Proteomic analysis of Rickettsia akari proposes a 44 kDa-OMP as a potential biomarker for Rickettsialpox diagnosis.

BACKGROUND: Rickettsialpox is a febrile illness caused by the mite-borne pathogen Rickettsia akari. Several cases of this disease are reported worldwide annually. Nevertheless, the relationship between the immunogenicity of R. akari and disease development is still poorly understood. Thus, misdiagnosis is frequent. Our study is aiming to identify immunogenic proteins that may improve disease recognition and enhance subsequent treatment. To achieve this goal, two proteomics methodologies were applied, followed by immunoblot confirmation. RESULTS: Three hundred and sixteen unique proteins were identified in the whole-cell extract of R. akari. The most represented protein groups were found to be those involved in translation, post-translational modifications, energy production, and cell wall development. A significant number of proteins belonged to amino acid transport and intracellular trafficking. Also, some proteins affecting the virulence were detected. In silico analysis of membrane enriched proteins revealed 25 putative outer membrane proteins containing beta-barrel structure and 11 proteins having a secretion signal peptide sequence. Using rabbit and human sera, various immunoreactive proteins were identified from which the 44 kDa uncharacterized protein (A8GP63) has demonstrated a unique detection capability. It positively distinguished the sera of patients with Rickettsialpox from other rickettsiae positive human sera. CONCLUSION: Our proteomic analysis certainly contributed to the lack of knowledge of R. akari pathogenesis. The result obtained may also serve as a guideline for a more accurate diagnosis of rickettsial diseases. The identified 44 kDa uncharacterized protein can be certainly used as a unique marker of rickettsialpox or as a target molecule for the development of more effective treatment.

Photosynthetic and Stress Responsive Proteins Are Altered More Effectively in Nicotiana benthamiana Infected with Plum pox virus Aggressive PPV-CR versus Mild PPV-C Cherry-Adapted Isolates.

Plum pox virus (PPV, family Potyviridae) is one of the most important viral pathogens of Prunus spp. causing considerable damage to stone-fruit industry worldwide. Among the PPV strains identified so far, only PPV-C, PPV-CR, and PPV-CV are able to infect cherries under natural conditions. Herein, we evaluated the pathogenic potential of two viral isolates in herbaceous host Nicotiana benthamiana. Significantly higher accumulation of PPV capsid protein in tobacco leaves infected with PPV-CR (RU-30sc isolate) was detected in contrast to PPV-C (BY-101 isolate). This result correlated well with the symptoms observed in the infected plants. To further explore the host response upon viral infection at the molecular level, a comprehensive proteomic profiling was performed. Using reverse-phase ultra-high-performance liquid chromatography followed by label-free mass spectrometry quantification, we identified 38 unique plant proteins as significantly altered due to the infection. Notably, the abundances of photosynthesis-related proteins, mainly from the Calvin-Benson cycle, were found more aggressively affected in plants infected with PPV-CR isolate than those of PPV-C. This observation was accompanied by a significant reduction in the amount of photosynthetic pigments extracted from the leaves of PPV-CR infected plants. Shifts in the abundance of proteins that are involved in stimulation of photosynthetic capacity, modification of amino acid, and carbohydrate metabolism may affect plant growth and initiate energy formation via gluconeogenesis in PPV infected N. benthamiana. Furthermore, we suggest that the higher accumulation of H2O2 in PPV-CR infected leaves plays a crucial role in plant defense and development by activating the glutathione synthesis.

Spodoptera frugiperda (J. E. Smith) Aminopeptidase N1 Is a Functional Receptor of the Bacillus thuringiensis Cry1Ca Toxin.

