Functional analysis of the Mycobacterium bovis AF2122/97 PhoPR system.

The PhoPR system is a master regulator in Mycobacterium tuberculosis. A key difference between M. tuberculosis and Mycobacterium bovis is a G71I substitution in the M. bovis PhoR orthologue. Functional studies of the M. bovis PhoPR system have generated conflicting findings, with some research suggesting that the M. bovis PhoPR is defective while others indicate it is functional. We sought to revisit the functionality of the M. bovis PhoPR system. To address this, we constructed a phoPR mutant in the reference strain M. bovis AF2122/97. We employed a combination of growth assays and transcriptomics analyses to assess the phenotype of the mutant vs wild type and complemented strains. We found that the M. bovis AF2122/97 ΔphoPR mutant showed a growth defect on solid and liquid media compared to the wild type and complemented strains. The transcriptome of the M. bovis AF2122/97 ΔphoPR mutant was also altered as compared to wild type, including differential expression of genes involved in lipid metabolism and secretion. Our work provides further insight into the activity of PhoPR in M. bovis and underlines the importance of the PhoPR system as a master regulator of gene expression in the Mycobacterium tuberculosis complex.

Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

Screening of Hydrophilic Polymers Reveals Broad Activity in Protecting Phages during Cryopreservation.

Bacteriophages have many biotechnological and therapeutic applications, but as with other biologics, cryopreservation is essential for storage and distribution. Macromolecular cryoprotectants are emerging for a range of biologics, but the chemical space for polymer-mediated phage cryopreservation has not been explored. Here we screen the cryoprotective effect of a panel of polymers against five distinct phages, showing that nearly all the tested polymers provide a benefit. Exceptions were poly(methacrylic acid) and poly(acrylic acid), which can inhibit phage-infection with bacteria, making post-thaw recovery challenging to assess. A particular benefit of a polymeric cryopreservation formulation is that the polymers do not function as carbon sources for the phage hosts (bacteria) and hence do not interfere with post-thaw measurements. This work shows that phages are amenable to protection with hydrophilic polymers and opens up new opportunities for advanced formulations for future phage therapies and to take advantage of the additional functionality brought by the polymers.

MtrA modulates Mycobacterium tuberculosis cell division in host microenvironments to mediate intrinsic resistance and drug tolerance.

The success of Mycobacterium tuberculosis (Mtb) is largely attributed to its ability to physiologically adapt and withstand diverse localized stresses within host microenvironments. Here, we present a data-driven model (EGRIN 2.0) that captures the dynamic interplay of environmental cues and genome-encoded regulatory programs in Mtb. Analysis of EGRIN 2.0 shows how modulation of the MtrAB two-component signaling system tunes Mtb growth in response to related host microenvironmental cues. Disruption of MtrAB by tunable CRISPR interference confirms that the signaling system regulates multiple peptidoglycan hydrolases, among other targets, that are important for cell division. Further, MtrA decreases the effectiveness of antibiotics by mechanisms of both intrinsic resistance and drug tolerance. Together, the model-enabled dissection of complex MtrA regulation highlights its importance as a drug target and illustrates how EGRIN 2.0 facilitates discovery and mechanistic characterization of Mtb adaptation to specific host microenvironments within the host.

The ΔCysK2 mutant of Mycobacterium tuberculosis is sensitive to vancomycin associated with changes in cell wall phospholipid profile.

Cysteine plays a versatile role in cellular physiology and has previously been shown to be instrumental to Mycobacterium tuberculosis (M.tb) pathophysiology. In this study, we have generated mutants deficient in CysK2 and CysH, the key Cysteine, biosynthetic enzymes. In contrast to the ΔcysH mutant, the ΔcysK2 mutant is not an auxotroph and as such not essential for cysteine biosynthesis. Interestingly, the ΔcysK2 mutant shows increased sensitivity to cumene hydroperoxide, vitamin C, diamide, rifampicin and Vancomycin and shows alterations in phospholipid profile of Mtb cell wall. Our findings suggest that alteration in phospholipids content of M.tb cell wall by CysK2 may form a mode of defence against selected antibiotics and oxidative stress.

The mycobacterial desaturase DesA2 is associated with mycolic acid biosynthesis.

Mycolic acids are critical for the survival and virulence of Mycobacterium tuberculosis, the causative agent of tuberculosis. Double bond formation in the merochain of mycolic acids remains poorly understood, though we have previously shown desA1, encoding an aerobic desaturase, is involved in mycolic acid desaturation. Here we show that a second desaturase encoded by desA2 is also involved in mycolate biosynthesis. DesA2 is essential for growth of the fast-growing Mycobacterium smegmatis in laboratory media. Conditional depletion of DesA2 led to a decrease in mycolic acid biosynthesis and loss of mycobacterial viability. Additionally, DesA2-depleted cells also accumulated fatty acids of chain lengths C19-C24. The complete loss of mycolate biosynthesis following DesA2 depletion, and the absence of any monoenoic derivatives (found to accumulate on depletion of DesA1) suggests an early role for DesA2 in the mycolic acid biosynthesis machinery, highlighting its potential as a drug target.

