TGF-ß limits IL-33 production and promotes the resolution of colitis through regulation of macrophage function.

Mϕs promote tissue injury or repair depending on their activation status and the local cytokine milieu. It remains unclear whether the immunosuppressive effects of transforming growth factor ß (TGF-ß) serve a nonredundant role in Mϕ function in vivo. We generated Mϕ-specific transgenic mice that express a truncated TGF-ß receptor II under control of the CD68 promoter (CD68TGF-ßDNRII) and subjected these mice to the dextran sodium sulfate (DSS) model of colitis. CD68TGF-ßDNRII mice have an impaired ability to resolve colitic inflammation as demonstrated by increased lethality, granulocytic inflammation, and delayed goblet cell regeneration compared with transgene negative littermates. CD68TGF-ßDNRII mice produce significantly less IL-10, but have increased levels of IgE and numbers of IL-33+ Mϕs than controls. These data are consistent with associations between ulcerative colitis and increased IL-33 production in humans and suggest that TGF-ß may promote the suppression of intestinal inflammation, at least in part, through direct effects on Mϕ function.

Dendritic cells restrict the transformation of Histoplasma capsulatum conidia into yeasts.

Infections due to Histoplasma capsulatum occur as a result of the inhalation of airborne microconidia of the mold into the alveoli of the lungs. In this study we quantified the transformation over time of conidia into yeast-like cells within macrophages (MΦ) and dendritic cells (DC). Conidia from strain G217B which had been surface labeled with carboxy-fluorescein succinimidyl ester (CFSE), or conidia from strain G217B that expresses green fluorescent protein (GFP) only in the yeast phase, were used to infect MΦ and DC. At various time points, numbers of intracellular conidia or yeasts were quantified via phase-contrast and fluorescent microscopy. Transformation of conidia from non-GFP-expressing G217B also was quantified by their incorporation of ³H-leucine. In both human and murine MΦ, numerous yeast-like cells appeared by day 3 post-infection. The time course of conidia transformation into yeasts in culture medium was the same as in MΦ. However, transformation of conidia to yeasts was significantly restricted in human DC and murine lung DC. In DC, significant numbers of yeasts did not appear until 5 days post-infection. Further, MΦ monolayers were destroyed by day 6-7 post-infection, whereas DC monolayers remained intact throughout the study period. These data suggest that in vivo, conidia may transform into yeast-like cells efficiently whether or not they are phagocytosed by MΦ, but not when ingested by DC.

Insertional mutagenesis enables cleistothecial formation in a non-mating strain of Histoplasma capsulatum.

BACKGROUND: Histoplasma capsulatum is a pathogenic ascomycete fungus that rapidly loses mating ability in culture. Loss of mating ability, as well as the organism's low rate of targeted gene replacement, limits techniques available for genetic studies in H. capsulatum. Understanding molecular mechanisms regulating mating in this organism may allow us to reverse or prevent loss of mating in H. capsulatum strains, introducing a variety of classical genetics techniques to the field. We generated a strain, UC1, by insertional mutagenesis of the laboratory strain G217B, and found that UC1 acquired the ability to form mating structures called cleistothecia. The aim of this study was to determine the mechanism by which UC1 gained the ability to form cleistothecia. We also present initial studies demonstrating that UC1 can be used as a tool to determine molecular correlates of mating in H. capsulatum. RESULTS: The strain UC1 was found to have increased RNA levels of the mating locus transcription factor (MAT1-1-1), and the putative alpha pheromone (PPG1) compared to G217B. Agrobacterium-mediated transformation and integration of T-DNA from the vector pCB301-GFP-HYG were found to be partially responsible for the increased RNA levels of these genes; however, the site of integration appeared to play the largest role in the strain's ability to form cleistothecia. Silencing HMK1, a putative FUS3/KSS1 homolog, had no effect on cleistothecial production by UC1. Protein kinase C (PKC1) RNA and protein levels were increased in UC1 compared to G217B, and pheromone production was found to be linked with Pkc1 activity in H. capsulatum. CONCLUSIONS: The site of the T-DNA integration event appears to play the largest role in UC1's ability to form cleistothecia. We show that the UC1 strain can be used as a tool to study cleistothecia production in H. capsulatum by manipulating the strain, or by identifying differences between UC1 and G217B. Using these approaches, we were able to link Pkc1 activity with pheromone production in H. capsulatum; however, further studies are required to determine molecular mechanisms behind this. These studies may reveal regulatory mechanisms that can be manipulated to restore mating ability in H. capsulatum laboratory strains.

