Monitoring F1651 P-like fimbria expression at the single-cell level reveals a highly heterogeneous phenotype.

F1651 and the pyelonephritis-associated pili (Pap) are two members of the type P family of adhesive factors. They play a key role in establishing disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) strains in animals and humans. Both F1651 and Pap are under the control of an epigenetic and reversible switch that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Using the Gfp reporter system, we monitored in vitro the level of fluorescence intensity corresponding to the F1651 and Pap fimbrial synthesis. Monitoring individual Escherichia coli cells by flow cytometry and by real-time fluorescence microscopy, we identified cells associated with a low or high level of fluorescence intensity and a large amount of cells with partial levels of fluorescence, mostly present in the F1651 system. This mixed population identified through fluorescence intensity could be attributed to the high switching rate previously observed in F1651-positive bacteria. The fimbrial heterogeneous phenotype for these ExPEC could represent increased fitness in unpredictable environments. Our study illustrates that within the large repertoire of fimbrial variants such as the well-characterized Pap, F1651 is an exquisite example of regulatory expression that arms the bacterium with strategies for surviving in more than one particular environment.

Defective IL-17- and IL-22-dependent mucosal host response to Candida albicans determines susceptibility to oral candidiasis in mice expressing the HIV-1 transgene.

BACKGROUND: The tissue-signaling cytokines IL-17 and IL-22 are critical to host defense against oral Candida albicans infection, by their induction of oral antimicrobial peptide expression and recruitment of neutrophils. Mucosal Th17 cells which produce these cytokines are preferentially depleted in HIV-infected patients. Here, we tested the hypothesis that defective IL-17- and IL-22-dependent host responses to C. albicans determine the phenotype of susceptibility to oropharyngeal candidiasis (OPC) in transgenic (Tg) mice expressing HIV-1. RESULTS: Naïve CD4+ T-cells and the differentiated Th1, Th2, Th17, Th1Th17 and Treg lineages were all profoundly depleted in cervical lymph nodes (CLNs) of these Tg mice. However, naive CD4+ cells from Tg mice maintained the capacity to differentiate into these lineages in response to polarizing cytokines in vitro. Expression of Il17, Il22, S100a8 and Ccl20 was enhanced in oral mucosal tissue of non-Tg, but not of Tg mice, after oral infection with C. albicans. Treatment of infected Tg mice with the combination of IL-17 and IL-22, but not IL-17 or Il-22 alone, significantly reduced oral burdens of C. albicans and abundance of Candida hyphae in the epithelium of tongues of infected Tg mice, and restored the ability of the Tg mice to up-regulate expression of S100a8 and Ccl20 in response to C. albicans infection. CONCLUSIONS: These findings demonstrate that defective IL-17- and IL-22-dependent induction of innate mucosal immunity to C. albicans is central to the phenotype of susceptibility to OPC in these HIV transgenic mice.

Altered immune response differentially enhances susceptibility to Cryptococcus neoformans and Cryptococcus gattii infection in mice expressing the HIV-1 transgene.

Cryptococcus neoformans var. grubii is the most frequent cause of AIDS-associated cryptococcosis worldwide, while Cryptococcus gattii usually infects immunocompetent people. To understand the mechanisms which cause differential susceptibility to these cryptococcal species in HIV infection, we established and characterized a model of cryptococcosis in CD4C/HIV(MutA) transgenic (Tg) mice expressing gene products of HIV-1 and developing an AIDS-like disease. Tg mice infected intranasally with C. neoformans var. grubii strain H99 or C23 consistently displayed reduced survival compared to non-Tg mice at three graded inocula, while shortened survival of Tg mice infected with C. gattii strain R265 or R272 was restricted to a single high inoculum. HIV-1 transgene expression selectively augmented systemic dissemination to the liver and spleen for strains H99 and C23 but not strains R265 and R272. Histopathologic examination of lungs of Tg mice revealed large numbers of widely scattered H99 cells, with a minimal inflammatory cell response, while in the non-Tg mice H99 was almost completely embedded within extensive mixed inflammatory cell infiltrates. In contrast to H99, R265 was dispersed throughout the lung parenchyma and failed to induce a strong inflammatory response in both Tg and non-Tg mice. HIV-1 transgene expression reduced pulmonary production of CCL2 and CCL5 after infection with H99 or R265, and production of these two chemokines was lower after infection with R265. These results indicate that an altered immune response in these Tg mice markedly enhances C. neoformans but not C. gattii infection. This model therefore provides a powerful new tool to further investigate the immunopathogenesis of cryptococcosis.

