HLB is the most devastating citrus disease and is associated with vector-borne Liberibacter. Currently there is no cure for HLB. Visual disease symptoms appear in only a few leaves months after initial Liberibacter exposure compromising disease management by tree removal. Since infected but non-symptomatic trees are inoculum sources, a robust platform for early diagnosis of HLB is urgently needed. Here, we describe our studies on the discovery of HLB pre-symptomatic RNA biomarkers. For this, we infected greenhouse citrus using Liberibacter inoculum delivered by psyllid vectors confined to branch cages for a single week. Total RNA was extracted from leaves at three distances from the point of inoculation and for a range of early post-exposure times. Gene expression data by RNA-seq was collected for all leaf samples. We expect citrus genes belonging to pattern recognition, plant hormone, and stress pathways to be expressed early during infection. The RNA-seq data will be analyzed to identify which citrus genes are altered in leaves during early infection. It is our hypothesis that pre-symptomatic citrus biomarkers will be systemically expressed during the early stages of infection even in leaves distant from the site of Liberibacter inoculation. We will validate the expression of the candidate pre-symptomatic biomarkers by qPCR. The candidate biomarkers will reflect citrus genes that are systemically expressed and the Liberibacter genes that are indicative of bacterial growth.

Immunomodulation is a promising therapeutic approach for Alzheimer's disease (AD); however, major drawbacks are cerebral microhemorrhages associated with increased cerebral amyloid angiopathy (CAA) and excessive inflammation. Our initial findings indicate that stimulation of TLR9 signaling with CpG oligodeoxynucleotide (ODN) is effective against CAA without inducing toxicity in AD mouse models. To further assess potential human use of CpG ODN we advanced our studies using a well-established non-human primate model of sporadic CAA, squirrel monkey (Saimiri Boliviensis). Safety and efficacy assessment studies were first performed in young squirrel monkeys (SQM). Elderly female monkeys were subcutaneously injected either with the most effective and non-toxic dosages of the class B CpG ODN containing a primate specific immunostimulatory sequence or saline. Both age groups were subjected to behavioral testing. Plasma taken during the course of treatment was analyzed to identify immune responses and AD biomarkers. Fluidigm RT-PCR was used to evaluate mRNA levels of cytokines in SQM PBMCs. CpG ODN elevated the levels of various Th1/Th2 cytokines in plasma from old monkeys. Upregulation of cytokines in CpG ODN group was further confirmed by RT-PCR. Pre-treatment behavioral assessment in our aged monkeys demonstrated cognitive deficits on the Inhibitory Control of Behavior and Delayed Response tests. Age effect on cognitive abilities was observed as the young group performed with overall lower session error rates compared to old animals. Post-treatment behavioral testing in our aged monkeys is ongoing. Here we report the first pyroglutamate (pE3) immunohistochemistry of aged Saimiri Boliviensis. In addition to 6E10/4G8 Aß-positive plaques, pyroglutamate Aß-positive deposits in the form of CAA and parenchymal plaques were detected. Our preliminary biomarker analyses revealed a noticeable increase in Aß40, Aß42 and AßpE3 plasma levels in CpG ODN-treated group. Further longitudinal assessment of potential AD biomarkers is currently in progress. The presented studies represent the first trial of specifically targeting CAA in non-human primates. We hope that our research will validate this novel approach of immunomodulation as a safer method to successfully ameliorate AD related pathologies and provide critical data for potential clinical use of CpG ODN in AD patients.

Abstract Pathogens circumvent host immune defense to propagate their lifecycle. We have been studying the mechanisms of pathogen-induced host cell death to develop a robust strategy for pathogen clearance. The strategy involves designing and developing a protein chimera with one domain for recognizing conserved pathogen membrane elements and another for lysing the pathogen membrane. This dualistic synergy of recognition and lysis permits rapid pathogen clearance thereby preventing host cell death and disease development. This strategy has been successfully applied to counteract bacterial infections and diseases in grape, citrus, and tobacco. We have engineered a chimera consisting of protease (recognition domain) and cecropin (lysis domain) to prevent Pierce’s disease, a deadly disease in grape caused by Xylella fastidiosa. We have shown that transgenic plants expressing the chimera of protease and cecropin effectively clear Xylella fastidiosa from sites of colonization. We have also designed a protein chimera of thionin (with both recognition and lysis domains) and a synthetic lytic peptide. We have shown that this same chimera prevents citrus canker which is caused by Xanthamonas axonopodis citri and wildfire disease in tobacco which is caused by Pseudomonas syringe pv. tobaci. This protein engineering strategy appears to be an effective therapy against a broad spectrum of plant pathogens. A similar approach is now being evaluated against multiple human pathogens.

