The Role of Nucleases and Nucleic Acid Editing Enzymes in the Regulation of Self-Nucleic Acid Sensing.

Detection of microbial nucleic acids by the innate immune system is mediated by numerous intracellular nucleic acids sensors. Upon the detection of nucleic acids these sensors induce the production of inflammatory cytokines, and thus play a crucial role in the activation of anti-microbial immunity. In addition to microbial genetic material, nucleic acid sensors can also recognize self-nucleic acids exposed extracellularly during turn-over of cells, inefficient efferocytosis, or intracellularly upon mislocalization. Safeguard mechanisms have evolved to dispose of such self-nucleic acids to impede the development of autoinflammatory and autoimmune responses. These safeguard mechanisms involve nucleases that are either specific to DNA (DNases) or RNA (RNases) as well as nucleic acid editing enzymes, whose biochemical properties, expression profiles, functions and mechanisms of action will be detailed in this review. Fully elucidating the role of these enzymes in degrading and/or processing of self-nucleic acids to thwart their immunostimulatory potential is of utmost importance to develop novel therapeutic strategies for patients affected by inflammatory and autoimmune diseases.

TatD DNases of African trypanosomes confer resistance to host neutrophil extracellular traps.

African trypanosomatid parasites escape host acquired immune responses through periodic antigenic variation of their surface coat. In this study, we describe a mechanism by which the parasites counteract innate immune responses. Two TatD DNases were identified in each of Trypanosoma evansi and Trypanosoma brucei. These DNases are bivalent metal-dependent endonucleases localized in the cytoplasm and flagella of the parasites that can also be secreted by the parasites. These enzymes possess conserved functional domains and have efficient DNA hydrolysis activity. Host neutrophil extracellular traps (NETs) induced by the parasites could be hydrolyzed by native and recombinant TatD DNases. NET disruption was prevented, and the survival rate of parasites was decreased, in the presence of the DNase inhibitor aurintricarboxylic acid. These data suggest that trypanosomes can counteract host innate immune responses by active secretion of TatD DNases to degrade NETs.

Cross-sectional study of population-specific streptococcal antibody titres in Uganda.

OBJECTIVE: Despite substantial variation of streptococcal antibody titres among global populations, there is no data on normal values in sub-Saharan Africa. The objective of this study was to establish normal values for antistreptolysin O (ASO) and antideoxyribonuclease B (ADB) antibodies in Uganda. DESIGN: This was an observational cross-sectional study. SETTING: This study was conducted at Mulago National Referral Hospital, which is located in the capital city, Kampala, and includes the Uganda Heart Institute. PATIENTS: Participants (aged 0-50 years) were recruited. Of 428 participants, 22 were excluded from analysis, and 183 (44.4%) of the remaining were children aged 5-15 years. MAIN OUTCOME MEASURES: ASO was measured in-country by nephelometric technique. ADB samples were sent to Australia (PathWest) for analysis by enzyme inhibition assay: 80% upper limit values were established. RESULTS: The median ASO titre in this age group was 220 IU/mL, with the 80th percentile value of 389 IU/mL. The median ADB titre in this age group was 375 IU/mL, with the 80th percentile value of 568 IU/mL. CONCLUSIONS: The estimated Ugandan paediatric population standardised 80% upper-limit-of-normal ASO and ADB titres is higher than many global populations. Appropriateness of using population-specific antibody cutoffs is yet to be determined and has important implications for the sensitivity and specificity of rheumatic fever diagnosis.

High Nuclease Activity of Long Persisting Staphylococcus aureus Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing.

Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.

Cloning, expression and purification of 3'-nucleotidase/nuclease, an enzyme responsible for the Leishmania escape from neutrophil extracellular traps.

