Improved understanding of biorisk for research involving microbial modification using annotated sequences of concern.

Regulation of research on microbes that cause disease in humans has historically been focused on taxonomic lists of 'bad bugs'. However, given our increased knowledge of these pathogens through inexpensive genome sequencing, 5 decades of research in microbial pathogenesis, and the burgeoning capacity of synthetic biologists, the limitations of this approach are apparent. With heightened scientific and public attention focused on biosafety and biosecurity, and an ongoing review by US authorities of dual-use research oversight, this article proposes the incorporation of sequences of concern (SoCs) into the biorisk management regime governing genetic engineering of pathogens. SoCs enable pathogenesis in all microbes infecting hosts that are 'of concern' to human civilization. Here we review the functions of SoCs (FunSoCs) and discuss how they might bring clarity to potentially problematic research outcomes involving infectious agents. We believe that annotation of SoCs with FunSoCs has the potential to improve the likelihood that dual use research of concern is recognized by both scientists and regulators before it occurs.

Chemotactic and inflammatory responses in the liver and brain are associated with pathogenesis of Rift Valley fever virus infection in the mouse.

Rift Valley fever virus (RVFV) is a major human and animal pathogen associated with severe disease including hemorrhagic fever or encephalitis. RVFV is endemic to parts of Africa and the Arabian Peninsula, but there is significant concern regarding its introduction into non-endemic regions and the potentially devastating effect to livestock populations with concurrent infections of humans. To date, there is little detailed data directly comparing the host response to infection with wild-type or vaccine strains of RVFV and correlation with viral pathogenesis. Here we characterized clinical and systemic immune responses to infection with wild-type strain ZH501 or IND vaccine strain MP-12 in the C57BL/6 mouse. Animals infected with live-attenuated MP-12 survived productive viral infection with little evidence of clinical disease and minimal cytokine response in evaluated tissues. In contrast, ZH501 infection was lethal, caused depletion of lymphocytes and platelets and elicited a strong, systemic cytokine response which correlated with high virus titers and significant tissue pathology. Lymphopenia and platelet depletion were indicators of disease onset with indications of lymphocyte recovery correlating with increases in G-CSF production. RVFV is hepatotropic and in these studies significant clinical and histological data supported these findings; however, significant evidence of a pro-inflammatory response in the liver was not apparent. Rather, viral infection resulted in a chemokine response indicating infiltration of immunoreactive cells, such as neutrophils, which was supported by histological data. In brains of ZH501 infected mice, a significant chemokine and pro-inflammatory cytokine response was evident, but with little pathology indicating meningoencephalitis. These data suggest that RVFV pathogenesis in mice is associated with a loss of liver function due to liver necrosis and hepatitis yet the long-term course of disease for those that might survive the initial hepatitis is neurologic in nature which is supported by observations of human disease and the BALB/c mouse model.

Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 µg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities.

Comparative Global Gene Expression Profiles of Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant at Flea and Human Body Temperatures.

Braun/murein lipoprotein (Lpp) is involved in inflammatory responses and septic shock. We previously characterized a Deltalpp mutant of Yersinia pestis CO92 and found that this mutant was defective in surviving in macrophages and was attenuated in a mouse inhalation model of plague when compared to the highly virulent wild-type (WT) bacterium. We performed global transcriptional profiling of WT Y. pestis and its Deltalpp mutant using microarrays. The organisms were cultured at 26 and 37 degrees Celsius to simulate the flea vector and mammalian host environments, respectively. Our data revealed vastly different effects of lpp mutation on the transcriptomes of Y. pestis grown at 37 versus 26 degrees C. While the absence of Lpp resulted mainly in the downregulation of metabolic genes at 26 degrees C, the Y. pestis Deltalpp mutant cultured at 37 degrees C exhibited profound alterations in stress response and virulence genes, compared to WT bacteria. We investigated one of the stress-related genes (htrA) downregulated in the Deltalpp mutant relative to WT Y. pestis. Indeed, complementation of the Deltalpp mutant with the htrA gene restored intracellular survival of the Y. pestis Deltalpp mutant. Our results support a role for Lpp in Y. pestis adaptation to the host environment, possibly via transcriptional activation of htrA.

Deletion of Braun lipoprotein gene (lpp) attenuates Yersinia pestis KIM/D27 strain: role of Lpp in modulating host immune response, NF-kappaB activation and cell death.

