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.
- Tonjum T, Koomey M: The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships--a review. Gene. 1997, 192: 155-163. 10.1016/S0378-1119(97)00018-8.View ArticleGoogle Scholar
- Alonso JM, Guiyoule A, Zarantonelli ML, Ramisse F, Pires R, Antignac A, Deghmane AE, Huerre M, van der Werf S, Taha MK: A model of meningococcal bacteremia after respiratory superinfection in influenza A virus-infected mice. FEMS Microbiol Lett. 2003, 222: 99-106. 10.1016/S0378-1097(03)00252-0.View ArticleGoogle Scholar
- Nassif X, So M: Interaction of pathogenic neisseriae with nonphagocytic cells. Clin Microbiol Rev. 1995, 8: 376-388.Google Scholar
- Spinosa MR, Progida C, Tala A, Cogli L, Alifano P, Bucci C: The Neisseria meningitidis capsule is important for intracellular survival in human cells. Infect Immun. 2007, 75: 3594-3603. 10.1128/IAI.01945-06.View ArticleGoogle Scholar
- Frosch M, Muller D, Bousset K, Muller A: Conserved outer membrane protein of Neisseria meningitidis involved in capsule expression. Infect Immun. 1992, 60: 798-803.Google Scholar
- Taha MK: Molecular detection and characterization of Neisseria meningitidis. Expert Rev Mol Diagn. 2002, 2: 143-150. 10.1586/1473722.214.171.124.View ArticleGoogle Scholar
- Swartley JS, Marfin AA, Edupuganti S, Liu LJ, Cieslak P, Perkins B, Wenger JD, Stephens DS: Capsule switching of Neisseria meningitidis. Proc Natl Acad Sci USA. 1997, 94: 271-276. 10.1073/pnas.94.1.271.View ArticleGoogle Scholar
- Zarantonelli ML, Lancellotti M, Deghmane AE, Giorgini D, Hong E, Ruckly C, Alonso JM, Taha MK: Hyperinvasive genotypes of Neisseria meningitidis in France. Clin Microbiol Infect. 2008, 14: 467-472. 10.1111/j.1469-0691.2008.01955.x.View ArticleGoogle Scholar
- Lancellotti M, Guiyoule A, Ruckly C, Hong E, Alonso JM, Taha MK: Conserved virulence of C to B capsule switched Neisseria meningitidis clinical isolates belonging to ET-37/ST-11 clonal complex. Microbes Infect. 2006, 8: 191-196. 10.1016/j.micinf.2005.06.012.View ArticleGoogle Scholar
- Kriz P, Musilek M, Skoczynska A, Hryniewicz W: Genetic and antigenic characteristics of Neisseria meningitidis strains isolated in the Czech Republic in 1997-1998. Eur J Clin Microbiol Infect Dis. 2000, 19: 452-459. 10.1007/s100960000293.View ArticleGoogle Scholar
- Alcala B, Salcedo C, de la Fuente L, Arreaza L, Uria MJ, Abad R, Enriquez R, Vazquez JA, Motge M, de Batlle J: Neisseria meningitidis showing decreased susceptibility to ciprofloxacin: first report in Spain. J Antimicrob Chemother. 2004, 53: 409-10.1093/jac/dkh075.View ArticleGoogle Scholar
- Alcala B, Salcedo C, Arreaza L, Berron S, De La Fuente L, Vazquez JA: The epidemic wave of meningococcal disease in Spain in 1996-1997: probably a consequence of strain displacement. J Med Microbiol. 2002, 51: 1102-1106.View ArticleGoogle Scholar
- Perez-Trallero E, Vicente D, Montes M, Cisterna R: Positive effect of meningococcal C vaccination on serogroup replacement in Neisseria meningitidis. Lancet. 2002, 360: 953-View ArticleGoogle Scholar
- Taha MK, Bichier E, Perrocheau A, Alonso JM: Circumvention of herd immunity during an outbreak of meningococcal disease could be correlated to escape mutation in the porA gene of Neisseria meningitidis. Infect Immun. 2001, 69: 1971-1973. 10.1128/IAI.69.3.1971-1973.2001.View ArticleGoogle Scholar
- Zarantonelli ML, Antignac A, Lancellotti M, Guiyoule A, Alonso JM, Taha MK: Immunogenicity of meningococcal PBP2 during natural infection and protective activity of anti-PBP2 antibodies against meningococcal bacteraemia in mice. J Antimicrob Chemother. 2006, 57: 924-930. 10.1093/jac/dkl066.View ArticleGoogle Scholar
- Grecco AC, Paula RF, Mizutani E, Sartorelli JC, Milani AM, Longhini AL, Oliveira EC, Pradella F, Silva VD, Moraes AS: Up-regulation of T lymphocyte and antibody production by inflammatory cytokines released by macrophage exposure to multi-walled carbon nanotubes. Nanotechnology. 22: 265103Google Scholar
- Hanahan D, Meselson M: Plasmid screening at high colony density. Methods Enzymol. 1983, 100: 333-342.View ArticleGoogle Scholar
- Hollanda LM, Cury GC, Pereira RF, Ferreira GA, Sousa A, Sousa EM, Lancellotti M: Effect of mesoporous silica under Neisseria meningitidis transformation process: environmental effects under meningococci transformation. J Nanobiotechnology. 2011, 9: 28-10.1186/1477-3155-9-28.View ArticleGoogle Scholar
