Mechanism And Therapeutic Strategy Of Staphylococcus Aureus Infection Of Epidermal Keratinocytes

Staphylococcus aureus(S.aureus)is the most virulent pathogen causing various diseases,including skin abscesses,pneumonia,endocarditis,and osteomyelitis,in humans and animals.As the leading cause of skin and skin structure infections,S.aureus have the ability to invade and survive in the epithelial barrier,which is mainly composed of distinctly differentiated keratinocytes.Furthermore,S.aureus is resistant to many antibiotics,especially to methicillin.The emergence and prevalence of methicillin-resistant S.aureus(MRSA)underscore the need for the development of effective therapeutic alternatives.To study the intracellular survival mechanism of MRSA,the iTRAQ comparative proteomics technique was used to identify 197 distinct proteins of MRSA after intracellular infection.Bioinformatics analysis revealed that these distinct proteins are mainly involved in cell wall synthesis,carbohydrate metabolism,and infection of the eukaryotic cells of S.aureus.To explore the function of distinct proteins in intracellular infection by MRSA,the mutant strains were constructed by selecting three enzyme proteins that played a key role in the bacterial metabolism of ribitol.Studies found that the strain that deleted the rbsK gene could lead to a significant decrease in the transcriptional level of genes related to wall teichoic acid(WTA)synthesis.Knockout of the rbsK gene could slow down the growth of the MRSA strain in a medium in which ribose is the lone carbon source,and lead to a significant reduction in the ability of the bacteria to survive in keratinocytes.This result indicated that RbsK helps MRSA adhere to keratinocytes,as well as affects the bacterial uptake of carbon sources in cells.In addition,the deletion of the bacterial WTA synthetases(TarI and TarJ)could influence the adhesion and invasive ability of S.aureus to keratinocytes,indicating that the WTA of bacteria played a key role in the process of adhesion and invasion of keratinocytes.As an alternative antibacterial agent,a lytic phage,designated SLPW,was isolated from fecal sewage in a pig farm.SLPW showed a broad host range and high efficiency of plating against MRSA strains.To develop a strategy against intracellular MRSA infections,phage-loaded bacteria(PLB)were used to treat intracellular infections caused by MRSA.The PLB were transported into keratinocytes,demonstrated improved ability to kill intracellular MRSA,and promoted the recovery of the keratinocytes.Furthermore,the research revealed that a shorter time of interaction(10 min)between phages and bacteria before they were added to the keratinocytes could contribute to increased intracellular phages and the eradication of intracellular S.aureus.To explore the intracellular bactericidal method,a cell wall hydrolase(also called lysin)derived from Staphylococcus phage JD007,short for JDlys,was identified.JDlys showed strong lytic activity against MRSA strains from different sources and multilocus sequence typing types.Furthermore,a fusion protein called CPP-JDlys,which consisted of a cell-penetrating peptide(CPP)fused to JDlys,was used against MRSA infection.CPP-JDlys,which the fusion of CPP almost has not affected the bacteriolytic activities of JDlys,was able to effectively eliminate intracellular MRSA and alleviate the inflammatory response and cell damage caused by MRSA.To evaluate the therapeutic potential of phage and lysine in vivo,assays were performed using mice after infection with high concentrations of S.aureus.The results showed that mice treated with phage and lysin displayed significantly higher survival rates than untreated mice were.In addition,treatment efficacy was evaluated by examining bacterial colonization in the organs and blood of mice after phage therapy with a non-lethal dosage of MRSA infections.The mice treated with phage showed significantly lower CFU burdens and levels of cytokines in the spleen and lung at every time point,a result suggesting that the phage was therapeutically effective against systemic infection caused by MRSA.In addition,lysin was capable of combatting MRSA-induced murine skin infections and consequently expediting the healing of cutaneous abscesses.Specifically,the combination therapies of phage–antibiotics and lysin–antibiotics are effective in reducing the numbers of bacteria and somatic cells,as well as the levels of pro-inflammatory cytokines,in the milk of mastitis cows,indicating that phage and lysin could prevent mastitis in dairy cows while improving milk quality.In summary,this research confirmed that MRSA strains could efficiently survive in keratinocytes.On the basis of the phage and the powerful bactericidal effect of lysin in vitro,the PLB and CPP-JDlys protein were designed.The study demonstrated that both of them could effectively prevent intracellular MRSA infection.Meanwhile,in vivo studies showed that phage and lysin had the ability to effectively treat local and systemic infections of mice caused by MRSA,and they enabled the prevention of mastitis caused by MRSA.Furthermore,the comparative proteome technique demonstrated that ribokinase and WTA played a key role in the MRSA infection and colonization of keratinocytes,which provided theories for the further exploration of therapeutic strategies for MRSA intracellular infection.