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��C0�W,�5W,W,W,���������7�������'jW,/0r60�6W,�;+��;W,W,,�;�,�W,���'5'5�+�����6�������������������������������������������������������������������������;����������	R�:Use and characterization of outer membrane vesicles from Neisseria meningitidis as anti-tumoral treatment for glioblastoma cells NG97

Izidoro M. S. Jr.1, Alves D. A.1, Pereira R.F.C.1, Almeida, W.P.2, Hollanda L. M.1, Lancellotti M.1*
1 - LABIOTEC� Biotechnology Laboratory � Biology institute, UNICAMP- State University of Campinas � S�o Paulo � Brazil
2 - LAFAME � Laboratory of Drug Design and Development � Institute of Chemistry, UNICAMP- State University of Campinas � S�o Paulo � Brazil





* Corresponding author: Rua Monteiro Lobato s/n, Department of Biochemistry, Institute of Biology CP6109, State University of Campinas � UNICAMP, 13083-970, Campinas, SP, Brazil. Tel: +55 19 35 21 61 50. FAX: +55 19 35 21 6129. 
e-mail: mlancell@unicamp.br









Abstract
Neisseria meningitidis is a human nasopharyngeal commensal, however, some strains occasionally exceed the respiratory mucosa and the blood brain barrier causing diseases such as meningitis and septicemia. This study produced OMVs from different strains of Neisseria meningitidis and analyzed physicochemical characteristics of these vesicles such as size and surface charge, and also, the main interactions between this nanoparticle and NG97 cell line by in vitro morphological tests and the analyze of the production of inflammatory cytokines by relative quantification Real Time PCR. The major finding of this study is that the use of ultrafiltration substituting ultracentrifugation on OMVs production makes the extraction viable, less laborous and faster than previous methods. Furthermore, the vesicles extracted by this process exhibited similar sizes and surface charges when compared to the literature and each strain produces different amounts of OMVs when comparing the same production time. In this work, we also observed that the OMVs generated by attenuated strains for virulence factors induced less morphological changes in the NG97 cells than OMVs generated from wild type strains. The virulence factors which assist the parasite-host cells interaction was also observed in the analysis of cytokines produced. It was observed in the absence of pilin (protein responsible for adherence of the bacteria to epithelial and endothelial cells) the production of cytokines was much lower when compared with the different strains where this virulence factor is present. For a possible use of these lipossome-like vesicles as delivery systems for drugs, it is believed that the more innocuous the greater the bioavailability as an antitumor molecule. The use of OMV produced by the strain C2135 �pilE show an important use of this organic nanoparticle in the new tumor treatment form.

Key words: Neisseria meningitidis, piline, astrocytoma, NG97, outer membrane vesicles, OMV