MadR mediates acyl CoA-dependent regulation of mycolic acid desaturation in mycobacteria.

Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR-null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of ß-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1/desA2 is relieved upon binding of saturated acyl-CoAs of chain length C16 to C24, while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation.

Structure-function analysis of MmpL7-mediated lipid transport in mycobacteria.

Mycobacterial membrane protein Large (MmpL7) is a Resistance-Nodulation-Division (RND) family transporter required for the export of the virulence lipid, phthiocerol dimycocerosate (PDIM), in Mycobacterium tuberculosis. Using a null mutant of the related, vaccine strain Mycobacterium bovis BCG, we show that MmpL7 is also involved in the transport of the structurally related phenolic glycolipid (PGL), which is also produced by the hypervirulent M. tuberculosis strain HN878, but absent in M. tuberculosis H37Rv. Furthermore, we generated an in silico model of M. tuberculosis MmpL7 that revealed MmpL7 as a functional outlier within the MmpL-family, missing a canonical proton-relay signature sequence, suggesting that it employs a yet-unidentified mechanism for energy coupling for transport. In addition, our analysis demonstrates that the periplasmic porter domain 2 insert (PD2-insert), which doesn't share any recognisable homology, is highly alpha-helical in nature, suggesting an organisation similar to that seen in the hopanoid PD3/4 domains. Using the M. bovis BCG mmpL7 mutant for functional complementation with mutated alleles of mmpL7, we were able to identify residues present in the transmembrane domains TM4 and TM10, and the PD2 domain insert that play a crucial role in PDIM transport, and in certain cases, biosynthesis of PDIM.

Insights into the ancestry evolution of the Mycobacterium tuberculosis complex from analysis of Mycobacterium riyadhense.

Current evolutionary scenarios posit the emergence of Mycobacterium tuberculosis from an environmental saprophyte through a cumulative process of genome adaptation. Mycobacterium riyadhense, a related bacillus, is being increasingly isolated from human clinical cases with tuberculosis-like symptoms in various parts of the world. To elucidate the evolutionary relationship between M. riyadhense and other mycobacterial species, including members of the M. tuberculosis complex (MTBC), eight clinical isolates of M. riyadhense were sequenced and analyzed. We show, among other features, that M. riyadhense shares a large number of conserved orthologs with M. tuberculosis and shows the expansion of toxin/antitoxin pairs, PE/PPE family proteins compared with other non-tuberculous mycobacteria. We observed M. riyadhense lacks wecE gene which may result in the absence of lipooligosaccharides (LOS) IV. Comparative transcriptomic analysis of infected macrophages reveals genes encoding inducers of Type I IFN responses, such as cytosolic DNA sensors, were relatively less expressed by macrophages infected with M. riyadhense or M. kansasii, compared to BCG or M. tuberculosis. Overall, our work sheds new light on the evolution of M. riyadhense, its relationship to the MTBC, and its potential as a system for the study of mycobacterial virulence and pathogenesis.

Biochemical and phenotypic characterisation of the Mycobacterium smegmatis transporter UspABC.

Mycobacterium tuberculosis (Mtb) is an intracellular human pathogen that has evolved to survive in a nutrient limited environment within the host for decades. Accordingly, Mtb has developed strategies to acquire scarce nutrients and the mycobacterial transporter systems provide an important route for the import of key energy sources. However, the physiological role of the Mtb transporters and their substrate preference(s) are poorly characterised. Previous studies have established that the Mtb UspC solute-binding domain recognises amino- and phosphorylated-sugars, indicating that the mycobacterial UspABC transporter plays a key role in the import of peptidoglycan precursors. Herein, we have used a wide array of approaches to investigate the role of UspABC in Mycobacterium smegmatis by analysis of mutant strains that either lack the solute binding domain: ΔuspC or the entire transport complex: ΔuspABC. Analysis of mycobacterial transcripts shows that the uspABC system is functionally expressed in mycobacteria as a contiguous reading frame. Topology mapping confirms an Nin-Cin orientation of the UspAB integral membrane spanning domains. Phenotypic microarray profiling of commercially available sugars suggests, unexpectedly, that the uspC and ΔuspABC mutants had different carbon utilisation profiles and that neither strain utilised glucose-1-phosphate. Furthermore, proteomics analysis showed an alteration in the abundance of proteins involved in sugar and lipid metabolism, crucial for cell envelope synthesis, and we propose that UspABC has an important role in determining the interplay between these pathways.

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis.

