SummaryTuberculosis is a massive public health problem on a global scale and the success of Mycobacterium tuberculosis is linked to its ability to persist within humans for long periods without causing any overt disease symptoms. Hypoxia is predicted to be a key host-induced stress limiting growth of the pathogen in vivo. However, multiple studies in vitro and in vivo indicate that M. tuberculosis adapts to oxygen limitation by entering into a metabolically altered state, while awaiting the opportunity to reactivate. Molecular signatures of bacteria adapted to hypoxia in vitro are accumulating, although correlations to human disease are only now being established. Similarly, defining the mechanisms that control this adaptation is an active area of research. In this review we discuss the historical precedents linking hypoxia and latency, and the gathering knowledge of M. tuberculosis hypoxic responses. We also examine the role of these responses in tuberculosis latency, and identify promising avenues for future studies.

Mycobacterium tuberculosis (MTB) enters a non-replicating state when exposed to low oxygen tension, a condition the bacillus encounters in granulomas during infection. Determining how mycobacteria enter and maintain this state is a major focus of research. However, from a public health standpoint the importance of latent TB is its ability to reactivate. The mechanism by which mycobacteria return to a replicating state upon re-exposure to favorable conditions is not understood. In this study, we utilized reaeration from a defined hypoxia model to characterize the adaptive response of MTB following a return to favorable growth conditions. Global transcriptional analysis identified the ∼100 gene Reaeration Response, induced relative to both log-phase and hypoxic MTB. This response includes chaperones and proteases, as well as the transcription factor Rv2745c, which we characterize as a Clp protease gene regulator (ClgR) orthologue. During reaeration, genes repressed during hypoxia are also upregulated in a wave of transcription that includes genes crucial to transcription, translation and oxidative phosphorylation and culminates in bacterial replication. In sum, this study defines a new transcriptional response of MTB with potential relevance to disease, and implicates ClgR as a regulator involved in resumption of replication following hypoxia.

Rosiglitazone is an insulin-sensitizing agent that has recently been shown to exert beneficial effects on atherosclerosis. In addition to peroxisome proliferator-activated receptor (PPAR)-␥, rosiglitazone can affect other targets, such as directly inhibiting recombinant long-chain acylCoA synthetase (ACSL)-4 activity. Because it is unknown if ACSL4 is expressed in vascular cells involved in atherosclerosis, we investigated the ability of rosiglitazone to inhibit ACSL activity and fatty acid partitioning in human and murine arterial smooth muscle cells (SMCs) and macrophages. Human and murine SMCs and human macrophages expressed Acsl4, and rosiglitazone inhibited Acsl activity in these cells. Furthermore, rosiglitazone acutely inhibited partitioning of fatty acids into phospholipids in human SMCs and inhibited fatty acid partitioning into diacylglycerol and triacylglycerol in human SMCs and macrophages through a PPAR-␥-independent mechanism. Conversely, murine macrophages did not express ACSL4, and rosiglitazone did not inhibit ACSL activity in these cells, nor did it affect acute fatty acid partitioning into cellular lipids. Thus, rosiglitazone inhibits ACSL activity and fatty acid partitioning in human and murine SMCs and in human macrophages through a PPAR-␥-independent mechanism likely to be mediated by ACSL4 inhibition. Therefore, rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis.

CSdeconv is a novel method for determining the location of transcription factor binding from ChIP-seq data that discriminates closely-spaced sites.

Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis, shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis, we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1, a polyketide synthase gene. When fused with pks15, as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1. In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence.

Host defense against infection can broadly be categorized into systemic immunity and cell-autonomous immunity. Systemic immunity is crucial for all multicellular organisms, increasing in importance with increasing cellular complexity of the host. The systemic immune response to Listeria monocytogenes has been studied extensively in murine models; however, the clinical applicability of these findings to the human newborn remains incompletely understood. Furthermore, the ability to control infection at the level of an individual cell, known as “cell-autonomous immunity,” appears most relevant following infection with L. monocytogenes; as the main target, the monocyte is centrally important to innate as well as adaptive systemic immunity to listeriosis. We thus suggest that the overall increased risk to suffer and die from L. monocytogenes infection in the newborn period is a direct consequence of age-dependent differences in cell-autonomous immunity of the monocyte to L. monocytogenes. We here review what is known about age-dependent differences in systemic innate and adaptive as well as cell-autonomous immunity to infection with Listeria monocytogenes.

