title: Neutrophil responses to Vibrio cholerae autoinducer-1 and structural analogues
Creators: name: Lee, Jason J
Publisher: name: Griffith University
Year: 2021
Subjects: subject: Vibrio cholerae
subject: pathogen
subject: cholera
subject: V. cholerae autoinducer-1
subject: immunomodulatory effects

description: Vibrio cholerae is a pathogen responsible for cholera, an infectious disease that usually manifests as severe diarrhea. V. cholerae cells can regulate population-wide gene expression changes in a density-dependent manner, in a process known as quorum sensing (QS). QS involves communication between bacterial cells using secreted signalling molecules. V. cholerae autoinducer-1 (CAI-1) is the dominant signalling molecule in the V. cholerae QS circuit and has roles in regulating biofilm formation/degradation and expression of virulence genes. Interactions between bacterial-produced QS molecules and eukaryotic cells have been documented. This is known as interkingdom or cross-kingdom signalling. CAI-1 has been reported to act as a chemoattractant for the nematode, Caenorhabditis elegans which feeds on V. cholerae cells as a food source. Legionella autoinducer-1 (LAI-1), which is structurally similar to CAI-1, is a signalling molecule produced by Legionella pneumophila. LAI-1 has been reported to impede the migration of Dictyostelium discoideum amoebae and macrophage-like RAW 264.7 cells, and has also been shown to destabilise the cytoskeleton of RAW 264.7 cells. Structural analogues of CAI-1 with more potent activity within the V. cholerae QS circuit have been developed as potential novel therapeutics against cholera. These QS agonists would force the bacterial cells to express high cell density behaviours, impairing colonisation and promoting detachment, therefore reducing pathogenesis. These previous findings led to the hypothesis that CAI-1 and structural analogues may have immunomodulatory effects on host cells during V. cholerae infection, particularly recruited immune cells which may be exposed to CAI-1 during cholera. There are several lines of evidence the neutrophil recruitment is prominent during cholera and that these granular leukocytes play a role in controlling infection. Thus, the key aims of this study were to characterise interactions between CAI-1, as well as structural analogues of CAI-1, and neutrophils. In vitro HL-60 cell culture revealed an upregulation of CD11b expression when cells were differentiated with DMSO in the presence of CAI-1. This increased differentiation marker expression was at the expense of both cell viability and total cell count. Additionally, two 3-acyl pyrrole analogues of CAI-1 also increased CD11b expression greater than CAI-1, when cells were differentiated with DMSO in the presence of either analogue. Again, this resulted in significantly reduced cell viability and total cell count, although at similar levels to CAI-1. HL-60 cells differentiated in the presence of CAI-1 or either analogue were generally more granular than cells differentiated with DMSO alone. The effects of CAI-1 and structural analogues on neutrophil effector functions were assessed, namely chemotaxis and oxidative burst. CAI-1 did not act as a chemoattractant for DMSO-differentiated HL-60 cells, nor did it reduce or enhance migration towards fMLP, a known chemoattractant. Pre-treatment of differentiated HL-60 cells with CAI-1 or one of the 3-acyl pyrrole analogues for 3 h resulted in decreased reactive oxygen species production. However, concomitant reduction in cell viability was observed over 3 h. Preliminary experiments assessed the effect of CAI-1 on primary human neutrophils. Isolated neutrophils appeared larger and less round with CAI-1 treatment. In contrast, CAI-1 treatment of whole blood resulted in apparent reductions in cell size as assessed by flow cytometry. Expression of activation markers (CD11b, CD64, CD66b) on granulocytes in whole blood appeared unaffected by CAI-1. Overall, the results within this study shed light onto the cross-kingdom interactions that may exist between host cells and bacterial signalling molecules. Identifying these interactions may lead to a deeper understanding of additional mechanisms that may be involved in V. cholerae pathogenesis. Additionally, these interactions may be important in revealing off-target effects that developing novel therapies, which interfere with bacterial QS, may have on host cells. These data highlight an advance that reveals many opportunities for further investigations surrounding host-microbe interactions.