Influence of mycobacterial trehalose 6,6'-dimycolate (TDM) on macrophage responses

Mycobacterium tuberculosis, the causative agent of tuberculosis, survives within macrophages by altering host cell activation and by manipulating phagosomal trafficking and acidification. Part of the success of M. tuberculosis as a major human pathogen has been attributed to its cell wall, a unique structure largely comprised of mycolic acids. Trehalose 6,6′-dimycolate (TDM) is the major glycolipid component on the surface of the mycobacterial cell wall. This study examines the contribution of TDM during mycobacterial infection of murine macrophages. Virulent M. tuberculosis was chemically depleted of surface-exposed TDM using petroleum ether extraction. Compared to their native counterparts, delipidated M. tuberculosis showed similar growth in broth culture. Bone marrow-derived macrophages (BMM) or the murine macrophage-like cell line J774A.1 were infected with delipidated M. tuberculosis, and responses were compared to cells infected with native M. tuberculosis. Delipidated M. tuberculosis demonstrated significantly decreased viability in macrophages by seven days after infection. Reconstitution of delipidated organisms with pure TDM restored viability. Infection with native M. tuberculosis led to high cellular production of cytokines (IL-1β, IL-6, IL-12, and TNF-α) and chemokines (MCP-1 and MIP-1α); infection with delipidated M. tuberculosis significantly abrogated responses. Cytokine and chemokine production were restored when delipidated organisms were reconstituted with TDM. Responses were specifically induced by TDM; all measured cytokines were elicited from macrophages incubated with TDM-coated beads, while control beads coated with bovine serum albumin (BSA) did not induce cytokine production. Visualization of mycobacterial localization in J774A.1 cells using fluorescence microscopy revealed that delipidated M. tuberculosis were significantly more likely to traffic to acidic vesicles (lysosomes) than native organisms. Reconstitution with TDM restored trafficking to non-acidic vesicles. Similarly, TDM-coated beads demonstrated significantly delayed localization to acidic vesicles compared to BSA-coated beads. In summary, the interaction of TDM with macrophages may regulate the outcome of M. tuberculosis infection by influencing cellular cytokine production and intracellular localization of organisms. This research has elucidated a novel and necessary role for TDM in survival of virulent M. tuberculosis in host macrophages during in vitro infection. ^

Identification and characterization of virulence determinants in the Lyme disease spirochete Borrelia burgdorferi

Borrelia burgdorferi is the etiological agent of Lyme disease, the most common tick-borne disease in the United States. Although the most frequently reported symptom is arthritis, patients can also experience severe cardiac, neurologic, and dermatologic abnormalities. The identification of virulence determinants in infectious B. burgdorferi strains has been limited by their slow growth rate, poor transformability, and general lack of genetic tools. The present study demonstrates the use of transposon mutagenesis for the identification of infectivity-related factors in infectious B. burgdorferi, examines the potential role for chemotaxis in mammalian infection, and describes the development of a novel method for the analysis of recombination events at the Ids antigenic variation locus. A pool of Himar1 mutants was isolated using an infectious B. burgdorferi clone and the transposon vector pMarGent. Clones exhibiting reduced infectivity in mice possessed insertions in virulence determinants putatively involved in host survival and dissemination. These results demonstrated the feasibility of extensive transposon mutagenesis studies for the identification of additional infectivity-related factors. mcp-5 mutants were chosen for further study to determine the role of chemotaxis during infection. Animal studies indicated that mcp-5 mutants exhibited a reduced infectivity potential, and suggested a role for mcp-5 during the early stages of infection. An in vitro phenotype for an mcp-5 mutant was not detected. Genetic complementation of an mcp-5 mutant resulted in restoration of Mcp-5 expression in the complemented clone, as demonstrated by western blotting, but the organisms were not infectious in mice. We believe this result is a consequence of differences in expression between genes located on the linear chromosome and genes present on the circular plasmid used for trans-complementation. Overall, this work implicates mcp-5 as an important determinant of mammalian infectivity. Finally, the development of a computer-assisted method for the analysis of recombination events occurring at the B. burgdorferi vls antigenic variation locus has proven highly valuable for the detailed examination of vls gene conversion. The studies described here provide evidence for the importance of chemotaxis during infection in mice and demonstrate advances in both genetic and computational approaches for the further characterization of the Lyme disease spirochete. ^

A review of the Malaria Chemoprophylaxis Compliance Program at ExxonMobil Corporation

ExxonMobil, a Fortune 500 oil and gas corporation, has a global workforce with employees assigned to projects in areas at risk for infectious diseases, particularly malaria. As such, the corporation has put in place a program to protect the health of workers and ensure their safety in malaria endemic zones. This program is called the Malaria Control Program (MCP). One component of this program is the more specific Malaria Chemoprophylaxis Compliance Program (MCCP), in which employees enroll following consent to random drug testing for compliance with the company's chemoprophylaxis requirements. Each year, data is gathered on the number of employees working in these locations and are selected randomly and tested for chemoprophylaxis compliance. The selection strives to test each eligible worker once per year. Test results that come back positive for the chemoprophylaxis drug are considered "detects" and tests that are negative for the drug and therefore show the worker is non-compliant at risk for severe malaria infection are considered "non-detect". ^ The current practice report used aggregate data to calculate statistics on test results to reflect compliance among both employees and contractors in various malaria-endemic areas. This aggregate, non-individualized data has been compiled and reflects the effectiveness and reach of ExxonMobil's Malaria Chemoprophylaxis Compliance Program. In order to assess compliance, information on the number of non-detect test results was compared to the number of tests completed per year. The data shows that over time, non-detect results have declined in both employee and contractor populations, and vary somewhat by location due to size and scope of the MCCP implemented in-country. Although the data indicate a positive trend for the corporation, some recommendations have been made for future implementation of the program.^