The pathogenic species of yersiniae potently blocks immune responses in host cells by using the type III secretion apparatus and its effector proteins. In this study, we characterized potential mechanisms associated with the Braun lipoprotein (Lpp) that contributed to a further attenuation of a pigmentation locus-minus Yersinia pestis KIM/D27 mutant strain and its ability to generate immune responses in mice. The lpp gene encodes one of the major outer membrane lipoproteins that is involved in inflammatory responses and septic shock. We found that sera and splenocytes from Deltalpp mutant-immunized mice, when transferred to naïve animals, provided protection to the latter against challenge with a lethal dose of the Y. pestis parental strain. Further, the Deltalpp mutant promoted ex vivo a significantly higher interleukin (IL)-2 and interferon-gamma production from T cells of immunized mice, when compared to those from animals infected with the sub-lethal dose of the parental Y. pestis KIM/D27 strain. Likewise, murine primary macrophages infected with the mutant, when compared to those infected with the parental strain in vitro, produced significantly higher IL-12 levels. Importantly, increased nuclear factor-kappa B activation and decreased apoptosis were noted in splenocytes and primary macrophages of mice challenged with the Deltalpp mutant, when compared to those in animals infected with the parental Y. pestis KIM/D27 strain. Finally, significantly higher levels of antibodies specific for the parental Y. pestis antigens were developed in mice first immunized with the Deltalpp mutant and then challenged with the parental strain, compared to the antibody levels in animals that were immunized and then infected with the parental KIM/D27 strain. To our knowledge, this is the first report of a mechanistic basis for attenuation and immunological responses associated with deletion of the lpp gene from the Y. pestis KIM/D27 strain.

Evaluation of a Yersinia pestis mutant impaired in a thermoregulated type VI-like secretion system in flea, macrophage and murine models.

Type VI secretion systems (T6SSs) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499-YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26 degrees C in comparison to 37 degrees C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499-YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 h post-infection for bacterial cells pre-grown at 26 degrees C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499-YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37 degrees C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain.

Deletion of Braun lipoprotein gene (lpp) and curing of plasmid pPCP1 dramatically alter the virulence of Yersinia pestis CO92 in a mouse model of pneumonic plague.

Deletion of the murein (Braun) lipoprotein gene, lpp, attenuates the Yersinia pestis CO92 strain in mouse models of bubonic and pneumonic plague. In this report, we characterized the virulence of strains from which the plasminogen activating protease (pla)-encoding pPCP1 plasmid was cured from either the wild-type (WT) or the Deltalpp mutant strain of Y. pestis CO92 in the mouse model of pneumonic infection. We noted a significantly increased survival rate in mice infected with the Y. pestis pPCP(-)/Deltalpp mutant strain up to a dose of 5000 LD(50). Additionally, mice challenged with the pPCP(-)/Deltalpp strain had substantially less tissue injury and a strong decrease in the levels of most cytokines and chemokines in tissue homogenates and sera when compared with the WT-infected group. Importantly, the Y. pestis pPCP(-)/Deltalpp mutant strain was detectable in high numbers in the livers and spleens of some of the infected mice. In the lungs of pPCP(-)/Deltalpp mutant-challenged animals, however, bacterial numbers dropped at 48 h after infection when compared with tissue homogenates from 1 h post-infection. Similarly, we noted that this mutant was unable to survive within murine macrophages in an in vitro assay, whereas survivability of the pPCP(-) mutant within the macrophage environment was similar to that of the WT. Taken together, our data indicated that a significant and possibly synergistic attenuation in bacterial virulence occurred in a mouse model of pneumonic plague when both the lpp gene and the virulence plasmid pPCP1 encoding the pla gene were deleted from Y. pestis.

Comparative Analyses of Transcriptional Profiles in Mouse Organs Using a Pneumonic Plague Model after Infection with Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant.

We employed Murine GeneChips to delineate the global transcriptional profiles of the livers, lungs, and spleens in a mouse pneumonic plague infection model with wild-type (WT) Y. pestis CO92 and its Braun lipoprotein (Deltalpp) mutant with reduced virulence. These organs showed differential transcriptional responses to infection with WT Y. pestis, but the overall host functional processes affected were similar across all three tissues. Gene expression alterations were found in inflammation, cytokine signaling, and apoptotic cell death-associated genes. Comparison of WT and Deltalpp mutant-infected mice indicated significant overlap in lipopolysaccharide- (LPS-) associated gene expression, but the absence of Lpp perturbed host cell signaling at critical regulatory junctions resulting in altered immune response and possibly host cell apoptosis. We generated a putative signaling pathway including major inflammatory components that could account for the synergistic action of LPS and Lpp and provided the mechanistic basis of attenuation caused by deletion of the lpp gene from Y. pestis in a mouse model of pneumonic plague.

Characterization of the rat pneumonic plague model: infection kinetics following aerosolization of Yersinia pestis CO92.

