Thiol reductive stress induces cellulose-anchored biofilm formation in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) forms biofilms harbouring antibiotic-tolerant bacilli in vitro, but the factors that induce biofilm formation and the nature of the extracellular material that holds the cells together are poorly understood. Here we show that intracellular thiol reductive stress (TRS) induces formation of Mtb biofilms in vitro, which harbour drug-tolerant but metabolically active bacteria with unchanged levels of ATP/ADP, NAD+/NADH and NADP+/NADPH. The development of these biofilms requires DNA, RNA and protein synthesis. Transcriptional analysis suggests that Mtb modulates only ∼7% of its genes for survival in biofilms. In addition to proteins, lipids and DNA, the extracellular material in these biofilms is primarily composed of polysaccharides, with cellulose being a key component. Our results contribute to a better understanding of the mechanisms underlying Mtb biofilm formation, although the clinical relevance of Mtb biofilms in human tuberculosis remains unclear.

. Transcriptional changes associated with TRS. Mtb cultures at an OD of 600 0.8 were exposed to 1 mM DTT for 3 hrs, and RNA was isolated and subjected to high-density oligonucleotide array analysis. Heat maps of significantly differentially expressed genes (SDEGs) with average fold changes of 1.5 or greater in all the replicates were created. Rv. NO.

Rv1854c ndh
Transfer of electrons from NADH to the respiratory chain.

Rv0088
Possible Thought to be involved in active transport of glutamine across the membran Rv0080 Function unknown Rv0081 Involved in transcriptional mechanism Rv0088 Possible polyketide cyclase/dehydrase Rv0122 Hypothetical protein

Rv0170 mce1B
Unknown, but thought to be involved in host cell invasion.

Rv0172 mce1D
Unknown, but thought to be involved in host cell invasion Rv0188 Probable conserved transmembrane protein Rv0190 Function unknown Rv0219 Probable conserved transmembrane protein Rv0232 Involved in transcriptional mechanism

Rv0291 mycP3
Thought to have proteolytic activity

Rv0469 umaA
Involved in mycolic acid modification or synthesis Rv0589 mce2A Unknown, but thought to be involved in host cell invasion

Rv0638 secE1
Essential for protein export

Rv0655 mkl
Thought to be involved in active transport of ribonucleotide across the membrane. Responsible for energy coupling to the transport system.

Rv0708 rplP
This protein binds directly to 23S ribosomal RNA and is located at the a site of the peptidyltransferase center.

Rv0709 rpmC
Involved in translation mechanisms

Rv0718 rpsH
Binds directly to the central domain of 16S ribosomal RNA.

Rv1182 papA3
Function unknown; thought to be involved in lipid metabolism.

Rv1183 mmpL10
Unknown. Thought to be involved in fatty acid transport.

Rv1284 canA
Catalyzes reversible dehydration of CO2 to form bicarbonate

Rv1285 cysD
Involved in sulfate activation pathway

Rv1802 PPE30
Function unknown Rv1887 Function unknown Rv1904 Function unknown

Rv2246 kasB
Involved in fatty acid biosynthesis

Rv2247 accD6
Involved in fatty acid biosynthesis

Rv2358 smtB
Involved in transcriptional mechanism

Rv2405
Function unknown Rv2623 Function unknown Rv2710 sigB RNA polymerase sigma factor Rv2905 Function unknown Rv2952 Function unknown

Rv3118 sseC
Thought to be involved in sulphur metabolism Rv3123 Function unknown Rv3131 Function unknown Rv3145 Function unknown

Rv3157 nuoM
Involved in aerobic|anaerobic respiration Rv3224 Function unknown Rv3269 Function unknown Rv3376 Function unknown

Rv1908c KatG
Multifunctional enzyme, exhibiting both a catalase, a broad-spectrum peroxidase, and a peroxynitritase activities Rv0096 PPE Function unknown

Rv2710
Sig B May control the regulons of stationary phase and general stress resistance.

Rv1623c appC
Involved in the respiratory chain (at the terminal step): aerobic respiration.

Rv3411c guaB2
Catalyses the first reaction unique to GMP biosynthesis.

Rv2150c
FtsZ Essential for cell division. It is thought that the intracellular concentration of FTSZ protein is critical for productive septum formation in mycobacteria.

Rv1177 fdxC
Ferredoxins are iron-sulfur proteins that transfer electrons in a wide variety of metabolic reactions. Rv0053 rpsF 30S ribosomal protein S6 RpsF.

Rv0709 rpmC
Involved in translation mechanisms.

Rv0302
Involved in transcriptional mechanism.

Rv0708 rplP
This protein binds directly to 23S ribosomal RNA and is located at the a site of the peptidyltransferase center.