- Dolan-Livengood JM, Miller YK, Martin LE, Urwin R, Stephens DS: Genetic basis for nongroupable Neisseria meningitidis. J Infect Dis. 2003, 187: 1616-1628. 10.1086/374740.View ArticleGoogle Scholar
- Sun L, Sun Y, Xu F, Zhang Y, Yang T, Guo C, Liu Z, Li Z: Atomic force microscopy and surface-enhanced Raman scattering detection of DNA based on DNA-nanoparticle complexes. Nanotechnology. 2009, 20: 125502-10.1088/0957-4484/20/12/125502.View ArticleGoogle Scholar
- Palma BF, Ferrari AB, Bitar RA: DNA extraction systematics for spectroscopic studies. Sensors. 2008, 9-Google Scholar
- Davis J, Smith AL, Hughes WR, Golomb M: Evolution of an autotransporter: domain shuffling and lateral transfer from pathogenic Haemophilus to Neisseria. J Bacteriol. 2001, 183: 4626-4635. 10.1128/JB.183.15.000-000.2001.View ArticleGoogle Scholar
- Li MS, Farrant JL, Langford PR, Kroll JS: Identification and characterization of genomic loci unique to the Brazilian purpuric fever clonal group of H. influenzae biogroup aegyptius: functionality explored using meningococcal homology. Mol Microbiol. 2003, 47: 1101-1111. 10.1046/j.1365-2958.2003.03359.x.View ArticleGoogle Scholar
- Kroll JS, Wilks KE, Farrant JL, Langford PR: Natural genetic exchange between Haemophilus and Neisseria: intergeneric transfer of chromosomal genes between major human pathogens. Proc Natl Acad Sci USA. 1998, 95: 12381-12385. 10.1073/pnas.95.21.12381.View ArticleGoogle Scholar
- Ribeiro H, Assunção JV: Efeitos das queimadas na saúde humana. Estudos Avançados. 2002, 16: 24-View ArticleGoogle Scholar
- Alonso JM, Bertherat E, Perea W, Borrow R, Chanteau S, Cohet C, Dodet B, Greenwood B, LaForce FM, Muros-Le Rouzic E: From genomics to surveillance, prevention and control: new challenges for the African meningitis belt. Bull Soc Pathol Exot. 2006, 99: 404-408.Google Scholar
- Caugant DA, Nicolas P: Molecular surveillance of meningococcal meningitis in Africa. Vaccine. 2007, 25 (Suppl 1): A8-11.View ArticleGoogle Scholar
- Zombre S, Hacen MM, Ouango G, Sanou S, Adamou Y, Koumare B, Konde MK: The outbreak of meningitis due to Neisseria meningitidis W135 in 2003 in Burkina Faso and the national response: main lessons learnt. Vaccine. 2007, 25 (Suppl 1): A69-71.View ArticleGoogle Scholar
- Wilder-Smith A: Meningococcal vaccine in travelers. Curr Opin Infect Dis. 2007, 20: 454-460. 10.1097/QCO.0b013e3282a64700.View ArticleGoogle Scholar
- Wilder-Smith A, Barkham TM, Chew SK, Paton NI: Absence of Neisseria meningitidis W-135 electrophoretic Type 37 during the Hajj, 2002. Emerg Infect Dis. 2003, 9: 734-737.View ArticleGoogle Scholar
- Wilder-Smith A, Barkham TM, Earnest A, Paton NI: Acquisition of W135 meningococcal carriage in Hajj pilgrims and transmission to household contacts: prospective study. Bmj. 2002, 325: 365-366. 10.1136/bmj.325.7360.365.View ArticleGoogle Scholar
- Wilder-Smith A, Barkham TM, Ravindran S, Earnest A, Paton NI: Persistence of W135 Neisseria meningitidis carriage in returning Hajj pilgrims: risk for early and late transmission to household contacts. Emerg Infect Dis. 2003, 9: 123-126.View ArticleGoogle Scholar
- Wilder-Smith A, Goh KT, Barkham T, Paton NI: Hajj-associated outbreak strain of Neisseria meningitidis serogroup W135: estimates of the attack rate in a defined population and the risk of invasive disease developing in carriers. Clin Infect Dis. 2003, 36: 679-683. 10.1086/367858.View ArticleGoogle Scholar
- Wilder-Smith A, Memish Z: Meningococcal disease and travel. Int J Antimicrob Agents. 2003, 21: 102-106. 10.1016/S0924-8579(02)00284-4.View ArticleGoogle Scholar
- Wilder-Smith A, Paton NI, Barkham TM, Earnest A: Meningococcal carriage in Umra pilgrims returning from Saudi Arabia. J Travel Med. 2003, 10: 147-149.View ArticleGoogle Scholar
- Wilder-Smith A: W135 meningococcal carriage in association with the Hajj pilgrimage 2001: the Singapore experience. Int J Antimicrob Agents. 2003, 21: 112-115. 10.1016/S0924-8579(02)00355-2.View ArticleGoogle Scholar
- Rojas-Chapana JA, Giersig M: Multi-walled carbon nanotubes and metallic nanoparticles and their application in biomedicine. J Nanosci Nanotechnol. 2006, 6: 316-321.Google Scholar
- Dubnau D: DNA uptake in bacteria. Annu Rev Microbiol. 1999, 53: 217-244. 10.1146/annurev.micro.53.1.217.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.