• Cloning and Characterization of Hypothetical Exported Proteins from Community Associated Staphylococcus Aureus

      Kaur, Haninder (2013-01-30)
      Methicillin 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.
    • Cloning and Characterization of SAS1738, a Hypothetical Exported Protein from Community-Associated Strain of Staphylococcus Aureus

      Vijaya Kumar, Deepak Kumar (2010-09-22)
      Methicillin resistant Staphylococcus aureus (MRSA) is a group of S. aureus strains that has acquired resistance to a class of beta lactam antibiotics and is the major cause of hospital associated infections. Their discovery goes back to 1960 when the first cases were identified. Recently community associated MRSA infections have emerged and are caused by strains that are independent of those from the hospital environment, related only because they carry some of the same antibiotic resistance genes. Community associated infections (CA) are more severe, producing pus filled lesions that are painful and capable of invasion of deep tissues. Virulence factors comprised of exported proteins are associated with the invasiveness of CA strains. Most of these proteins are hypothetical in nature with unknown function. The aim of this study is to identify and characterize potential virulence factor proteins that may be involved in the infection pathway of CA-MRSA. This study focuses on a unique gene that encodes an exported protein, SAS1738, found on the chromosome of the CA strain MSSA476. The protein SAS1738 was chosen because it is unique to CA strains and has homology to some proteins identified in other S. aureus strains known for their virulence and host immune evasion. The goal of this work is to characterize SAS1738 and to determine its role in the infection pathway of the organism. The gene of interest has been successfully cloned, expressed, and tested for toxicity in Caenorhabditis elegans, a nematode. The toxicity tests showed that SAS1738 is inhibitory to the growth and development of C. elegans. The actual mode of action of this protein in C. elegans is yet to be established. However, location of SAS1738 using a GFP fusion showed that the highest concentration of the fusion protein was in the gut of the worms. The purified protein when tested in a killing assay against C. elegans, resulted in the death of the worms at an average time point of 8 min after treatment. Microbiological assay results showed that the purified SAS1738 possessed antibacterial activity towards Micrococcus luteus and Proteus vulgaris. This suggests that SAS1738 may play a dual role of antagonizing the commensal flora of the human skin such as Micrococcus luteus and also induce a toxic effect on the human cells as suggested by its toxic effect on C. elegans. Determination of the role of this protein in the infection cycle of CA-MRSA will lead to a better understanding of the pathogenicity of the organism and possible development of new treatment strategies.