Journal of Life Sciences Volume 6 Number (9)
J LS
Journal of Life Sciences
Volume 6, Number 12, December 2012 (Serial Number 56)
Contents
Molecular Biology
1307 Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in
Azerbaijan
Irada Huseynova and Jalal Aliyev 1317 Helicobacter pylori Biofilm Formation and Gene Expression on Abiotic Surfaces Using a
Cyanobacterial Extract
Alba E. Vega, Fabio A. Persia, Gabriel A. Salinas-Ibañez, Teresa Alarcón, Stella M.C. Lucero Estrada, Teresa I. Cortiñas and Humberto J. Silva
1328 RAPD-PCR Based Marker Approach for the Genetic Differentiation of Two Species of Cockroach
(Order-Dictyoptera)
Bharat Neekhra, Divya Pandey and Subodh Kumar Jain 1334 The Determination of Geographical Origin of Foodstuffs by Using Innovative Biological Bar-Code
Aly El Sheikha and Didier Montet 1343 Molecular Characterization of Olive Cultivars in Iraq Using SSR Markers and Compare with
Phenotypic Characterization
Iqbal Harbi, Salwa Jaber Al-Awadi and Ali Imad Mohammad Moner 1351 Arg-X Protease-Sensitive in Supramolecular Structures of Interphase Cell Nucleus during Growth
Morphogenesis Mature Germs of Wheat
Ivanov Ruslan, Vafina Gulnara and Ivanova Evilina
Zoology and Botany
1356 What Do Cattle Prefer in a Tropical Climate: Water Immersion or Artificial Shade? Ana Carina Alves Pereira de Mira Geraldo, Alfredo Manuel Franco Pereira, Cristiane Gonçalves Titto
and Evaldo Antonio Lencioni Titto
1363 Genetic Parameters for Udder Traits in Slovak Dairy Sheep and Their Crosses with Specialized
Breeds
Milan Margetín, Marta Oravcová1, Dušan Apolen and Michal Milerski 1371 Amino Acid and Fatty Acid Profile in Epidermal Mucus of Bluestreak Cleaner Wrasse (Labroides
dimidiatus ): Possible Role as Defense Mechanism against Pathogens
Maziidah Ab Rahman, Roslan Arshad, Faizah Shaharom and Nur Asma Ariffin 1378 Validation of Monomeric Anthocianin Determination Method for Bilberry Juice and Marc
Extracts
Ance Dandena, Ieva Leimane and U ģis Klētnieks 1383 Necrotic Incompatibility in Crosses of Bread Wheats in Argentina
Francisco Javier Di Pane and Gilberto Kraan 1387 Effect of Natural Surface Secretes of Some Common Ornamental Plants Leaves on Pathogenic
Micro-organisms
Fouad H. Kamel, Hero M. Ismael and Akhter A. Ahmed
Interdisciplinary Researches
1391 Enabling Sustainable Management of Non Wood Forest Products in South East Europe–Albania’s
Experience
Elvira Bazina 1396 Verbal Communication in HIV-1 Patients: A New Perspective on the Study of Cognitive Disorders
Valeria Abusamra, Lorena Abusamra, Bárbara Sampedro, María Macaya, Mercedes Güemes, Micaela Difalcis and Aldo Ferreres
1408 Cortical Arousal Strategies in Left-Handers during the Aural Perception and Manual Playback of
Mono- and Polyphonic Rhythmical Patterns
Pavlovych Olga Sergiyivna, Morenko Alevtyna Grigorivna, Kotyk Olena Adamivna and Vlasjuk Svitlana Sergiyivna
1414 Impact of Omega-3 Nutritional Protocol on the Occurrence of Preeclampsia among High Risk
Pregnant Women
Abeer Mokhtar Abu Khatwa and Shadia Hassan Abdel Kader 1420 Wildlife Crossing Zones along the Ring Changbai Mountain Scenic Highway, China
Yun Wang, Zhengji Piao, Lei Guan, Haifeng Li, Qilin Li, Yayi Lu, Lei Gao and Jiding Chen
Journal of Life Sciences 6 (2012) 1307-1316
Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
Irada Huseynova and Jalal Aliyev Department of Fundamental Problems of Biological Productivity, Institute of Botany, Azerbaijan National Academy of Sciences, Baku AZ1073, Azerbaijan
Received: July 28, 2012 / Accepted: October 10, 2012 / Published: December 30, 2012.
Abstract: Viral diseases are an important limiting factor in many crop production systems in Azerbaijan. Symptomatic plants in main crop-producing areas were tested by ELISA (enzyme-linked immunosorbent assay) using specific monoclonal and polyclonal antibodies. Then RCA (rolling circle amplification) of circular DNA and PCR using different specific primer pairs have indicated that the tested symptomatic plant samples were completely infected by the following viruses: Luteovirus [BLRV (Bean leaf roll virus)], Potyviruses [BCMV (Bean common mosaic virus), BYMV (Bean yellow mosaic virus)], Bromovirus [(AMV) Alfa-alfa mosaic virus], Geminiviruses [CpCDV (Cickpea chlorotic dwarf virus) and TYLCV (Tomato yellow leaf curl virus)] and Nanoviruses [two different FBNYV (Faba bean necrotic yellow virus) and FBNSV (Faba bean necrotic stunt virus)]. At the same time generation sites of superoxide and hydrogen peroxide radicals and activity of antioxidant enzymes were studied in the naturally infected plants.