Leishmaniasis is one of the most significant of the neglected tropical diseases, with 350 million people in 98 countries worldwide living at risk of developing one of the many forms of the disease. During the transmission of the parasite from its vector to the vertebrate host, neutrophils are rapidly recruited to the site of the sandfly bite. Using different strategies, neutrophils can often kill a large number of parasites. However, some parasites can resist neutrophil-killing mechanisms and survive until macrophage arrival at the infection site. One of the strategies for neutrophil-mediated killing is the production of neutrophil extracellular traps (NETs). Because of its ecto-localized nuclease activity, the enzyme 3'-nucleotidase/nuclease (3'NT/NU), present in different Leishmania species, was recently identified as part of a possible parasite escape mechanism from NET-mediated death. Previous studies showed that 3'NT/NU also plays an important role in the establishment of Leishmania infection by generating extracellular adenosine that favors the parasite and macrophage interaction. This study aims to deepen the knowledge about 3'NT/NU, mainly with respect to its nuclease activity that is little studied in the current literature. For this, we cloned, expressed and purified the recombinant La3'NT/NU and have confirmed its contribution to the parasite escape from NET-mediated killing.

Streptococcal Serology in Acute Rheumatic Fever Patients: Findings From 2 High-income, High-burden Settings.

BACKGROUND: Globally, there is wide variation in streptococcal titer upper limits of normal (ULN) for antistreptolysin O (ASO) and anti-deoxyribonuclease B (ADB) used as an evidence of recent group A streptococcal infection to diagnose acute rheumatic fever (ARF). METHODS: We audited ASO and ADB titers among individuals with ARF in New Zealand (NZ) and in Australia's Northern Territory. We summarized streptococcal titers by different ARF clinical manifestations, assessed application of locally recommended serology guidelines where NZ uses high ULN cut-offs and calculated the proportion of cases fulfilling alternative serologic diagnostic criteria. RESULTS: From January 2013 to December 2015, group A streptococcal serology results were available for 350 patients diagnosed with ARF in NZ and 182 patients in Northern Territory. Median peak streptococcal titers were similar in both settings. Among NZ cases, 267/350 (76.3%) met NZ serologic diagnostic criteria, whereas 329/350 (94.0%) met Australian criteria. By applying Australian ULN titer cut-off criteria to NZ cases, excluding chorea, ARF definite cases would increase by 17.6% representing 47 cases. CONCLUSIONS: ASO and ADB values were similar in these settings. Use of high ULN cut-offs potentially undercounts definite and probable ARF diagnoses. We recommend NZ and other high-burden settings to use globally accepted, age-specific, lower serologic cut-offs to avoid misclassification of ARF.

Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity.

Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus CsmcrRNA binary, CsmcrRNA-target RNA and CsmcrRNA-target RNAanti-tag ternary complexes in the 3.1 Å range. The topological features of the crRNA 5'-repeat tag explains the 5'-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5'-repeat serving as sensors for avoidance of autoimmunity. The Csm3 thumb elements introduce periodic kinks in the crRNA-target RNA duplex, facilitating cleavage of the target RNA with 6-nt periodicity. Key Glu residues within a Csm1 loop segment of CsmcrRNA adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into CsmcrRNA complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.

DNA as a self-antigen: nature and regulation.

High-affinity antibodies to double-stranded DNA are a hallmark of systemic lupus erythematosus (SLE) and are thought to contribute to disease flares and tissue inflammation such as nephritis. Notwithstanding their clinical importance, major questions remain about the development and regulation of these pathogenic anti-DNA responses. These include the mechanisms that prevent anti-DNA responses in healthy subjects, despite the constant generation of self-DNA and the abundance of DNA-reactive B cells; the nature and physical form of antigenic DNA in SLE; the regulation of DNA availability as an antigen; and potential therapeutic strategies targeting the pathogenic DNA in SLE. This review summarizes current progress in these directions, focusing on the role of secreted DNases in the regulation of antigenic extracellular DNA.

A nuclease-toxin and immunity system for kin discrimination in Myxococcus xanthus.

The use of toxin to attack neighbours and immunity proteins to protect against toxin has been observed in bacterial conflicts, including kin discrimination. Here, we report a novel nuclease-toxin and its immunity protein function in the colony-merger incompatibility, a kind of bacterial kin discrimination, in Myxococcus xanthus DK1622. The MXAN_0049 gene was determined to be a genetic determinant for colony-merger incompatibility, and the incompatibility could be eliminated by deletion of the upstream co-transcribed MXAN_0050 gene. We demonstrated that the MXAN_0050 protein was a nuclease, and MXAN_0049 protein was able to bind to MXAN_0050 to block nuclease activity in vitro. Expression of MXAN_0050 in Escherichia coli inhibited cellular growth, and the inhibition effect could be recovered by co-expression of MXAN_0049. We found that deletion of the PAAR-encoding gene (MXAN_0044) or the type VI secretion system led to the colony-merger and co-existence with the ΔMXAN_0049 mutant, suggesting that they were associated with colony-merger incompatibility. Homologues of the nuclease-toxin and cognate immunity pair are widely distributed in bacteria. We propose a simplified model to explain the kin discrimination mechanism mediated by the nuclease-toxin and immunity protein.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Molecular characterization of shrimp harbinger transposase derived 1 (HARBI1)-like and its role in white spot syndrome virus and Vibrio alginolyticus infection.

