Mingqi Deng

Gene Expression and Antimicrobial Activity of Bovine Macrophages in Response to Mycobacterium avium subsp. paratuberculosis

We evaluated gene expression and antimicrobial responses of bovine monocyte—derived macrophages incubated with Mycobacterium avium subsp. paratuberculosis (M. a. ptb), the causative agent of Johnes disease. Gene expression was evaluated by the use of human noncompetitive high-density oligonucleotide microarrays. Bovine messenger RNA hybridized with 14.2–18.2% of the 12,600 oligonucleotide probe sets. When macrophages incubated with M. a. ptb were compared with nonactivated control macrophages, macrophages activated by addition of interferon-γ and lipopolysaccharide, and macrophages incubated with Mycobacterium avium subspecies avium (M. a. a), 47, 79, and 27 genes, respectively, were differentially expressed. Differential expression of six of these genes was confirmed using reverse transcriptase polymerase chain reaction. Several functional assays were performed to evaluate the potential relevance of differentially expressed genes to host defense. Macrophages phagocytizing M. a. a had a greater capacity to kill the organisms and to acidify phagosomes and a greater degree of apoptosis than did macrophages incubated with M. a. ptb. The results of these studies indicate that multiple genes and metabolic pathways are differentially expressed by macrophages ingesting mycobacterial organisms. Although the intracellular fate of mycobacterial organisms appears to be dependent on a complex interaction between macrophage and organism, phagosome acidification and apoptosis may play central roles in organism survival.

Cryptosporidium parvum Genes Containing Thrombospondin Type 1 Domains

ABSTRACT Cryptosporidium parvum is recognized as an enteropathogen of great worldwide medical and veterinary importance, yet understanding of its pathogenesis has been hampered in part by limited knowledge of the invasion machinery of this parasite. Recently, genes containing thrombospondin type 1 (TSP1) domains have been identified in several genera of apicomplexans, including thrombospondin-related adhesive proteins (TRAPs) that have been implicated as key molecules for parasite motility and adhesion onto host cell surfaces. Previously, a large-scale random survey of the C. parvum genome conducted in our laboratory revealed the presence of multiple genomic DNA sequences with a high degree of similarity to known apicomplexan TRAP genes. In the present study, TBLASTN screening of available C. parvum genomic sequences by using TSP1 domains as queries identified a total of 12 genes possessing TSP1-like domains. All genes have putative signal peptide sequences, one or more TSP1-like domains, plus additional extracellular protein modules such as Kringle, epidermal growth factor, and Apple domains. Two genes, putative paralogs CpTSP8 and CpTSP9, contain predicted introns near their amino termini, which were verified by comparing PCR products from cDNA versus genomic DNA templates. Reverse transcription-PCR analysis of transcript levels reveals that C. parvum TSP genes were developmentally regulated with distinct patterns of expression during in vitro infection. TRAPC1, CpTSP3, and CpTSP11 were expressed at high levels during both early and late stages of infection, whereas CpTSP2, CpTSP5, CpTSP6, CpTSP8, and CpTSP9 were maximally expressed during the late stages of infection. Only CpTSP4 was highly expressed solely at an early stage of infection.

Biphasic Modulation of Apoptotic Pathways in Cryptosporidium parvum-Infected Human Intestinal Epithelial Cells

ABSTRACT The impact of Cryptosporidium parvum infection on host cell gene expression was investigated by microarray analysis with an in vitro model using human ileocecal HCT-8 adenocarcinoma cells. We found changes in 333 (2.6%) transcripts at at least two of the five (6, 12, 24, 48, and 72 h) postinfection time points. Fifty-one of the regulated genes were associated with apoptosis and were grouped into five clusters based on their expression patterns. Early in infection (6 and 12 h), genes with antiapoptotic roles were upregulated and genes with apoptotic roles were downregulated. Later in infection (24, 48, and 72 h), proapoptotic genes were induced and antiapoptotic genes were downregulated, suggesting a biphasic regulation of apoptosis: antiapoptotic state early and moderately proapoptotic state late in infection. This transcriptional profile matched the actual occurrence of apoptosis in the infected cultures. Apoptosis was first detected at 12 h postinfection and increased to a plateau at 24 h, when 20% of infected cells showed nuclear condensation. In contrast, experimental silencing of Bcl-2 induced apoptosis in 50% of infected cells at 12 h postinfection. This resulted in a decrease in the infection rate and a reduction in the accumulation of meront-containing cells. To test the significance of the moderately proapoptotic state late in the infection, we inhibited apoptosis using pancaspase inhibitor Z-VAD-FMK. This treatment also affected the progression of C. parvum infection, as reinfection, normally seen late (24 h to 48 h), did not occur and accumulation of mature meronts was impaired. Control of host apoptosis is complex and crucial to the life of C. parvum. Apoptosis control has at least two components, early inhibition and late moderate promotion. For a successful infection, both aspects appear to be required.

Comparative gene expression by WC1+ γδ and CD4+ αβ T lymphocytes, which respond to Anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell‐associated genes by WC1+ γδ T cells

The functions of γδ T cells are enigmatic, and these cells are often considered as evolutionary remnants of well‐characterized αβ T cells. However, their conservation throughout evolution suggests that γδ T cells are biologically unique. In ruminants, γδ T cells expressing the workshop cluster 1 (WC1) scavenger receptor comprise a large proportion of circulating lymphocytes, suggesting these cells are biologically relevant and functionally different from αβ T cells. In fact, bovine WC1+ γδ T cells can act as APC for αβ T cells, indicating they may express genes encoding proteins associated with innate immunity. The present study was designed to compare immune function gene expression profiles of clonal populations of WC1+ γδ and CD4+ αβ T cells derived from the same animal, which respond to major surface protein 2 (MSP2) of the intraerythrocytic rickettsial pathogen of cattle, Anaplasma marginale. Gene expression profiles of activated T cell clones were compared using a microarray format, and differential gene expression was confirmed by real‐time RT‐PCR and protein analyses. We demonstrate that although MSP2‐specific αβ and γδ T cell clones express many of the same genes, γδ T cell clones express high levels of genes associated with myeloid cells, including chemokines CCL2, CXCL1, CXCL2, CXCL6, and surface receptors CD68, CD11b, macrophage scavenger receptor 1, macrophage mannose receptor, and galectin‐3. It is important that many of these genes were also expressed at higher levels in polyclonal WC1+ γδ T cells when compared with CD4+ αβ T cells selected from peripheral blood.

Microarray-based Expression Analysis of Human Epithelial Cell Response to Cryptosporidium parvum Infection

The apicomplexan Crypfosporidiwn parvwn infects primarily the epithelial cells of the small intestine of mammals and causes gastrointestinal disease that is characterized by acute watery diarrhea. The severity of the disease is largely dependent on the immune status of the h a t and could become persistent and life threatening in patients with deficient immune system. There is currently no effective therapy to treat the disease and efforts to develop novel therapeutical strategies are hampered by the limited understanding of the mechanism by which C. parvum causes disease. especially the host-parasite interaction [l]. As of today, little is known about the responses of host epithelial cells to C. parvum infection. such as biochemical pathways that are affected or genes that are specifically regulated. in the past few years, the development of DNA microarray technology has allowed mRNA expression to be assessed on a global scale [2]. By measuring transcription levels of genes in an organism under various conditions, differential expression profiles for tens of thousands of genes could be assessed in parallel. Systematic application of th is approach has been widely used for the identification of candidate genes involved in a variety of processes. This study was designed to develop and apply a human cDNA microarray in investigating human epithelial cell response to cryptosporidial infection and to identify host genes regulated during intracellular C. parvum development.