Yersinia pestis, the causative agent of human bubonic and pneumonic plague, is spread during natural infection by the fleas of rodents. Historically associated with infected rat fleas, studies on the kinetics of infection in rats are surprisingly few, and these reports have focused mainly on bubonic plague. Although the natural route of primary infection results in bubonic plague in humans, it is commonly thought that aerosolized Y. pestis will be utilized during a biowarfare attack. Accordingly, based on our previous characterization of the mouse model of pneumonic plague, we sought to examine the progression of infection in rats exposed in a whole-body Madison chamber to aerosolized Y. pestis CO92. Following an 8.6 LD(50) dose of Y. pestis, injury was apparent in the rat tissues based on histopathology, and chemokines and cytokines rose above control levels (1h post infection [p.i.]) in the sera and organ homogenates over a 72-h infection period. Bacteria disseminated from the lungs to peripheral organs, with the largest increases in the spleen, followed by the liver and blood at 72h p.i. compared to the 1h controls. Importantly, rats were as sensitive to pneumonic plague as mice, having a similar LD(50) dose by the intranasal and aerosolized routes. Further, we showed direct transmission of plague bacteria from infected to uninfected rats. Taken together, the data allowed us to characterize for the first time a rat pneumonic plague model following aerosolization of Y. pestis.

Mutation in the S-ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila.

A diarrheal isolate SSU of Aeromonas hydrophila produces a cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. Our laboratory has characterized from the above Aeromonas strain, in addition to Act, the type 3- and T6-secretion systems and their effectors, as well as the genes shown to modulate the production of AI-1-like autoinducers, N-acylhomoserine lactones (AHLs) involved in quorum sensing (QS). In this study, we demonstrated the presence of an S-ribosylhomocysteinase (LuxS)-based autoinducer (AI)-2 QS system in A. hydrophila SSU and its contribution to bacterial virulence. The luxS isogenic mutant of A. hydrophila, which we prepared by marker exchange mutagenesis, showed an alteration in the dynamics and architecture of the biofilm formation, a decrease in the motility of the bacterium, and an enhanced virulence in the septicemic mouse model. Moreover, these effects of the mutation could be complemented. Enhanced production of the biofilm exopolysaccharide and filaments in the mutant strain were presumably the major causes of the observed phenotype. Our earlier studies indicated that the wild-type A. hydrophila with overproduction of DNA adenine methyltransferase (Dam) had significantly reduced motility, greater hemolytic activity associated with Act, and an enhanced ability to produce AI-1 lactones. Furthermore, such a Dam-overproducing strain was not lethal to mice. On the contrary, the luxS mutant with Dam overproduction showed an increased motility and had no effect on lactone production. In addition, the Dam-overproducing luxS mutant strain was not altered in its ability to induce lethality in a mouse model of infection when compared to the parental strain which overproduced Dam. We suggested that an altered gene expression in the luxS mutant of A. hydrophila SSU, as it related to biofilm formation and virulence, might be linked with the interruption of the bacterial metabolic pathway, specifically of methionine synthesis.

Characterization of a mouse model of plague after aerosolization of Yersinia pestis CO92.

Yersinia pestis is a Gram-negative bacterium, and the causative agent of bubonic plague and pneumonic plague. Because of its potential use as a biological warfare weapon, the plague bacterium has been placed on the list of category A select agents. The dynamics of pneumonic infection following aerosolization of the highly virulent Y. pestis CO92 strain have been poorly studied; therefore, the purpose of this study was to determine the LD(50) dose, bacterial dissemination, cytokine/chemokine production and tissue damage in Swiss-Webster mice over a 72 h course of infection. We exposed mice in a whole-body Madison chamber to various doses of Y. pestis CO92 aerosolized by a Collison nebulizer, and determined that the LD(50) presented dose (Dp) of the bacterium in the lungs was 2.1 x 10(3) c.f.u. In a subsequent study, we infected mice at a Dp of 1.3 x 10(4) c.f.u., and harvested organs and blood at 1, 24, 48 and 72 h post-infection. Histopathological examination, in addition to measurement of bacterial dissemination and cytokine/chemokine analysis, indicated progressive tissue injury, and an increased number of animals succumbing to infection over the course of the experiment. Using these data, we were able to characterize the mouse plague model following aerosolization of Y. pestis CO92.

Alteration in the activation state of new inflammation-associated targets by phospholipase A2-activating protein (PLAA).

