Pili contribute to biofilm formation in vitro in Mycobacterium tuberculosis
- 1k Downloads
Organized bacterial communities, or biofilms, provide an important reservoir for persistent cells that are inaccessible or tolerant to antibiotics. Curli pili are cell-surface structures produced by certain bacteria and have been implicated in biofilm formation in these species. In order to determine whether these structures, which were suggested to be encoded by the Rv3312A (mtp) gene, have a similar role in Mycobacterium tuberculosis, we generated a Δmtp mutant and a mtp-complemented strain of a clinical isolate of M. tuberculosis and analyzed these strains for their ability to produce pili in comparison to the wild-type strain. Phenotypic analysis by transmission electron microscopy proved the essentiality of mtp for piliation in M. tuberculosis. We then compared biofilm formation of the derived strains in detergent-free Sauton’s media. Biofilm mass was quantified spectrophotometrically using crystal violet. Furthermore, we examined mtp gene expression by quantitative real-time PCR in wild-type cells grown under biofilm versus planktonic growth conditions. We found a 68.4 % reduction in biofilm mass in the mutant compared to the wild-type strain (P = 0.002). Complementation of the mutant resulted in a restoration of the wild-type biofilm phenotype (P = 0.022). We, however, found no significant difference between mtp expression in cells of the biofilm to those growing planktonically. Our findings highlight a crucial, but non-specific, role of pili in the biofilm lifestyle of M. tuberculosis and indicate that they may represent an important target for the development of therapeutics to attenuate biofilm formation, thereby potentially reducing persistence.
KeywordsMycobacterium tuberculosis Curli pili Gene deletion Complementation Adhesin Persistence
We thank Mr Mhlengi Vella Ncube (UKZN) for his contribution to the generation of the Δmtp mutant; Ms Charissa Naidoo (UKZN) for help with the statistical analysis; and the National Research Foundation (NRF), SA, Medical Research Council (MRC), SA, and College of Health Sciences (CHS), UKZN, for financial support. Mr S. Ramsugit gratefully acknowledges scholarship from the NRF and Canon Collins Trust.
Conflict of interest
The authors declare that they have no conflict of interest.
- Alteri CJ (2005) Novel pili of Mycobacterium tuberculosis. Ph.D. Thesis, The University of ArizonaGoogle Scholar
- Ausubel FM, Brent R, Kingston RE, Moore DD, Smith JA, Seidman JG, Struhl K (1989) Current protocols in molecular biology. Greene Publishing and Wiley Interscience, New YorkGoogle Scholar
- Bardarov S, Bardarov S Jr, Pavelka MS Jr, Sambandamurthy V, Larsen M, Tufariello J, Chan J, Hatfull G, Jacobs WR Jr (2002) Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148:3007–3017PubMedGoogle Scholar
- Canetti G (1955) Tubercle Bacillus in the pulmonary lesion of man: histobacteriology and its bearing on the therapy of pulmonary tuberculosis. Springer, New YorkGoogle Scholar
- Larsen MH, Biermann K, Tandberg S, Hsu T, Jacobs WR Jr (2007) Genetic manipulation of Mycobacterium tuberculosis. Curr Protoc Microbiol 6:10A.2Google Scholar
- Marsollier L, Aubry J, Coutanceau E, André JP, Small PL, Milon G, Legras P, Guadagnini S, Carbonnelle B, Cole ST (2005) Colonization of the salivary glands of Naucoris cimicoides by Mycobacterium ulcerans requires host plasmatocytes and a macrolide toxin, mycolactone. Cell Microbiol 7:935–943PubMedCrossRefGoogle Scholar
- Ojha AK, Hatfull GF (2012) Biofilms of Mycobacterium tuberculosis: new perspectives of an old pathogen. In: Cardona P (ed) Understanding tuberculosis—deciphering the secret life of the bacilli. Intech Open Access Publisher, Reijek, pp 181–192Google Scholar
- Rost B, Yachdav G, Liu J (2004) The PredictProtein Server. Nucleic Acids Res 32(Web Server issue):W321–W326Google Scholar
- van Soolingen D, Hermans PW, de Haas PE, Soll DR, van Embden JD (1991) Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 29:2578–2586PubMedGoogle Scholar
- Velayati AA, Farnia P, Masjedi MR (2012) Pili in totally drug resistant Mycobacterium tuberculosis (TDR-TB). Int J Myco 1:57–58Google Scholar
- World Health Organization (2012) Global tuberculosis report 2012. http://apps.who.int/iris/bitstream/10665/75938/1/9789241564502_eng.pdf. Accessed 6 March 2013