Key words: Food crops, virus-like symptoms, viral diseases, molecular methods, reactive oxygen species, antioxidant enzymes.
1. Introduction The last four years i nfection incidences were determined for nine viruses on major food crops such
The ability to accurately detect and identify a as chickpea, lentil, faba bean, tomato, pea and alfa-alfa . potential plant pathogenic organism is fundamental to The probable cause of decay of virus infected plants plant pathogen diagnostics and plant disease is not only the virus activity itself, but also the management for food quality. Increasing international reduced tolerance to repeated unfavorable travel and trade of plant materials enhances the risk of environmental situations [4]. Therefore, any little but introducing new viruses and their vectors into long lasting defect in the biochemical process might production systems. In addition, changing climate have determinant role in limiting the lifetime of conditions can contribute to a successful spread of vegetables. Plants have evolved complex antioxidant newly introduced viruses or their vectors and systems in order to protect cellular membranes and establishment of these organisms in areas that were organelles from the damaging effects of ROS (reactive previously unfavorable [1, 2]. oxygen species) [5]. Antioxidant enzymes and Viral diseases are also an important limiting factor metabolites are located in different plant cell in many crop production systems in our country. It compartments to fulfill their protective function. The causes extensive leaf yellowing, stem and leaf key enzymes, SODs (superoxide dismutases; EC deformation, reduced fruit quality, substantial crop 1.15.1.1), are a family of metalloenzymes catalyzing loss and shortening the life-span of vegetable crops [3]. the dismutation of O − 2 to H 2 O 2 . SODs can be found in
chloroplasts, mitochondria, peroxisomes, and in Corresponding author: Jalal Aliyev, professor, cytoplasm. CATs (Catalases; EC 1.11.1.6), heme
academician, research field: plant physiology. E-mail: aliyev-j@botany-az.org.
proteins that catalyze the removal of H 2 O 2 , are located
1308 Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
in peroxisomes. Enzymes and metabolites of the infection, the main crop-producing areas of Azerbaijan, ascorbate-glutathione cycle (APO (ascorbate fields of faba bean (Vicia faba L.), pea (Pisum sativum peroxidase; EC 1.11.1.11); GR (glutathione reductase,
L.), chickpea (Cicer arietinum L.), tomato (Solanum
lycop еrsicum L.) and lentil (Lens culinaris L.) were are located in organelles and cytoplasm [5, 6].
EC 1.8.1.7)) which is important in H 2 O 2 scavenging
surveyed in different regions (including Goychay, Antioxidant enzymes were often studied at sites of
Nakhchivan, Masalli and Lerik) during the period of attempted pathogen attack and in connection with
2009-2011. Samples were collected from plants immediate responses of invaded cells [7].
showing virus-like symptoms, such as leaf rolling, The main aim of this study is focused on detection
yellowing, mosaic, stunting, wilting, and shortening of of viral infections of vegetable crops by different
the internodes, phloem discoloration, necrosis and molecular techniques, to find out generation sites of
stunted growth (Fig. 1).
superoxide and hydrogen peroxide radicals in the The number of samples collected in each field naturally infected plants and to investigate the
depended on the number of symptomatic plants possible role of antioxidant systems against stress, in
observed. Virus-free plants for negative control were order to deepen our knowledge of the plant-virus
collected under same field conditions. Each field was interaction.
evaluated using a standard format, recording location,
2. Materials and Methods conditions, development stage, virus disease
symptoms, and presence or absence of the insect
2.1 Field Visits and Sample Collections populations. Virus disease incidence in each field To determine the presence or absence of virus
was determined on the basis of visual symptoms and by
(a) (b)
(c) (d)
Fig. 1 Symptomatic Solanum lycop еrsicum plants collected from Masalli fields associated with virus infestation and showing virus-like symptoms such as (a) leaf deformation and discoloration; (b) shortening the life-span, extensive leaf yellowing and stunting; (c) leaf curling; (d) destroyed and reduced fruit quality.
Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
counting the percentage of infected plants at different, amplified by PCR using different specific primer pairs, randomly selected locations in the field. Collected
which yielded PCR products of the expected size. The plant samples with symptoms of potential virus
amplifications were carried out in an Applied
Biosystems 2720 Thermal Cycler© Biorad stored at -20 °C.
infection were immediately frozen in liquid N 2 and
(SingAPOre) using the special program. Amplification products were resolved by electrophoresis in a 1.2%
2.2 Detection of Viruses Using ELISA agarose gel with TAE buffer and stained with ethidium
Virus detection was performed with double and bromide (0.5 μg/mL). The gels were photographed triple antibody sandwich ELISA (DAS-ELISA and
under UV light by “Gel Documentation System TAS-ELISA) [8] using polyclonal and monoclonal
UVITEK” (UK).
antibodies (diluted 1:500 and 1:1000) for following
2.4 Histochemical Staining of Superoxide Anion viruses: BLRV (Bean leaf roll virus), BCMV (Bean
Radical
common mosaic virus), BYMV (Bean yellow mosaic virus), AMV (Alfa-alfa mosaic virus), CpCDV
Histochemical staining for ROS accumulation was (Cickpea chlorotic dwarf virus) and TYLCV (Tomato
conducted as described previously [12-14] with yellow leaf curl virus). All antibodies were kindly
some modifications. For superoxide determination provided by Dr. S. Winter (DSMZ, Braunschweig,
Petri dishes were used, the leaf samples were Germany). ELISA result was measured by recording
immersed in 6 mM NBT solution containing 50 mM its absorbance value using an ELISA plate reader (Stat
sodium phosphate (pH 7.5) and 10 mM sodium azide Fax) at A 405 . Samples with absorbance values higher
for 12 h in the dark. ROS reaction was stopped by than the mean value for non-infected control plants
soaking the leaves with lacto-glycerol-ethanol (1:1:4 plus two or three standard deviations were considered
by vol.) and boiling in water for 5 min, and the positive.
cleared leaves were preserved in 50% ethanol and photographed.
2.3 DNA Extraction and Molecular Analyses
2.5 Histochemical Staining of H 2 O 2 Plant samples that reacted serologically with the
virus antibody were selected for further testing by To detect hydrogen peroxide, traverse sections of RCA (rolling circle amplification) and PCR the leaf stem were cut by hand, and the detached (polymerase chain reaction). Frozen plant samples
leaves of the virus infected plants were deposited in were ground in liquid nitrogen, and DNA was
Petri dishes containing a solution of 5 mM DAB and prepared using the method of Edwards [9], modified
10 mM MES at pH 3.8 for 12 h in darkness. To detect as described by Grigoras [10]. The circular viral DNA
hydrogen peroxide, reaction was stopped by soaking was amplified by RCA using the TempliPhi
the leaves with lacto-glycerol-ethanol (1:1:4 by vol) Amplification Kit (GE HealthCare, UK). φ29 DNA
and boiling in water for 5 min, and the cleared leaves polymerase amplifies single- or double-stranded were preserved in 50% ethanol and photographed circular DNA templates by rolling circle amplification.
[12-14].
Viral DNA for detection nanovirus infection was
2.6 Enzyme Extractions and Determination of the amplified by PCR using primer pairs F103/R101 and
Activity
C5F/C5R, which yielded PCR products of the expected size (770 bp and 660 bp, respectively) [11].
Plants were excised and rapidly weighed (1 g fr wt). Viral DNA extracted from infected plants was also
For all enzyme extracts leaf material was ground with
1310 Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
a pestle and ice-cold mortar using different specific using φ29 DNA polymerase and restricted by enzyme buffers. The homogenates were filtered
endonucleases AatII, xBaI, Sau3A, BamHI or HindIII through four layers of cheesecloth and then [18-19]. All amplified products were resolved by centrifuged at 4 °C for 20 min at 15,000× g. The
agarose gel electrophoresis. Detection of virus supernatant was collected and used for analyses of
infection in symptomatic samples was also performed enzymatic activities.
by PCR method using different specific primer pairs
2.6.1 CAT [11]. The use of different molecular methods indicated The activity of catalase was determined as a
that the tested symptomatic plant samples were decrease in absorbance at 240 nm for 1 min
completely infected by the following viruses:
Luteovirus [BLRV (Bean leaf roll virus)], Potyviruses Kumar and Knowles [15]. The reaction mixture
following the decomposition of H 2 O 2 as described by
[BCMV (Bean common mosaic virus), BYMV (Bean contained 50 mM phosphate buffer (pH 7.0) and 15
yellow mosaic virus)], Bromovirus [AMV (Alfa-alfa mM H 2 O 2 and reaction was initiated by adding
mosaic virus)], Geminiviruses [CpCDV (Cickpea enzyme extract.
chlorotic dwarf virus) and TYLCV (Tomato yellow
2.6.2 APO leaf curl virus)] and Nanoviruses [two different The activity of ascorbate peroxidase was assayed
FBNYV (Faba bean necrotic yellow virus) and according to Nakano and Asada [16]. The assay
FBNSV (Faba bean necrotic stunt virus)]. It is
mixture consisted of 0.05 mM ASA, 0.1 mM H 2 O 2 ,
important to note that additional pathogens can be
0.1 mM EDTA, 50 mM sodium phosphate buffer (pH
expected on these crops.