Abstract Multi-drug resistance, resistance-nodulation-differentiation efflux pumps in gram-negative bacteria are membrane protein complexes that extrude a wide variety of drugs before they can act on their bacterial target. However, they are not merely pumps for physical transport of drugs but their expression level is also up-regulated by the presence of drugs inside the bacteria, which, in turn, facilitates the release of quorum-sensing molecules and subsequent induction of genes responsible for biofilm formation and virulence. It is our hypothesis that efflux mediated biofilm formation and virulence expression offer gram-negative bacteria additional resistance not only against drugs but also against host innate immune defense. In order to test our hypothesis, we performed real-time qPCR studies on Fluidigm platform to monitor the expression of (i) the efflux genes and their regulators, (ii) the genes for synthesis of quorum sensing molecules and their regulators, and (iii) the genes for biofilm formation and virulence. For this, we used Pseudomonas aeruginosa and its efflux and quorum sensing mutants. The gene expression studies were performed for planktonic cells and nascent and mature biofilms in the presence and absence of a drug, ciprofloxacin. We were able to show efflux mediated expression of biofilm and virulence genes, which resulted in the abrogation of host innate immune defense in a cellular assay.

Background: Multi-drug resistance (MDR) efflux pumps belonging to the nodulation cell division (RND) family in Pseudomonas aeruginosa are able to extrude out a wide variety of drugs and antibiotics. These complexes of inner-membrane, periplasm, and outer-membrane protein components are not merely molecular machines that pump out drugs. The very drugs that are pumped out by these efflux pumps can also induce expression of the efflux genes/proteins. In addition, the MDR efflux pumps transport out quorum-sensing molecules, which can induce expression of several genes including those related to biofilm formation, virulence, and efflux, thereby conferring additional resistance. Therefore, a complete understanding of the function of the MDR efflux pumps requires measurement and modeling of three processes: (a) how a given drug is extruded through these pumps; (b) how the same drug induces the expression of these pumps; and (c) how the release of quorum-sensing molecules cause the expression of biofilm, virulence, and efflux genes. Methods & Materials: We measured and modeled the survival kinetics and intra- and extra-cellular concentration of ciprofloxacin in the wild-type P. aeruginosa strain PAO1 and the mutants deficient in efflux and quorum sensing. We also measured the formation of biofilm, antibiotic-induced expression of virulence, and efflux genes by real-time qPCR (Fluidigm) and modeled how the expression of these genes confers additional drug resistance via biofilm formation and enhanced virulence. Results: We showed that (i) the P. aeruginosa tripartite MexA-MexB-OprM protein complex plays an important role in the efflux of ciprofloxacin and in the induction of the quorum-sensing pathways (ii) a sharp increase in the efflux within 5–10 minutes after the treatment of ciprofloxacin after which the efflux levels off (iii) the quorum sensing mediated biofilm, virulence, and efflux genes are expressed at higher level by mature biofilm than the nascent one providing additional resistance. Conclusion: Combination of experimental and modeling studies provides an insight into how MDR efflux and quorum sensing systems work together to confer drug resistance. A similar approach can be extended to understand the structural, genetic, and cellular processes underlying the function of MDR efflux pumps.