Histoplasma capsulatum proteome response to decreased iron availability.

BACKGROUND: A fundamental pathogenic feature of the fungus Histoplasma capsulatum is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. H. capsulatum can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis. RESULTS: To investigate the proteomic response by H. capsulatum to decreasing iron availability we have created H. capsulatum protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the H. capsulatum G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 H. capsulatum proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown. CONCLUSION: We have created a bioinformatics platform for H. capsulatum and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in H. capsulatum at the protein level. We also identified H. capsulatum proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by H. capsulatum to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for H. capsulatum proteomic analysis.

Global analysis of cellular factors and responses involved in Pseudomonas aeruginosa resistance to arsenite.

The impact of arsenite [As(III)] on several levels of cellular metabolism and gene regulation was examined in Pseudomonas aeruginosa. P. aeruginosa isogenic mutants devoid of antioxidant enzymes or defective in various metabolic pathways, DNA repair systems, metal storage proteins, global regulators, or quorum sensing circuitry were examined for their sensitivity to As(III). Mutants lacking the As(III) translocator (ArsB), superoxide dismutase (SOD), catabolite repression control protein (Crc), or glutathione reductase (Gor) were more sensitive to As(III) than wild-type bacteria. The MICs of As(III) under aerobic conditions were 0.2, 0.3, 0.8, and 1.9 mM for arsB, sodA sodB, crc, and gor mutants, respectively, and were 1.5- to 13-fold less than the MIC for the wild-type strain. A two-dimensional gel/matrix-assisted laser desorption ionization-time of flight analysis of As(III)-treated wild-type bacteria showed significantly (>40-fold) increased levels of a heat shock protein (IbpA) and a putative allo-threonine aldolase (GlyI). Smaller increases (up to 3.1-fold) in expression were observed for acetyl-coenzyme A acetyltransferase (AtoB), a probable aldehyde dehydrogenase (KauB), ribosomal protein L25 (RplY), and the probable DNA-binding stress protein (PA0962). In contrast, decreased levels of a heme oxygenase (HemO/PigA) were found upon As(III) treatment. Isogenic mutants were successfully constructed for six of the eight genes encoding the aforementioned proteins. When treated with sublethal concentrations of As(III), each mutant revealed a marginal to significant lag period prior to resumption of apparent normal growth compared to that observed in the wild-type strain. Our results suggest that As(III) exposure results in an oxidative stress-like response in P. aeruginosa, although activities of classic oxidative stress enzymes are not increased. Instead, relief from As(III)-based oxidative stress is accomplished from the collective activities of ArsB, glutathione reductase, and the global regulator Crc. SOD appears to be involved, but its function may be in the protection of superoxide-sensitive sulfhydryl groups.

Serologic responses to epitopes of the major surface glycoprotein of Pneumocystis jiroveci differ in human immunodeficiency virus-infected and uninfected persons.

The major surface glycoprotein (Msg) of Pneumocystis jiroveci (P. jiroveci) is important in the immunopathogenesis of Pneumocystis pneumonia (PcP), but is difficult to study in humans. We generated 3 overlapping recombinant Msg fragments (MsgA, MsgB and MsgC), and analyzed their reactivity with serum samples from 95 healthy blood donors and 94 human immunodeficiency virus (HIV)-infected persons. Reactivity to the Msg fragments varied with HIV infection and prior episodes of PcP but not with geographic origin. Recognition of MsgA was lower-and recognition of MsgB was significantly lower-in HIV(+) serum compared with donor serum. Serum samples from HIV-positive patients with prior PcP recognized MsgC more frequently than did serum samples from those without PcP. None of the serum samples drawn from 9 patients before they had developed PcP recognized MsgC. These data suggest that these novel recombinant proteins are useful for the analysis of antibody responses to Msg.