Phospholipase A(2) (PLA(2))-activating protein (PLAA) is a novel signaling molecule that regulates the production of prostaglandins (PGE(2)) and tumor necrosis factor (TNF)-alpha. To characterize the function of native PLAA in situ, we generated HeLa (Tet-off) cells overexpressing plaa (plaa(high)) and control (plaa(low)) cells, with the plaa gene in opposite orientation in the latter construct. The plaa(high) cells produced significantly more PGE(2) and interleukin (IL)-6 compared to plaa(low) cells in response to TNF-alpha. There was an increased activation and/or expression of cytosolic PLA(2), cyclooxgenase-2, and NF-kappaB after induction of plaa(high) cells with TNF-alpha compared to the respective plaa(low) cells. Microarray analysis of plaa(high) cells followed by functional assays revealed increased production of proinflammatory cytokine IL-32 and a decrease in the production of annexin A4 and clusterin compared to plaa(low) cells. We demonstrated the role of annexin A4 as an inhibitor of PLA(2) and showed that addition of exogeneous clusterin limited the production of PGE(2) from plaa(high) cells. To understand regulation of plaa gene expression, we used a luciferase reporter system in HeLa cells and identified one stimulatory element, with Sp1 binding sites, and one inhibitory element, in exon 1 of the plaa gene. By using decoy DNA oligonucleotides to Sp1 and competitive binding assays, we showed that Sp1 maintains basal expression of the plaa gene and binds to the above-mentioned stimulatory element. We demonstrated for the first time that the induction of native PLAA by TNF-alpha can perpetuate inflammation by enhancing activation of PLA(2) and NF-kappaB.

Braun lipoprotein (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague.

Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 x 10(7) CFU of the Deltalpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these Deltalpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD(50)). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the Deltalpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the Deltalpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the Deltalpp mutant than in those infected with WT CO92. Additionally, the Deltalpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the Deltalpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.

Immunological responses against Salmonella enterica serovar Typhimurium Braun lipoprotein and lipid A mutant strains in Swiss-Webster mice: potential use as live-attenuated vaccines.

We generated and characterized Salmonella enterica serovar Typhimurium mutants that were deleted for the genes encoding Braun lipoprotein (lpp) alone or in conjunction with the msbB gene, which codes for an enzyme required for the acylation of the lipid A moiety of lipopolysaccharide. Two copies of the lpp gene, designated as lppA and lppB, exist on the chromosome of S. Typhimurium. These mutants were highly attenuated in a mouse infection model and induced minimal histopathological changes in mouse organs compared to those seen in infection with wild-type (WT) S. Typhimurium. The lppB/msbB and the lppAB/msbB mutants were maximally attenuated, and hence further examined in this study for their ability to induce humoral and cellular immune responses. Importantly, infection of out-bred Swiss-Webster mice with the mutant S. Typhimurium generated superior T helper cell type 2 (Th2) responses compared to WT S. Typhimurium, as determined by measuring IgG subclasses and cytokines. WT S. Typhimurium induced higher levels of IgG2a in sera of infected mice, while the lppB/msbB and lppAB/msbB mutants mounted higher levels of IgG1 as determined by an enzyme-linked immunosorbent assay. Mice immunized with lppB/msbB and lppAB/msbB mutants rapidly cleared WT S. Typhimurium upon subsequent rechallenge, and naïve mice passively immunized with sera from animals infected with S. Typhimurium mutants were protected against subsequent challenge with WT S. Typhimurium. Splenic T cells produced higher levels of interferon-gamma following ex vivo exposure to WT S. Typhimurium, while splenic T cells infected with the above-mentioned two mutants evoked higher levels of interleukin-6. Further, mice infected with lppB/msbB and lppAB/msbB mutants showed much higher levels of splenic T cell activation as measured by CD44(+) expression on CD4(+) T cells by flow cytometry and by incorporation of (3)H-thymidine compared to mice that were infected with WT S. Typhimurium. We expect the lppB/msbB and lppAB/msbB mutants to be excellent live-attenuated vaccine candidates, because they induced minimal inflammatory responses and evoked stronger and specific antibody and cellular immune responses.

Cytotoxic herpes simplex type 2-specific, DQ0602-restricted CD4 T+-cell clones show alloreactivity to DQ0601.

Alloreactivity is one of the most serious problems in organ transplantation. It has been hypothesized that pre-existing alloreactive T cells are actually cross-reacting cells that have been primed by the autologous major histocompatibility complex (MHC) and a specific peptide. CD8+ cytotoxic T lymphocytes that are alloreactive and recognize a virus-peptide that is presented by the autologous MHC have been reported. Here we demonstrate a cross-reactivity that exists between DQ0602 restricted, herpes simplex type 2 VP16 40-50 specific CD4+ T-cell clones, which can be alloreactive to DQ0601. Though most of the DQ0602 restricted T-cell clones we isolated from two different donors were not alloreactive, weakly cross-reacting T-cell clones could be isolated from both donors. Two strongly cross-reacting T-cell clones with high affinity interaction of their T-cell receptor (TCR) with both DQ0602/VP16 40-50 and DQ0601 could be isolated from one donor. DNA sequencing of the a fragment of the Vbeta gene used in their TCR confirmed that these two T cells indeed are two independent clones. These clones are cytotoxic and produce cytokines of a T helper 2-like pattern. Possible implications in a DR-matched transplantation setting are discussed.