Bacillus thuringiensis Cry1Ca is toxic to different Spodoptera species. The aims of this work were to identify the Cry1Ca-binding proteins in S. frugiperda, to provide evidence on their participation in toxicity, and to identify the Cry1Ca amino acid residues involved in receptor binding. Pulldown assays using Spodoptera frugiperda brush border membrane vesicles (BBMV) identified aminopeptidase N (APN), APN1, and APN2 isoforms as Cry1Ca-binding proteins. Cry1Ca alanine substitutions in all residues of domain III ß16 were characterized. Two ß16 nontoxic mutants (V505A and S506A) showed a correlative defect on binding to the recombinant S. frugiperda APN1 (SfAPN1). Finally, silencing the expression of APN1 transcript, by double-stranded RNA (dsRNA) feeding, showed that silenced larvae are more tolerant of the Cry1Ca toxin, which induced less than 40% mortality in silenced larvae whereas nonsilenced larvae had 100% mortality. Overall, our results show that Cry1Ca relies on APN1 binding through domain III ß16 to impart toxicity to S. frugiperdaIMPORTANCEBacillus thuringiensis Cry toxins rely on receptor binding to exert toxicity. Cry1Ca is toxic to different populations of S. frugiperda, a major corn pest in America. Nevertheless, the S. frugiperda midgut proteins that are involved in Cry1Ca toxicity have not been identified. Here we identified aminopeptidase N1 (APN1) as a functional receptor of Cry1Ca. Moreover, we showed that Cry1Ca domain III ß16 is involved in APN1 binding. These results give insights on potential target sites for improving Cry1Ca toxicity to S. frugiperda.

Identification of Coxiella burnetii surface-exposed and cell envelope associated proteins using a combined bioinformatics plus proteomics strategy.

The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within the phagolysosomal vacuoles of eukaryotic cells. This pathogen can infect a wide range of hosts, and is the causative agent of Q fever in humans. Surface-exposed and cell envelope associated proteins are thought to be important for both pathogenesis and protective immunity. Herein, we propose a complementary strategy consisting of (i) in silico prediction and (ii) inventory of the proteomic composition using three enrichment approaches coupled with protein identification. The efficiency of classical Triton X-114 phase partitioning was compared with two novel procedures; isolation of alkaline proteins by liquid-phase IEF, and cell surface enzymatic shaving using biofunctional magnetic beads. Of the 2026 protein sequences analyzed using seven distinct bioinformatic algorithms, 157 were predicted to be outer membrane proteins (OMP) and/or lipoproteins (LP). Using the three enrichment protocols, we identified 196 nonredundant proteins, including 39 predicted OMP and/or LP, 32 unknown or poorly characterized proteins, and 17 effectors of the Type IV secretion system. We additionally identified eight proteins with moonlighting activities, and several proteins apparently peripherally associated with integral or anchored OMP and/or LP.

Detection of Coxiella antibodies in ruminants using a SucB recombinant antigen.

The detection of Coxiella burnetii in ruminants remains challenging despite the use of new technology and the accumulation of novel knowledge. Serology tools, the primary methods of infection surveillance in veterinary medicine, have limitations. We used recombinant antigen production to develop an ELISA based on the SucB protein, one of the major immunodominant antigens described in humans and laboratory animals. We produced the antigen successfully in an Escherichia coli heterologous system, confirmed by sequencing and mass spectrometry, and seen as a band of ~50 kDa in SDS-PAGE and on western blot analysis. We compared the performance of the recombinant ELISA with a commercial ELISA. We observed agreement of 83.5% and a substantial Cohen κ value of 0.67 in our pilot study.

In silico biosynthesis of virenose, a methylated deoxy-sugar unique to Coxiella burnetii lipopolysaccharide.

BACKGROUND: Coxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has an obligate intracellular growth habit, it is able to persist for extended periods outside of a host cell and can resist environmental conditions that would be lethal to most prokaryotes. It is these extracellular bacteria that are the infectious stage encountered by eukaryotic hosts. The intracellular form has evolved to grow and replicate within acidified parasitophorous vacuoles. The outer coat of C. burnetii comprises a complex lipopolysaccharide (LPS) component that includes the unique methylated-6-deoxyhexose, virenose. Although potentially important as a biomarker for C. burnetii, the pathway for its biosynthesis remains obscure. RESULTS: The 6-deoxyhexoses constitute a large family integral to the LPS of many eubacteria. It is believed that precursors of the methylated-deoxyhexoses traverse common early biosynthetic steps as nucleotide-monosaccharides. As a prelude to a full biosynthetic characterization, we present herein the results from bioinformatics-based, proteomics-supported predictions of the pathway for virenose synthesis. Alternative possibilities are considered which include both GDP-mannose and TDP-glucose as precursors. CONCLUSION: We propose that biosynthesis of the unique C. burnetii biomarker, virenose, involves an early pathway similar to that of other C-3'-methylated deoxysugars which then diverges depending upon the nucleotide-carrier involved. The alternatives yield either the D- or L-enantiomers of virenose. Both pathways require five enzymatic steps, beginning with either glucose-6-phosphate or mannose-6-phosphate. Our in silico results comprise a model for virenose biosynthesis that can be directly tested. Definition of this pathway should facilitate the development of therapeutic agents useful for treatment of Q fever, as well as allowing improvements in the methods for diagnosing this highly infectious disease.