Introduction 
N. meningitidis or meningococcus is a nonsporulated, non-flagellate and encapsulated gram-negative diplococcus, belonging to the class of �-proteobacteria member of the Neisseriaceae Family. The amount of bacteria that belongs to the genus Neisseria is quite high and the main components of this group are pathogenic microorganisms species meningitidis and gonorrhoeae. The majority of the world's population has the species Neisseria meningitidis as a commensal microorganism colonizing the upper respiratory mucosa thus, it can colonize the host without, however, cause some kind of pathology. This phenomenon is known, from the viewpoint of the host as carrier  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Caugant et al. 1994). Moreover, as a casual pathogen, in susceptible individuals the asymptomatic colonization can lead to an invasive infection  ADDIN EN.CITE <EndNote><Cite><Author>Taha</Author><Year>2002</Year><RecNum>9020</RecNum><DisplayText>(Taha, Parent Du Chatelet et al. 2002)</DisplayText><record><rec-number>9020</rec-number><foreign-keys><key app="EN" db-id="0swvdszxlr0d9oexsvk5exebtffd29frrpx0">9020</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Taha, M. K.</author><author>Parent Du Chatelet, I.</author><author>Schlumberger, M.</author><author>Sanou, I.</author><author>Djibo, S.</author><author>de Chabalier, F.</author><author>Alonso, J. M.</author></authors></contributors><auth-address>Neisseria Unit, National Reference Centre for Meningococci, Institut Pasteur, 75724 Paris Cedex 15, France.</auth-address><titles><title>Neisseria meningitidis serogroups W135 and A were equally prevalent among meningitis cases occurring at the end of the 2001 epidemics in Burkina Faso and Niger</title><secondary-title>J Clin Microbiol</secondary-title><alt-title>Journal of clinical microbiology</alt-title></titles><periodical><full-title>J Clin Microbiol</full-title><abbr-1>Journal of clinical microbiology</abbr-1></periodical><alt-periodical><full-title>J Clin Microbiol</full-title><abbr-1>Journal of clinical microbiology</abbr-1></alt-periodical><pages>1083-4</pages><volume>40</volume><number>3</number><edition>2002/03/07</edition><keywords><keyword>Humans</keyword><keyword>Meningitis, Meningococcal/epidemiology/*microbiology</keyword><keyword>Neisseria meningitidis/*classification/genetics/isolation &amp; purification</keyword><keyword>Polymerase Chain Reaction</keyword><keyword>Serotyping</keyword></keywords><dates><year>2002</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>0095-1137 (Print)&#xD;0095-1137 (Linking)</isbn><accession-num>11880446</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/11880446</url></related-urls></urls><custom2>120283</custom2><language>eng</language></record></Cite></EndNote>(Taha et al. 2002), which occurs after a period of proliferation, leading to the entry of the organism into the circulatory system through its mucosa  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Brandtzaeg et al. 1995). This invasion leads to meningococcemia and/or meningitis, caused by the entry of bacteria in the meninges and clinically triggers fever, vomiting, stiff neck, among other symptoms  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Zarantonelli et al. 2008; DeVoe 1982; Lancellotti et al. 2006).
The first observations of bacteria that serve as potential antitumor agents were held for nearly 300 years. However, the most significant works date from 1868 and 1882, when the doctors W. Busch and F. Fehleisen observed that certain inoperable cancers regressed when the patients were infected with Streptococcus pyogenes and developed erysipelas (cutaneous disease result from bacterial infection by Streptococcus type A). In addition, another study in 1890 conducted by William Coley demonstrated that a mixture of heat-killed strains of Streptococcus pyogenes with Serratia marcescens were effective in treating cancer patients. This combination of bacteria known as Coley toxin and has been used in the treatment of sarcomas until 1963  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Wei et al. 2008).
In 2009, MacDiarmid demonstrated that minicells or EDVs (EnGenelC Delivery Vehicles) were effective on lowering the dose of chemotherapy needed for the treatment of specific types of cancers which was only possible because the medicine was being delivered more specifically to the tumor mass instead of all the patient cells. Therefore in this work we analyze the possibility of using outer membrane vesicles (OMVs) as a more specific delivery system for tumors, despite the morphological differences between minicells and OMVs, this last one is easily extracted from any gram negative bacterial strain.  ADDIN EN.CITE  ADDIN EN.CITE.DATA (MacDiarmid et al. 2009)
Outer membrane vesicles is a type of secretion from gram negative bacteria that allows them to interact with the environment without spending energy on movement, for a better understanding of its morphology first we need to take a look at the structure of the outer membranes of the gram negative bacteria. This envelope is formed by two membranes one internal and one external, a layer of peptidoglycan, and  a periplasm  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Bos et al. 2007b, a), and the membranes differ in the composition of lipids and proteins. In most Gram-negative bacteria, the outer leaflet of the outer membrane (OM) is mainly composed of lipopolysaccharide (LOS), whereas the inner leaflet and both leaflets of the inner membrane are composed of phospholipids. Next we show the scheme of the membranes of a Gram negative bacteria (figure 1).
The first step in obtaining an OMV is the projection of the outer membrane of the bacteria into the extracellular medium. This implies that such projections are formed in areas where the proteins that bind the OM with the peptidoglycan layer are absent and, sometimes, there is an accumulation of periplasmic proteins which forces the inner layer of the outer membrane towards the extracellular medium causing the production of an outer membrane vesicle.
In order to evaluate the possibility of using outer membrane vesicles as a more specific drug delivery system, we analyzed the interaction between this nanoparticle and NG97 cells. These are glioblastoma cells which are the most common forms of primary brain tumor and the most aggressive variety of glioma. Despite recent advances in the understanding of the physiopathology of this neoplasm, little progress has been achieved in treatment. Here, we describe our results concerning the action of N. meningitidis in NG97 cells line were described by Pereira et al. in continuous studies of bacterial action in solid tumors.  ADDIN EN.CITE <EndNote><Cite><Author>Pereira</Author><Year>2011</Year><RecNum>25490</RecNum><record><rec-number>25490</rec-number><foreign-keys><key app="EN" db-id="p9save5adfr528et2r2x9zzia0vvpw2trzdr">25490</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Pereira, RFC</author><author>Alves DA</author><author>Renan Koseki Jacinto</author><author>Luciana Maria de Hollanda</author><author>Liana Maria Cardoso Verinaud</author><author>Camila Maria Longo Machado</author><author>Marcelo Lancellotti</author></authors></contributors><titles><title>Effects of Neisseria meningitidis Infection in Tumor Glioblastoma Cell Line NG97: Respiratory Pathogen Inducing Apoptosis</title><secondary-title>Journal of Bacteriology and Parasitology</secondary-title></titles><periodical><full-title>Journal of Bacteriology and Parasitology</full-title></periodical><volume>2</volume><section>114</section><dates><year>2011</year></dates><urls></urls><electronic-resource-num>doi: 10.4172/2155-9597.1000114</electronic-resource-num></record></Cite></EndNote>(Pereira et al. 2011).

