Transcriptional portrait of M. bovis BCG during biofilm production shows genes differentially expressed during intercellular aggregation and substrate attachment

Abstract

Mycobacterium tuberculosis and M. smegmatis form drug-tolerant biofilms through dedicated genetic programs. In support of a stepwise process regulating biofilm production in mycobacteria, it was shown elsewhere that lsr2 participates in intercellular aggregation, while groEL1 was required for biofilm maturation in M. smegmatis. Here, by means of RNA-Seq, we monitored the early steps of biofilm production in M. bovis BCG, to distinguish intercellular aggregation from attachment to a surface. Genes encoding for the transcriptional regulators dosR and BCG0114 (Rv0081) were significantly regulated and responded differently to intercellular aggregation and surface attachment. Moreover, a M. tuberculosis H37Rv deletion mutant in the Rv3134c-dosS-dosR regulon, formed less biofilm than wild type M. tuberculosis, a phenotype reverted upon reintroduction of this operon into the mutant. Combining RT-qPCR with microbiological assays (colony and surface pellicle morphologies, biofilm quantification, Ziehl-Neelsen staining, growth curve and replication of planktonic cells), we found that BCG0642c affected biofilm production and replication of planktonic BcG, whereas ethR affected only phenotypes linked to planktonic cells despite its downregulation at the intercellular aggregation step. our results provide evidence for a stage-dependent expression of genes that contribute to biofilm production in slow-growing mycobacteria. In nature, microbial species are often found within a matrix, forming multicellular communities that attach to surfaces or air-liquid interfaces, called biofilms 1. Biofilms are relevant to human health, as a majority of bacterial pathogens employ these structures to modify the host response 2 contributing to persistence 3. In this regard, a link exists between in vitro biofilm production and in vivo persistence for BCG 4 and M. tuberculosis 5. Biofilm formation occurs via a series of well-defined steps. These include the attachment of single-cell planktonic microbes onto a substratum; aggregation and growth of the adherent cells into three-dimensionally organized structures; and encapsulation of the structures by a self-produced matrix of extracellular polymeric substance 1 .

Three-dimensional low shear culture of Mycobacterium bovis BCG induces biofilm formation and antimicrobial drug tolerance

“…In summary, biofilm formation in free-fall suspension caused adaptations to cell surface control systems, exemplified by induction of sigE and BCG_0559c , in comparison to stationary phase culture in the absence of detergent. Significantly, sigE and elements of the ESX-5 secretion system ( esxJ , esxM , PE19 found here, plus esxN , BCG_1826 and esxK ) were also found to be induced when M. bovis BCG cells first began to aggregate in a Sauton no detergent model of pellicle formation 18 , highlighting these systems as likely mediators of the dynamic mycobacterial phenotypes generated in biofilm-like growth.…”

“…1a ). Unlike other biofilm models, this system enabled the natural cord-forming mycobacterial phenotype to develop without attachment to an artificial solid medium, nor reliance on an air/liquid interface for pellicle formation 16 , 18 , 19 , 29 . Mycobacterial biofilms were macroscopically visible in rotating RCCS vessels, as was the accumulation of biomass in the bottom of non-rotating vessels by day 21 (Fig.…”

“…The addition of detergents to mycobacterial media is common practice to encourage homogenous bacterial culture, and while this facilitates reproducible in vitro research, it does not accurately reflect the growth state of mycobacteria in vivo. The development of new tools to engineer, characterise and screen mycobacterial biofilms in detergent-free systems 18 , 47 may lead to the discovery of new transformative drugs targeted where they are required, at drug-tolerant sub-populations of bacilli residing in pulmonary lung lesions.…”

“…Since Ojha et al 16 demonstrated that M. tb biofilms formed at the liquid-air interface harbour antimicrobial drug tolerant bacilli, research into the role of biofilms in TB pathogenesis has expanded. Mycobacterial biofilm models, often based on the formation of a pellicle at the liquid-air interface, have demonstrated that mycobacteria in biofilm-like structures are less sensitive to antimicrobial drug exposure, show biofilm architecture and an extracellular matrix readily identifiable by microscopy, and express distinct transcriptional profiles when compared to planktonic growth [16][17][18][19] . These systems often rely on attachment to artificial surfaces or exposure to stringent environmental conditions to create biofilm-like structures.…”

Dissecting Host-Pathogen Interactions in TB Using Systems-Based Omic Approaches

“…The discovery of next-generation sequencing (NGS) facilitates the genomic and transcriptomic research of mycobacteria. The genomic or transcriptomic analysis focuses on the variation of genomic features, such as gene expression level, DNA sequencing or regulatory elements annotation, which enables the identification of essential genetic or regulatory targets under certain conditions (16)(17)(18). Resistance mutation of mycobacterium to drugs can be detected by genomic analysis of clinical isolates (19).…”