The role of the nuclease, HARBI1-like protein (mjHARBI1-like) in the innate immunity of Marsupenaeus japonicus was explored in this study. The 1361 bp cDNA sequence of mjHARBI1-like was cloned from M. japonicus using RACE. RT-qPCR analysis results showed that the gills and hepatopancreas of M. japonicus were the main tissues where mjHARBI1-like is expressed. In addition, it was also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could stimulate mjHARBI1-like expression. After mjHARBI1-likewas inhibited, expression of immune genes such as toll, p53, myosin, and proPO were significantly downregulated (P < 0.01). However, in shrimp hemocytes, hemocyanin and tumor necrosis factor-α (TNF-α) were up-regulated significantly (P < 0.01). This study demonstrated that mjHARBI1-like plays a key role in the progression of WSSV and V. alginolyticus infection. Specifically, the cumulative mortality of WSSV-infected and V. alginolyticus-infected shrimp was significantly advanced by double-strand RNA interference (dsRNAi) of mjHARBI1-like. Apoptosis studies indicated that mjHARBI1-dsRNA treatment caused a reduction in hemocyte apoptosis in bacterial and viral groups. In addition, phagocytosis experiments illustrated that mjHARBI1-dsRNA treatment led to a lower phagocytosis rate in hemocytes of V. alginolyticus-challenged shrimp. It was also found that knockdown of mjHARBI1-like inhibited shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, and total hemocyte count (THC) after WSSV or V. alginolyticus infection. These data indicate a regulative role of mjHARBI1-likein the immunity of shrimp in response to pathogen infection. Resultantly, it was concluded that mjHARBI1-like might have a positive effect on the anti-WSSV immune response of shrimp by regulating apoptosis, THC, PO activity, and SOD activity. Additionally, mjHARBI1-like might promote anti-V. alginolyticus infection by participating in regulating phagocytosis, apoptosis, SOD activity, PO activity, and THC.

The major membrane nuclease MnuA degrades neutrophil extracellular traps induced by Mycoplasma bovis.

Mycoplasma bovis has been increasingly recognised worldwide as an economically important pathogen of cattle, causing a range of diseases, including pneumonia, mastitis, polyarthritis and otitis media. It is believed that M. bovis utilises a range of cell surface proteins, including nucleases, to evade the host immune response and survive. However, despite the importance of neutrophils in controlling pathogenic bacteria, the interaction between these cells and M. bovis is not well-characterised. In addition to phagocytosis, neutrophils combat pathogens through the release of neutrophil extracellular traps (NETs), which are composed of their nuclear and granular components, including DNA. Here we investigated the effect of the major membrane nuclease MnuA of M. bovis, which in vitro is responsible for the majority of the nuclease activity of M. bovis, on NET formation. We quantified NET formation by bovine neutrophils 4 h after stimulation with wild-type M. bovis, an mnuA mutant and a mnuA-pIRR45 complemented mnuA mutant. NETs were detected following stimulation of neutrophils with the mnuA mutant but not after exposure to either the wild-type or the mnuA-pIRR45 complemented mutant, and NETs were degraded in the presence of even low concentrations of wild type M. bovis. Surprisingly, there was no increase in levels of intracellular reactive oxygen species (ROS) production in neutrophils stimulated with M. bovis, even though these neutrophils produced NETs. These results clearly demonstrate that M. bovis can induce NET formation in bovine neutrophils, but that the major membrane nuclease MnuA is able to rapidly degrade NETs, and thus is likely to play a significant role in virulence. In addition, M. bovis appears to induce NETs even though ROS production seems to be suppressed.

Type I interferon-mediated autoinflammation due to DNase II deficiency.

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.