Materials and Methods
Bacterial growth and colony-forming units (CFU) determination
The N. meningitidis strains used in this work were grown on chocolate blood agar at 37�C with 5 % CO2. For CFU determination, several colonies grown were picked up with a sterile loop and re-suspended in saline solution 0.9 % (w/v) to a final optical density of 1.0, following the list of Neisseria meningitidis strains used in this work:
Maintenance of NG97 cell culture
NG97 cells were grown in plastic flasks (25cm2) with RPMI 1640 medium (Cultilab, Campinas, SP, Brazil), supplemented with 2 % L-glutamine, 120 ug/mL kanamycin and 13 % inactivated fetal bovine serum (complete medium). The cultures were incubated at 37�C in an atmosphere containing 5 % of CO2. Medium was changed every 48 h and when the culture reached confluence, the subculture was performed by treatment with trypsin (Cultilab, Campinas, SP, Brazil). 



Construction of the N. meningitidis "synA  knockout strains.
The "synA strain was produced by replacing each coding sequence with a similar sequence but with the erythromycin resistance cassette in its middle. The N. meningitidis knockout strains were made through the methodology illustrated below (figure 2). The production of the "pilE has followed the same methodology (figure 3).

OMV extraction
The OMV extraction was performed using ultrafiltration. Strains of N. meningitidis were previously grown and transferred into Roux bottles containing culture medium. Bacteria grown in the bottles were suspended in PBS and subsequently centrifuged at 1.180g/10 min. The supernatant resulting from the centrifugation was transferred and vacuum filtered through a Millipore� cellulose membrane with a porosity of 0.022 micrometers. The filtrate was discarded and the membrane was washed with PBS. The membrane was discarded and PBS containing OMVs was stored at -80 � C.
The quantification of proteins were made following the Bradford protocol. For the Bradford assay (Bradford, 1976), we used the bovine serum albumin (BSA) to make the standard curve. To do so was used  Concentrated Dye BioAgency � (cod. 500-0006N) and the reading was performed at 595 nm. All results are the average of measurements performed in triplicate. The concentration of OMVs obtained by this process relies on its protein concentration, all the samples were diluted to reach the same concentration of protein equivalent to OMV's concentration. Particle size, polydispersion and zeta potential of the samples were analyzed using a Malvern Instruments Zetasizer Nano ZS operated at 25�C using triplicate readings and a two minutes of equilibration time.


