T Cell Interactions in Mycobacterial Granulomas: Non-Specific T Cells Regulate Mycobacteria-Specific T Cells in Granulomatous Lesions

Infections with pathogenic mycobacteria are controlled by the formation of a unique structure known as a granuloma. The granuloma represents a host–pathogen interface where bacteria are killed and confined by the host response, but also where bacteria persist. Previous work has demonstrated that the T cell repertoire is heterogenous even at the single granuloma level. However, further work using pigeon cytochrome C (PCC) epitope-tagged BCG (PCC-BCG) and PCC-specific 5CC7 RAG−/− TCR transgenic (Tg) mice has demonstrated that a monoclonal T cell population is able to control infection. At the chronic stage of infection, granuloma-infiltrating T cells remain highly activated in wild-type mice, while T cells in the monoclonal T cell mice are anergic. We hypothesized that addition of an acutely activated non-specific T cell to the monoclonal T cell system could recapitulate the wild-type phenotype. Here we report that activated non-specific T cells have access to the granuloma and deliver a set of cytokines and chemokines to the lesions. Strikingly, non-specific T cells rescue BCG-specific T cells from anergy and enhance the function of BCG-specific T cells in the granuloma in the chronic phase of infection when bacterial antigen load is low. In addition, we find that these same non-specific T cells have an inhibitory effect on systemic BCG-specific T cells. Taken together, these data suggest that T cells non-specific for granuloma-inducing agents can alter the function of granuloma-specific T cells and have important roles in mycobacterial immunity and other granulomatous disorders.

Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.

Genetic Analysis of Mitochondrial Sorting from the MSC3 Mosaic Mutant of Cucumber

Cucumber (Cucumis sativus) plants regenerated from cell cultures occasionally possess mosaic (MSC) phenotypes on cotyledons and leaves. Lines MSC3 and MSC16 have distinct MSC phenotypes and originated from plants regenerated from different cell-culture experiments established using a highly inbred wild-type cucumber. Both the mitochondrial (mt) DNA and MSC phenotype of cucumber show paternal transmission, and MSC3 and MSC16 have different mt coding regions at significantly lower copy numbers relative to wild-type plants. A nuclear locus, Paternal sorting of mitochondria (Psm), conditions a high proportion of wild-type progenies, specifically when MSC16 is crossed as the male with wild-type female plants. During this research, we identified plants that produced a high proportion of wild-type progenies in crosses with MSC3 as the male parent. Plants from an F2 family were crossed with MSC3 as the male, progenies were scored for numbers of MSC vs. wild-type plants, and single-nucleotide polymorphisms (SNP) were identified for genetic mapping. A major quantitative trait locus on chromosome 3 was associated with a higher frequency of wild-type progenies from MSC3 as the male parent, and the 1.5-logarithm-of-odds interval for the most significant SNP was located 627 kb from Psm. These results reveal that separate genetic factors control sorting to the wild-type phenotype in progenies from crosses with different MSC parents. The identification of causal genes controlling mitochondrial sorting in cucumber should provide insight regarding nuclear-mitochondrial interactions affecting the prevalence of specific mitochondrial DNA in plants.

Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence

The pathology associated with malaria infection is largely due to the ability of infected human erythrocytes to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin.Taken together, our data reveal a role for PFA66 in host cell modification, implicate human HSP70 as also being essential in this process, and uncover a KAHRP-independent mechanism for correct knob biogenesis. Our observations open up exciting new avenues for the development of new anti-malarials.

A New Generation of Functional Tagged Proteins for HIV Fluorescence Imaging

During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity.

