Sortase A Mediated Bioconjugation of Common Epitopes Decreases Biofilm Formation in Staphylococcus aureus.

Staphylococcus aureus is one of the most notorious pathogens and is frequently associated with nosocomial infections imposing serious risk to immune-compromised patients. This is in part due to its ability to colonize at the surface of indwelling medical devices and biofilm formation. Combating the biofilm formation with antibiotics has its own challenges like higher values of minimum inhibitory concentrations. Here, we describe a new approach to target biofilm formation by Gram positive bacteria. Sortase A is a transpeptidase enzyme which is responsible for tagging of around ∼22 cell surface proteins onto the outer surface. These proteins play a major role in the bacterial virulence. Sortase A recognizes its substrate through LPXTG motif. Here, we use this approach to install the synthetic peptide substrates onS. aureus. Sortase A substrate mimic, 6His-LPETG peptide was synthesized using solid phase peptide chemistry. Incorporation of the peptide on the cell surface was measured using ELISA. Effect of peptide incubation on Staphylococcus aureus biofilm was also studied. 71.1% biofilm inhibition was observed with 100 µM peptide while on silicon coated rubber latex catheter, 45.82% inhibition was observed. The present work demonstrates the inability of surface modified S. aureus to establish biofilm formation thereby presenting a novel method for attenuating its virulence.

Essential role of the ESX-3 associated eccD3 locus in maintaining the cell wall integrity of Mycobacterium smegmatis.

Mycobacterial pathogens have evolved a unique secretory apparatus called the Type VII secretion system (T7SS) which comprises of five gene clusters designated as ESX1, ESX2, ESX3, ESX4, and ESX5. Of these the ESX3 T7SS plays an important role in the regulatory uptake of iron from the environment, thereby enabling the bacteria to establish successful infection in the host. However, ESX3 secretion system is conserved among all the mycobacterial species including the fast-growing nonpathogenic species M. smegmatis. Although the function of ESX3 T7SS is known to be absolutely critical for establishing infection by M. tuberculosis, its conserved nature in all the pathogenic and nonpathogenic mycobacterial species intrigues to explore the additional functional roles in Mycobacterium species through which potent targets for drugs can be identified and developed. In the present study, we investigated the possible role of EccD3, a transmembrane protein of the ESX3 T7SS in M. smegmatis by deleting the entire eccD3 gene by efficient allelic exchange method. The preliminary investigations through the creation of knockout mutant of the eccD3 gene indicate that this secretory apparatus has an important role in maintaining the cell wall integrity which was evident from the abnormal colony morphology, lack of biofilm formation and difference in cell wall permeability.

Extraction and characterization of microcrystalline cellulose from fodder grass; Setaria glauca (L) P. Beauv, and its potential as a drug delivery vehicle for isoniazid, a first line antituberculosis drug.

Microcrystalline cellulose (MCC) is generally produced through acid hydrolysis of woody plants and agro sources. MCC synthesized from a common wild grass Setaria glauca (L) P. Beauv was characterized to explore the possibility of application in pharmaceutical industry especially as a drug delivery vehicle. The SEM, TGA, XRD and FTIR investigations of the prepared MCC reveal that the 5-30µm long, non aggregated MCC rods have high crystallinity index of 80% and were stable at 286°C. The preliminary investigation of the MCC incorporated micro beads containing isoniazid, one of the first line drugs for treatment of tuberculosis was carried out in the simulated intestinal fluid (SIF). The MCC incorporated micro beads with isoniazid drug load showed sustained release upto 24h with release of 0.521µg of isoniazid equivalent drug in the SIF system. No cytotoxicity of the MCC was observed in the haemolytic assay. The MCC also showed good antioxidant activity. Thus, the study reveals that the MCC can be prepared from an inexpensive and abundant grass species. The MCC have properties advantageous for application in the pharmaceutical industry and may be explored further in drug delivery research.