Abstract Different influenza A virus subtypes display different pathogenicity during host infection. It is our hypothesis that the host innate immune defense inversely correlates with the viral pathogenicity, i.e., the higher the viral pathogenicity the lower is the host innate immune defense and vice versa. In order to test our hypothesis, we monitored infection of normal human bronchial epithelial cells due to high pathogenicity H1N1 and low pathogenicity H3N2 subtypes during the early stages (0 to 24 hours) of infection. Specifically, we performed whole genome microarray analysis of the infected cells, identified the genes significantly altered in their expression, and validated the expression patterns of these genes by real-time qPCR. Of particular importance were the inducer and effector genes in the IFN-α/β pathway, a prominent innate immune response against viral infection. The prominent inducers include: (i) the OAS genes involved in the production of small viral RNA, (ii) the viral RNA sensors RIG-I, MDA-5, and TLR3, and (iii) the IRF transcription factors involved in the expression of IFN-α/β. The prominent effectors include: (i) ISG15, which activates RIG-I by ubiquitination, (ii) RSAD2 which inhibit the budding of influenza A virus, and (iii) GBPs, which inhibit viral replication. We validate our hypothesis by demonstrating that the release and expression of inducers and effectors of IFN-α/β are lower in high pathogenicity H1N1 than those in low pathogenicity H3N2. .

Abstract A few years ago, we introduced a concept that a protein chimera of pathogen recognition and lysis domains would be able to rapidly clear a broad-spectrum of pathogens [Crit Rev Immunol 2007;27(3):233-245]. For a wide variety of viral, bacterial, and fungal pathogens, appropriate recognition and lysis domains can be chosen from the host innate immune repertoire. A chimera of the recognition and lysis domains would be designed with the aid of a flexible linker to ensure synergy of the two functions and therefore, the rapid clearance of the targeted pathogen. In this work, we demonstrate the design of such a chimera and the efficacy of this chimera in clearing a plant pathogen Xylella fastidiosa (Xf) that causes diseases in multiple plants of economic importance. The most notable ones are Pierce’s disease (PD) in grape and variegated chlorosis (CVC) in citrus. Specifically, we show the construction of the transgenic grapevines expressing a protein chimera of recognition and lysis domains specific for Xf. This chimera clears Xf from the xylem (the site of colonization) and blocks the development of PD [Proc Natl Acad Sci U S A. 2012;109(10):3721-3725]. The same chimera can be applied to block CVC. Finally, we indicate how such chimeras of recognition and lysis domains can be developed to target multiple human pathogens.

We postulated that a synergistic combination of two innate immune functions, pathogen surface recognition and lysis, in a protein chimera would lead to a robust class of engineered antimicrobial therapeutics for protection against pathogens. In support of our hypothesis, we have engineered such a chimera to protect against the Gram-negative Xylella fastidiosa (Xf), which causes diseases in multiple plants of economic importance. Here we report the design and delivery of this chimera to target the Xf subspecies fastidiosa (Xff), which causes Pierce disease in grapevines and poses a great threat to the wine-growing regions of California. One domain of this chimera is an elastase that recognizes and cleaves MopB, a conserved outer membrane protein of Xff. The second domain is a lytic peptide, cecropin B, which targets conserved lipid moieties and creates pores in the Xff outer membrane. A flexible linker joins the recognition and lysis domains, thereby ensuring correct folding of the individual domains and synergistic combination of their functions. The chimera transgene is fused with an amino-terminal signal sequence to facilitate delivery of the chimera to the plant xylem, the site of Xff colonization. We demonstrate that the protein chimera expressed in the xylem is able to directly target Xff, suppress its growth, and significantly decrease the leaf scorching and xylem clogging commonly associated with Pierce disease in grapevines. We believe that similar strategies involving protein chimeras can be developed to protect against many diseases caused by human and plant pathogens.

The rapid and unabated spread of vector-borne diseases within US specialty crops threatens our agriculture, our economy, and the livelihood of growers and farm workers. Early detection of vector-borne pathogens is an essential step for the accurate surveillance and management of vector-borne diseases of specialty crops. Currently, we lack the tools that would detect the infectious agent at early (primary) stages of infection with a high degree of sensitivity and specificity. In this paper, we outline a strategy for developing an integrated suite of platform technologies to enable rapid, early disease detection and diagnosis of huanglongbing (HLB), the most destructive citrus disease. The research has two anticipated outcomes: i) identification of very early, disease-specific biomarkers using a knowledge base of translational genomic information on host and pathogen responses associated with early (asymptomatic) disease development; and ii) development and deployment of novel sensors that capture these and other related biomarkers and aid in presymptomatic disease detection. By combining these two distinct approaches, it should be possible to identify and defend the crop by interdicting pathogen spread prior to the rapid expansion phase of the disease. We believe that similar strategies can also be developed for the surveillance and management of diseases affecting other economically important specialty crops.

