Complete Genome Sequences of Cluster A Mycobacteriophages BobSwaget, Fred313, KADY, Lokk, MyraDee, Stagni, and StepMih.

Seven mycobacteriophages from distinct geographical locations were isolated, using Mycobacterium smegmatis mc2155 as the host, and then purified and sequenced. All of the genomes are related to cluster A mycobacteriophages, BobSwaget and Lokk in subcluster A2; Fred313, KADY, Stagni, and StepMih in subcluster A3; and MyraDee in subcluster A18, the first phage to be assigned to that subcluster.

Macrophage cell death and transcriptional response are actively triggered by the fungal virulence factor Cbp1 during H. capsulatum infection.

Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally, Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis.

Rapid quantification of pathogenic fungi by Cellometer image-based cytometry.

The objective of this study was to develop an image-based cytometric methodology for the quantification of viable pathogenic yeasts, which can offer increased sensitivity and efficiency when compared to the traditional colony forming unit (CFU) assay. Live/dead yeast quantification by flow cytometry has been previously demonstrated, however, adoption of flow cytometric detection of pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome. Here, we quantitatively assess the growth of H. capsulatum yeasts within RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with Cellometer image-based cytometry; this method faithfully recapitulates growth trends as measured by traditional CFU enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of bone marrow-derived macrophages with a GFP-expressing strain of C. albicans. To demonstrate that image-based cytometry can be used as a tool to assess the susceptibility of fungi to antifungal drugs, we perform dose response experiments with the antifungal drugs amphotericin B and itraconazole and show that image-based cytometry allows rapid assessment of the kinetics of cytotoxicity induced by these antifungals. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens, either alone or in association with host cells.

Conidia but not yeast cells of the fungal pathogen Histoplasma capsulatum trigger a type I interferon innate immune response in murine macrophages.

Histoplasma capsulatum is the most common cause of fungal respiratory infections and can lead to progressive disseminated infections, particularly in immunocompromised patients. Infection occurs upon inhalation of the aerosolized spores, known as conidia. Once inside the host, conidia are phagocytosed by alveolar macrophages. The conidia subsequently germinate and produce a budding yeast-like form that colonizes host macrophages and can disseminate throughout host organs and tissues. Even though conidia are the predominant infectious particle for H. capsulatum and are the first cell type encountered by the host during infection, very little is known at a molecular level about conidia or about their interaction with cells of the host immune system. We examined the interaction between conidia and host cells in a murine bone-marrow-derived macrophage model of infection. We used whole-genome expression profiling and quantitative reverse transcription-PCR (qRT-PCR) to monitor the macrophage signaling pathways that are modulated during infection with conidia. Our analysis revealed that type I interferon (IFN)-responsive genes and the beta type I IFN (IFN-beta) were induced in macrophages during infection with H. capsulatum conidia but not H. capsulatum yeast cells. Further analysis revealed that the type I IFN signature induced in macrophages in response to conidia is independent of Toll-like receptor (TLR) signaling and the cytosolic RNA sensor MAVS but is dependent on the transcription factor interferon regulatory factor 3 (IRF3). Interestingly, H. capsulatum growth was restricted in mice lacking the type I IFN receptor, indicating that an intact host type I IFN response is required for full virulence of H. capsulatum in mice.

MyoD synergizes with the E-protein HEB beta to induce myogenic differentiation.

The MyoD family of basic helix-loop-helix transcription factors function as heterodimers with members of the E-protein family to induce myogenic gene activation. The E-protein HEB is alternatively spliced to generate alpha and beta isoforms. While the function of these molecules has been studied in other cell types, questions persist regarding the molecular functions of HEB proteins in skeletal muscle. Our data demonstrate that HEB alpha expression remains unchanged in both myoblasts and myotubes, whereas HEB beta is upregulated during the early phases of terminal differentiation. Upon induction of differentiation, a MyoD-HEB beta complex bound the E1 E-box of the myogenin promoter leading to transcriptional activation. Importantly, forced expression of HEB beta with MyoD synergistically lead to precocious myogenin expression in proliferating myoblasts. However, after differentiation, HEB alpha and HEB beta synergized with myogenin, but not MyoD, to activate the myogenin promoter. Specific knockdown of HEB beta by small interfering RNA in myoblasts blocked differentiation and inhibited induction of myogenin transcription. Therefore, HEB alpha and HEB beta play novel and central roles in orchestrating the regulation of myogenic factor activity through myogenic differentiation.

Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters.

We used a combination of genome-wide and promoter-specific DNA binding and expression analyses to assess the functional roles of Myod and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that Myod and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, Myod can bind and recruit histone acetyltransferases. At early targets, Myod is sufficient for near full expression, whereas, at late expressed genes, Myod initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without Myod; however, transcriptional activation requires the combined activity of Myod and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by Myod.

MyoD and the transcriptional control of myogenesis.

The basic helix-loop-helix myogenic regulatory factors MyoD, Myf5, myogenin and MRF4 have critical roles in skeletal muscle development. Together with the Mef2 proteins and E proteins, these transcription factors are responsible for coordinating muscle-specific gene expression in the developing embryo. This review highlights recent studies regarding the molecular mechanisms by which the muscle-specific myogenic bHLH proteins interact with other regulatory factors to coordinate gene expression in a controlled and ordered manner.

MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex.

The activation of muscle-specific gene expression requires the coordinated action of muscle regulatory proteins and chromatin-remodeling enzymes. Microarray analysis performed in the presence or absence of a dominant-negative BRG1 ATPase demonstrated that approximately one-third of MyoD-induced genes were highly dependent on SWI/SNF enzymes. To understand the mechanism of activation, we performed chromatin immunoprecipitations analyzing the myogenin promoter. We found that H4 hyperacetylation preceded Brg1 binding in a MyoD-dependent manner but that MyoD binding occurred subsequent to H4 modification and Brg1 interaction. In the absence of functional SWI/SNF enzymes, muscle regulatory proteins did not bind to the myogenin promoter, thereby providing evidence for SWI/SNF-dependent activator binding. We observed that the homeodomain factor Pbx1, which cooperates with MyoD to stimulate myogenin expression, is constitutively bound to the myogenin promoter in a SWI/SNF-independent manner, suggesting a two-step mechanism in which MyoD initially interacts indirectly with the myogenin promoter and attracts chromatin-remodeling enzymes, which then facilitate direct binding by MyoD and other regulatory proteins.

Pbx marks genes for activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential.

Skeletal muscle differentiation is initiated by the transcription factor MyoD, which binds directly to the regulatory regions of genes expressed during skeletal muscle differentiation and initiates chromatin remodeling at specific promoters. It is not known, however, how MyoD initially recognizes its binding site in a chromatin context. Here we show that the H/C and helix III domains, two domains of MyoD that are necessary for the initiation of chromatin remodeling at the myogenin locus, together regulate a restricted subset of genes, including myogenin. These domains are necessary for the stable binding of MyoD to the myogenin promoter through an interaction with an adjacent protein complex containing the homeodomain protein Pbx, which appears to be constitutively bound at this site. This demonstrates a specific mechanism of targeting MyoD to loci in inactive chromatin and reveals a critical role of homeodomain proteins in marking specific genes for activation in the muscle lineage.