Proteomic analysis revealed the survival strategy of Coxiella burnetii to doxycycline exposure.

Antibiotic resistance is a global threat with a top concern in healthcare. Doxycycline is an antibiotic highly permeable to cell membrane used for treating a broad variety of bacteria, including Coxiella burnetii. This intracellular pathogen is the causative agent of Q fever, a re-emerging zoonosis found worldwide. Hence, C. burnetii has a considerable impact on the farming industry and public health, it is essential to explore its antibiotic adaptation/tolerance strategy to ensure effective therapy. Herein, we tracked changes in the bacterium induced by doxycycline exposure. Our proteomic analysis detected fifteen significantly altered proteins. Adjustments of some key proteins were verified by gene expression analysis. We also observed an increasing in hydrogen peroxide as a consequence of treatment, indicating deregulation of redox balance. Thus, our data suggests the reduction of protein synthesis to minimal levels, activation of the defense mechanism against oxidative stress and maintenance of cell envelope integrity as the key processes ensuring C. burnetii survival under doxycycline exposure. SIGNIFICANCE: Infection by intracellular microorganisms like C. burnetii requires long periods of treatment, thus antibiotic resistance development is a risk. In this report, 2-DE quantitative proteomics was used to identify changes in the proteome that occurs when C. burnetii is exposed to high concentrations of doxycycline. The identification of pathways impacted by doxycycline could be helpful to understand the mechanism of how C. burnetii is dealing with antibiotic stress.

Comparative proteomics of the vector Dermacentor reticulatus revealed differentially regulated proteins associated with pathogen transmission in response to laboratory infection with Rickettsia slovaca.

BACKGROUND: Tick-borne rickettsial diseases are caused by pathogens acquired from hard ticks. In particular, Rickettsia slovaca, a zoonotic infectious bacterium causing tick-borne lymphadenopathy (TIBOLA), is transmitted by the vectors Dermacentor spp. that can be found all over Europe. Although recent studies point out the extreme complexity of bacteria-induced effects in these blood-feeding vectors, the knowledge of individual molecules involved in the preservation and transmission of the pathogen is still limited. System biology tools, including proteomics, may contribute greatly to the understanding of pathogen-tick-host interactions. METHODS: Herein, we performed a comparative proteomics study of the tick vector Dermacentor reticulatus that was experimentally infected with the endosymbiotic bacterium R. slovaca. Rickettsia-free ticks, collected in the southern region of Slovakia, were infected with the bacterium by a capillary tube-feeding system, and the dynamics of infection was assessed by quantitative PCR method after 5, 10, 15 and 27 days. RESULTS: At the stage of controlled proliferation (at 27 dpi), 33 (from 481 profiled) differentially abundant protein spots were detected on a two-dimensional gel. From the aforementioned protein spots, 21 were successfully identified by tandem mass spectrometry. CONCLUSIONS: Although a few discovered proteins were described as having structural or housekeeping functions, the vast majority of the affected proteins were suggested to be essential for tick attachment and feeding on the host, host immune system evasion and defensive response modulation to ensure successful pathogen transmission.

Mass production of a S-layer protein of Bacillus thuringiensis and its toxicity to the cattle tick Rhipicephalus microplus.

The most commonly used biopesticides to control agricultural, forest and insect vectors of human diseases are derived from the bacterium Bacillus thuringiensis, which begins to produce Cry and Cyt insecticidal proteins during the onset of the sporulation phase. Some B. thuringiensis strains also produce S-layer proteins that are toxic to certain pests. S-layer proteins are the most abundant proteins in bacteria and archaea. This proteins' key trait to design high performace processes for mass production is their continuous expression during the vegetative phase, unlike Cry and Cyt, which are restricted to the sporulation phase. In this work, a S-layer protein expressed by the GP543 strain of B. thuringiensis that is toxic to the cattle tick Rhipicephalus microplus was mass produced using the batch culture fermentation technique. In addition, the spore-protein complex showed a mortality rate of 75% with a dose of 300 µg·mL-1 on adult females of R. microplus after fourteen days. The lethal concentration 50 was 69.7 µg·mL-1. The treatment also caused a decrease of 13% in the weight of the mass of oviposited eggs with 200 µg·mL-1 of the spore-protein complex and inhibition of the hatching of eggs from 80 to 92%. Therefore, this could be a good option for controlling this parasite. The advantages of S-layer protein synthesis are focused on the production of a new generation of proteins in pest control. This is the first report on the mass production of an S-layer protein that is responsible for toxicity.

