Modulation of haemocyte motility by chemical and biological stresses in Mytilus edulis and Dreissena polymorpha.

Mussels are constantly exposed to various pollutants in the environment, which can impair their immune defences against microbes and thus threaten their survival. In this study, we expand the insight into a key parameter of immune response in two mussel species by exploring the impact of exposure to pollutants or bacteria or simultaneous chemical and biological exposure on haemocyte motility. Basal haemocyte velocity in primary culture was high and increasing over time in Mytilus edulis (mean cell speed of 2.32 µm/min ± 1.57) whereas Dreissena polymorpha showed a constant and rather low cell motility with time (mean cell speed of 0.59 µm/min ± 0.1). In the presence of bacteria, the motility of haemocytes was instantly enhanced and slowed down after 90 min for M. edulis. In contrast, in vitro exposure of haemocytes to chemicals, either Bisphenol A, oestradiol, copper, or caffeine, induced an inhibition of cell motility in both mussel species. Finally, the cellular activation observed during bacterial challenges was inhibited by simultaneous exposure to bacteria and pollutants. Overall, our results indicate that chemical contaminants can alter haemocyte migration in mussels which can weaken their response to pathogens and therefore increase their susceptibility to infectious diseases.

Comparative immune responses of blue mussel and zebra mussel haemocytes to simultaneous chemical and bacterial exposure.

Biomonitoring at the scale of the aquatic continuum and based on biomarkers, requires various representative species and a knowledge of their sensitivity to contaminants. Mussel immunomarkers are established tools for evaluating immunotoxic stress, but little is known about the consequences of an immune activation by local microorganisms on their response to pollution. This study aims to compare the sensitivity of cellular immunomarkers in two mussel species from different environments, the marine mussel Mytilus edulis (blue mussel) and the freshwater mussel Dreissena polymorpha (zebra mussel), to chemical stressors combined with bacterial challenge. Haemocytes were exposed ex vivo to the contaminants (bisphenol A, caffeine, copper chloride, oestradiol, ionomycin) for 4 h. The chemical exposures were coupled with simultaneous bacterial challenges (Vibrio splendidus and Pseudomonas fluorescens) to trigger activation of the immune response. Cellular mortality, phagocytosis efficiency and phagocytosis avidity were then measured by flow cytometry. The two mussel species had different basal levels since D. polymorpha showed higher cell mortality than M. edulis (23.9 ± 11% and 5.5 ± 3% dead cells respectively), and lower phagocytosis efficiency (52.6 ± 12% and 62.2 ± 9%), but similar phagocytosis avidity (17.4 ± 5 and 13.4 ± 4 internalised beads). Both bacterial strains led to an increase in cellular mortality (+8.4% dead cells in D. polymorpha, +4.9% in M. edulis), as well an activation of phagocytosis (+9.2% of efficient cells in D. polymorpha, +6.2% efficient cells and +3 internalised beads per cell in M. edulis). All chemicals triggered an increase in haemocyte mortality and/or phagocytotic modulations, except for bisphenol A. The two species differed in the amplitude of their response. The addition of a bacterial challenge significantly altered cell responses to chemicals with synergetic and antagonistic variations compared to a single exposure, depending on the compound used and the mussel species. This work highlights the species-specific sensitivity of mussel immunomarkers to contaminants, with or without bacterial challenge, and the necessity of considering the presence of in natura non-pathogenic microorganisms for future in situ applications of immunomarkers.

Pleiotropic effects of rfa-gene mutations on Escherichia coli envelope properties.

Mutations in the rfa operon leading to severely truncated lipopolysaccharide (LPS) structures are associated with pleiotropic effects on bacterial cells, which in turn generates a complex phenotype termed deep-rough. Literature reports distinct behavior of these mutants in terms of susceptibility to bacteriophages and to several antibacterial substances. There is so far a critical lack of understanding of such peculiar structure-reactivity relationships mainly due to a paucity of thorough biophysical and biochemical characterizations of the surfaces of these mutants. In the current study, the biophysicochemical features of the envelopes of Escherichia coli deep-rough mutants are identified from the molecular to the single cell and population levels using a suite of complementary techniques, namely microelectrophoresis, Atomic Force Microscopy (AFM) and Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) for quantitative proteomics. Electrokinetic, nanomechanical and proteomic analyses evidence enhanced mutant membrane destabilization/permeability, and differentiated abundances of outer membrane proteins involved in the susceptibility phenotypes of LPS-truncated mutants towards bacteriophages, antimicrobial peptides and hydrophobic antibiotics. In particular, inner-core LPS altered mutants exhibit the most pronounced heterogeneity in the spatial distribution of their Young modulus and stiffness, which is symptomatic of deep damages on cell envelope likely to mediate phage infection process and antibiotic action.