Enzyme-linked immunosorbent assay for group A Streptococcal anti-DNase B in human sera, using recombinant proteins - Comparison to the DNA methyl green micromethod.

Among the four known Streptococcal nucleases comprising of DNase A, B, C and D; DNase B is the most common, and determination of the levels of antibody to DNase B (ADB) is often used to confirm a clinical diagnosis of Streptococcus pyogenes/group A Streptococcal (GAS) infection. The commonly used assays for antibodies that neutralize DNase B or streptolysin O activity use partially purified antigens that often fail to detect antibody changes subsequent to culture documented infections. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed employing his-tagged recombinant DNase B as plate antigen for comparison to the commonly used DNA methyl green micromethod (DMGM). DNAs from various Streptococcal species were screened for presence of dnaseB gene by PCR. Measurements of ADB in sera collected from subjects belonging to different ages, and ethnic groups were used to compare the two methods. dnaseB was not detected by PCR in DNA samples isolated from different strains of group B (GBS), C (GCS) and G (GGS) Streptococci. The ADB based ELISA proved to be highly sensitive and more responsive to changes in antibody concentration than DMGM. Use of recombinant DNase B eliminates the variability associated with the enzyme, partially purified from Streptococcal culture supernatants from various commercial sources and may provide a more reliable source of antigen to a wider group of laboratories concerned with GAS diagnosis.

Deletion of ssnA Attenuates the Pathogenicity of Streptococcus suis and Confers Protection against Serovar 2 Strain Challenge.

Streptococcus suis serotype 2 (SS2) is a major porcine and human pathogen which causes arthritis, meningitis, and septicemia. Streptococcus suis nuclease A (SsnA) is a recently discovered deoxyribonuclease (DNase), which has been demonstrated to contribute to escape killing in neutrophil extracellular traps (NETs). To further determine the effects of ssnA on virulence, the ssnA deletion mutant (ΔssnA) and its complemented strain (C-ΔssnA) were constructed. The ability of ΔssnA mutant to interact with human laryngeal epithelial cell (Hep-2) was evaluated and it exhibited dramatically decreased ability to adhere to and invade Hep-2 cells. This mutation was found to exhibit significant attenuation of virulence when evaluated in CD1 mice, suggesting ssnA plays a critical role in the pathogenesis of SS2. Finally, we found that immunization with the ΔssnA mutant triggered both antibody responses and cell-mediated immunity, and conferred 80% protection against virulent SS2 challenge in mice. Taken together, our results suggest that ΔssnA represents an attractive candidate for designing an attenuated live vaccine against SS2.

Deficient Activity of the Nuclease MRE11A Induces T Cell Aging and Promotes Arthritogenic Effector Functions in Patients with Rheumatoid Arthritis.

Immune aging manifests with a combination of failing adaptive immunity and insufficiently restrained inflammation. In patients with rheumatoid arthritis (RA), T cell aging occurs prematurely, but the mechanisms involved and their contribution to tissue-destructive inflammation remain unclear. We found that RA CD4+ T cells showed signs of aging during their primary immune responses and differentiated into tissue-invasive, proinflammatory effector cells. RA T cells had low expression of the double-strand-break repair nuclease MRE11A, leading to telomeric damage, juxtacentromeric heterochromatin unraveling, and senescence marker upregulation. Inhibition of MRE11A activity in healthy T cells induced the aging phenotype, whereas MRE11A overexpression in RA T cells reversed it. In human-synovium chimeric mice, MRE11Alow T cells were tissue-invasive and pro-arthritogenic, and MRE11A reconstitution mitigated synovitis. Our findings link premature T cell aging and tissue-invasiveness to telomere deprotection and heterochromatin unpacking, identifying MRE11A as a therapeutic target to combat immune aging and suppress dysregulated tissue inflammation.

Characterization of the immune response and evaluation of the protective capacity of rSsnA against Streptococcus suis infection in pigs.