Reliable tool for detection of novel Coxiella burnetii antigens, using immobilized human polyclonal antibodies.

Coxiella burnetii (C. burnetii) is the etiological agent of a Q fever-the re-emerging disease with considerable economic impact. Due to many similar symptoms with commonly occurring infections, its clinical diagnosis is very difficult. Thus, a strong effort should be taken to raise the awareness and develop a robust strategy for an accurate diagnosis. The identification of specific C. burnetii biomarkers could be valuable for a sensitive and selective diagnosis of the disease. Herein, we described a workflow to identify immunoreactive proteins of C. burnetii with a high confidence. It is based on immunocapturing of bacterial antigens by biofunctionalized magnetic microspheres, followed by tandem mass spectrometry (MS/MS) identification. We detected dozens of previously reported antigens and proposed 15 novel biomarkers, which specificity was confirmed by in silico epitope analysis. Among them, the cardiolipin synthetase participating in the synthesis of cardiolipin was recognized. This biomarker could play a critical role in the early management of acute Q fever and prevention of Q fever endocarditis.

Partially resistant Cucurbita pepo showed late onset of the Zucchini yellow mosaic virus infection due to rapid activation of defense mechanisms as compared to susceptible cultivar.

Zucchini yellow mosaic virus (ZYMV) is an emerging viral pathogen in cucurbit-growing areas wordwide. Infection causes significant yield losses in several species of the family Cucurbitaceae. To identify proteins potentially involved with resistance toward infection by the severe ZYMV-H isolate, two Cucurbita pepo cultivars (Zelena susceptible and Jaguar partially resistant) were analyzed using a two-dimensional gel electrophoresis-based proteomic approach. Initial symptoms on leaves (clearing veins) developed 6-7 days post-inoculation (dpi) in the susceptible C. pepo cv. Zelena. In contrast, similar symptoms appeared on the leaves of partially resistant C. pepo cv. Jaguar only after 15 dpi. This finding was confirmed by immune-blot analysis which showed higher levels of viral proteins at 6 dpi in the susceptible cultivar. Leaf proteome analyses revealed 28 and 31 spots differentially abundant between cultivars at 6 and 15 dpi, respectively. The variance early in infection can be attributed to a rapid activation of proteins involved with redox homeostasis in the partially resistant cultivar. Changes in the proteome of the susceptible cultivar are related to the cytoskeleton and photosynthesis.

Proteomic comparison of virulent phase I and avirulent phase II of Coxiella burnetii, the causative agent of Q fever.

Coxiella burnetii, a category B biological warfare agent, causes multiple outbreaks of the zoonotic disease Q fever world-wide, each year. The virulent phase I and avirulent phase II variants of the Nine Mile RSA 493 and 439 strains of C. burnetii were propagated in embryonated hen eggs and then purified by centrifugation through Renografin gradients. Total protein fractions were isolated from each phase and subjected to analysis by one-dimensional electrophoresis plus tandem mass spectrometry. A total of 235 and 215 non-redundant proteins were unambiguously identified from the phase I and II cells, respectively. Many of these proteins had not been previously reported in proteomic studies of C. burnetii. The newly identified proteins should provide additional insight into the pathogenesis of Q fever. Several of the identified proteins are involved in the biosynthesis and metabolism of components of the extracellular matrix. Forty-four of the proteins have been annotated as having distinct roles in the pathogenesis or survival of C. burnetii within the harsh phagolysosomal environment. We propose that nine enzymes specifically involved with lipopolysaccharide biosynthesis and metabolism, and that are distinctively present in phase I cells, are virulence-associated proteins.

The effect of amino acid deletions and substitutions in the longest loop of GFP.

BACKGROUND: The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region. RESULTS: In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Delta I129 and sgGFP-Delta D130. Interestingly, both mutants were thermosensitive and at 30 degrees C sgGFP-Delta D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein. CONCLUSION: The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.