Induction of macrophage necrosis is an important strategy used by virulent Mycobacterium tuberculosis (Mtb) to avoid innate host defense. In contrast, attenuated Mtb causes apoptosis, which limits bacterial replication and promotes T cell cross priming by antigen presenting cells. Here we demonstrated that Mtb infection causes plasma membrane microdisruptions. Resealing of these lesions—a process crucial for preventing necrosis and promoting apoptosis—required the translocation of lysosome and Golgi apparatus-derived vesicles to the plasma membrane. Plasma membrane repair depended on prostaglandin E2 (PGE2), which regulates synaptotagmin 7, the Ca++ sensor involved in the lysosome-mediated repair mechanism. By inducing production of lipoxin A4 (LXA4), which blocks PGE2 biosynthesis, virulent Mtb prevented membrane repair and induced necrosis. Thus, virulent Mtb impairs macrophage plasma membrane repair to evade host defenses.

Summary Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M. tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to death mechanisms, leaving the mechanisms underlying this observation unresolved. We find that following apoptosis, M. tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M. tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M. tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism.

Summary The lipidic envelope of Mycobacterium tuberculosis promotes virulence in many ways, so we developed a lipidomics platform for broad survey of cell walls. Here we report two new databases (MycoMass, MycoMap), 30 lipid fine maps and mass spectrometry datasets that comprise a static lipidome. Further, by rapidly regenerating lipidomic datasets during biological processes, comparative lipidomics provides statistically valid, organism-wide comparisons that broadly assess lipid changes during infection or among clinical strains of mycobacteria. Using stringent data filters, we tracked more than 5,000 molecular features in parallel with few or no false positive molecular discoveries. The low error rates allowed the first chemotaxonomic analyses of mycobacteria, which describe the extent of chemical change in each strain and identified particular strain-specific molecules for use as biomarkers.

The fate of infected macrophages has an essential role in protection against Mycobacterium tuberculosis by regulating innate and adaptive immunity. M. tuberculosis exploits cell necrosis to exit from macrophages and spread. In contrast, apoptosis, which is characterized by an intact plasma membrane, is an innate mechanism that results in lower bacterial viability. Virulent M. tuberculosis inhibits apoptosis and promotes necrotic cell death by inhibiting production of prostaglandin E2. Here we show that by activating the 5-lipoxygenase pathway, M. tuberculosis not only inhibited apoptosis but also prevented cross-presentation of its antigens by dendritic cells, which impeded the initiation of T cell immunity. Our results explain why T cell priming in response to M. tuberculosis is delayed and emphasize the importance of early immunity.

Wolbachia are widespread maternally-transmitted intracellular bacteria that infect most insect species and are able to alter the reproduction of innumerous hosts. The cellular bases of these alterations remain largely unknown. Here we report that Drosophila mauritiana infected with a native Wolbachia wMau strain produces about four times more eggs than the non-infected counterpart. Wolbachia infection leads to an increase in the mitotic activity of germline stem cells (GSCs) as well as a decrease in programmed cell death in the germarium. Our results suggest that upregulation of GSCs division is mediated by a tropism of Wolbachia for the germline stem cell niche (GSCN), the cellular microenvironment that supports GSCs.

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

4 suggesting that CMYA3 is directly regulated by Ang II signaling. This gene, since named Xirp2 (also known as mXin␤ and myomaxin), is a direct target of the MEF2A transcription factor and is markedly downregulated in hearts lacking MEF2A. 5,6 Xirp2 belongs to the ancient, muscle-specific, actin-binding Xin gene family whose expression can be traced to ancestral vertebrates with a 2-chambered heart. 7-9 Xirp2 is expressed in cardiac and skeletal muscle where it interacts with filamentous actin and ␣-actinin through the novel actin-binding motif, the Xin repeat. 5,8 In striated muscle, Xirp2 localizes to the peripheral Z-disc region, or costamere, 5 and the intercalated disk. 10,11 The subcellular localization of Xirp2 is significant in that the costamere and intercalated disk harbor mechanical stress sensors that are critical for normal muscle function. [12][13][14] Antisense knockdown of Xin in developing chick embryos, the sole Xin family member in this species, results in a severe disruption of cardiac looping morphogenesis. 9 In mice, a lossof-function mutation of mXin␣, the mammalian ortholog of Xin, results in cardiomyopathy and conduction defects. 11 In the present study we sought to determine the role of Xirp2 in cardiac development and/or function. Mice harboring a hypomorphic Xirp2 allele are viable but display cardiac hypertrophy. As Xirp2 is regulated by Ang II, we also examined cardiac pathology in hypomorphic mice with long-term administration of this hormone. In contrast to wild type mice exposed to a chronic Ang II infusion, hypomorphic mice displayed diminished cardiac hypertrophy, fibrosis, and apoptosis. Furthermore, we demonstrate that regulation of Xirp2 gene expression in response to Ang II signaling is mediated by MEF2A. Our results suggest that Original

The Mef2 family of transcription factors regulates muscle differentiation, but the specific gene programs controlled by each member remain unknown. Characterization of Mef2A knockout mice has revealed severe myofibrillar defects in cardiac muscle indicating a requirement for Mef2A in cytoarchitectural integrity. Through comprehensive expression analysis of Mef2A-deficient hearts, we identified a cohort of dysregulated genes whose products localize to the peripheral Z-disc/ costamere region. Many of these genes are essential for costamere integrity and function. Here we demonstrate that these genes are directly regulated by Mef2A, establishing a mechanism by which Mef2A controls the costamere. In an independent model system, acute knockdown of Mef2A in primary neonatal cardiomyocytes resulted in profound malformations of myofibrils and focal adhesions accompanied by adhesion-dependent programmed cell death. These findings indicate a role for Mef2A in cardiomyocyte survival through regulation of costamere integrity. Finally, bioinformatics analysis identified over-represented transcription factor-binding sites in this network of costamere promoters that may provide insight into the mechanism by which costamere genes are regulated by Mef2A. The global control of costamere gene expression adds another dimension by which this essential macromolecular complex may be regulated in health and disease.