Harry B. Hines

Nitric oxide and redox mechanisms in the immune response

The role of redox molecules, such as NO and ROS, as key mediators of immunity has recently garnered renewed interest and appreciation. To regulate immune responses, these species trigger the eradication of pathogens on the one hand and modulate immunosuppression during tissue‐restoration and wound‐healing processes on the other. In the acidic environment of the phagosome, a variety of RNS and ROS is produced, thereby providing a cauldron of redox chemistry, which is the first line in fighting infection. Interestingly, fluctuations in the levels of these same reactive intermediates orchestrate other phases of the immune response. NO activates specific signal transduction pathways in tumor cells, endothelial cells, and monocytes in a concentration‐dependent manner. As ROS can react directly with NO‐forming RNS, NO bioavailability and therefore, NO response(s) are changed. The NO/ROS balance is also important during Th1 to Th2 transition. In this review, we discuss the chemistry of NO and ROS in the context of antipathogen activity and immune regulation and also discuss similarities and differences between murine and human production of these intermediates.

Type VI secretion is a major virulence determinant in Burkholderia mallei

Burkholderia mallei is a host‐adapted pathogen and a category B biothreat agent. Although the B. mallei VirAG two‐component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown. Here we show with expression profiling that overexpression of virAG resulted in transcriptional activation of ∼60 genes, including some involved in capsule production, actin‐based intracellular motility, and type VI secretion (T6S). The 15 genes encoding the major sugar component of the homopolymeric capsule were up‐expressed > 2.5‐fold, but capsule was still produced in the absence of virAG. Actin tail formation required virAG as well as bimB, bimC and bimE, three previously uncharacterized genes that were activated four‐ to 15‐fold when VirAG was overproduced. Surprisingly, actin polymerization was found to be dispensable for virulence in hamsters. In contrast, genes encoding a T6S system were up‐expressed as much as 30‐fold and mutations in this T6S gene cluster resulted in strains that were avirulent in hamsters. SDS‐PAGE and mass spectrometry demonstrated that BMAA0742 was secreted by the T6S system when virAG was overexpressed. Purified His‐tagged BMAA0742 was recognized by glanders antiserum from a horse, a human and mice, indicating that this Hcp‐family protein is produced in vivo during infection.

Design and Testing for a Nontagged F1-V Fusion Protein as Vaccine Antigen against Bubonic and Pneumonic Plague

A two‐component recombinant fusion protein antigen was re‐engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine‐tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1‐V fusion protein absent extraneous coding sequences. Soluble F1‐V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea‐denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N‐terminal methionine. Purity, quality, and higher‐order structure were compared between lots using RP‐HPLC, intrinsic fluorescence, CD spectroscopy, and multi‐angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1‐V protein also protected mice from wild‐type and non‐encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1‐V as the active pharmaceutical ingredient of the next plague vaccine.

Quorum Sensing: A transcriptional Regulatory System Involved in the Pathogenicity of Burkholderia mallei

ABSTRACT Numerous gram-negative bacterial pathogens regulate virulence factor expression by using a cell density mechanism termed quorum sensing (QS). An in silico analysis of the Burkholderia mallei ATCC 23344 genome revealed that it encodes at least two luxI and four luxR homologues. Using mass spectrometry, we showed that wild-type B. mallei produces the signaling molecules N-octanoyl-homoserine lactone and N-decanoyl-homoserine lactone. To determine if QS is involved in the virulence of B. mallei, we generated mutations in each putative luxIR homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models. Disruption of the B. mallei QS alleles, especially in RJ16 (bmaII) and RJ17 (bmaI3), which are luxI mutants, significantly reduced virulence, as indicated by the survival of mice who were aerosolized with 104 CFU (10 50% lethal doses [LD50s]). For the B. mallei transcriptional regulator mutants (luxR homologues), mutation of the bmaR5 allele resulted in the most pronounced decrease in virulence, with 100% of the challenged animals surviving a dose of 10 LD50s. Using a Syrian hamster intraperitoneal model of infection, we determined the LD50s for wild-type B. mallei and each QS mutant. An increase in the relative LD50 was found for RJ16 (bmaI1) (>967 CFU), RJ17 (bmaI3) (115 CFU), and RJ20 (bmaR5) (151 CFU) compared to wild-type B. mallei (<13 CFU). These findings demonstrate that B. mallei carries multiple luxIR homologues that either directly or indirectly regulate the biosynthesis of an essential virulence factor(s) that contributes to the pathogenicity of B. mallei in vivo.