7.6), and 0.3 mL enzyme extract. The activity was
3.2 Determination of Superoxide Anion measured as a decrease in absorbance at 290 nm for
30 s. As known, the level of plant resistance to viral
2.6.3 GR diseases provides many physiological and biochemical Glutathione reductase activity was determined at
parameters responsible for maintaining the viability 340 nm for 10 min in 1 mL reaction mixture
and alterations in plant metabolism under stress containing 100 mM potassium phosphate buffer (pH
conditions. On this basis, histochemical study of the 7.8), 1 mM EDTA, 0.2 mM NADPH and 0.5 mM
possible presence of superoxide anion, H 2 O 2 and GSSG [17].
activity of antioxidant enzymes in virus infected plant
2.6.4 SOD leaves were performed in the present work. Superoxide dismutase activity was estimated by
ROS generation is a common feature in both using SOD Assay Kit-WST (Sigma-Aldrich, USA).
incompatible and compatible plant-pathogen The absorbance was recorded at 450 nm and one
interactions. The oxidative burst observed in the enzyme unit of SOD activity was defined as the
initial stages of incompatible interactions [20] is amount of enzime recuired to cause 50% inhibition of
responsible for the induction of defense reactions the rate of NBT reduction.
leading to hypersensitive responses and the
3. Results and Discussion
development of SAR (systemic acquired resistance) [21]. A major defense mechanism in plants is HR
3.1 Virus Infection (hypersensitive response) whereby cells infected
All collected plant samples firstly were tested by with pathogens are instructed to self-destruction by enzyme-linked immunosorbent assay. Then DNA was
the host plant. This is thought to deny nutrients to the extracted from the fresh leaves, amplified by RCA
invading pathogen. It is proposed that in plants the
Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
process is initiated by a reaction between NO (nitric
oxide) and H 2 O 2 , which is formed by O − 2 (superoxide)
dismutation by SOD during the HR.
(a)
Plant-virus interaction may result in a host hypersensitive response or in systemic symptoms [22,
23]. One of the earliest responses of plant cells to pathogens is the production of ROS. The typical ROS
detected are O .
2 (superoxide radicals) and H 2 O 2 [24,
(b)
25]. ROS play a crucial role during pathogenesis. They are involved in the hypersensitive response
typical for plant-pathogen incompatible interactions. They can limit the spread of pathogen by strengthening plant cell walls and/or by killing
(c)
pathogens directly [26, 27]. However, ROS act as cytotoxic compounds, too.
In this work, the authors detected the presence of Fig. 2 Detection of superoxide anion radical with NBT
staining. Leaves were infiltrated after being submerging in
superoxide anion O 2 in places of infection with the
a reaction mixture containing 6 mM NBT in 50 mM sodium
use of NBT. Accumulation of insoluble blue-colored
phosphate buffer (pH 7.6) plus 10 mM sodium azide. (a)
formazan complex (reduced NBT) is an indicator of
Cucumis sativus L.; (b) Zea mays L.; (c) Solanum lycop еrsicum L. Healthy leaves shown on the right panels,
generation of ROS, in particular of superoxide anion.
infected leaves shown on the left panels. The experiment
This accumulation was observed in infected leaves
was repeated two times.
after infiltration. Then staining declined rapidly, preceding the apparition of necrosis (Fig. 2). Histochemical staining for superoxide production in
leaves tissues was based on the ability of O – 2 to
(a)
reduce NBT and used to detect in situ the production of superoxide radicals [28]. Detached leaflets from
plants subjected to the viral diseases above described and their respective controls were immersed in sodium
phosphate buffer (pH 7.8) containing 0.1% NBT and (b)
10 mM sodium azide. Leaflets of healthy plants were also infiltrated with
50 mM sodium phosphate buffer (pH 7.8) containing only 10 mM sodium azide and used as control. (c)
Superoxide was visualized as a purple discoloration of NBT. Discoloration of leaf was quantified using a
Fig. 3 Detection of H 2 O 2 using DAB (3,3 ’ - digital imaging system (Fig. 3). In typical diaminobenzidine tetrahydrochloride) staining method. The
incompatible interactions, one of the early events of
reaction mixture contained 5 mM DAB in 10 mM MES buffer (pH 3.8). (a) Cucumis sativus L.; (b) Zea mays L.; (c)
HR is an oxidative burst with the generation of
− Solanum lycop еrsicum L. Healthy leaves shown on the right
superoxide (O 2 ) and the subsequent accumulation of
panels, infected leaves shown on the left panels. The
H 2 O 2 [21].
experiment was repeated two times.
1312 Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
3.3 Determination of H 2 O 2 APO or CAT activities in cells is crucial for determining the steady-state level of O –
2 and H 2 O 2 .
