STUDIES ON PHYSIOLOGICAL IMPORTANCE OF HFQ AND SRNA TARGETS IN ACINETOBACTER BAUMANNII

Abstract

Acinetobacter baumannii is one of the major causes of hospital acquired infections all over the world and one the most frequently isolated nosocomial pathogen in India. This Gram-negative coccobacillus causes a myriad of diseases like hospital acquired pneumonia, meningitis, bacteraemia, urinary tract infections, wound infections and other soft tissue infections. However, the bacterium has gained worldwide notoriety for rapidly developing resistance to most of the antimicrobials and global dissemination of pan-drug resistant strains. Much of the information on determinants of antimicrobial resistance, virulence factors and survival strategies of this bacterium has poured in recently which includes our foray into identification of small RNA in this highly successful nosocomial pathogen. Small RNAs are short non-coding regulatory RNA molecules that interact with their target mRNAs in antisense manner, thereby affecting their translation, and adding an extra posttranscriptional layer to the overall gene regulation scheme in bacteria. This sRNA-mRNA interaction is often assisted by chaperone proteins, of which Hfq is a major player throughout the Gram-negative genera. We identified 31 putative sRNA in A. baumannii and characterized a novel sRNA, AbsR25, in our previous study. This subsequently led to the identification of Hfq protein in A. baumannii. The A. baumannii Hfq is unusually long due to an unstructured Cterminal extension. Our experiments prove that this seemingly unimportant part is required for efficient sRNA interaction and plays a significant role in maintaining the phenotype that is dependent on the presence of functional Hfq. In an earlier endeavor of our research group, AbsR25 was identified as a novel sRNA that regulates the expression of efflux pump genes including A1S_1331. Such efflux pumps are determinants of antibiotic resistance and A1S_1331, rechristened as AbaF, was indeed found in this study to be responsible for intrinsic fosfomycin resistance in A. baumannii. Apart from antibiotic efflux, AbaF was also determined to be involved in extruding biofilm material and important for virulence of A. baumannii. From the aforementioned 31 candidate sRNA, AbsR1, a novel small RNA was validated by Northern blotting in the present work. This 89 nt long sRNA is present in the intergenic region between 50S ribosome subunit coding genes and conserved throughout the A. baumannii strains. The over expression of AbsR1 at acidic challenge and decreased viability of A. baumannii ΔAbsR1 cells in acidic conditions indicates that AbsR1 might be involved in regulation of cellular response to acidic stress. ii From the same list of candidate sRNAs, AbsR10 coding region was identified to be exclusive to A. baumannii. PCR primers designed to amplify this unique target region could identify all the clinical strains present in the lab with 100% accuracy. Moreover, the detection was specific as no amplification was achieved when the template DNA from other Gram negative and Grampositive pathogens was used. The PCR based detection system was shown to rapidly detect A. baumannii from simulated clinical surfaces without any enrichment. Quantitative detection of A. baumannii could also be made using qPCR, which also led to enhancement in the sensitivity of the detection system.