Analysis Of Biofilm Structure And Molecular Mechanisms Related With Biofilm Formation Of Staphylococcus Epidermidis And Structure-based Screening Potential Leading-compounds As Histidine Kinase YycG Inhibitors Against Staphylococcus Infections

Staphylococcus epidermidis is a normal inhabitant of human skin and mucous membranes that rarely causes pyogenic infections in healthy individuals. However, during the past two decades S. epidermidis has emerged as one of the major pathogens in nosocomial infections. The primary pathogenicity trait of S. epidermidis is associated with its ability to form biofilms on surfaces of medical devices, limiting severely the efficacy of many conventional antibiotics, and biofilms may also protect the bacteria against attacks from the host defence system. In parallel, the appearance of multi-drug resistant S. epidermidis strains has increased quickly due to the increasing use of antibiotics in hospitals, which require an urgent need to design novel antibiotics against staphylococcus infections, especially in relation to biofilm development.The crystal violet staining represents a simple and rapid method to measure the biofilm formation ability of S. epidermidis strains in 96-well microtiter plates. However, based on this method it can not tell us the detailed structure of bacterial biofilms, nor the components distribution or bacterial physiological features in biofilms. The flow-chamber cultivation system combined with fluorescent staining and confocal laser scanning microscopy technology have been successfully used to study all kinds of features mentioned above in some bacterial biofilms (such as in Pseudomonas aeruginosa). However, there is few data describing its application in S. epidermidis biofilm formation, so it needs to be found the appropriate cultivation conditions for S. epidermidis biofilm formation in this system.S. epidermidis biofilm formation has been described as a two-step process. The first stage involves attachment of cells to a surface (initial attachment phase). The second stage includes cell-cell aggregation and the formation of a multilayered architecture (accumulative phase). Much attention has been focused on the Polysaccharide Intercellular Adhesion (PIA) component of the extracellular polymeric substance (EPS) matrix of S. epidermidis, which is encoded by ica operon and considered as a major cell-to-cell interconnecting compound during biofilm formation. However, it is possible that other matrix components may be important for biofilm development of S. epidermidis, such as extracellular DNA which was shown to be important for biofilm formation of Pseudomonas aeruginosa and other bacteria (but remains unknown functions in S. epidermidis biofilm formation).In the present work, two cultivation systems flow-chamber and static-chamber were established to study S. epidermidis biofilm development in vitro. Based on these systems, two types of clinical isolates (ica^-/BF⁺ and ica⁺/BF⁺) were investigated including their biofilm development process, structural components, resistance to antibiotics treatment etc. The role of extracellular DNA and the molecular mechanism of release were also further investigated in S. epidermidis biofilm. At last, a series of novel inhibitors of the histidine kinase YycG protein of S. epidermidis were discovered first using structure-based virtual screening from a small molecular leading-compound library, followed by experimental validation of their antibacterial activities.Chapterâ… Establishment of S. epidermidis biofilm cultivation systems and investigation of different S. epidermidis clinical isolates biofilm developmentTwo cultivation systems flow-chamber and static-chamber were established to study the features of S. epidermidis biofilms in vitro (such as dynamic structural development, Live/Dead cells distribution). Based on these systems, several S. epidermidis clinical isolates were chosen to be investigated. First, two ica^-/BF⁺ S. epidermidis clinical strains SE1 and SE4 exhibit their heterogeneity in biofilm architecture under static and flow conditions, compared with the ica⁺/BF⁺ RP62A strain. This kind of biofilms absence of PIA displays resistant to NaIO₄ but not Proteinase K, whereas the biofilm formed by RP62A can be disrupted by NaIO₄ but resistant to Proteinase K. More importantly, the cells of both SE1 and SE4 stains show more tolerant than those of RP62A after exposure to lysostaphin and vancomycin, which is associated with the decreased autolysis ability of SE1 and SE4 cells. Based on these results, it suggests that the ica^-/BF⁺ strains may represent a newly emergent subpopulation of S. epidermidis clinical strains by the selection of antibiotics in the nosocomial milieu, which displays survival advantage in host environment.The long-term biofilm development process of 4 ica⁺/BF⁺ S. epidermidis clinical isolates (SE698, SE847, SE886 and SEG203-2) and the laboratory standard strain RP62A (ica⁺/BF⁺) were recorded in the flow-chamber system. The common feature of biofilms formed by these 5 strains is the rebirth ability shown by the cells embedded in biofilm microcolonies: At the 1st day, there are lots of dead cells in the center of microcolony structure especially in those clinical isolates; at the 2nd