Accumulation of H 2 O 2 was observed using DAB
Redox metabolites, such as ASC and the tripeptide staining method. The reaction mixture contained 5
GSH (glutathione), also protect plant cells against mM DAB in 10 mM MES (pH 3.8). Leaves were
ROS-induced damage, either by directly removing incubated overnight at 25 °C, and then cleared twice
reactive chemical species or blocking the oxidative in 50% (v⁄v) ethanol for 10 min. DAB polymerizes to
chain reactions triggered by ROS. Results reported in
the literature indicate that alteration in the the presence of peroxidase, and, thus, provides a
produce a brown precipitate on contact with H 2 O 2 in
expression/activity of ROS-scavenging enzymes useful marker of peroxide accumulation [29]. could also be a key step in the activation of
Superoxide anions are thought to be produced outside phytopathogen defence. On this base, the authors the plant cell by a plasma membrane-associated
also studied the activities of the antioxidant enzymes, NAD(P)H oxidase, and are usually rapidly converted
CAT, APO, SOD and GR, in viral infected plant to H 2 O 2 by superoxide dismutase. To examine whether
leaves. These enzymes are known to be involved in
H 2 O 2 is also accumulated at the site of elicited HR, an immediate plant defense response. Samples for infected leaves were dipped in a solution of DAB. Fig. 3
activity measurements of the antioxidant enzymes
were collected during the early stage of the infection, caused by virus infection. In higher plants, production
clearly shows that H 2 O 2 accumulated during the HR
when the first visible symptoms of the virus infection of H 2 O 2 is thought to be driven by increases in the
appeared on the leaves (in early June). As shown in concentrations of superoxide anions. However, a
Fig. 4, the activity of antioxidant enzymes in infected slightly lower level of DAB staining was observed in
leaves generally was higher than that of comparable healthy leaves compared with infected leaves (Fig. 4).
healthy leaves.
Thus, the results indicate that an alteration in the Analysis of CAT activity in infected leaves chloroplastic metabolism is produced during the early
showed that this enzyme in all the samples studied response to virus infection favoring the accumulation
had a significant difference compared with the of ROS in the plants.
control. CAT activity was 1.4-fold higher (up to 41%) in infected leaves of Solanum lycopersicum and
3.4 Analyses of the Antioxidant Enzymes Activity 1.27-fold higher (up to 32%) in infected leaves of
Plants have evolved complex antioxidant systems in Vicia faba compared to the healthy plants. The most order to protect cellular membranes and organelles
significant differences between the values of CAT from the damaging effects of ROS [30, 31]. Increase
activity were observed in infected Cicer arietinum in peroxidase activity is also a response to viral
samples, where the activity was 2.6-fold higher (up infection, and has been reported in tobacco [32],
to 163%) compared to the control plants. In infected peaches, apricots [33] and beans [34].
Lens culinaris and Pisum sativum samples, CAT In plant cells, enzymes and redox metabolites act
activity only was slightly compared with the control. in synergy to carry out ROS detoxification. SOD
Analysis of CAT activity in infected Lens culinaris
leaves showed that this enzyme activity did not differ dismutates H 2 O 2 to oxygen and water, and APO
catalyses the dismutation of O – 2 to H 2 O 2 , CAT
significantly (only up to 17%) compared to the reduces H 2 O 2 to water by utilising ASC (ascorbate)
control and accounting 0.42 mmol/mg min, as specific electron donor. These are considered the
respectively. As shown in Fig. 4, CAT activity in main enzymatic systems for protecting cells against
infected Pisum sativum leaves did not seem to be oxidative damage. The balance between SOD and
affected significantly by viral stress.
Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
C (mMol/ 0.2 0.1
A (mMol/
D 0.1 SO (mMol/
Fig. 4 Activities of catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase in viral infected plant leaves.
APO activity was 1.2-fold higher in infected leaves significantly differed compared to the control. The of Solanum lycopersicum compared to the healthy
most significant differences between the activities of plants and accounting 0.44 mmoL/mg min. As shown
APO and CAT were observed in infected Lens in Fig. 4, APO activity in infected leaves of Vicia faba
culinaris and Pisum sativum samples, where the APO and Cicer arietinum slightly increased (up to 24% and
activity was 2.2 and 1.5-fold higher compared to the 16%, respectively), although the activity of CAT
control plants. This may indicate the existing
1314 Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
functional relationship and competition between the accounting 0.28 mmoL/mg min in infected leaves of studied enzymes under these viral diseases. Other
Solanum lycopersicum compared to the healthy plants. works also suggest that, along with the activation of
GR activity in infected samples of Vicia faba increased SOD and APO in the leaves, a sharp decrease in
up to 17% compared to the control. In contrast, the GR activity of CAT is observed, which may be due to
activity in infected samples of Cicer arietinum and inhibition of the enzyme substrate—H 2 O 2 Lens culinaris did not significantly differ from the
Interestingly, it has been reported that in a compatible control plants. As shown in Fig. 4, the most significant response between barley and powdery mildew the
difference between the GR activities were observed in cytosolic isoenzyme of APO is up-regulated in both
infected Pisum sativum samples, where activity epidermal and mesophyll cells. In these cells, that are
increased approximately 2-fold higher, i.e. up to 96% not able to trigger a response to stop pathogens, the
compared to the control plants.