Rv2069 sigC
Involved in promoter recognition, transcription initiation.

Rv0824c desA1
Catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids.

Rv0655 mkl
Thought to be involved in active transport of ribonucleotide across the membrane. Responsible for energy coupling to the transport system.

Rv0715 rplX
This protein is found in the ribonucleoprotein core and is involved in the early assembly of the 50S subunit.

Rv0718 rpsH
Binds directly to the central domain of 16S ribosomal RNA.

Rv0714 rplN
This protein binds directly to 23S ribosomal RNA.

Rv2412 rpsT
Involved in translation mechanisms. Binds directly to 16S ribosomal RNA.

Rv0172 mce1D
Unknown, but thought to be involved in host cell invasion.

Rv0815c cysA2
May be a sulfotransferase involved in the formation of thiosulfate.

Rv0703 rplW
Binds to a specific region on the 23S rRNA.

Rv0170 mce1B
Unknown, but thought to be involved in host cell invasion.

Rv0701 rplC
This protein binds directly to 23S ribosomal RNA and may participate in the formation of the peptidyltransferase center of the ribosome.         Fig. 3a). Upregulation of furA (ferric uptake regulator protein), bfrB  Fig. 3d). Furthermore, the genes involved in cysteine biosynthesis (cysA2, cysA3), fatty acid biosynthesis (kasA and kasB), and cholesterol metabolism (mce1 operon members yrbE1B, mce1B, and mce1D) were downregulated. Interestingly, the transcriptional regulator WhiB2 (a regulator of cell shape and growth) was also downregulated.

Supplementary Note 2: Monitoring the kinetics of transcriptional changes during biofilm formation
After analysing the transcriptional changes associated with the mild/moderate thiol reductive stress that does not result in biofilm formation, we exposed Mtb cultures to biofilm inducing concentrations of DTT (5 mM DTT) for 3 hrs and isolated RNA and followed by high-density oligonucleotide array analysis to explore the Mtb transcriptional response. Significance analysis of microarray identified 229 genes that are modulated at least 1.5-fold upon exposure to 5 mM DTT for 3 hrs. We observed that 210 genes (Supplementary NADH dehydrogenase I is expressed under aerobic conditions 8,9 and is often downregulated under hypoxic conditions. NADH dehydrogenase I is considered a key virulence factor of Mtb due to its ability to modulate apoptosis of the host cells 10,11 . We also detected a change in the expression of genes coding antigenic proteins belonging to PE/PPE proteins (PE13, PE15, PE31, PE34, PE60) was also decreased, indicating a gross change in the antigenic profile of the bacterial cells.

Supplementary Note 3: Transcriptome of bacteria residing in biofilms
After analysing the transcriptional response of Mtb to TRS, we analysed the transcriptional profile of the Mtb residing in biofilms. RNA was isolated from the Mtb biofilms, and high-density oligonucleotide array analysis was performed. Only 284 genes (<1% of total genes) were differentially modulated in biofilm bacteria. Of these, 114 were upregulated (Supplementary Table 7) and 170 were downregulated (Supplementary Table 8). Interestingly, the upregulated genes included those encoding the type VII secretion system ESX-3 (mycP3, eccB3, eccA3, eccD3, espG3, eccE3, eccC3, PPE4, and others). As the ESX-3 system plays an important role in iron uptake 12 , its induction suggests that the bacteria residing in the mycobacterial biofilms suffer from iron depletion. This finding was further supported by the upregulation of genes encoding the iron uptake regulator FurA (furA) and the iron-regulated nucleoid-associated protein (lsr2) 13 . In addition to ESX-3, several genes related to ESX-1 (Rv3865, Rv3866, Rv3868) and EsxR were induced in mycobacterial communities. However, some genes coding for ESX-5 (esxM, esxN, Rv1794, mycP5) were downregulated. Microarray data also suggest catabolic changes associated with the phenotypic switch towards biofilms. These changes include increased expression of genes involved in the biosynthesis of histidine (hisB and hisC), nucleotide biosynthesis (nrdB), glycogen biosynthesis (glgB), peptidoglycan biosynthesis (murF and murX), and molybdopterin biosynthesis (mog).
Interestingly, the tRNA synthesis of alanyl-tRNA (alaS) and cysteinyl-tRNA (cysS) were increased, suggesting that a specific pool of tRNAs is required for survival in biofilms. It was interesting to note that biofilm formation was associated with several metabolic changes, as evidenced by increased expression of pdhA and pdhC, The microarray data also suggest an increase in the expression of the cell division protein FtsW and the chromosome partitioning protein ParA. In addition to these genes, a number of genes encoding PPE/PGRS proteins (Rv0742, Rv2490c,