Outer membrane proteins are essential for maintaining the integrity and selective permeability of bacterial membranes . They also play an important role in antibiotic resistance . Due to its cellular location, OM is highly sensitive to environmental conditions prevailing in the extra-cellular medium. This is important for bacterial pathogenesis because it enhances the adaptability of pathogens to various environments [20, 23]. We have previously reported a SMF effect on cell viability and gene expression of S. Hadar ; we now address the question of the SMF impact on the OMPs pattern of this pathogen.
Impact of SMF on cell envelope integrity. The peptidoglycan-associated lipoprotein (Pal) and the TolB protein were found to be up-regulated after SMF exposure. TolB, a periplasmic protein, is partially associated with the outer membrane through a specific interaction with Pal . The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane . Two possible functions have emerged for the Tol/Pal system: its involvement in cell envelope biogenesis and/or as a tether in cell division that maintains the appropriate juxtaposition of the two membranes relative to the peptidoglycan layer . It could be hypothesized that the SMF increased the expression of the system involving the TolB and the peptidoglycan-associated lipoprotein Pal in order to maintain the envelope integrity and thus cell division. This is confirmed by a renewal of the cell viability observed following 9 h of SMF exposure . These data suggest a contribution of TolB and Pal proteins in mechanisms of defense in order to overcome the stress effects of the SMF.
Proteins involved in oxidative stress. Three overexpressed proteins may be related to the oxidative stress response. The OmpW protein belongs to a family of small OMPs involved in the transport of small hydrophobic molecules across the outer membrane . It also may be involved in the protection of bacteria against various forms of environmental stress. Recent studies have indicated that its expression is activated in response to oxidative stress . The up-regulated dihydrolipoamide dehydrogenase (Lpd) is a component of the pyruvate dehydrogenase complex (PDC) that connects glycolysis and tricarboxylic acid cycle enzymes. PDC carries out the conversion of pyruvate to acetyl-CoA. Haramaki et al. (1997)  suggested an antioxidant function for Lpd, related to the role of free lipoic acid as an antioxidant. The up-regulated STY1416 protein, identified as the universal stress protein UspF, has been described to confer resistance against oxidative stress. It has, however, a minor role compared to other members of the Usp family . Therefore, as shown in eukaryotes [2–4, 31], SMF exposure might induce an oxidative stress in Salmonella. The down-regulation of bacterioferritin in cells under magnetic field exposure may lead to a decrease of iron storage and thus to the impairment of iron detoxification. This observation suggests a contribution of the bacterioferritin down-regulation in the oxidative stress status, a correlation being established between iron homeostasis involving bacterioferritin and the oxidative stress response [32–34].
Impact of SMF on the Salmonella virulence. Two proteins involved in virulence, OmpX and YfgL, were down-regulated under SMF. The integral OmpX belongs to a family of virulence-related membrane proteins which promotes mammalian cell adhesion and invasion and helps to defend against the human complement system . This small channel could also play a role in the antibiotic resistance as it is up-regulated in conditions where the Omp35 and Omp36 major porins in Enterobacter aerogenes have a decreased expression . However, we did not detect an increasing expression of the Salmonella major porins, as we could expect concomitantly to the under-expression of OmpX. In other respects, the outer membrane lipoprotein YfgL is involved in virulence of Salmonella as it plays a crucial role in the regulation of the expression of all Salmonella TTSS systems (Type Three Secretion Systems) that occurs during host infection. Indeed, three TTSS (TTSS-1, TTSS-2, and flagella) allow the bacterium to cross the intestinal barrier and to disseminate systemically . The deletion of yfgL gene in S. enterica serovar Enteritidis led to the transcriptional down-regulation of the flagellar genes encoding the TTSS structural proteins and of the effector proteins secreted by these TTSS . This down-expression of both ompX and YfgL suggests a possible impairment of the virulence under a SMF exposure (200 mT, 9 h).
Impact of SMF on the Salmonella motility. For a lot of pathogens, virulence and motility are often intimately linked by complex regulatory networks . Thus, we observed a down-regulation of the hook flagellar protein FlgE by exposed cells. In accordance with this result, SMF-exposed cells exhibited a decrease in the swarming motility, a flagella-driven movement. The flagellar hook, a constituent of the bacterial motile flagellum, is a short connection between the flagellar motor and the long filament acting as a helical propeller. It is made of about 120 copies of a single protein, FlgE, and its function is essential for dynamic and efficient bacterial motility and taxis . Confirming this bacterial motility decrease was the up-regulation of the STY1416 protein, which was shown to exhibit a negative effect on bacterial motility .
Effects of SMF on antibiotic susceptibility. As pointed out by proteomic data, SMF exposure induced an overexpression of the TolC protein. This protein is the outer membrane component of the efflux pump AcrAB-TolC, an efflux system known to play an important role in the multidrug resistance in Salmonella . TolC is required for the functioning of seven drug efflux systems in S. enterica serovar Typhimurium . In this context, we compared the susceptibility of S. Hadar to different antibiotics before and after SMF exposure. Our data showed no influence of SMF on antibiotic susceptibility, except for gentamicin that underwent a significant decrease of its effectiveness and for ticarcillin + clavulanic acid mixture that acts in an unexpected increasing manner (Figure 2). Efflux pumps have already been described as involved in the resistance to aminoglycoside in Pseudomonas aeruginosa . These pumps usually play an essential role in export of different structurally unrelated substrates ranging from antimicrobials, heavy metals, and detergents to large toxins . If we cannot exclude a role of TolC accumulation in the decrease of susceptibility of exposed cells to gentamicin, it was actually not sufficient to promote an effect on other antibiotic susceptibility. We can consequently imagine that the decrease of susceptibility to gentamicin by SMF exposure might also result from other mechanisms. Indeed, gentamicin is a cationic antibiotic which binds reversibly to anionic sites of the bacterial cell membrane dependent on concentration . Its binding to anionic phospholipids (such as cardiolipids) of the OM facilitates the rupture of this membrane . The membrane lipid composition and modification, especially cardiolipid's proportion, should therefore be examined after SMF exposure.
Electromagnetic fields have been hypothesized to affect the membrane permeability through modifications on channel-forming proteins . Since we did not detect any differential expression of the OmpC and OmpF porins, we could hypothesize that the SMF may alter kinetics of beta-lactamase inhibition by clavulanic acid, thus reducing beta-lactamase activity.
Accumulation of major outer membrane protein OmpA. We observed the up-regulation of OmpA by SMF exposed cells. This OMP is a multifaceted protein which can function as an adhesin and invasin, participate in biofilm formation, act as both an immune target and evasin, and serve as a receptor for several bacteriophages . The structure of OmpA has given many insights into the possible porous nature of this protein. However, the definition of molecules that utilize this channel into the cell has not been resolved . OmpA is the predominant cell surface antigen in Enterobacteria, found in about 100,000 copies per cell . Furthermore, OmpA-sal modulates the adaptive immune responses to S. enterica serovar Typhimurium by activating dendritic cells and initiates an adaptive immune response, two important properties to be considered in the development of effective S. enterica serovar Typhimurium vaccines . OmpA expression is controlled by two stress-responsive ribonucleolytic mechanisms, and the environmental stimuli regulating OmpA expression could be transduced through these pathways in response to membrane stress . Concomitantly to the disappearance of the flagellum, the overexpression of this adhesion involved in the first steps of biofilm formation raises questions about a possible enhancement of the adhesion capacities of the bacterium.