APO increase limits the propagation of oxidative As shown in Fig. 4, the change of SOD activity in processes allowing cells to maintain their viability, a
infected plant leaves was different, the actives of condition required for the penetration of biotrophic
Cu/Zn-SOD decreased (up to 46% and 22%) in powdery mildew in plant tissues [35]. This infected samples of Cicer arietinum and Lens culinaris up-regulation of APO confirms previous results
compared to the healthy plants. The most interesting reporting an increase in APO activity during value of SOD activity observed in infected Pisum successful infection of barley leaves by biotrophic
sativum samples, where the activity did not differ from compatible pathogens [36-38] and has also been
the control and accounted 0.22 unit/mg proteins. reported to occur in leaves of susceptible apricot
Obviously, the activation of antioxidant defence infected by plum pox virus [39]. CAT activity has also
systems in plants by abiotic and biotic stresses is a been reported to decrease in cells undergoing HR.
general phenomenon and probably contributes to However, the suppression mechanisms of these two
increased resistance against a subsequent stress.
H 2 O 2 scavenging enzymes are different. CAT is
4. Conclusion
down-regulated at the transcription level [40], whereas, APO regulation in HR involves both transcription and
The following viruses on major food crops translation (or posttranslation) processes. In tobacco
collected from different regions of Azerbaijan: leaves, inoculated with TMV (tobacco mosaic virus), a
Luteovirus [BLRV (Bean leaf roll virus)], Potyviruses rise in APO mRNA occurs [41], probably as an
[BCMV (Bean common mosaic virus), BYMV (Bean antioxidant response triggered by the increasing
yellow mosaic virus)], Bromovirus [AMV (Alfa-alfa presence of H 2 O 2 within cells and similar to that
mosaic virus)], Geminiviruses [CpCDV (Cickpea activated under abiotic stress [42]. In spite of the
chlorotic dwarf virus) and TYLCV (Tomato yellow increase in its expression, the activity of the enzyme is
leaf curl virus)] and Nanoviruses [two different strongly suppressed in the TMV-infected cells by a
FBNYV (Faba bean necrotic yellow virus) and mechanism, still not well characterized, that acts at the
FBNSV (Faba bean necrotic stunt virus)] were transcriptional or post-transcriptional level [43]. In
detected in this study. ROS generation is a common this case, the high activity of antioxidant enzymes can
feature in both incompatible and compatible probably be one of the markers of resistance to the
plant-pathogen interactions. On this basis, pathogen.
histochemical evaluation of the possible presence of The effects of a viral infection caused an increase in
superoxide anion, H 2 O 2 and activity of antioxidant GR activity about 1.86-fold higher (up to 72%) and
enzymes in virus infected plant leaves were performed
Evaluation of Free Radicals and Antioxidant Properties of Virus Infected Food Crops in Azerbaijan
in the present work. Results reported in this study also assay for the detection of plant viruses, Journal of General Virology 34 (1977) 475-483.
indicate that the alteration in the expression/activity of [9] K. Edwards, C. Johnstone, C. Thompson, A simple and
ROS-scavenging antioxidant enzymes could also be a rapid method for the preparation of plant genomic DNA key step in the activation of phytopathogen defence.
for PCR analysis, Nucleic Acids Research 19 (1991) The ability to maintain high physiological function
13-49.
[10] in the presence of viruses may be a more important Grigoras, T. Timchenko, L. Katul, A. Grande-Pérez, H.J. Vetten, B. Gronenborn, Reconstitution of authentic resistance or tolerance mechanism than actual
nanovirus from multiple cloned DNAs, Journal of avoidance of infection. The information presented in
Virology 83 (2009) 10778-10787. the current study is important because the better
[11] I.M. Huseynova, N.F. Sultanova, A.Ch. Mammadov, Detection of single-stranded DNA plant viruses in
understanding of the mechanisms behind the viral vegetable plants using PCR method, Proceedings of
impact on host plant physiology can lead to the
ANAS 66 (2) (2011) 5-12.
development of improved cultivars that either resist [12] M.J. Fryer, L. Ball, K. Oxborough, S. Karpinski, P.M. viral infection or can better tolerate infection by
Mullineaux, N.R. Baker, Control of ascorbate peroxidase 2 expression by hydrogen peroxide and leaf water status
experiencing less severe symptoms. during excess light stress reveals a functional organisation of Arabidopsis leaves, Plant Journal 33
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Journal of Life Sciences 6 (2012) 1317-1327
Helicobacter pylori Biofilm Formation and Gene Expression on Abiotic Surfaces Using
a Cyanobacterial Extract
1 1 1 2 Alba E. Vega 1 , Fabio A. Persia , Gabriel A. Salinas-Ibañez , Teresa Alarcón , Stella M.C. Lucero Estrada , Teresa
I. Cortiñas 1 and Humberto J. Silva 1
1. Microbiology Laboratory, Department of Biochemistry and Biological Sciences, Faculty of Chemistry, Biochemistry and Pharmacy, National University of San Luis, San Luis 5700, Argentina 2. Department of Microbiology, University Hospital La Princesa, Princess Health Research Institute (IP), Madrid 28006, Spain
Received: July 10, 2012 / Accepted: September 17, 2012 / Published: December 30, 2012.
