Jeffery S. Cox

Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase

The transcription of genes encoding soluble proteins that reside in the endoplasmic reticulum (ER) is induced when unfolded proteins accumulate in the ER. Thus, an intracellular signal transduction pathway must exist that mediates communication between the ER lumen and the nucleus. We have identified a gene in S. cerevisiae, IRE1, that is required for this pathway: ire1- mutants cannot activate transcription of KAR2 and PDI1, which encode the ER resident proteins BiP and protein disulfide isomerase. Moreover, IRE1 is essential for cell viability under stress conditions that cause unfolded proteins to accumulate in the ER. IRE1 encodes a transmembrane serine/threonine kinase that we propose transmits the unfolded protein signal across the ER or inner nuclear membrane. IRE1 is also required for inositol prototrophy, suggesting that the induction of ER resident proteins is coupled to the biogenesis of new ER membrane.

A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response

Cells respond to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing transcription of genes encoding ER-resident proteins. The information is transmitted from the ER lumen to the nucleus by an intracellular signaling pathway, the unfolded protein response (UPR). We have identified a basic-leucine zipper transcription factor, Hac1p, that is required for the UPR and binds to the UPR element in the promoter of UPR-regulated genes. Surprisingly, Hac1p is found in UPR-activated cells only, and its level is controlled by regulated splicing of its mRNA. Splicing replaces the C-terminal tail of Hac1p with a different peptide that renders Hac1p more resistant to an otherwise extremely rapid ubiquitin-dependent degradation. We propose that the complex regulation of Hac1p expression serves to provide multiple levels at which the UPR can be controlled.

Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice

Tuberculosis is the leading cause of death in the world resulting from a single bacterial infection. Despite its enormous burden on world health, little is known about the molecular mechanisms of pathogenesis of Mycobacterium tuberculosis. Bacterial multiplication and concomitant tissue damage within an infected host, including experimentally infected mice, occurs primarily in the lungs—the favoured niche of M. tuberculosis. Although it has been proposed that the distinctive cell wall of M. tuberculosis is important for virulence, rigorous genetic proof has been lacking. Here, using signature-tagged mutagenesis, we isolated three attenuated M. tuberculosis mutants that cannot synthesize or transport a complex, cell wall-associated lipid called phthiocerol dimycocerosate (PDIM) which is found only in pathogenic mycobacteria. Two mutants have transposon insertions affecting genes implicated in PDIM synthesis; the third has a disruption in a gene encoding a large transmembrane protein required for proper subcellular localization of PDIM. Synthesis and transport of this complex lipid is only required for growth in the lung; all three mutants are unaffected for growth in the liver and spleen. This clearly shows that a lipid is required for M. tuberculosis virulence.

A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis.

Colonial morphology of pathogenic bacteria is often associated with virulence. For M. tuberculosis, the causative agent of tuberculosis (TB), virulence is correlated with the formation of serpentine cords, a morphology that was first noted by Koch. We identified a mycobacterial gene, pcaA, that we show is required for cording and mycolic acid cyclopropane ring synthesis in the cell wall of both BCG and M. tuberculosis. Furthermore, we show that mutants of pcaA fail to persist within and kill infected mice despite normal initial replication. These results indicate that a novel member of a family of cyclopropane synthetases is necessary for lethal chronic persistent M. tuberculosis infection and define a role for cyclopropanated lipids in bacterial pathogenesis.

Type VII secretion — mycobacteria show the way

Recent evidence shows that mycobacteria have developed novel and specialized secretion systems for the transport of extracellular proteins across their hydrophobic, and highly impermeable, cell wall. Strikingly, mycobacterial genomes encode up to five of these transport systems. Two of these systems, ESX-1 and ESX-5, are involved in virulence — they both affect the cell-to-cell migration of pathogenic mycobacteria. Here, we discuss this novel secretion pathway and consider variants that are present in various Gram-positive bacteria. Given the unique composition of this secretion system, and its general importance, we propose that, in line with the accepted nomenclature, it should be called type VII secretion.

Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system

Although many bacterial pathogens use specialized secretion systems for virulence, no such systems have been described for Mycobacterium tuberculosis, a major pathogen of humans that proliferates in host macrophages. In a screen to identify genes required for virulence of M. tuberculosis, we have discovered three components and two substrates of the first Sec-independent secretion pathway described in M. tuberculosis, which we designate the Snm pathway. Here we demonstrate that the proteins Snm1, -2, and -4 are required for the secretion of ESAT-6 and CFP-10, small proteins previously identified as major T cell antigens. Snm2, a member of the AAA ATPase family, interacts with substrates and with Snm1, another AAA ATPase. We show that M. tuberculosis mutants lacking either the Snm system or these substrates exhibit defects in bacterial growth during the acute phase of a mouse infection and are attenuated for virulence. Strikingly, snm mutants fail to replicate in cultured macrophages and to inhibit macrophage inflammatory responses, two well established activities of wild-type M. tuberculosis bacilli. Thus, the Snm secretion pathway works to subvert normal macrophage responses and is a major determinant of M. tuberculosis virulence.

