Antonie Van Leeuwenhoek. 2013 Nov;104(5):725-35. DOI 10.1007/s10482-013-9981-6.

Pili contribute to biofilm formation in vitro in Mycobacterium tuberculosis

Saiyur Ramsugit, Sinenhlanhla Guma, Balakrishna Pillay, Paras Jain, Michelle H. Larsen, Siva Danaviah, and Manormoney Pillay

 

Abstract

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 68.43 % 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.

PMID: 23907521

 

Supplementary information

Whilst there has been major progress in reducing global tuberculosis (TB) cases and mortality rates in the past two decades, the global burden of TB remains high (1). Effective TB therapy is hindered by the presence of Mycobacterium tuberculosis bacilli capable of persisting against challenges posed by the host immune system and drug therapy. Understanding the mechanisms by which persistence is achieved by M. tuberculosis is, therefore, key to improved TB control.

The persistence of microbes is well known to be largely due to their ability to aggregate into community structures, termed biofilms. Laboratory-cultivated in vitro M. tuberculosis cultures are well known for their ability to spontaneously aggregate into macroscopic structures. However, the concept of M. tuberculosis biofilms has for long been met with much scepticism. The striking similarity between M. tuberculosis infections and those caused by biofilming pathogens, coupled with the global urgency for improved TB therapeutics, has resulted in a resurgence of research interest into plausible M. tuberculosis biofilm formation.

Ojha et al. provided evidence that M. tuberculosis forms biofilms in vitro, which harbour drug-tolerant populations of cells that persist despite exposure to high levels of antibiotics (2). However, it remains unclear whether this pathogen forms biofilms in its host. The ability of microbes to assemble into biofilms is dependent on several genetically-determined factors. The initial stages of biofilm development are dependent on the production of extracellular factors (or adhesins) that promote surface colonization or cell-to-cell contact (3), as indicated in figure 1.

Figure 1Figure 1. Stages of biofilm formation. The expression of surface adherence molecules (termed adhesins) on the bacterial surface enables the attachment of the bacterium to a surface. The organism will grow and divide and other organisms may attach via their adhesins, leading to the maturation of the biofilm. The production of extrapolymeric substances encapsulates the microbes and physically protects them from environmental challenges, including antibiotics. The biofilm is a heterogenous environment, with organisms at the core exposed to less nutrients and oxygen than those on the surface. These non-uniform adaptive responses may facilitate phenotypic diversity and stress-tolerance within the population.

In this study, we showed, as a consequence of mtp gene deletion, that the absence of surface localized pili fibres abolishes the ability of M. tuberculosis to form biofilms in vitro (Figure 2).

 

Figure 2Figure 2. The requirement of M. tuberculosis pili (MTP) in biofilm formation. The upper panel shows transmission electron micrographs of negatively-stained M. tuberculosis V9124 wild-type (a), Δmtp mutant (b), and mtp-complemented (c) strains. Black arrows point to pili fibres. Scale bars are 0.2 µm. The lower panel shows biofilm growth of the respective strains in Sauton’s media in 6-well plates, after incubation for 5 weeks at 37 °C and 5 % CO2 without shaking. From the results, it is evident that the mtp gene is essential for piliation, and piliation in-turn is necessary for M. tuberculosis biofilm formation (modified with kind permission from Springer+Business Media).

This study could have three important implications. Firstly, it adds to the ever-growing body of evidence that M. tuberculosis exists as biofilms in its host. The finding by Alteri et al. that M. tuberculosis pili (MTP) are expressed in vivo (4) and their essentiality for biofilm formation in vitro, as identified in this study, together supports the notion that pili could also be actively-engaged in surface attachment and biofilm formation in vivo.

Secondly, MTP may represent a molecular signature of multicellular structures of bacilli. Whilst gene expression analysis of planktonic versus biofilm in vitro wild-type cultures revealed similar mtp expression between these lifestyles, this may be different in vivo and needs to be confirmed experimentally. No doubt, a pili-deficient mutant strain, as derived in this study, may be useful as a negative control for such in vivo studies, to determine possible biofilm formation in the host, which has yet to be conclusively determined.

Finally, it indicates that MTP may represent an important target for the development of therapeutics to attenuate biofilm formation, thereby potentially reducing TB persistence. Improved TB control is dependent on the development of newer drugs that can clear infection efficiently and within a shorter duration. The fact that pili are cell-surface structures of adherence involved in the initial stages of biofilm formation, make them promising drug targets to dismantle the biofilm structure, as their role in biofilm formation would be structural and not likely on the adaptation of the resident bacteria within the structure. Pilicides and curlicides (5) are a well-known class of compounds that block the formation of pili and could prove useful as an anti-biofilming agent.

 

References

  1. World Health Organization (2012) Global Tuberculosis Report 2012. http://apps.who.int/iris/bitstream/10665/75938/1/9789241564502_eng.pdf  Accessed 6 March 2013
  2. Ojha AK, Baughn AD, Sambandan D, Hsu T, Trivelli X, Guerardel Y, Alahari A, Kremer L, Jacobs WR Jr, Hatfull GF (2008) Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol Microbiol 69:164-174
  3. Barnhart MM, Chapman MR (2006) Curli biogenesis and function. Annu Rev Microbiol 60:131-147
  4. Alteri CJ, Xicohténcatl-Cortes J, Hess S, Caballero-Olín G, Girón JA, Friedman RL (2007) Mycobacterium tuberculosis produces pili during human infection. Proc Natl Acad Sci U S A 104:5145-5150
  5. Cegelski L, Pinkner JS, Hammer ND, Cusumano CK, Hung CS, Chorell E, Åberg V, Walker JN, Seed PC, Almqvist F, Chapman MR, Hultgren SJ (2009) Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation. Nat Chem Biol 5:913-919

 

Acknowledgments

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.

 

Contact

Manormoney Pillay, PhD

Mailing address: Medical Microbiology and Infection Control, School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, 1st floor Doris Duke Medical Research Institute, Private Bag 7, Congella, 4013, Durban, South Africa. Phone: +27 312 604 765. E-mail: pillayc@ukzn.ac.za

 

Copyright notice

The abstract and figure 2 were reproduced/modified with kind permission from Springer Business Media: Article title: Pili contribute to biofilm formation in vitro in Mycobacterium tuberculosis; Volume: 104; Year of publication: 2013; Pages: 725-735; Authors: Ramsugit S, Guma S, Pillay B, Jain P, Larsen MH, Danaviah S & Pillay M; Copyright material used: Abstract and figures 2 and 3.

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