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

Mycobacterium abscessus biofilms produce an extracellular matrix and have a distinct mycolic acid profile.

A non-tuberculous mycobacterium, Mycobacterium abscessus is an emerging opportunistic pathogen associated with difficult to treat pulmonary infections, particularly in patients suffering from cystic fibrosis. It is capable of forming biofilms in vitro that result in an increase of already high levels of antibiotic resistance in this bacterium. Evidence that M. abscessus forms biofilm-like microcolonies in patient lungs and on medical devices further implicated the need to investigate this biofilm in detail. Therefore, in this study we characterized the M. abscessus pellicular biofilm, formed on a liquid-air interface, by studying its molecular composition, and its transcriptional profile in comparison to planktonic cells. Using scanning electron micrographs and fluorescence microscopy, we showed that M. abscessus biofilms produce an extracellular matrix composed of lipids, proteins, carbohydrates and extracellular DNA. Transcriptomic analysis of biofilms revealed an upregulation of pathways involved in the glyoxylate shunt, redox metabolism and mycolic acid biosynthesis. Genes involved in elongation and desaturation of mycolic acids were highly upregulated in biofilms and, mirroring those findings, biochemical analysis of mycolates revealed molecular changes and an increase in mycolic acid chain length. Together these results give us an insight into the complex structure of M. abscessus biofilms, the understanding of which may be adapted for clinical use in treatment of biofilm infections, including strategies for dispersing the extracellular matrix, allowing antibiotics to gain access to bacteria within the biofilm.

Cyclic di-GMP sensing histidine kinase PdtaS controls mycobacterial adaptation to carbon sources.

Cell signaling relies on second messengers to transduce signals from the sensory apparatus to downstream signaling pathway components. In bacteria, one of the most important and ubiquitous second messenger is the small molecule cyclic diguanosine monophosphate (c-di-GMP). While the biosynthesis, degradation, and regulatory pathways controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP controls these processes are not entirely understood. Herein we present the report of a c-di-GMP sensing sensor histidine kinase PdtaS (Rv3220c), which binds to c-di-GMP at submicromolar concentrations, subsequently perturbing signaling of the PdtaS-PdtaR (Rv1626) two-component system. Aided by biochemical analysis, genetics, molecular docking, FRET microscopy, and structural modelling, we have characterized the binding of c-di-GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in Mycobacterium smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. The perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes recorded by a multiparametric systems' approach of transcriptomics, unbiased metabolomics, and lipid analyses.

Generation of Liposomes to Study the Effect of Mycobacterium Tuberculosis Lipids on HIV-1 cis- and trans-Infections.

Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.

Fundamental Cell Morphologies Examined With Cryo-TEM of the Species in the Novel Five Genera Robustly Correlate With New Classification in Family Mycobacteriaceae.

A recent study proposed the novel classification of the family Mycobacteriaceae based on the genome analysis of core proteins in 150 Mycobacterium species. The results from these analyses supported the existence of five distinct monophyletic groups within the genus Mycobacterium. That is, Mycobacterium has been divided into two novel genera for rapid grower Mycobacteroides and Mycolicibacterium, and into three genera for slow grower Mycolicibacter, Mycolicibacillus, and an emended genus Mycobacterium, which include all the major human pathogens. Here, cryo-TEM examinations of 1,816 cells of 31 species (34 strains) belonging to the five novel genera were performed. The fundamental morphological properties of every single cell, such as cell diameter, cell length, cell perimeter, cell circularity, and aspect ratio were measured and compared between these genera. In 50 comparisons on the five parameters between any two genera, only five comparisons showed "non-significant" differences. That is, there are non-significant differences between slow grower genus Mycolicibacillus and genus Mycobacterium in average cell diameter (p = 0.15), between rapid grower genus Mycobacteroides and slow grower genus Mycobacterium in average cell length (p > 0.24), between genus Mycobacteroides and genus Mycobacterium (p > 0.68) and between genus Mycolicibacter and genus Mycolicibacillus (p > 0.11) in average cell perimeter, and between genus Mycolicibacterium and genus Mycobacterium in circularity (p > 0.73). The other 45 comparisons showed significant differences between the genera. Genus Mycobacteroides showed the longest average cell diameter, whereas the genus Mycolicibacter showed the shortest average diameter. Genus Mycolicibacterium showed the most extended average cell length, perimeter, and aspect ratio, whereas the genus Mycolicibacillus showed the shortest average cell length, perimeter, and aspect ratio. Genus Mycolicibacillus showed the highest average cell circularity, whereas genus Mycobacterium showed the lowest average cell circularity. These fundamental morphological data strongly support the new classification in the family Mycobacteriaceae, and this classification is rational and effective in the study of the members of the family Mycobacteriaceae. Because both the genus Mycolicibacterium and the genus Mycobacterium contain many species and showed larger significant standard deviations in every parameter, these genera may be divided into novel genera which show common genotype and phenotypes in morphology and pathogenicity.