The Rice Plastidial Phosphorylase Participates Directly in Both Sink and Source Processes

The plastidial starch phosphorylase (Pho1) functions in starch metabolism. A distinctive structural feature of the higher Pho1 is a 50–82-amino-acid long peptide (L50–L82), which is absent in phosphorylases from non-plant organisms. To study the function of the rice Pho1 L80 peptide, we complemented a pho1− rice mutant (BMF136) with the wild-type Pho1 gene or with a Pho1 gene lacking the L80 region (Pho1ΔL80). While expression of Pho1 in BMF136 restored normal wild-type phenotype, the introduction of Pho1ΔL80 enhanced the growth rate and plant productivity above wild-type levels. Mass spectrometry analysis of proteins captured by anti-Pho1 showed the surprising presence of PsaC, the terminal electron acceptor/donor subunit of photosystem I (PSI). This unexpected interaction was substantiated by reciprocal immobilized protein pull-down assays of seedling extracts and supported by the presence of Pho1 on isolated PSI complexes resolved by blue-native gels. Spectrophotometric studies showed that Pho1ΔL80 plants exhibited modified PSI and enhanced CO2 assimilation properties. Collectively, these findings indicate that the higher plant Pho1 has dual roles as a potential modulator of source and sink processes.

Micro-RNA Clusters Integrate Evolutionary Constraints on Expression and Target Affinities: The miR-6/5/4/286/3/309 Cluster in Drosophila

A striking feature of micro-RNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. Here, we investigated a micro-RNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this micro-RNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of micro-RNA cluster members were also constructed. Expression of individual micro-RNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient micro-RNAs together (miR-5/4/286/3/309) or more recently evolved clustered micro-RNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed downregulation of leg-patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of micro-RNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct micro-RNAs. Considered together, the micro-RNA targets and the evolutionary ages of each micro-RNA in the cluster demonstrate the importance of micro-RNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing micro-RNAs. Key words: micro-RNA, cluster, evolution.

Role of p53-Mediated Apoptosis in Limiting Metastasis

p53 is a transcription factor known to play important roles in limiting tumorigenesis, including controlling the induction of pro-apoptotic genes and apoptosis in response to oncogenic stress. Using ChIP-Seq and RNA-Seq, our lab has identified p53 binding sites potentially responsible for p53-mediated induction of pro-apoptotic genes following DNA damage. Using CRISPR Cas9, we have generated fly strains with deletions or mutations within the p53 binding motif located in the experimentally verified p53 binding site. This deletion blocked DNA damage induced apoptosis. To study the functionality of the motif in limiting tumorigenesis, we introduced the deletion into a genetic tumorigenesis and metastasis model, where the tumor-suppressor cell polarity gene scribble was knocked down via shRNAi in the non-essential tissue compound eye and introduced together with strong oncogenic mutation that can lead to neoplasia and metastasis. We developed a scoring scale to quantify the severity of neoplasia and metastasis, where 0 indicates a wild type phenotype and 5 indicates the most severe metastatic phenotype. Results show that the p53BSKO predominantly displayed a score of 5 (39%). However, our preliminary results also suggested that the severity of this model may also subject to genetic background besides the p53 binding motif.

ES5 is involved in the regulation of phosphatidylserine synthesis and impacts on early senescence in rice (Oryza sativa L.)

Leaf senescence, which affects plant growth and yield in rice, is an ideal target for crop improvement and remarkable advances have been made to identify the mechanism underlying this process. We have characterized an early senile mutant es5 (early leaf senescence 5) in rice exhibiting leaf yellowing phenotype after the 4-leaf stage. This phenotype was confirmed by the higher accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), the disintegration of chloroplasts, reduction in chlorophyll content and photosynthetic rate and up-regulation of senescence-associated genes (SAGs) like Osh36, OsI57, and OsI85. Positional cloning revealed that the es5 phenotype is the result of one base substitution in ES5, encoding phosphatidylserine synthase (PSS) family protein, which is involved in the base-exchange type reaction to synthesize the minor membrane phospholipid phosphatidylserine. Functional complementation of ES5 in the es5 plants completely restored the wild-type phenotype. Ultra-high-performance liquid chromatography (UHPLC) analysis showed that es5 plants had increased levels of phosphatidylserine (PS) and decreased level of phosphatidylcholine (PC). These results provide evidence about the role of PS in rice leaf senescence.

C. elegans expressing D76N β2-microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β2-m expressing worms. We also demonstrated the specificity of the β2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates.