Effects of multi-walled carbon nanotubes (MWCNT) under Neisseria meningitidis transformation process
© Mattos et al; licensee BioMed Central Ltd. 2011
Received: 24 March 2011
Accepted: 16 November 2011
Published: 16 November 2011
This study aimed at verifying the action of multi-walled carbon nanotubes (MWCNT) under the naturally transformable Neisseria meningitidis against two different DNA obtained from isogenic mutants of this microorganism, an important pathogen implicated in the genetic horizontal transfer of DNA, causing the escape of the principal vaccination measured worldwide by the capsular switching process.
Materials and methods
The bacterium receptor strain C2135 was cultivated and had its mutant DNA donor M2 and M6, which received a receptor strain and MWCNT at three different concentrations. The inhibition effect of DNAse on the DNA in contact with nanoparticles was evaluated.
The results indicated an in increase in the transformation capacity of N. meninigtidis in different concentrations of MWCNT when compared with negative control without nanotubes. A final analysis of the interaction between DNA and MWCNT was carried out using Raman Spectroscopy.
These increases in the transformation capacity mediated by MWCNT, in meningococci, indicate the interaction of these particles with the virulence acquisition of these bacteria, as well as with the increase in the vaccination escape process.
Neisseria meningitidis is a commensal bacterium of the human upper respiratory tract that may occasionally provoke invasive infections such as septicemia and meningitis. It is also naturally competent and therefore can exchange genetic information with each other by this process. This natural competence has been directly correlated to pilliation of these organisms, as well as a specific uptake sequence, within the genome of these bacterium .
The use of mutations for the study of the capsular polysaccharide of N. meningitidi s is the aim of several studies of the meningococci pathogenesis [2–4]. The capsular polysaccharide is the major virulence factor and a protective antigen. Meningococcal strains are classified into 12 different serogroups according to their capsular immune specificity, along with serogroups A, B, C, Y and W135 are the most frequently found in invasive infections. The capsule of serogroups B, C, Y and W135 strains is composed of either homopolymers (B and C) or heteropolymers (Y and W135) of sialic acid-containing polysaccharides that are specifically linked, depending on the serogroup [5, 6]. This polymerization is mediated by the polysialyltransferase, encoded by the siaD gene in strains of serogroups B and C (also called synD and synE, respectively) and by synG in serogroup W135. Capsule switching after replacement of synE, in a serogroup C strain, by synG may result from the conversion of capsule genes by transformation and allelic recombination [7–10]. Such capsule switching from serogroup C to B N. meningitidis was observed in several countries, either spontaneously or after vaccination campaigns [7–13]. It might explain the emergence and the clonal expansion of strains of serogroup W135 of N. meningitidis in the year 2000 among Hajj pilgrims who had been vaccinated against meningococci of serogroups A and C . These W135 strains belong to the same clonal complex ET-37/ST-11 as prominent serogroup C strains involved in outbreaks worldwide [8, 9, 15]. Hence, the emergence of these W135 strains in epidemic conditions raised the question about a possible capsule switching as an escape mechanism to vaccine-induced immunity. Also, these events are expected to occur continuously and can be selected by immune response against a particular capsular polysaccharide .
However, the interference of immune response with transformation efficacy has not been yet evaluated. Specific capsular antibodies are expected to bind to the bacterial surface and hence they interfere in DNA recognition and uptake. Also, environmental interference under the transformation process of this bacterium is unknown.
This work aimed at the use of multi-walled carbon nanotubes (MWCNT) for the study of the nanostructures action on the transformation process of meningococci, specifically their functions under the capsular switching process. The methods used in this work aimed at the action of MWCNT in the transformation of serogroup C N. meningitidis against two different DNA obtained from isogenic mutants of this microorganism.
Synthesis of multi-walled carbon nanotubes
Bacterial Strains and Media
Bacterial Strains used in this work
Escherichia coli F-, endA1, hsdR17 c, supE44, thi-1, gir A96, relA1
Plasmid containing ΔNMB0065::ΩaaDA derivated from pGEMTEasy
Plasmid containing the fusion of synG::ermAM
Neisseria meningitidis serogroup C, BIOMERIEUX
INCQS - FIOCRUZ
Neisseria meningitidis serogroup W135, ATCC35559
INCQS - FIOCRUZ
N.meningitidis isogenic mutant ΔNMB0065::ΩaaDA
N.meningitidis W135ATCC transformed with pLAN13 to generate a fusioned strain synG:ermAM
Oligonucleotides used in this work
Construction of NMB0065 mutant by polar mutation
Construction of serogroup W135 mutants in transcriptional fusion synG::ermAM
Analysis of transformation frequency up to MWCNT contact
At 1.108 colony-forming units - CFU - of the receptor strain C2135, we added 1 μg genomic DNA from M2 and M6 mutants and 10, 20 and 50 μg of different MWCNT. A negative control was also performed without MWCNT. The suspension was incubated for three hours at 37°C in atmosphere of 5% of CO2 by three hours. The counts of total CFU were performed in GCB spectinomycin or erythromycin plates in triplicate analysis (for M2 and M6 isogenic mutants, respectively). The CFU obtained in plates containing specific antibiotic were analyzed by PCR for the presence of target gene transfer in the transforming units (ΩaaDA cassette for the M2 DNA and synG for M6 donor DNA). In order to verify the interaction between DNA, MWCNT and DNAse action, the same amounts of DNA(1 μg) from M2 and M6 mutants, MWCNT (20 μg) and bacterial cells were submitted to action 5 U of DNase (New England Biolabs, UK) and further transformation process. Also, the counts of cfu were performed in GCB spectinomycin or erythromycin plates in triplicate analysis (for M2 and M6 isogenic mutants respectively).