Biofilms of the non-tuberculous Mycobacterium chelonae form an extracellular matrix and display distinct expression patterns.

Mycobacterium chelonae is an environmental, non-tuberculous mycobacterial species, capable of causing infections in humans. Biofilm formation is a key strategy used by M. chelonae in colonising niches in the environment and in the host. We studied a water-air interface (pellicle) biofilm of M. chelonae using a wide array of approaches to outline the molecular structure and composition of the biofilm. Scanning electron micrographs showed that M. chelonae biofilms produced an extracellular matrix. Using a combination of biochemical analysis, Raman spectroscopy, and fluorescence microscopy, we showed the matrix to consist of proteins, carbohydrates, lipids and eDNA. Glucose was the predominant sugar present in the biofilm matrix, and its relative abundance decreased in late (established) biofilms. RNA-seq analysis of the biofilms showed upregulation of genes involved in redox metabolism. Additionally, genes involved in mycolic acid, other lipid and glyoxylate metabolism were also upregulated in the early biofilms.

A mycobacterial DivIVA domain-containing protein involved in cell length and septation.

Mycobacterial cells elongate via polar deposition of cell wall material, similar to the filamentous Streptomyces species, which contain a tip-organizing centre. Coiled-coiled proteins such as DivIVA play an important role in this process. The genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes many coiled-coil proteins that are homologous to DivIVA with a potential role in mycobacterial cell elongation. Here we describe studies on Mycobacterium smegmatis MSMEG_2416, a homologue of M. tuberculosis Rv2927c. Two previous independent studies showed that MSMEG_2416 was involved in septation (subsequently referred to as sepIVA). Contrary to these previous reports, we found sepIVA to be dispensable for growth in laboratory media by generating a viable null mutant. The mutant strain did, however, show a number of differences, including a change in colony morphology and biofilm formation that could be reversed on complementation with sepIVA as well as Rv2927c, the sepIVA homologue from M. tuberculosis. However, analysis of cell wall lipids did not reveal any alterations in lipid profiles of the mutant strain. Microscopic examination of the mutant revealed longer cells with more septa, which occurred at irregular intervals, often generating mini-compartments, a profile similar to that observed in the previous studies following conditional depletion, highlighting a role for sepIVA in mycobacterial growth.

Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1ß production.

Genetic diversity of Mycobacterium tuberculosis affects immune responses and clinical outcomes of tuberculosis (TB). However, how bacterial diversity orchestrates immune responses to direct distinct TB severities is unknown. Here we study 681 patients with pulmonary TB and show that M. tuberculosis isolates from cases with mild disease consistently induce robust cytokine responses in macrophages across multiple donors. By contrast, bacteria from patients with severe TB do not do so. Secretion of IL-1ß is a good surrogate of the differences observed, and thus to classify strains as probable drivers of different TB severities. Furthermore, we demonstrate that M. tuberculosis isolates that induce low levels of IL-1ß production can evade macrophage cytosolic surveillance systems, including cGAS and the inflammasome. Isolates exhibiting this evasion strategy carry candidate mutations, generating sigA recognition boxes or affecting components of the ESX-1 secretion system. Therefore, we provide evidence that M. tuberculosis strains manipulate host-pathogen interactions to drive variable TB severities.

The mycolic acid reductase Rv2509 has distinct structural motifs and is essential for growth in slow-growing mycobacteria.

The final step in mycolic acid biosynthesis in Mycobacterium tuberculosis is catalysed by mycolyl reductase encoded by the Rv2509 gene. Sequence analysis and homology modelling indicate that Rv2509 belongs to the short-chain fatty acid dehydrogenase/reductase (SDR) family, but with some distinct features that warrant its classification as belonging to a novel family of short-chain dehydrogenases. In particular, the predicted structure revealed a unique α-helical C-terminal region which we demonstrated to be essential for Rv2509 function, though this region did not seem to play any role in protein stabilisation or oligomerisation. We also show that unlike the M. smegmatis homologue which was not essential for growth, Rv2509 was an essential gene in slow-growing mycobacteria. A knockdown strain of the BCG2529 gene, the Rv2509 homologue in Mycobacterium bovis BCG, was unable to grow following the conditional depletion of BCG2529. This conditional depletion also led to a reduction of mature mycolic acid production and accumulation of intermediates derived from 3-oxo-mycolate precursors. Our studies demonstrate novel features of the mycolyl reductase Rv2509 and outline its role in mycobacterial growth, highlighting its potential as a new target for therapies.

Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle.

BACKGROUND: BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG's efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host's immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria. RESULTS: To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host. CONCLUSION: These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host's innate immune system to allow it to persist, a property that is important for its protective efficacy.