Cell viability and in vitro cytotoxicity assay
The test has been previously described by Borenfreund in 1985  ADDIN EN.CITE <EndNote><Cite><Author>Borenfreund</Author><Year>1985</Year><RecNum>8905</RecNum><DisplayText>(Borenfreund and Puerner 1985)</DisplayText><record><rec-number>8905</rec-number><foreign-keys><key app="EN" db-id="0swvdszxlr0d9oexsvk5exebtffd29frrpx0">8905</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Borenfreund, E.</author><author>Puerner, J. A.</author></authors></contributors><titles><title>Toxicity determined in vitro by morphological alterations and neutral red absorption</title><secondary-title>Toxicol Lett</secondary-title><alt-title>Toxicology letters</alt-title></titles><periodical><full-title>Toxicol Lett</full-title><abbr-1>Toxicology letters</abbr-1></periodical><alt-periodical><full-title>Toxicol Lett</full-title><abbr-1>Toxicology letters</abbr-1></alt-periodical><pages>119-24</pages><volume>24</volume><number>2-3</number><edition>1985/02/01</edition><keywords><keyword>Animals</keyword><keyword>Cell Line</keyword><keyword>Fibroblasts/cytology/drug effects/metabolism</keyword><keyword>Mice</keyword><keyword>Mice, Inbred BALB C</keyword><keyword>Neutral Red/*diagnostic use</keyword><keyword>Phenazines/*diagnostic use</keyword><keyword>Toxicology/*methods</keyword></keywords><dates><year>1985</year><pub-dates><date>Feb-Mar</date></pub-dates></dates><isbn>0378-4274 (Print)&#xD;0378-4274 (Linking)</isbn><accession-num>3983963</accession-num><work-type>Comparative Study&#xD;Research Support, Non-U.S. Gov&apos;t</work-type><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/3983963</url></related-urls></urls><language>eng</language></record></Cite></EndNote>(Borenfreund and Puerner 1985). After incubation of NG97 cells with same volumes of OMVs  for 3 hours the culture medium was removed and washed with phosphate buffered saline (PBS) pH 7.4. To each well was added 0.2 mL of RPMI 1640  medium containing 50 mg/mL of neutral red dye and the plate incubated for 3 h at 37 � for uptake of the dye by the lysosomes of viable cells. After incubation, the medium containing the dye was removed and the wells were washed briefly with calcium-formalin solution for fixation. Immediately after, 0.2 mL of ethanol-acetic acid was added to each well and the plate was kept for 15 minutes. The solubilized dye was quantified by spectrophotometer at 540 nm.

Morphological assay
The morphological analysis of the cells was carried out using microscope slides containing NG97 cells after infection with OMVs from different N. meningitidis strains, the cell culture medium was discarded and plates were washed 5 times with sterile RPMI 160. Then, the microscope slides were subjected to the Wright's Stain Solution (Nalgon, Itupeva, S�o Paulo, Brazil).

Inflammatory Chemokines Quantification
The analysis of the production of inflammatory chemokines such as TNF�, TGF�, IFN�, IL6, IL8 e IL10 were performed by Real Time PCR after the NG97 cell infection with OMVs. The cells were infected as described before and incubated for 5 hours. Then, the total RNA was extracted using the Trizol Reagent (Invitrogen, Calsbag, CA, USA). RNA yield was estimated by Nanodrop (Thermo Scientific). A minimum of 0.2 mg RNA was submitted to reverse transcriptase followed by detection reaction by qRT PCR. Real-time PCR primers are listed below. qRT-PCR was performed using a StepONE Plus thermocycler (Applied Biosystems). Each 10uL reaction contained 400 nM of each primer, 5uL Master Mix and 1/60 000 Fast EVA Green Master Mix (both from Biotium), 0,25uL of Super Script III Platinum One-Step qRT-PCR System (Invitrogen Calsbad, CA, USA), according to the recommendations for cDNA production described in the kit. Crossing threshold (CT) values were averaged. Fold change in expression was calculated according to the standard formula 2(En-Rn)-(Et-Rt), where En is the ct of the experimental gene in the normal sample, Rn is the CT of the reference gene in the normal sample, Et is the ct of the experimental gene in the treated sample and Rt is the ct of the reference gene in the treated sample. qRT-PCRs were repeated on three different biological replicates using as inner control the GAPDH gene and fold expression changes were averaged  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Overbergh et al. 2003; Schmittgen and Livak 2008).

Results 
The extraction of outer membrane vesicles from Neisseria meningitidis by ultrafiltration was able to detect OMVs of sizes smaller than 200nm (table 3), demonstrating that the method works. The polydispersion and size of OMVs varies between the strains, even between the same strain with different knockout genes (table 3). Also, the morphological changes in the NG97 cells vary according to the strain that originated the OMV (figure 5 and 6) and  the same strain that produced the greatest amount of alterations in the NG97 cell line was also the strain that produced the greatest amount of cytokines (figure  7).
The expression of chemokines in several meningococci strain is described in the figures 7 and 8. These figures showed the differences of chemokines expression level of IL6, IL8, IL10, IFN�, TNF� and TGF� in relationship to endogenous gene GAPDH in a relative RT-qPCR. Also, these results showed a evidence decrease of chemokines under expression with a piline and capsule knock -out mutants as viewed in the figure 8.
In this work the OMV characterization performed by transmission electronic microscopy was performed to determine the size of the OMVs, later the precise sizes and superficial charges were measured by Zetasizer Nano  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Park et al. 2010). As proposed by MacDiarmid et al.  ADDIN EN.CITE  ADDIN EN.CITE.DATA (MacDiarmid et al. 2009) all sizes of nanostructures studied here are small enough to penetrate the junctions between the cells of the blood vessels that feed the tumors, which are approximately 400nm.
In the figure 5, the morphological assay, the C2135 strain and its knockout strains showed the lower percentage of cells changed. The pilin protein (absent in the strain C2135 "pilE) is primarily responsible for the adhesion of this bacterium to the epithelial and endothelial cells of the host and in its absence, strain C2135 �pilE, there is the decreased ability of the bacteria to cause damage to the host cell that must be explained by the difficulty of this first interaction described above  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Virji et al. 1993). In Figure 6 are examples of changes found in the assay: Change of the size and shape of the cell with condensation of cytoplasm, increased presence of vacuolization and blebs.
Concerning the OMVs cytotoxicity over the NG97 cells, the figure 5 demonstrates that they have shown little or no cytotoxicity. Further analysis about the expression of inflammatory cytokines by NG97 cells exposed to different types of OMVs significantly (p <0.05) and are shown in the Figures 7 and 8 as average. 
The process of infection by N. meningitidis is associated with expression of certain inflammatory cytokines, e.g., expression of TNF� by meningococcal disease is well described in the literature. Also, a pro-inflammatory molecule involved in the regulation of the immune response, induction of cell death via apoptosis and inflammation. It is known that TNF� is one of the most important cytokines related to septic shock and its release occurs early in this pathophysiological process. IL8 is also a pro-inflammatory molecule involved in tissue inflammation and sepsis it induces the attraction of specific cells, such as neutrophils  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Tsiotou et al. 2005).
The inflammatory response during meningitis as well as the release of TNF� may increase the permeability of the BBB (blood brain barrier)  ADDIN EN.CITE  ADDIN EN.CITE.DATA (Kolb-Maurer et al. 2001). Causing damage to neural cell types, such as the NG97 cells. Thus, it is also possible to relate the high expression of TNF� in NG97 after infection with OMVs, and according to Figure 7, the highest expression of interleukin when compared to GAPDH expression was the P2143 strain, followed by the P374 and C2135 strains. This pattern is found in all pro-inflammatory cytokines, which are TNF�, IFN�, IL06 and IL08 and was not observed in anti-inflammatory cytokines, such as IL10 and TGF�. Also, the induction of inflammation in this case is independent of the strain, since both responsible for the highest increase and lowest increase in expression are strains of the same serogroup C.
Still in the figure 8, where we compare the OMVs produced by knockout strains of  N. meningitidis C2135 we have some interesting results. The strain that does not produce the piline, bacterial membrane protein, a virulence factor considered important to the process of adherence to epithelial cells, the results show that it produced less mRNA for the cytokines than the endogenous control (GAPDH), the only exception being IFN�. On the other hand, the strain that does not produce the capsule showed a large amounts of inflammatory cytokines, almost as pronounced as its parent strain, the C2135. 
Also, the use of OMVs as carriers of drugs, RNAi or even plasmids would only be possible with innocuous nanoparticles and, by keeping some of the features of the bacterial cell membrane itself, they would lead its content directly to the tumor cells. However, when this does not happen there is also the possibility of working with the nanoparticle in order to make them more selective to the tumor cells, for example, using antibodies where a portion of it would bind to the particle and the other part to a receptor over expressed by the tumor cells  ADDIN EN.CITE  ADDIN EN.CITE.DATA (MacDiarmid et al. 2009).
In conclusion, we can state that the piline, protein responsible for the adhesion of bacterial cells to human endothelial and epithelial cells, plays an important role in the inflammatory process and, therefore, its absence considerably decreases the quantity of cytokines produced by the human cell in response to its outer membrane vesicle.
For the use of nanoparticles as a new therapeutic method against this type of cancer, it is not allowed to trigger the inflammatory system once it will eventually reaches the blood stream in order to get to the tumor cells. Furthermore, this work introduces a new possibility of treatment, since the outer membrane vesicle of the knockout strain for the protein pilin showed lower production of inflammatory cytokines by the NG97 cells. 