Molecular and genetic analysis of Myxococcus xanthus early *development

To identify more mutations that can affect the early development of Myxococcus xanthus, the synthetic transposon TnT41 was designed and constructed. By virtue of its special features, it can greatly facilitate the processes of mutation screening/selection, mapping, cloning and DNA sequencing. In addition, it allows for the systematic discovery of genes in regulatory hierarchies using their target promoters. In this study, the minimal regulatory region of the early developmentally regulated gene 4521 was used as a reporter in the TnT41 mutagenesis. Both positive (P) mutations and negative (N) mutations were isolated based on their effects on 4521 expression.^ Four of these mutations, i.e. N1, N2, P52 and P54 were analyzed in detail. Mutations N1 and N2 are insertion mutations in a gene designated sasB. The sasB gene is also identified in this study by genetic and molecular analysis of five UV-generated 4521 suppressor mutations. The sasB gene encodes a protein without meaningful homology in the databases. The sasB gene negatively regulates 4521 expression possibly through the SasS-SasR two component system. A wild-type sasB gene is required for normal M. xanthus fruiting body formation and sporulation.^ Cloning and sequencing analysis of the P52 mutation led to the identification of an operon that encodes the M. xanthus high-affinity branched-chain amino acid transporter system. This liv operon consists of five genes designated livK, livH, livM, livC, and livF, respectively. The Liv proteins are highly similar to their counterparts from other bacteria in both amino acid sequences, functional motifs and predicted secondary structures. This system is required for development since liv null mutations cause abnormality in fruiting body formation and a 100-fold decrease in sporulation efficiency.^ Mutation P54 is a TnT41 insertion in the sscM gene of the ssc chemotaxis system, which has been independently identified by Dr. Shi's lab. The sscM gene encodes a MCP (methyl-accepting chemotaxis protein) homologue. The SscM protein is predicted to contain two transmembrane domains, a signaling domain and at least one putative methylation site. Null mutations of this gene abolish the aggregation of starving cells at a very early stage, though the sporulation levels of the mutant can reach 10% that of wild-type cells. ^

Retinoid -induced regression of human head and neck squamous cell carcinomas is associated with the induction of a pro -inflammatory response

Retinoids are Vitamin A derivatives that are effective chemopreventative and chemotherapeutic agents for head and neck squamous cell carcinomas (HNSCC). Despite the wide application of retinoids in cancer treatment, the mechanism by which retinoids inhibit head and neck squamous cell carcinomas is not completely understood. While in vitro models show that drugs affect cell proliferation and differentiation, in vivo models, such as tumor xenografts in nude mice drugs affect more complex parameters such as extracellular matrix formation, angiogenesis and inflammation. Therefore, we studied the effects of retinoids on the growth of the 22B HNSCC tumors using a xenograft model. In this system, retinoids had no effect on tumor cell differentiation but caused invasion of the tumor by inflammatory cells. Retinoid induced inflammation lead to tumor cell death and tumor regression. Therefore, we hypothesized that retinoids stimulated the 22B HNSCC xenografts to produce a pro-inflammatory signal such as chemokines that in turn activated host inflammatory responses. ^ We used real time quantitative RT-PCR to measure cytokine and chemokine expression in retinoid treated tumors. Treatment of tumors with an RAR-specific retinoid, LGD1550, had no effect on the expression of TNFα, IL-1α, GROα, IP-10, Rantes, MCP-1 and MIP-1α but induced IL-8 mRNA 5-fold. We further characterized the retinoid effect on IL-8 expression on the 22B HNSCC and 1483 HNSCC cells in vitro. Retinoids increased IL-8 expression and enhanced TNFα-dependent IL-8 induction. In addition, retinoids increased the basal and TNFα-dependent expression of MCP-1 but decreased the basal and TNFα dependent expression of IP-10. The effect of retinoids on IL-8 and MCP-1 expression was very rapid with increased levels of mRNA detected within 1–2 hours. This effect did not require new protein synthesis and did not result from mRNA stabilization. Both RAR and RXR ligands increased IL-8 expression whereas only RAR ligands activated MCP-1 expression. ^ We identified a functional retinoid response element in the IL-8 promoter that was located adjacent to the C/EBP-NFkB response element. TNFα treatment of the 22B cells caused rapid, transient and selective acetylation of regions of the IL-8 promoter associated with the NFkB response element. Co-treatment of the cells with retinoids plus TNF increased the acetylation of chromatin in this region without altering the kinetics of acetylation. These results demonstrate that ligand activated retinoid receptors can cooperate with NFkB in histone acetylation and chromatin remodeling. We believe that in certain HNSCC tumors this cooperation and the resulting enhancement of IL-8 expression can induce an inflammatory response that leads to tumor regression. We believe that the induction of inflammation in susceptible tumors, possibly coupled with cytotoxic interventions may be an important component in the use of retinoids to treat human squamous cancers. ^