During latent infection of humans with Mycobacterium tuberculosis, bacteria persist in the asymptomatic host within granulomas, organized collections of differentiated macrophages, and other immune ...cells. The mechanisms for persistence remain poorly understood, as is the metabolic and replicative state of the microbes within granulomas. We analyzed the gene expression profile of Mycobacterium marinum, the cause of fish and amphibian tuberculosis, during its persistence in granulomas. We identified genes expressed specifically when M. marinum persists within granulomas. These granuloma-activated genes were not activated in vitro in response to various conditions postulated to be operant in tuberculous granulomas, suggesting that their granuloma-specific activation was caused by complex conditions that could not be mimicked in vitro. In addition to the granuloma-activated genes, the bacteria resident in granulomas expressed a wide range of metabolic and synthetic genes that are expressed during logarithmic growth in laboratory medium. Our results suggest a dynamic host-pathogen interaction in the granuloma, where metabolically active bacteria are kept in check by the host immune system and where the products of granuloma-specific bacterial genes may thwart the host's attempt to completely eradicate the bacteria.
The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed ...in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein. Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TTG. C. albicans cells expressing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C. albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.
The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 ...auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.
Gastric B-cell lymphoma of mucosa-associated lymphoid tissue type is closely linked to chronic
Helicobacter pylori
infection. Most clinical and histopathological features of the tumor can be ...reproduced by prolonged
Helicobacter
infection of BALB/c mice. In this study, we have addressed the role of antigenic stimulation in the pathogenesis of the lymphoma by experimental infection with
Helicobacter felis
, followed by antibiotic eradication therapy and subsequent re-infection. Antimicrobial therapy was successful in 75% of mice and led to complete histological but not “molecular” tumor remission. Although lympho-epithelial lesions disappeared and most gastric lymphoid aggregates resolved, transcriptional profiling revealed the long-term mucosal persistence of residual B cells. Experimental re-introduction of
Helicobacter
led to very rapid recurrence of the lymphomas, which differed from the original lesions by higher proliferative indices and more aggressive behavior. Immunophenotyping of tumor cells revealed massive infiltration of lesions by CD4
+ T cells, which express CD28, CD69, and interleukin-4 but not interferon-γ, suggesting that tumor B-cell proliferation was driven by Th 2-polarized, immunocompetent, and activated T cells. Tumors were also densely colonized by follicular dendritic cells, whose numbers were closely associated with and predictive of treatment outcome.
Long-term colonization of humans with Helicobacter pylori can cause the development of gastric B cell mucosa-associated lymphoid tissue lymphoma, yet little is known about the sequence of molecular ...steps that accompany disease progression. We used microarray analysis and laser microdissection to identify gene expression profiles characteristic and predictive of the various histopathological stages in a mouse model of the disease. The initial step in lymphoma development is marked by infiltration of reactive lymphocytes into the stomach and the launching of a mucosal immune response. Our analysis uncovered molecular markers of both of these processes, including genes coding for the immunoglobulins and the small proline-rich protein Sprr 2A. The subsequent step is characterized histologically by the antigen-driven proliferation and aggregation of B cells and the gradual appearance of lymphoepithelial lesions. In tissues of this stage, we observed increased expression of genes previously associated with malignancy, including the laminin receptor-1 and the multidrug-resistance channel MDR-1. Finally, we found that the transition to destructive lymphoepithelial lesions and malignant lymphoma is marked by an increase in transcription of a single gene encoding calgranulin A/Mrp-8.
Salmonella typhimurium invades host macrophages and can either induce a rapid cell death or establish an intracellular niche within the phagocytic vacuole. Rapid cell death requires the Salmonella ...pathogenicity island (SPI)1 and the host protein caspase‐1, a member of the pro‐apoptotic caspase family of proteases. Salmonella that do not cause this rapid cell death and instead reside in the phagocytic vacuole can trigger macrophage death at a later time point. We show here that the human pathogen Salmonella typhi also triggers both rapid, caspase‐1‐dependent and delayed cell death in human monocytes. The delayed cell death has previously been shown with S. typhimurium to be dependent on SPI2‐encoded genes and ompR. Using caspase‐1–/– bone marrow‐derived macrophages and isogenic S. typhimurium mutant strains, we show that a large portion of the delayed, SPI2‐dependent death is mediated by caspase‐1. The two known substrates of activated caspase‐1 are the pro‐inflammatory cytokines interleukin‐1β (IL‐1β) and IL‐18, which are cleaved to produce bioactive cytokines. We show here that IL‐1β is released during both SPI1‐ and SPI2‐dependent macrophage killing. Using IL‐1β–/– bone marrow‐derived macrophages and a neutralizing anti‐IL‐18 antibody, we show that neither IL‐1β nor IL‐18 is required for rapid or delayed macrophage death. Thus, both rapid, SPI1‐mediated killing and delayed, SPI2‐mediated killing require caspase‐1 and result in the secretion of IL‐1β, which promotes inflammation and may facilitate the spread of Salmonella beyond the gastrointestinal tract in systemic disease.
Bacterial pathogens manipulate host cells to promote pathogen survival and dissemination. We used a 22,571 human cDNA microarray to identify host pathways that are affected by the Salmonella enterica ...subspecies typhimurium phoP gene, a transcription factor required for virulence, by comparing the expression profiles of human monocytic tissue culture cells infected with either the wild-type bacteria or a phoP::Tn10 mutant strain. Both wild-type and phoP::Tn 10 bacteria induced a common set of genes, many of which are proinflammatory. Differentially expressed genes included those that affect host cell death, suggesting that the phoP regulatory system controls bacterial genes that alter macrophage survival. Subsequent experiments showed that the phoP::Tn 10 mutant strain is defective for killing both cultured and primary human macrophages but is able to replicate intracellularly. These experiments indicate that phoP plays a role in Salmonella-induced human macrophage cell death.
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