Acknowledgements
Thanks for FAPESP (Grants 2011/14079-2, 2011/21685-6 and 2012/15046-3) and CAPES (fellowship grants for Izidoro Jr) for the financial support for the LABIOTEC.

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10.1111/j.1469-0691.2008.01955.x





















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Figure 1: the outer membrane (OM), the periplasmic space (PS) and inner membrane (IM). Featured are some important proteins present in Neisseria meningitidis and its LOS (lipoligosacar�deo) highly immunogenic molecule, porine (porA), surfaces antigen (ionic channel, NadA)   ADDIN EN.CITE <EndNote><Cite><Author>Varela</Author><Year>2012</Year><RecNum>25491</RecNum><record><rec-number>25491</rec-number><foreign-keys><key app="EN" db-id="p9save5adf9PSz������������������	E	g	������z�z�z�z�z�z�l[l[J!h)�h�n�B*OJQJ^Jph!h�UPh�n�B*OJQJ^Jphh�n�B*OJQJ^Jph,h�UPh�n�B*H*OJQJ^JmHphsH)h�UPh�n�B*OJQJ^JmHphsH$h�UPh��5�B*OJQJ^Jphh��5�B*OJQJ^Jph'h�UPh�n�5�6�B*OJQJ^Jph$h�UPh�n�5�B*OJQJ^Jphh�n�5�B*OJQJ^Jph���c	�	�	�	�	�	�	�
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q6r�����ùïùùåùùùù�Î�ùùvivih�UPh|g�OJQJ^Jh|g�OJQJ^Jh�ph�n�6�OJQJ^Jh�ph�n�OJQJ^Jh��OJQJ^Jhf�OJQJ^JhU�OJQJ^Jh�n�OJQJ^Jh�UPh�n�OJQJ^Jh�zOJQJ^J'�jh�UPh�n�OJQJU^J!jh�UPh�n�OJQJU^J#6r7rFrGr[r\r]r^r_rwrxrzr�r�r�r�r�t�teu�u�u�u�uvv8v:vEvFv��������ᢘ����������v�iYH!jhmish�n�OJQJU^Jh�:�h�n�5�OJQJ\�^Jh�n�5�OJQJ\�^Jh6OJQJ^Jh9<�h�n�5�OJQJ^Jh��OJQJ^Jh�n�OJQJ^Jh�UPh�n�OJQJ^Jh�zOJQJ^J'�j$h�UPh|g�OJQJU^J'�j�h�UPh|g�OJQJU^Jh�UPh|g�OJQJ^J!jh�UPh|g�OJQJU^JFvXvYvZ�[�\�a�c�f�o�p�~���������&�b�����K��
������¸®£��~qdVdL>L<LUjh�n�OJQJU^Jh�n�OJQJ^Jh�-h�n�6�OJQJ^Jh�-h�n�OJQJ^Jh��5�OJQJ\�^Jh�J�5�OJQJ\�^Jh�n�5�OJQJ\�^Jh�J�5�OJQJ^Jh�n�5�OJQJ^JhU�OJQJ^Jh�n�OJQJ^Jh�zOJQJ^Jh�zh�z^Jh��h�zOJQJ^J!jhmish�n�OJQJU^Jhmish�n�OJQJ^JYzV{<|�}^~V{��'���c�_�9�Z�[�]�^�_�`�a�b�c�d�e�������������������������$���0�^��`�0�a$gd�z$a$gd�z$���0�d�^��`�0�a$gd��e�f�g�h�i�j�k�l�m�n�o�p�������%�&���7�8�����,�-�������������������������
$d�a$gdB�$���0�d�^��`�0�a$gdmis$���0�^��`�0�a$gd�zr528et2r2x9zzia0vvpw2trzdr">25491</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Varela, J�lia</author><author>Izidoro Jr, M�rio S�rvulo</author><author>Hollanda, Luciana Maria de</author><author>Lancellotti, M</author></authors></contributors><titles><title>Membrane Protein as Novel Targets for Vaccine Production in Haemophilus influenzae and Neisseria meningitidis</title><secondary-title>Journal of Vaccines &amp; Vaccinology</secondary-title></titles><periodical><full-title>Journal of Vaccines &amp; Vaccinology</full-title></periodical><volume>3</volume><section>152</section><dates><year>2012</year></dates><urls></urls><electronic-resource-num>10.4172/2157-7560.1000152</electronic-resource-num></record></Cite></EndNote>(Varela et al. 2012).