Macrophage-mediated responses to Candida albicans in mice expressing the human immunodeficiency virus type 1 transgene.

The critical impairments of innate and adaptive immunity that cause susceptibility to mucosal candidiasis in human immunodeficiency virus (HIV) infection have not been fully determined. We therefore conducted an analysis of macrophage-mediated responses to Candida albicans in transgenic (Tg) mice expressing Nef, Env, and Rev of HIV type 1 (HIV-1) in CD4(+) T cells, dendritic cells, and macrophages and developing an AIDS-like disease (CD4C/HIV(MutA) Tg mice). Macrophages were successfully recruited to the oral and gastric mucosae of these Tg mice in response to chronic carriage of C. albicans and displayed polarization toward an alternatively activated phenotype. Functionally, peritoneal macrophages from uninfected Tg mice exhibited increased phagocytosis of C. albicans and enhanced production of interleukin 6 and monocyte chemoattractant protein 1, demonstrating that the HIV-1 transgene independently activates selected macrophage functions. Production of H(2)O(2) by macrophages from Tg mice primed with gamma interferon and treated with phorbol 12-myristate 13-acetate or C. albicans was moderately reduced, but expression of the HIV-1 transgene did not alter production of nitric oxide or reduce killing of C. albicans. A knockout of the inducible nitric oxide synthase (NOS2) gene in these Tg mice did not augment oral or gastrointestinal burdens during chronic carriage of C. albicans or cause systemic dissemination, likely due to a redundancy provided by partially preserved production of H(2)O(2) and oxygen-independent candidacidal mechanisms. Thus, the macrophage response to C. albicans is largely preserved in these Tg mice, and no functional macrophage defect appears to primarily determine the susceptibility to mucosal candidiasis.

CD8+ T cells but not polymorphonuclear leukocytes are required to limit chronic oral carriage of Candida albicans in transgenic mice expressing human immunodeficiency virus type 1.

Candida albicans causes oropharyngeal candidiasis (OPC) but rarely disseminates to deep organs in human immunodeficiency virus (HIV) infection. Here, we used a model of OPC in CD4C/HIV(Mut) transgenic (Tg) mice to investigate the role of polymorphonuclear leukocytes (PMNs) and CD8+ T cells in limiting candidiasis to the mucosa. Numbers of circulating PMNs and their oxidative burst were both augmented in CD4C/HIV(MutA) Tg mice expressing rev, env, and nef of HIV type 1 (HIV-1), while phagocytosis and killing of C. albicans were largely unimpaired compared to those in non-Tg mice. Depletion of PMNs in these Tg mice did not alter oral or gastrointestinal burdens of C. albicans or cause systemic dissemination. However, oral burdens of C. albicans were increased in CD4C/HIV(MutG) Tg mice expressing only the nef gene of HIV-1 and bred on a CD8 gene-deficient background (CD8-/-), compared to control or heterozygous CD8+/- CD4C/HIV(MutG) Tg mice. Thus, CD8+ T cells contribute to the host defense against oral candidiasis in vivo, specifically in the context of nef expression in a subset of immune cells.

Altered CD4+ T cell phenotype and function determine the susceptibility to mucosal candidiasis in transgenic mice expressing HIV-1.

The impairments of protective mucosal immunity which cause susceptibility to oropharyngeal candidiasis (OPC) in HIV infection remain undefined. This study used a model of OPC in CD4C/HIV MutA transgenic (Tg) mice expressing Rev, Env, and Nef of HIV-1 to investigate the role of transgene expressing dendritic cells (DCs) and CD4+ T cells in maintenance of chronic oral carriage of Candida albicans. DCs were depleted in the Tg mice and had an immature phenotype, with low expression of MHC class II and IL-12. CD4+ T cells were quantitatively reduced in the oral mucosa, cervical lymph nodes (CLNs) and peripheral blood of the Tg mice, and displayed a polarization toward a nonprotective Th2 response. Proliferation of CLN CD4+ T cells from infected Tg mice in response to C. albicans Ag in vitro was abrogated and the cells failed to acquire an effector phenotype. Coculture of C. albicans-pulsed DCs with CD4+ T cells in vitro showed that Tg expression in either or both of these cell populations sharply reduced the proliferation of CD4+ T cells and their production of IL-2. Finally, transfer of naive non-Tg CD4+ T cells into these Tg mice restored proliferation to C. albicans Ag and sharply reduced oral burdens of C. albicans. Overall, these results indicate that defective CD4+ T cells primarily determine the susceptibility to chronic carriage of C. albicans in these Tg mice.