Browsing Biology by Subject "Gateway cloning."
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Cloning and Characterization of Hypothetical Exported Proteins from Community Associated Staphylococcus AureusMethicillin resistant Staphylococcus aureus (MRSA), a major cause of nosocomial infections, has acquired resistance to beta-lactam and other antibiotics. Recently, community associated MRSA (CA-MRSA) has developed independent of hospital associated MRSA (HA-MRSA). One of the major differences between the hospital and community strains is that the former is multi-resistant to antibiotics while the latter is not as resistant but is significantly more invasive. This increased invasiveness and the ability to cause life-threatening infections, even in immunocompetent individuals, makes CA-MRSA critically important as a public health problem. CA-MRSA is known to cause skin and soft tissue infections; bacteria interact with host skin cells and gain access to deeper tissues causing invasive infections. During this process the bacteria may secrete proteins that aid in the interaction with the host by adhering, invading or causing host cell death and lysis. To understand the virulence mechanisms involved in invasion, we investigated genes described as hypothetical proteins in MSSA476. The bioinformatics-selected proteins showed high probability of being secreted and most were unique to CA-MRSA. Our analysis showed 24 such genes. This study shows primer design for 15 of the genes (7 of the 24 had already been cloned in our laboratory). Using gateway cloning, the 15 genes were cloned into BL-21 expression clones. CA-MRSA’s are known for causing invasive skin infections. To further understand the involvement of our proteins of interest in invasion, human keratinocyte cell lines were used in a study of virulence and interaction with skin. To understand the involvement of our hypothetical secreted proteins, we investigated the mRNA expression level, using RT-qPCR and Livak method, of 20 hypothetical exported proteins in presence of human dermal keratinocyte cell line. Our investigation revealed two genes that showed increased mRNA expression in the presence of keratinocytes, which may be due to factors associated with keratinocytes that may have triggered increased mRNA expression. Keratinocytes are capable of forming cell-cell junctions and producing antimicrobial peptides and cytokines in response to microbes. The increased mRNA expression of two genes may be towards binding to junctions for invasion or may be expressed in response to antimicrobial peptides or cytokines.