To examine QS-1 effects on APEC CE129, the strain was complemented with the Y. enterocolitica derived yenI gene to endogenously produce AHLs(Atkinson et al. 2006). Additionally, the AHL receptor, sidA, was deleted in CE129 to form CE129△sidA, which lacks AHL detection ability. Construction of recombinant strains was confirmed by pSB401 bioluminescence and fluorescence RT-qPCR assays (Fig. 1). Both exogenous and endogenous AHL did not influence the growth curves of CE129, ensuring the feasibility of subsequent assays.
Changes in the possibly survival ability of CE129 under AHL were subsequently discussed, based on recombinant strains. The serum is a complement-sufficient bactericidal environment(Palaniyandi et al. 2013), and serum resistance in APEC is linked to different genes, such as iss. Both exogenous and endogenous AHL did not participate in the regulation of serum resistance or iss expression in CE129.
We previously found that EHEC O157/H7 86 − 24 and ETEC F18 107/86, which endogenously synthesize AHL, exhibited significantly enhanced AR responses(Yang et al. 2018). Robust AR is a classic feature of enterobacterium strains, by which strains are able to pass through acidic stomach environments and play pathogenic roles at low doses(Van Houdt et al. 2006). The results of this study were consistent with the findings of other papers that discuss the relationship between QS and the AR system in enteropathogenic E. coli. Moreover, it has not been clarified whether QS in APEC also affects acid-tolerant systems. Interestingly, the phenomenon that E. coli AR is regulated by AHL was not observed in APEC CE129, which differed considerably from ETEC and EHEC (Fig. 2A). To clarify whether a difference exists between APEC and enteropathogenic E. coli strains, or determine if the difference is derived from special genetic characteristics of CE129 itself, three APEC strains were used for the AR test with APEC under AHL influence. The three strains belong to the three dominant serotypes of APEC strains in most poultry farms in China (O78 serotype strain CE2, O1 serotype strain CE11, and O2 serotype strain CE12). In all four APEC strains, AR did not obviously change under 100 µM exogenous C6HSL, while more viable bacteria were observed in EHEC O157/H7 86 − 24 and ETEC 107/86 under acidic pressure, which increased by 3.1- and 2.6-fold, respectively (Fig. 2B). These results were consistent with the gadA mRNA expression experiments (Fig. 2C). gadA is one of the key genes by which E. coli exercises AR effects and has been reported to participate in the QS-induced AR upregulation of ETEC and EHEC. APEC infects the avian respiratory tract and first colonizes the air sacs and has different infection routes for EHEC and ETEC. It is not necessary to survive in the acidic environment of the digestive tract; therefore, its AR may exhibit different AHL regulation characteristics compared to enteropathogenic E. coli.
The growth curve assay revealed that there were no differences in the growth conditions between wild type CE129 and the recombinant strain. However, the growth curve assay was conducted using individual cultivation conditions and the assumption that the APEC strain may possess a competitive growth advantage under AHL needed to be verified. Because CE129△sdiA cannot perceive AHL signals, competitive viability assays between two different strains were targeted. The same amount of CE129△sdiA was added to CE129/pyenI, CE129/pBR, and CE129/pBR with 100 µM C6HSL for co-incubation. After 9 hrs, CE129/pyenI exhibited a clear disadvantage and decreased by 30% in the competitive environment, whereas CE129△sdiA and CE129/pyenI co-incubated, indicating that endogenous AHL inhibited the competitive growth of APEC. Additionally, CE129/pBR also exhibited a disadvantage and decreased by 28% in the competitive environment, whereas CE129△sdiA and CE129/ pBR co-incubated with 100 µm C6HSL, indicating that exogenous AHL also induced competitive growth disadvantages. AHL stimulation may activate some potential pathways of the APEC strain and consume energy required for its growth, resulting in a competitive growth disadvantage. This growth disadvantage is reflected in the planktonic and extracellular growth states of APEC, which may have possible implications in the survival ability of bacteria in a growth or biofilm state.
In the process of APEC infection in avian respiratory tracts, macrophages play an important antibacterial role with leukocytes. Whether APEC can evade or resist macrophages is crucial for APEC virulence. In the engulfment and survival assays, an 83% increase in survival was observed for CE129/pyenI compared to wild type and CE129/pBR (Fig. 2E), indicating that endogenous AHL upregulated the survival ability of CE129 in chicken macrophage cells. Bacteria need to evolve to evade and resist the killing activity of macrophages, which is important for its virulence. When CE129 is co-infected with AHL-positive strains in the host, pathways activated by AHL may weaken the competitive growth of extracellular bacteria. Additionally, it may also upregulate the intracellular viability of bacteria, obtaining an intracellular survival advantage from quorum eavesdropping.
In the DF-1 adherence assay, adherence of CE129/pyenI increased by 66% compared to CE129 and CE129/pBR (Fig. 3A). Subsequently, this study attempted to target the main adhesin that mediates this change in adherence under the influence of AHL. Flagella and type I fimbriae are two important adhesion factors in APEC. Therefore, motility assays were employed to explore whether flagella expression levels changed (Fig. 3B). RT-qPCR was also employed to examine the mRNA expression levels of the flagella gene, fliC, and the type I fimbriae gene, fimA (Fig. 3C). Results revealed that the increase in adherence did not come from fliC or type I fimbriae, indicating that there may be other adhesins being upregulated by AHL, which requires further investigation. Flagella are important bacterial virulence factors that provide bacterial motility and contribute to the bacterial colonization of host cells and penetration of the mucosal layer. It has been proposed that flagella allow enteric bacteria to exploit inflammation to compete with intestinal microbiota in vivo (Stecher et al. 2004; Stecher et al. 2008; Duan et al. 2013; Yang et al. 2013). Previous studies revealed a close relationship between QS and E. coli flagella expression, in which flagella expression is inhibited by AHL in several E. coli strains. The flagella expression of three APEC strains was therefore examined, and similar results were obtained (Fig. 3D), indicating that AHL regulated flagella expression in APEC is considerably different from enteropathogenic E. coli. This phenomenon may be derived from the infection route differences between APEC and enteropathogenic E. coli strains; however, the mechanism remains to be elucidated.
Biofilms are communities of microorganisms that have attached to a solid surface through extracellular polymeric substances and are related to many essential virulence factors that contribute to colonization, immune escape, and antibiotic resistance(Yang et al. 2013; Yang et al. 2014). AHL elicited CE129 disadvantages in the planktonic growth state but it brought advantages in the intracellular growth state, CE129 biofilm formation under AHL influence was subsequently examined. Results revealed that both exogenous and endogenous AHL suppressed biofilm formation in polystyrene 96-well plates (Fig. 4A) and glass surfaces (Fig. 4B).
In conclusion, this study demonstrated that AHL results in extracellular growth disadvantages in APEC CE129 in both a planktonic and biofilm state. AHL also upregulated the intracellular survival ability of CE129 in macrophage cells. AHL exerts an advantage on CE129 adherence, which is the first step in APEC infection and plays a key role in its virulence. Thus, for the first time, this study uncovered the differences between APEC and enteropathogenic E. coli strains in terms of AHL function upon flagella expression and AR. Earlier studies found that AHL inhibited the expression of E. coli flagella and increased AR, but similar phenotypes did not exist in APEC. This difference may be derived from the different infection routes of APEC and enteropathogenic E. coli; however, the mechanism requires further investigation. This study also provides a new perspective on the function of QS-1 in the regulation of APEC strains.