Abstract: The effects of a cyanobacterial extract (CE) on Helicobacter pylori biofilm formation onto hydrophobic and hydrophilic abiotic surfaces and the expression of luxS, flaA, omp18, lpxD and ureA genes associated to biofilm were studied. NCTC11638 reference strain and HP796, a resistant clinical isolate, were grown in Mueller-Hinton broth supplemented with 5% fetal calf serum (FCS) or 1% CE. The ability to form biofilm, viability, morphological changes and gene expression of adhered H. pylori cells were determined. The strains were able to form biofilm on both surfaces with the nutritional supplements analyzed. H. pylori conserved a characteristic bacillary morphology and viability with CE. Cells attachment was higher with CE than FCS regardless of strains and surfaces. The most remarkable increase in gene expression was observed with the omp18 gene using the CE supplement, indicating the important participation of outer membrane proteins in biofilm establishment. The clinical isolate showed similar and even greater gene expression than the reference strain. The results obtained indicated that the nutrients provided by CE favored biofilm formation with retained pathogenicity that under certain conditions can occur in natural aquatic environments.
Key words: Helicobacter pylori, biofilm, cyanobacterial extract, gene expression.
1. Introduction The biofilm mode of growth confers a protective advantage to the bacteria which are physiologically
Helicobacter pylori is a human pathogen that distinct from the free-swimming counterpart of the colonizes the gastric mucosa, resulting in an acute
same species, becoming more resistant to host defense inflammatory response and damage to epithelial cells, and adverse environmental conditions [3], and up to progressing to a number of disease states, including 1,000 times more resistant to antibiotics [4, 5]. gastritis, peptic ulceration, and gastric cancer [1]. The metabolic activity of biofilms is controlled by Recent studies indicate that H. pylori can exist both the environmental conditions found at the surfaces and in human gastric mucosa and on abiotic surface the expression of specific genes induced by adhesion. forming biofilms, explaining the ability of the In many bacteria biofilm formation of is a cell density organism to survive within and outside the host. H. dependent process that relies upon an intercellular pylori biofilm survival in drinking water is discussed communication system known as quorum sensing as a possible waterborne route of transmission [2, 3]. (QS), which is also important in the dissolution of
Corresponding author: Alba E. Vega, Ph.D., professor, biofilm communities [6, 7]. QS signal molecules research field: microbiology. E-mail: aevega@unsl.edu.ar.
Helicobacter pylori Biofilm Formation and Gene Expression on
Abiotic Surfaces Using a Cyanobacterial Extract
regulates a variety of physiological functions The presence of cyanobacteria is common in natural including the generation of bioluminescence, aquatic environments where they can generate blooms sporulation and the expression of virulence factors [8].
generally associated with a diverse community of The luxS gene codifies for the autoinducer 2 (AI-2)
heterothophic cultivable bacteria, some of them of QS system that is functional in stimulating the H.
considered putative pathogens [21-23]. Aditionally a pylori lux operon present in several gram-positive and
cyanobacterial extract (CE) obtained from the gram-negative bacteria [9, 10].
cyanobacterium Nostoc sp. have nutrients, including The expression of luxS gene is essential for H.
amino acid, soluble and crude proteins, carbohydrates, pylori colonization of the human stomach and
and different minerals, useful in the culture of represents a significant indicator of biofilm production
fastidious bacteria [24].
in which bacteria migrate and adhere forming The replacement of fetal calf serum (FCS) by CE in microcolonies [9, 11].
liquid culture media of H. pylori increased growth and Apart from the luxS gene, other genes are involved
cell viability with delays in the appearance of coccoid in biofilm formation including genes encoding for
forms considered viable but nonculturable cells flagella (flaA), type I and type IV pili and surface
(VBNC) [25, 26]. H. pylori adhesion and biofilm adhesins [12].
formation is strongly affected by the presence of The flagella play important roles in biofilm
certain nutrients [27]. In this work the effects of CE formation in several gram-negative bacteria, both as
on H. pylori biofilm formation and the expression of surface adhesins and as providers of force-generating
luxS, flaA, omp18, lpxD and ureA genes associated to motility [13]. The expression of H. pylori flaA is
biofilm is presented.
dependent on luxS and the flaA transcription increases
2. Material and Methods
with culture density [14]. Bacterial outer membrane proteins (OMPs) are
2.1 Strains and Media
important for ion transport, osmotic stability, bacterial
H. pylori NCTC11638 (reference strain) a kind gift virulence and adherence. Omp18 is a for Dr. Manuel López-Brea, Microbiology Service of
peptidoglycan-associated lipoprotein precursor, Hospital Universitario de la Princesa, Madrid, Spain present in H. pylori, which is involved in adhesion to
and HP796, characterized as clarithromicyn (CLA) and gastric cell [15]. The cell envelope gene (lpxD)
metronidazole (MTZ) resistant strain and cagA + and encoding the UDP-3-0-(3-hydroxymyristoyl) vacA s1m1, were used for this study. The bacterial
glucosamine N-acyltransferase is up-regulated after strains were routinely grown on Mueller-Hinton agar adhesion to gastric cell in vitro [16]. Therefore, omp18
(MHA), supplemented with 7% horse blood and lpxD genes could be involved in biofilm
(MHA-HB), and incubated in a microaerobic formation.
atmosphere for 48 h at 37 °C. The identity of H. pylori
H. pylori urease enzyme (ureA) is essential for pH was confirmed by the following criteria: regulation. The loss of urease activity acidifies the
microaerophilic growth requirement, morphology, biofilm, decreasing the stability of the biofilm
Gram’s stain, oxidase, catalase and urease reactions. community [17].