Recognition of the pathogen-associated molecular pattern (PAMP) by host Toll-like receptors (TLR) is an important component of the innate immune response for countering against invading viruses, bacteria, and fungi. Upon PAMP recognition, the TLR induces intracellular signaling cascades that involve adapter, signalosome, and transcription factor complexes and result in the production of both pro- and anti-inflammatory cytokines and chemokines. An inflammatory response for a short duration can be beneficial because it helps to clear the infectious agent. However, prolonged inflammation can be detrimental because it may cause host toxicity and tissue damage. Indeed, excessive production of inflammatory cytokines and chemokines via TLR pathways is often associated with many inflammatory and autoimmune diseases. Therefore, fine control of inflammation in the TLR pathway is highly desirable for effective host defense. In this article, we review intrinsic control mechanisms that include a balance between pro-inflammatory and anti-inflammatory cytokines and chemokines, production of host effectors, and regulation at the level of adapter, signalosome, and transcription factor complexes in the TLR pathways. We also discuss how understanding of the TLR signaling steps leads to the development of small-molecule drugs that can interfere with the formation of active adapter, signalosome, and adapter complexes.

This chapter summarizes our studies on the three toll-like receptor pathways, namely TLR4, TLR2, and TLR3, induced by lipopolysaccharides (LPS), peptidoglycan (PGN), and double-stranded RNA (dsRNA) in antigen presenting cells (APC). The particular emphasis is on the activation of human innate immune responses via cytokine and chemokine production. Three different measurements have been performed on monocytic and dendritic cells as model APCs: (i) the expression of various cytokine and chemokine genes by real-time PCR, (ii) the release of the cytokines and chemokines by ELISA, and (iii) gene expression analysis by cytokine and chemokine pathway-specific and whole genome microarrays. Real-time PCR and ELISA enable us to identify cytokines and chemokines that are produced specifically upon LPS, PGN, or dsRNA stimulation. Subsequently, microarray studies and appropriate validation experiments help us to identify genes involved in the upstream pathways that cause the induction of cytokines and chemokines. It is evident that TLR4-LPS, TLR2-PGN, and TLR3-dsRNA pathways are distinguished by the specific set of cytokines and chemokines they induce as well as by the upstream signaling events.

Exposure to beryllium (Be) induces a delayed-type hypersensitivity immune reaction in the lungs of susceptible individuals, which leads to the onset of Be sensitivity and Chronic Beryllium Disease (CBD). Although some mechanistic aspects of CBD have begun to be characterized, very little is known about the molecular mechanisms by which Be activates the host immune response. To gain insight into the cellular response to Be exposure, we have performed global microarray analysis using a mixture of peripheral blood mononuclear and dendritic cells (PBMC/DCs) from a non-CBD source to identify genes that are specifically upregulated in response to BeSO4 stimulation, compared to a control metal salt, Al2(SO4)3. We identified a number of upregulated immunomodulatory genes, including several chemokines in the MIP-1 and GRO families. Using PBMC/DCs from three different donors, we demonstrate that BeSO4 stimulation generally exhibits an increased rate of both chemokine mRNA transcription and release compared to Al2(SO4)3 exposure, although variations among the individual donors do exist. We show that MIP-1α and MIP-1β neutralizing antibodies can partially inhibit the ability of BeSO4 to stimulate cell migration of PBMC/DCs in vitro. Finally, incubation of PBMC/DCs with BeSO4 altered the binding of the transcription factor RUNX to the MIP-1α promoter consensus sequence, indicating that Be can regulate chemokine gene activation. Taken together, these results suggest a model in which Be stimulation of PBMC/DCs can modulate the expression and release of different chemokines, leading to the migration of lymphocytes to the lung and the formation of a localized environment for development of Be disease in susceptible individuals.

