The MIC of AP2 in vitro
The MIC of AP2 and AP IB against ST in vitro was first measured to explore their antibacterial activity. The results showed that the MIC of AP2 against Salmonella was 5 µg mL-1, which was better than that of AP IB with MIC of 10 µg mL-1 and kanamycin (Kan) and streptomycin (Strep) (Table 1).
Table 1
The MIC of antimicrobial peptides.
Strain | AP2 (µg mL-1) | AP IB (µg mL-1) | Kan (µg mL-1) | Strep (µg mL-1) |
ST | 5 | 10 | 12.5 | 32.5 |
ST: Salmonella Typhimurium CMCC 50115. AP IB: apidaecin IB. Kan: kanamycin. Strep: streptomycin.
AP2 attenuated the symptoms of ST infections in vivo
To investigate whether AP2 has a protective effect against ST infection in vivo, C57BL/6 mice were treated with or without AP2 before ST infection. Compared with the control group, none of the animals in the AP2 group showed obvious weight loss (Fig. 1), whereas mice challenged with ST exhibited significant body weight loss at day 2 (ST group) and day 3 (both ST and AP2 + ST groups). However, the body weight loss of mice in AP2 + ST group markedly alleviated at day 3 and day 4 compared with ST group (Fig. 1).
The results of the intestinal histopathology showed that ST infection induced an acute inflammation in the mucosa characterized by the swelling of the lamina propria (Fig. 2a), inflammatory infiltration and desquamation (Fig. 2b) and the shedding of microvilli (Fig. 2c). Furthermore, the ST infection also induced intestinal mitochondria swelling (Fig. 2c), which was significantly ameliorated by AP2 administration (Fig. 2).
The level of serum pro-inflammatory cytokines reflect the intensity of inflammation. Mice in the control mice and AP2-treat mice) groups had similar low levels of serum inflammatory cytokines (IL-1β, and IFN-γ (Fig. 3). While ST inoculation resulted in a significant increase in the serum pro-inflammatory cytokines IL-1β and IFN-γ, which were decreased by AP2 pretreatment (Fig. 3).
ST infection may result in bacterial translocation across the intestinal barrier, followed by migration to the spleen and liver [38]. Glutamic pyruvic transaminase (GPT) is mainly present in the cytoplasm of hepatic cell. When hepatocyte is injured, GPT will release into blood, and thus increasing the serum GPT activity[39]. Therefore, the level of this enzymes in serum could be used to assess the extent of damage[40]. Results showed that GTP activity were significantly increased in the ST group, which was markedly reduced (P < 0.05) in the AP2 + ST group (Fig. 3). These results suggested that AP2 administration ameliorated ST-induced liver damage.
AP2 does not inhibit ST growth in the intestinal tract
Since AP2 conferred protection against ST infection, next we want to verify whether AP2 could inhibit the growth of ST within intestinal tract directly. Since the microbiota confers colonization resistance to block Salmonella gut colonization [41], the streptomycin mouse model was used to remove gut microbial community and exclude its interference, and the amount of ST in feces was monitored every 3 h after treated with AP2 (Fig. 4a). Surprisingly, we found that AP2 treatment did not decrease the ST loads in the feces (Fig. 4b), which is inconsistent with the results obtained from in vitro experiments. Thus, the protective effect of AP2 against ST in vivo was not associated with its bactericidal effect directly.
Several studies have demonstrated that the gut microbiota and its metabolites provide colonization resistance to ST infection [13, 30, 42]. But ST exploits inflammation to compete with this colonization resistance [43–45]. A previous study also found that AMPs beneficially affected the intestinal health by shaping the microbial ecology [46]. This led us to focus our further analyses on the gut microbiota composition.
AP2 treatment modified the gut microbiota composition
Since AP2 exhibited strong antibacterial capacity, which may influence the composition of the microbiome, a 16S rRNA-based analysis was used to determine the microbiota from the cecum content. Results showed that there were no significant differences in alpha diversity of the microbiota as reflected by Shannon, Chao1, Faith’s phylogenetic, and observed species indexes among control, AP2, ST and AP2 + ST groups (Supplemental Fig. S1). However, beta diversity assessment with weighted UniFrac distance revealed that the microbial community significantly different (ANOSIM, p < 0.05) between control and AP2 groups and between AP2 + ST and ST groups both at the phylum (Fig. 5a) and the genus (Fig. 5b) level.
AP2 treatment did not significantly change the major microbial composition at the phylum level (Fig. 5c), but significantly increased the proportion of Actinobacteria, Alcaligenaceae, Allobaculum, Bifidobacteriales, Betaproteobacteria, Burkholderiales, Clostridiaceae, Lachnospiraxeae, Mogibacteriaceae and Sutterella, and decreased the proportion of Coprobacillus and Verrucomicrobia compared with control group. Meanwhile, the proportion of Alcaligenaceae, Betaproteobacteria, Burkholderiales, and Sutterella in the AP2 + ST mice were significantly higher than those in ST mice. Notably AP2 treatment also decreased the proportion of Verrucomicrobia independent of ST-inoculation. (Supplemental Fig. S2).
The linear discriminant analysis effect size (LEfSe) [35] was used to identify specific OTUs that differed between the control and AP2 with or without ST inoculation. 17 discriminative features (LDA score > 2) whose relative abundances varied significantly between the control and AP2 groups was identified. Furthermore, 11 bacterial taxa, such as Actinobacteria, Bifidobacterium, Allobaculum, and Sutterella were enriched in the AP2 group, while 6 bacterial taxa were increased in the control group, such as Verrucomicrobia, Coprobacillus, and Akkermansia. Based on the LEfSe, 17 bacterial taxa were significantly more abundant in the AP2 + ST group (e.g. Prevotella, AF12, Dehalobacterium, Oscillospira, Coprobacillus, Sutterella, Bilophila, and Desulfovibrio; p < 0.05), while only 9 taxa were overrepresented in the ST group (e.g. Verrucomicrobia, Clostridium, Coprococcus, and Akkermansia; p < 0.05) (Fig. 5d). Collectively, these data suggested that the gut microbiota modified by AP2 may be associated with its protective effect against ST infection in mice.
Fecal microbiota transplant (FMT) of AP2-treated mice influences the course of ST-induced caecal inflammation in mice
To further verify the hypothesis above, we performed FMT experiment (Fig. 6a). The mice that were pre-treated with streptomycin [30], received microbiotas from the control or AP2-treated mice through anorectal inoculation, respectively. 4 h later, mice were infected with ST by oral gavage to evaluate the effects of the AP2-treated microbiota on ST infection (Fig. 6a). Results showed that AP2-treated microbiota could significantly decrease levels of ST in the stool and MLN (Fig. 6b), and also decreased intestinal pathology (Fig. 7), providing evidence that this alerted microbial community induced by AP2 treatment was effective against ST infection. These findings suggest that the beneficial effect of AP2 treatment on the course of infection was transferable via FMT.