Chlamydiae exit via membrane-encased extrusion or through lysis of the host cell. Extrusions are novel, pathogen-containing structures that confer infectious advantages to Chlamydia, and are hypothesized to promote cell-to-cell spread, dissemination to distant tissues and facilitate immune evasion. The extrusion phenomenon has been characterized for several Chlamydia trachomatis serovars, but a thorough investigation of extrusion for additional clinically relevant C. trachomatis strains and Chlamydia species has yet to be performed. The key parameters investigated in this study were: (i) the conservation of extrusion across the Chlamydia genus, (ii) the functional requirement for candidate Chlamydia genes in extrusion formation i.e. IncA and CT228 and (iii) extrusion-mediated uptake, and consequent survival of Chlamydia inside macrophages. Inclusion morphology was characterized by live fluorescence microscopy, using an inverted GFP strategy, at early and mid-stages of infection. Enriched extrusions were used to infect bone marrow-derived macrophages, and bacterial viability was measured following macrophage engulfment. Our results demonstrate that extrusion is highly conserved across chlamydiae, including ocular, STD and LGV biovars and divergent Chlamydia species. Consequently, this exit mechanism for Chlamydia may fulfill common advantages important for pathogenesis.

The strategies utilized by pathogens to exit host cells are an area of pathogenesis which has received surprisingly little attention, considering the necessity of this step for infections to propagate. Even less is known about how exit through these pathways affects downstream host-pathogen interactions and the generation of an immune response. Chlamydia trachomatis exits host epithelial cells through two equally active mechanisms: lysis and extrusion. Studies have characterized the outcome of interactions between host innate immune cells, such as dendritic cells and macrophages, and free, extracellular Chlamydia bacteria, such as those resulting from lysis. Exit via extrusion generates a distinct, host-membranebound compartment of Chlamydia separate from the original infected cell. In this study, we assessed the effect of containment within extrusions upon the interaction between Chlamydia and host dendritic cells. Extrusion dramatically affected the outcome of Chlamydia-dendritic cell interactions for both the bacterium and the host cell. Dendritic cells rapidly underwent apoptosis in response to engulfment of an extrusion, while uptake of an equivalent dose of free Chlamydia had no such effect. Containment within an extrusion also prolonged bacterial survival within dendritic cells and altered the initial innate immune signaling by the dendritic cell.KEYWORDS Chlamydia, apoptosis, dendritic cell, extrusion C hlamydia trachomatis is a highly successful Gram-negative bacterial pathogen, being the leading bacterial cause of sexually transmitted infections and the leading cause of infectious blindness globally (1-3). In the absence of diagnosis and treatment, Chlamydia infections can lead to severe long-term outcomes, such as chronic pelvic pain, infertility, and ectopic pregnancy (4, 5). During infection, innate and adaptive immune responses are mounted against Chlamydia; however, approximately 50% of infections last for a year or more (6). Even after the resolution of infection, only partial protective immunity is achieved and reinfection is common (7-9). This suggests that Chlamydia is adept at establishing and sustaining infection in the face of immune recognition.Due to Chlamydia's obligate intracellular nature and a historically limited genetic toolbox, key attributes of the host-Chlamydia interaction are still unknown. Chlamydia infection is initiated by the uptake of metabolically inactive elementary bodies (EB) into mucosal epithelial cells, which transition into reticulate bodies (RB) within a vacuole called an inclusion. Here, the metabolically active RB replicates robustly to generate hundreds of Chlamydia bacteria that undergo transition back to EB (10). Chlamydia inhibits apoptosis of the host epithelial cell, ensuring its ability to complete this replication cycle before exit from the cell (11-13). Bacterial exit from host cells is a crucial but underscrutinized stage of the life cycle of this and other intracellular pathogens. Chlamydia possesses two distinct, equally prevalent exit mechan...