Mutational Analysis and Biochemical Characterization of the Burkholderia thailandensis DW503 Quorum-Sensing Network

Numerous gram-negative bacteria communicate and regulate gene expression through a cell density-responsive mechanism termed quorum sensing (QS), which involves the synthesis and perception of diffusible N-acyl-homoserine lactones (AHL). In this study we genetically and physiologically characterized the Burkholderia thailandensis DW503 QS network. In silico analysis of the B. thailandensis genome revealed the presence of at least three AHL synthases (AHS) and five transcriptional regulators belonging to the LuxIR family of proteins. Mass spectrometry demonstrated that wild-type B. thailandensis synthesizes N-hexanoyl-homoserine lactone (C6-HSL), N-octanoyl-homoserine lactone (C8-HSL), and N-decanoyl-homoserine lactone (C10-HSL). Mutation of the btaI1 (luxI) AHS gene prevented accumulation of C8-HSL in culture supernatants, enhanced beta-hemolysis of sheep erythrocytes, increased lipase production, and altered colony morphology on swarming and twitching motility plates. Disruption of the btaI3 (luxI) AHS prevented biosynthesis of C6-HSL and increased lipase production and beta-hemolysis, whereas mutagenesis of the btaI2 (luxI) allele eliminated C10-HSL accumulation and reduced lipase production. Complementation of the btaI1 and btaI3 mutants fully restored the synthesis of C8-HSL and C6-HSL to parental levels. In contrast, mutagenesis of the btaR1, btaR3, btaR4, and btaR5 (luxR) transcriptional regulators had no effect on AHL accumulation, enhanced lipase production, and resulted in extensive beta-hemolysis on sheep blood agar plates. Furthermore, interruption of the btaI1, btaR1, and btaR3 genes altered colony morphology on twitching and swarming motility plates and induced pigmentation. Additionally, phenotypic microarray analysis indicated that QS in B. thailandensis both positively and negatively affects the metabolism of numerous substrates, including citric acid, formic acid, glucose 6-phosphate, capric acid, gamma-hydroxybutyric acid, and d-arabinose. These results demonstrate that mutagenesis of the B. thailandensis QS system affects various cellular processes, including lipase production, swarming and twitching motility, beta-hemolysis of sheep erythrocytes, and carbon metabolism and/or transport.

Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

ABSTRACT Botulinum toxin analysis has renewed importance. This study included the use of nanochromatography-nanoelectrospray-mass spectrometry/mass spectrometry to characterize the protein composition of botulinum progenitor toxins and to assign botulinum progenitor toxins to their proper serotype and strain by using currently available sequence information. Clostridium botulinum progenitor toxins from strains Hall, Okra, Stockholm, MDPH, Alaska, and Langeland and 89 representing serotypes A through G, respectively, were reduced, alkylated, digested with trypsin, and identified by matching the processed product ion spectra of the tryptic peptides to proteins in accessible databases. All proteins known to be present in progenitor toxins from each serotype were identified. Additional proteins, including flagellins, ORF-X1, and neurotoxin binding protein, not previously reported to be associated with progenitor toxins, were present also in samples from several serotypes. Protein identification was used to assign toxins to a serotype and strain. Serotype assignments were accurate, and strain assignments were best when either sufficient nucleotide or amino acid sequence data were available. Minor difficulties were encountered using neurotoxin-associated protein identification for assigning serotype and strain. This study found that combined nanoscale chromatographic and mass spectrometric techniques can characterize C. botulinum progenitor toxin protein composition and that serotype/strain assignments based upon these proteins can provide accurate serotype and, in most instances, strain assignments using currently available information. Assignment accuracy will continue to improve as more nucleotide/amino acid sequence information becomes available for different botulinum strains.

Nitric oxide synthase and breast cancer: role of TIMP-1 in NO-mediated Akt activation.

Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation.

[3H]-Saxitoxinol metabolism and elimination in the rat

Tritiated saxitoxinol was used to obtain preliminary information on saxitoxin metabolism in the rat. Sublethal doses of tritiated saxitoxinol (18.9-microCi/kg; 3.8 micrograms/kg) were injected i.v. into each of six rats. Urine and fecal samples were collected up to 144 hr post-injection. Within 4 hr, 60% of injected radioactivity was excreted in urine. No radioactivity was found in feces. High performance liquid chromatography analyses of urine showed that saxitoxinol was not metabolized by the rats.

Nitric oxide-mediated regulation of β-amyloid clearance via alterations of MMP-9/TIMP-1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimers disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimers disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2−/− mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.

Urinary elimination of saxitoxin after intravenous injection

Paralytic shellfish poisoning is a serious public health concern throughout the world. An analytical method with diagnostic potential was used to isolate and measure saxitoxin, the most potent and studied paralytic shellfish poisoning toxin, in the urine of rats injected i.v. with sublethal doses (2 micrograms/kg) of saxitoxin. Urine was collected at intervals between 4 and 144 hr after injection. Saxitoxin was isolated from urine with an ion-exchange procedure, identified, and measured with a precolumn-oxidation-HPLC procedure coupled with fluorescence detection. The identity of oxidized saxitoxin was confirmed with electrospray ionization mass spectrometry. Four hours after injection, approximately 19% of the injected saxitoxin dose was excreted. By 24 hr, approximately 58% of the administered dose was excreted. Average total urinary excretion of administered saxitoxin was approximately 68% for the full study period. These results demonstrate that small quantities of unmetabolized saxitoxin can be detected in rat urine up to 144 hr after i.v. administration, and that the analytical method may have diagnostic potential for saxitoxin intoxication and paralytic shellfish poisoning.