Rapid, accurate, and sensitive detection of biothreat agents requires a broad-spectrum assay capable of discriminating between closely related microbial or viral pathogens. Moreover, in cases where a biological agent release has been identified, forensic analysis demands detailed genetic signature data for accurate strain identification and attribution. To date, nucleic acid sequences have provided the most robust and phylogentically illuminating signature information. Nucleic acid signature sequences are not often linked to genomic or extrachromosomal determinants of virulence, a link that would further facilitate discrimination between pathogens and closely related species. Inextricably coupling genetic determinants of virulence with highly informative nucleic acid signatures would provide a robust means of identifying human, livestock, and agricultural pathogens. By means of example, we present here an overview of two general applications of microarray-based methods for: (1) the identification of candidate virulence factors; and (2) the analysis of genetic polymorphisms that are coupled to Bacillus anthracis virulence factors using an accurate, low cost solid-phase mini-sequencing assay. We show that microarray-based analysis of gene expression can identify potential virulence associated genes for use as candidate signature targets, and, further, that microarray-based single nucleotide polymorphism assays provide a robust platform for the detection and identification of signature sequences in a manner independent of the genetic background in which the signature is embedded. We discuss the strategy as a general approach or pipeline for the discovery of virulence-linked nucleic acid signatures for biothreat agents.

ABSTRACT DNA from over 300 Bacillus thuringiensis , Bacillus cereus , and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis . All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.

Several findings have revealed a likely role for DNA ligase IV, and interacting protein XRCC4, in the final steps of mammalian DNA double-strand break repair. Recent evidence suggests that the human DNA ligase IV protein plays a critical role in the maintenance of genomic stability. To identify protein-protein interactions that may shed further light on the molecular mechanisms of DSB repair and the biological roles of human DNA ligase IV, we have used the yeast two-hybrid system in conjunction with traditional biochemical methods. These efforts have resulted in the identification of a physical association between the DNA ligase IV polypeptide and the human condensin subunit known as hCAP-E. The hCAP-E polypeptide, a member of the Structural Maintenance of Chromosomes (SMC) super-family of proteins, coimmunoprecipitates from cell extracts with DNA ligase IV. Immunofluorescence studies reveal colocalization of DNA ligase IV and hCAP-E in the interphase nucleus, whereas mitotic cells display colocalization of both polypeptides on mitotic chromosomes. Strikingly, the XRCC4 protein is excluded from the area of mitotic chromosomes, suggesting the formation of specialized DNA ligase IV complexes subject to cell cycle regulation. We discuss our findings in light of known and hypothesized roles for ligase IV and the condensin complex.

DNA-dependent protein kinase (DNA-PK) consists of three polypeptide subunits: Ku70, Ku80, and the DNA-PK catalytic subunit (DNA-PKcs). Mammalian mutants deficient in either Ku80 or DNA-PKcs function have been shown to be lacking in DNA double-strand break repair and V(D)J recombination, respectively. The precise role of the Ku70 gene in this process has not yet been determined, in part because no cell lines, animals, or human diseases involved with deficiencies in this gene have yet been identified. Both the human and the mouse Ku70 cDNAs have been cloned, and the human gene has been mapped to chromosome 22q13. The original mouse cDNA clones, however, lacked a complete 5'-region, and none of the mammalian Ku70 genomic sequences have been characterized. This report contains an analysis of the 5'-region of the mouse cDNA sequence, a characterization of the mouse Ku70 genomic structure, and fluorescence in situ hybridization data that map the mouse gene to chromosome 15. The deduced amino acid sequence of the mouse gene consists of 608 amino acids compared to 609 for the human gene. The genomic sequence is 24 kb and consists of 13 exons, including an untranslated first exon. Sequences from the upstream region of exon 1 revealed four consensus GC box sequences and a strong transcription initiation site at a reasonable location. The assignment of the mouse Ku70 gene to chromosome 15 is consistent with the syntenic relationship of this gene in human (chromosome 22q13) and mouse and adds to the comparative mapping data for the genes involved in the SCID phenotype.