The efforts made to develop vaccines against Streptococcus suis have failed because of lack of common antigens cross-reactive against different serotypes of this species. The cell wall-anchored proteins can be good vaccine candidates due to their high expression and accessibility to antibodies, among these, a cell-wall protein, DNA-nuclease (SsnA), present in most of the S. suis serotypes and clinical isolates collected from infected pigs, was selected. An experimental challenge against S. suis serotype 2 in a pig model was used to validate the efficacy of recombinant SsnA combined with aluminium hydroxide plus Quil A as adjuvants, previously tested in mice by our research group with good results. In our study, clinical characteristics, bacterial load and spread, haematological and immunological parameters and the antibody response, including the opsonophagocytosis analysis of the sera were evaluated. Moreover the composition of peripheral blood leukocyte populations was studied in infected animals. The results show that the immunization of piglets with rSsnA elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are challenged with a virulent strain in our conventional vaccination model. Further studies are necessary to evaluate the use of rSsnA as a vaccine candidate for swine.

Contamination of DNase Preparations Confounds Analysis of the Role of DNA in Alum-Adjuvanted Vaccines.

Aluminum salt (alum) adjuvants have been used for many years as adjuvants for human vaccines because they are safe and effective. Despite its widespread use, the means by which alum acts as an adjuvant remains poorly understood. Recently, it was shown that injected alum is rapidly coated with host chromatin within mice. Experiments suggested that the host DNA in the coating chromatin contributed to alum's adjuvant activity. Some of the experiments used commercially purchased DNase and showed that coinjection of these DNase preparations with alum and Ag reduced the host's immune response to the vaccine. In this study, we report that some commercial DNase preparations are contaminated with proteases. These proteases are responsible for most of the ability of DNase preparations to inhibit alum's adjuvant activity. Nevertheless, DNase somewhat reduces responses to some Ags with alum. The effect of DNase is independent of its ability to cleave DNA, suggesting that alum improves CD4 responses to Ag via a pathway other than host DNA sensing.

Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum.

Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease.

The TatD-like DNase of Plasmodium is a virulence factor and a potential malaria vaccine candidate.

Neutrophil extracellular traps (NETs), composed primarily of DNA and proteases, are released from activated neutrophils and contribute to the innate immune response by capturing pathogens. Plasmodium falciparum, the causative agent of severe malaria, thrives in its host by counteracting immune elimination. Here, we report the discovery of a novel virulence factor of P. falciparum, a TatD-like DNase (PfTatD) that is expressed primarily in the asexual blood stage and is likely utilized by the parasite to counteract NETs. PfTatD exhibits typical deoxyribonuclease activity, and its expression is higher in virulent parasites than in avirulent parasites. A P. berghei TatD-knockout parasite displays reduced pathogenicity in mice. Mice immunized with recombinant TatD exhibit increased immunity against lethal challenge. Our results suggest that the TatD-like DNase is an essential factor for the survival of malarial parasites in the host and is a potential malaria vaccine candidate.

Immunoprotective potential of in silico predicted Acinetobacter baumannii outer membrane nuclease, NucAb.

Acinetobacter baumannii is an emerging multi-drug resistant pathogen causing significant mortality in hospitalized ICU patients which demands developing new methods for prevention and treatment. A. baumannii 19606 proteome was analysed in silico through the online tool Vaxign for finding potential vaccine candidates. The selected nuclease (NucAb) was predicted to possess all the attributes of a promising vaccine candidate like outer membrane localization, high adhesin probability (0.53), one transmembrane helix only, non-homology to human proteins and presence of B-cell and T-cell epitopes binding with high affinity (percentile rank≤1) to HLA alleles prevalent at high frequency in North Indian populations. nucAb gene was highly prevalent (100%) among the clinical isolates (40/40) and conserved (>98%) among NCBI sequenced Acinetobacter strains. It was cloned in pET28a, purified and its immunoprotective potential was validated in murine pneumonia model. Immunization of BALB/c mice with recombinant NucAb (25µg) elicited high antibody titre (1-5×10(5)) which reduced bacterial load by 5 log cycles in lungs of mice challenged with optimized lethal dose (10(8)CFU). Lung histopathology revealed marked suppression of inflammation. Pro-inflammatory cytokines (TNF-α and IL-6) levels were reduced significantly and anti-inflammatory (IL-10) cytokine increased in lungs and serum leading to decreased severity and slow progression of disease. Though active immunization showed low survival rate (20%), passive immunization improved the survival (40%). This is the first study reporting an outer membrane nuclease as a vaccine candidate in Gram negative bacterium, A. baumannii through reverse vaccinology approach.