Analysis of interaction between DNA and MWCNT by Raman spectroscopy
The prior analysis of DNA from M2 and M6 mutant strains with MWCNT was performed under a mix of 1 μg of M6 genomic DNA and 20 μg of MWCNT. The samples were characterized by Raman spectroscopy [20, 21]. The spectra were recorded at room temperature using a Renishaw microprobe in Via system, employing an UV laser for excitation (λ = 325 nm) at about 10 mW. The samples M2 and M6 were dripped onto a quartz substrate for UV laser Raman spectroscopy.
Results and discussion
Values obtained from C21 35 transformation using the donor DNA from M2 and M6 mutants.
Donor DNA (1 μg)
Ratio (means obtained exposed to MWCNT/mean of negative control)
P values (one way Tukey's test)
Negative Control (without MWCNT) M2
1.02 ± 0.17
NT1 (10 μg)
0.89 ± 0.09
P = 0, 1631 (non significant)
NT1 (20 μg)
2.24 ± 0.70
P < 0, 05 (P = 0, 0496 significant)
NT1 (50 μg)
3.52 ± 0.50
P < 0, 05 (P = 0, 0073 very significant)
NC (10 μg)
0.85 ± 0.50
P = 0, 3166 (non significant)
NC (20 μg)
2.18 ± 0.90
P = 0, 0798 (non significant)
NC (50 μg)
4.36 ± 1.18
P < 0, 05 (P < 0, 0020 significant)
NT2 (20 μg)
1.42 ± 0.13
P < 0, 05 (P = 0, 0240 significant)
Negative Control (without mesoporous siliM6
1.09 ± 0.25
NT1 (10 μg)
1.71 ± 0.25
P < 0, 05 (P = 0, 0385 significant)
NT1 (20 μg)
2.03 ± 0.08
P < 0, 05 (P = 0, 0034 very significant)
NT1 (50 μg)
2.11 ± 0.30
P < 0, 05 (P = 0, 0106 significant)
NC (10 μg)
2.03 ± 0.35
P < 0, 05 (P = 0, 0193 significant)
NC (20 μg)
2.44 ± 0.88
P < 0, 05 (P = 0, 0490 significant)
NC (50 μg)
2.14 ± 0.49
P < 0, 05 (P = 0, 0403 significant)
NT2 (20 μg)
5.58 ± 0.86
P < 0, 05 (P = 0, 0065 very significant)
The intention of two different DNA donors was to certificate the independence of MWCNT action under the same bacterial strain - N. meningitidis C2135. Further analysis by PCR demonstrated the transfer of the tagged gene from M2 and M6 in transformed strains (data not shown). The Raman analysis showed the interaction of MWCNT with the DNA obtained from M6 mutant strains as viewed in Figure 4(a-b).
Data analyses were made by ratio values between the numbers of transformants cfu obtained with MWCNT by median values of transformants cfu obtained without nanotubes treatment (Figures 4c-d and table 3). The values were analyzed by one-way analysis of variance ANOVA (Tukey's test compared each treatment to control without nanoparticles in transformation, considering significant values of P > 0.05). Some values obtained with commercial MWCNT - NC and NT2 showed different results when compared with NT1 (table 3 and Figure 4).
The relations between the meningococci transformation and MWCNT action viewed in these results could mimic the presence of carbon nanoparticles in atmosphere and evoke the emergence of outbreaks of Brazilian purpuric fever (BPF) caused by another naturally competent bacteria, Haemophilus influenzae biogroup aegyptius [22, 23]. The Haemophilus influenzae biotype aegyptius causes BPF, a dangerous inflammatory disease known as purpura fulminans with a great mortality rate . Kroll et al.  described these Haemophilus influenzae strains, usually associated with conjunctivitis cases, as a product of horizontal transfer between N. meningitidis and Haemophilus influenzae. In the same geographic region of these outbreaks, the primitive agricultural practice, performed by burning sugar cane, generates an emission of carbon micro and nanoparticles in the atmosphere, potentially provoking respiratory disorders by particles inhalation . Our group has been studying these bacteria and testing them with MWCNT on its transformation process.
This process is similar to the phenomena of capsular switching as described in sub Saharan African [26–28] and Saudi Arabian regions (Hajj pilgrimage) [26, 29–35]. In desert zones, the ramarthan wind and the presence of silica nanostructures facilitates the capsular switching process in meningococci strains [26, 29–36]. Thus, new experiments using animal models that could confirm this hypothesis have been performed by our group. Also, the increases in the transformation capacity in bacteria have been verified in Escherichia coli by nanotube structures, as described by Rojas-Chapana et al. .
This work indicated, for the first time in scientific literature, that the action of atmospheric nanoparticles obtained from anthropic activities, such as primitive agriculture, influences the bacterial transformation process.
The increase in the transformation capacity mediated by MWCNT in meningococci indicates an interaction of these particles with the bacterial DNA leading to virulence acquisition and an increase in the escape to vaccination. The presence of these nanoparticles protects the DNA from DNAse action, increasing the recombination frequency. These results show that important measures for public health, in places where the MWCNT or carbon microparticles are produced, need to be carefully revised.
This study has been financed by CAPES, FAPESP and CNPq. These supports helped us to supply reagent and equipments for the entire research development. FAPESP (number 2008/56777-5) and CNPq (number 575313/2008-0) supported the Laboratory of Biotechnology (Coordinated by M.L.). CAPES supported NanoEng (Coordinated by VB, Nanobiotechnology Program - Effects of carbon nanotubes under biological systems) and the personal fellowships for LMH, HJC. Thanks for the English revision to Júlia N. Varela and Maria Cecília T. Amstalden.
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