Strains were stored in trypticase soy broth Epidemiological data suggest that contaminated
supplemented with 20% glycerol at -80 ºC until use. water is a potential reservoir for this microorganism
2.2 Cyanobacterial Extract
[18], even specific H. pylori DNA fragments have been detected in river water [19, 20].
The CE was obtained as previously described by
Helicobacter pylori Biofilm Formation and Gene Expression on
Abiotic Surfaces Using a Cyanobacterial Extract
Silva et al. [24]. microscopy (SEM) using a Zeiss LEO 1450VP microscope. Biofilms formed on abiotic surfaces were
2.3 Biofilm Assays washed with 0.9% saline solution, dried and
The ability of H. pylori to form biofilm on abiotic dehydrated using a graded ethanol series (70%, 95%, surfaces was determined by total bacterial count and
and 100% three times for 10 min each). Samples were
mounted on aluminium stubs and coated with a gold glass and polypropylene (PP) placed on 90 mm Petri
viable cell count methods using 2 cm 2 coverslips of
layer and processed in a standard sputter. dishes added with 12 mL of 0.3% glucose Observations were made at 20 KV. Mueller-Hinton Broth (MHB) supplemented with 5%
2.6 Gene Expression
FCS (MHB-FCS) or 1% CE (MHB-CE). The biofilm formation was initiated by inoculating H. pylori cells
Cells coming from biofilms developed on the
abiotic surfaces and planktonic cells were treated with units (CFUs)/mL in each plate. Cultures were TRIzol reagent (Invitrogen) for total RNA extraction. incubated under microaerophilic conditions for 196 h
at a final concentration of 1 × 10 6 colony forming
The QS luxS, virulence ureA, flaA, adhesion lpxD, at 37 ºC without shaking. In order to analyze biofilm
omp18 and housekeeping 16S rRNA genes were evolution, coverslips were sampled at different times,
analyzed. cDNA was performed with random hexamer rinsed three times with phosphate-buffered saline
and 200 U Moloney murine leukaemia virus reverse (PBS) to remove planktonic cells and biofilm debris
transcriptase (Invitrogen). The identification of and stained with 0.1% crystal violet (CV). Also rinsed
amplified fragments of 465-, 411-, 111-, 1001- 165- coverslips were vortexed for 3 min in PBS to allow
and 390- bp for, luxS, ureA, flaA, lpxD, omp18 and cell detachment from biofilm. Total bacterial count
16S rRNA genes respectively, was performed with was performed using the Breed Counting Method by
1.8% agarose gel electrophoresis at a constant voltage spreading 0.01 mL of resuspended biofilm cells over 1
of 80 V/cm for 45 min. The gels were stained with microscope slide, the smear was dried and stained
cm 2
GelRed nucleic acid gel stain (Biotium, Inc.), with 0.1% CV for 30 min and the cells counted with
visualized under UV light and photographed. The an optical microscope. For viable cell counts,
DNA fragment size was determined by comparison undiluted and 1:10 dilution resuspended biofilm cells
with molecular weight markers with a range of 50 bp were plated onto MHA-HB by duplicate. CFUs were
to 1,000 bp.
counted after incubation for three days at 37 ºC.
2.7 Statistical Analysis
2.4 Fluorescence Microscopy The statistical analysis was determined by
To assess the membrane integrity of sessile bacteria, Tuckey-Kramer multiple comparisons tests. the coverslips were washed with 0.9% saline and Differences were considered statistically significant at stained with fluorescent dyes of the Live/Dead
P ≤ 0.05.
BacLight kit (Molecular Probes, Invitrogen Semi-quantification of the bands was performed Corporation) and incubated for 20 min in dark. with an image analyzer (ImageJ WCIF) against the Coverlips were observed with a Zeiss Axioplan 2
constitutive gene 16S rRNA.
fluorescent microscope. Images were acquired by a camera using Axiovision 3.0 software.
3. Results
2.5 Scanning Electron Microscopy
3.1 Biofilm Formation
Biofilms were analyzed by scanning electron
H. pylori strains were able to form biofilm on both
Helicobacter pylori Biofilm Formation and Gene Expression on
Abiotic Surfaces Using a Cyanobacterial Extract
surfaces with the nutritional supplements analyzed 196 h, with CE or FCS supplements by optical (FCS or CE) (Table 1). The attachment of cells
microscopy is showed in Fig. 3.
increased up to 96 h and in all the cases was higher
The membrane integrity of biofilm cells developed
with CE (P ≤ 0.05) and with the HP796 strain (P ≤
on glass surface with MHB-FCS and MHB-CE media