tRNA Ligase Is Required for Regulated mRNA Splicing in the Unfolded Protein Response

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers an intracellular signaling pathway, the unfolded protein response (UPR), that leads to increased transcription of genes encoding ER-resident proteins. Transcriptional activation is mediated by a dedicated transcription factor, Hac1p, whose activity is controlled by regulated splicing of its mRNA. We have identified a mutation in tRNA ligase that disrupts the UPR in the yeast Saccharomyces cerevisiae. In this mutant, splicing of HAC1 mRNA, but not tRNA, is blocked. In contrast, HAC1 mRNA splicing is not impaired in cells that are blocked in spliceosome-mediated mRNA splicing. Furthermore, the splice junctions of HAC1 mRNA do not conform to the consensus sequences of other yeast pre-mRNAs. Our results suggest that the regulated splicing of HAC1 mRNA occurs by a novel pathway, involving tRNA ligase and bypassing the spliceosome.

Extracellular M. tuberculosis DNA Targets Bacteria for Autophagy by Activating the Host DNA-Sensing Pathway

Eukaryotic cells sterilize the cytosol by using autophagy to route invading bacterial pathogens to the lysosome. During macrophage infection with Mycobacterium tuberculosis, a vacuolar pathogen, exogenous induction of autophagy can limit replication, but the mechanism of autophagy targeting and its role in natural infection remain unclear. Here we show that phagosomal permeabilization mediated by the bacterial ESX-1 secretion system allows cytosolic components of the ubiquitin-mediated autophagy pathway access to phagosomal M. tuberculosis. Recognition of extracelluar bacterial DNA by the STING-dependent cytosolic pathway is required for marking bacteria with ubiquitin, and delivery of bacilli to autophagosomes requires the ubiquitin-autophagy receptors p62 and NDP52 and the DNA-responsive kinase TBK1. Remarkably, mice with monocytes incapable of delivering bacilli to the autophagy pathway are extremely susceptible to infection. Our results reveal an unexpected link between DNA sensing, innate immunity, and autophagy and indicate a major role for this autophagy pathway in resistance to M. tuberculosis infection.

Comprehensive Functional Analysis of Mycobacterium tuberculosis Toxin-Antitoxin Systems: Implications for Pathogenesis, Stress Responses, and Evolution

Toxin-antitoxin (TA) systems, stress-responsive genetic elements ubiquitous in microbial genomes, are unusually abundant in the major human pathogen Mycobacterium tuberculosis. Why M. tuberculosis has so many TA systems and what role they play in the unique biology of the pathogen is unknown. To address these questions, we have taken a comprehensive approach to identify and functionally characterize all the TA systems encoded in the M. tuberculosis genome. Here we show that 88 putative TA system candidates are present in M. tuberculosis, considerably more than previously thought. Comparative genomic analysis revealed that the vast majority of these systems are conserved in the M. tuberculosis complex (MTBC), but largely absent from other mycobacteria, including close relatives of M. tuberculosis. We found that many of the M. tuberculosis TA systems are located within discernable genomic islands and were thus likely acquired recently via horizontal gene transfer. We discovered a novel TA system located in the core genome that is conserved across the genus, suggesting that it may fulfill a role common to all mycobacteria. By expressing each of the putative TA systems in M. smegmatis, we demonstrate that 30 encode a functional toxin and its cognate antitoxin. We show that the toxins of the largest family of TA systems, VapBC, act by inhibiting translation via mRNA cleavage. Expression profiling demonstrated that four systems are specifically activated during stresses likely encountered in vivo, including hypoxia and phagocytosis by macrophages. The expansion and maintenance of TA genes in the MTBC, coupled with the finding that a subset is transcriptionally activated by stress, suggests that TA systems are important for M. tuberculosis pathogenesis.

The Type I IFN Response to Infection with Mycobacterium tuberculosis Requires ESX-1-Mediated Secretion and Contributes to Pathogenesis

The ESX-1 secretion system is a major determinant of Mycobacterium tuberculosis virulence, although the pathogenic mechanisms resulting from ESX-1-mediated transport remain unclear. By global transcriptional profiling of tissues from mice infected with either wild-type or ESX-1 mutant bacilli, we found that host genes controlled by ESX-1 in vivo are predominantly IFN regulated. ESX-1-mediated secretion is required for the production of host type I IFNs during infection in vivo and in macrophages in vitro. The macrophage signaling pathway leading to the production of type I IFN required the host kinase TANK-binding kinase 1 and occurs independently of TLR signaling. Importantly, the induction of type I IFNs during M. tuberculosis infection is a pathogenic mechanism as mice lacking the type I IFNR were more restrictive for bacterial growth in the spleen than wild-type mice, although growth in the lung was unaffected. We propose that the ESX-1 secretion system secretes effectors into the cytosol of infected macrophages, thereby triggering the type I IFN response for the manipulation of host immunity.