.0 × 10 12 CFU·mL − 1 , respectively. In contrast, when the incubation temperature was 42°C, F. novicida was not able to survive after the second day of incubation (Figure 1).

Bacterial Growth in Amoeba after
We can conclude that the infection of amoeba cells with MOI 100 induced the highest intracellular growth of F. novicida in Dictyostelium. e number of intracellular bacteria within D. discoideum increased with higher MOI and prolonged incubation times.   We can conclude that the increase of the LDH activity in the culture supernatant of the infected amoeba cells is proportional to the intracellular growth of bacteria within D. discoideum. Our results show that the MOI 10 induces the lowest level of cytotoxicity in Dictyostelium, so it will be used in our further experiments.

e Influence of Incubation Temperature on the Bacterial Growth within Amoeba.
e influence of different incubation temperatures on the F. novicida growth within D. discoideum was also examined. Based on our previous results, the optimal dose of infection for the linear growth of bacteria within amoeba with low cytotoxicity is MOI of 10. erefore, the amoeba cells were infected with MOI 10 and incubated at different temperatures (22°C, 25°C, 27°C, 30°C, and 37°C).
At all observed temperatures, F. novicida showed similar intracellular growth at 2 and 4 hours after infection and the bacterial number increased up to 10 4 CFU·mL − 1 . e bacterial cells continued to replicate within amoeba to 24 hours after infection. At 24 h after infection, the highest number of bacteria was determined at a temperature of 30°C (10 8 CFU·mL − 1 ) (Figure 4). In contrast, the lowest number of intracellular bacteria was observed 24 hours after infection at the incubation temperature of 22°C (10 5 CFU·mL − 1 ). At 72 hours after infection, the highest number of bacteria was determined after incubation on the temperature of 27 and 30°C (Figure 4).
Our results clearly show that the temperature of 30°C is an optimal temperature for Francisella intracellular growth within amoeba. In comparison with optimal temperature, the number of intracellular bacteria was statistically different at 24 hours after infection at 22°C (p � 0.001), 25°C (p � 0.001), 27°C (p � 0.002), and 37°C (0.001). Also, the statistically different number of bacteria was found at 48 hours after infection at 22°C (p � 0.001), 25°C (p � 0.001), 27°C (p � 0.002), and 37°C (0.002). At 72 hours after infection, only the number of intercellular bacteria observed

Discussion
Numerous studies have shown that the interaction between free-living amoeba and human pathogens, such as F. tularensis, Legionella pneumophila, and Mycobacterium spp., has significant implication in their environmental persistence. It was also shown that after growing within the amoeba, nonpathogenic bacteria gained their pathogenic features and that bacteria become more resistant to antibiotics [30][31][32][33]. In addition, many studies described amoeba like "Trojan horse" due to its protective role for bacteria in unfavorable environmental conditions and a role in the transmission of intracellular pathogens to humans [34,35]. Many factors may play a role in the interaction of free-living amoeba and bacteria, such as incubation temperature and bacterial concentration. Francisella tularensis is a unique pathogen that can survive different environmental conditions and animal reservoirs. To be able to understand this wide range of hosts and the diversity of virulence strategies, there is a need for establishing more models for studying mechanisms required for infecting diverse hosts. is was successfully accomplished using D. discoideum as a host model to study the pathogenesis of Klebsiella, Legionella, Pseudomonas, and Salmonella infections [21][22][23]36]. e effect of incubation temperature on the replication of Gram-negative bacteria in vitro was previously discussed in many researches. Although previous studies showed that the optimal temperature for studying the host-pathogen interaction within Dictyostelium cells is 21-23°C [21][22][23]36], Francisella shows a little bit different behavior in comparison to other bacterial pathogens. e optimal growth of bacteria and D. discoideum was achieved at temperature 27-30°C. At lower temperatures, the growth of bacteria was significantly lower. Giving the concern that lower temperate may influence the expression of virulence traits, probably F. novicida somehow needs higher incubation temperature to be able to successfully replicate in this host. Our data show that F. novicida does not survive at 42°C, but it grows at 25 and 37°C. However, previous studies showed that subspecies holartica is more tolerant to a higher temperature (42°C) [8]. Each species of the genus Francisella requires different incubation temperatures. Various researches indicated that the findings of incubation temperature might improve the understanding of pathogenicity potential of the pathogens with the fastidious nature, such as L. pneumophila [37].
In addition, incubation temperature is crucial for the interaction between bacteria and amoeba. For the first time, in this study, we investigated the influence of incubation temperature on the replication of F. novicida within D. discoideum.
e growth of bacteria within amoeba was temperature-dependent. Higher survival of Francisella within Dictyostelium was noted at a higher temperature (30°C) at the dose of infection 10. Due to F. novicida adaptation to environmental conditions, bacteria also have the ability to survive within amoeba at lower temperatures (22°C). In contrast, studies showed that Legionella pneumophila is unable to replicate intracellularly at room temperature, while it grew significantly within amoeba at 30, 32, and 37°C [38,39]. Since it was shown that the growth of Gram-negative bacteria within amoeba depends on incubation temperatures, understanding the temperature dependence of F. novicida growth within amoeba will result in a better understanding of F. novicida ecological niches.

Conclusion
Our results demonstrate that the higher MOI (100) induce higher replication of F. novicida within amoeba with a high level of cytotoxicity. e optimal dose of infection is 10 at the incubation temperature of 30°C since it induces linear growth of F. novicida with low cytotoxicity in Dictyostelium.
Altogether, the survival and replication of F. novicida within amoeba depend on temperature and bacterial concentration. Since the free-living amoeba may be a reservoir for bacterial pathogens, growth factors in the interaction between bacteria and amoeba need to be clarified in future research for different Francisella subspecies.

Data Availability
e Graph Pad data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.