day, most of dead cells detach from the biofim structure, leaving the vacuolar architecture in microcolonies; at the 3rd/4th day, the residual cells proliferate and occupy the vacuolar architecture, forming a "renew" biofilm in which few dead cells are present; at the 5th/6th day, reappearance of many dead cells in the center of microcolonies. This kind of "rebirth" phenomenon is associated with the repeated infection occurred in patients, which greatly increases the risk of bacteremia.Chapterâ…¡The role of extracellular DNA in S. epidermidis biofilm formation and associated mechanism of extracellular DNA releaseAs a component of extracellular polymeric substance (EPS) matrix, extracellular DNA has been shown to be important for biofilm formation of Pseudomonas aeruginosa and other bacteria, but in S. epidermidis its role remains unknown. In this work, DNaseâ… treatment is found to inhibit S. epidermidis initial attachment to surface and subsequent biofilm formation, but resisted by the established biofilms. The results indicate that extracellular DNA is a major component required for bacterial initial attachment to surfaces, as well as for the subsequent early phase of biofilm development by S. epidermidis. Moreover, evidence is presented that release of extracellular DNA from S. epidermidis mainly is caused by the activity of the autolysin AtlE. For instance, the atlE mutant strain displays dramatically decreased extracellular DNA release (～90%), initial attachment (～95%) and biofilm formation (97%), compared with the wild type (wt) strain, whereas the atlE complementary strain can recover all the features to the level of wt strain. In both flow and static chamber systems, the wt and atlE complementary strains form intact biofilm structure (microcolonies), in which abundant extracellular DNA (shown by DDAO staining) is present. In contrast, the atlE mutant strain only forms a few small cell-clumpings with much less extracellular DNA than its parental strain. In addition, almost no dead cells (shown by PI staining) are observed in cell-clumpings formed by the atlE mutant strain, which is in accord with the decreased autolysis in the atlE mutant cells.Chapterâ…¢Structure-based discovery of inhibitors of the YycG histidine kinase: New leading-compounds to combat Staphylococcus epidermidis infectionsMany two-component signal transduction systems (TCSTSs) are ubiquitous in bacteria and are integral components of the adaptive regulatory processes utilized by pathogenic bacteria to sense the environment and coordinate the expression of the genes encoding virulence factors, making them attractive targets for antimicrobial therapy. Based on bioinformatics analysis, 16 pairs of TCSTSs are found in the whole genome of S. epidermidis, most of which regulate multiple functions such as exoprotein production, adhesion and autolysis. Among these TCSTSs, two pairs (YycG/YycF and YhcS/YhcR) are required for bacterial growth. Subsequently, a series of novel inhibitors of the histidine kinase YycG protein of S. epidermidis were discovered first using structure-based virtual screening (SBVS) from a small molecular leading-compound library, followed by experimental validation. Of the 76 candidates derived by SBVS targeting of the homolog model of the YycG HATPase_c domain of S. epidermidis, seven compounds (compound 1-7) displayed significant activity in inhibiting S. epidermidis growth (MIC values are 0.2～100μM). Furthermore, five of them (compound 1-5) displayed bactericidal effects on S. epidermidis cells (MBC values are 25～200μM). Except for one (compound 6), the other six compounds were found to bind to the recombinant YycG protein (K_D values are 2.3～40.4) and to inhibit its auto-phosphorylation (IC₅₀ values are 6.5～48μM) in vitro, indicating that they are potential inhibitors of the YycG/YyeF TCSTS, which is essential in S. epidermidis. Importantly, all these compounds did not affect the stability of mammalian cells nor hemolytic activities at the concentrations used in our study, indicating they are of potential value for developing new antibiotics against infecting staphylococci. Based on a static-chamber system, we have assessed the bactericidal effect of two leading-compounds active as YycG inhibitors (compound 2 and 5) on biofilm cells of S. epidermidis laboratory and clinical strains by confocal laser scanning microscopy (CLSM) combined with viability staining. In young biofilms (6-h-old), the two compounds killed the majority of the embedded cells at concentrations of 100μM and 25μM, respectively (MBC values). In mature biofilms (24-h-old), compound 5 was still effectively killing biofilm cells, whereas compound 2 mainly killed cells located at the bottom of the biofilm. In contrast, vancomycin was found to stimulate biofilm development at the MBC (8μg/mL), especially to those clinical isolates. Even at a high concentration (128μg/mL), vancomycin exhibited poor killing on cells embedded in biofilms. The two compounds exhibited faster and more effective killing of S. epidermidis planktonic cells than vancomycin at the early stage of exposure (6 hours). The data suggest that the new inhibitors can serve as potential agents against S. epidermidis biofilms when added alone or in concert with other antimicrobial agents.