Figure 2: (1) Amplification of the synA gene which has a restriction site for the XhoI enzyme; (2) insertion of the amplified gene in the cloning vector pGem �-T Easy (Promega) and consequent breakage of the gene sequence at the synA Xho I site; (3) addition of the erythromycin resistance cassette (ermAM) in the plasmid by the action of T4 DNA ligase, (4) inserting of the plasmid in the C2135 strain and the selection of the mutated strains were made by adding erythromycin to the medium.

Figure 3: (1) Amplification of the gene in two parts with the insertion of the restriction site for the enzyme BamHI, (2) linearization of the two fragments by PCR using the primers of the first fragment and the second fragment; (3) linearized fragments with the BamHI restriction site, (4) inserting of the fragment into the cloning vector pGem �-T Easy (Promega) and sequent cleavage with the restriction enzyme previously mentioned, (5) inserting of  the erythromycin resistance cassette ermAM; (6) insertion of the plasmid in the C2135 strain.

Figure 4: Cell viability and in vitro cytotoxicity assay. Number of living cells after infection with OMV from different strains.

Figure 5: Morphological assay. The percentage of cells with morphological alterations after infection with OMVs.

Figure 6: (A) NG97 cells without the presence of OMVs (Negative Control), (B) NG97 cells showing increased cell vacuolization after the infection with OMV; (C) highlighted the change in cell shape and the presence of blebs (ii) (D) highlighted cytoplasmic condensation (iii).

Figure 7: Difference in gene expression of inflammatory cytokines compared to the endogenous control GAPDH in NG97 cells after interaction with OMVs from different strains of meningococci.

Figure 8: Difference in gene expression of cytokines when compared to the endogenous control GAPDH  (RQ in Y axe) in NG97 cells after interaction with OMVs from knockout strains of meningococcus. All the RQ values obtained from each chemokine of  OMV treatment were p<0,005 by one-way ANOVA test considered significant.








Table 1: N. meningitidis strains used in this work	
StrainCharacteristicsOriginN. meningitidis C2135Serogroup C, BIOMERIEUX C2135INCQSN. meningitidis P2143Serogroup C:NT: P1.18-1,3 Cluster A4, ST8, l�quorINCQSN. meningitidis P374Serogroup Y, l�quor, Campo Grande - RJ, BrasilINCQSE.coli DH5(NalR used as plasmid receptor. 
F-, endA1, hsdR17 (rk-, mk-), supE44, thi-1, gir A96, relA1Hanahan et al. ,1983INCQS � FIOCRUZ � National Institute of Quality Control - Funda��o Oswaldo Cruz, Rio de Janeiro, RJ


















Table 2: Oligonucleotides used in this work

GeneOligonucleotides (5  �! 3�)TNF�FW: TCTTCTCGAACCCCGAGTGA
RV: CCTCTGATGGCACCACCAGTGF�FW: CAGCAACAATTCCTGGCGATA
RV:AAGGCGAAAGCCCTCAATTTINF�FW: GGCTGTTACTGCCAGGACCCATATGT
RV: GATGCTCTTGCACCTCGAACAGCATIL-06FW: GAGGATACCACTCCCAACAGACC
RV: AAGTGCATCATCGTTGTTCATACAIL-08FW: ATGACTTCCAAGCTGGCCGTGGCT
RV: TTATGAATTCTCAGCCCTCTTCAAAAAIL-10FW: GTGA TGCCCCAAGCTGAGA
RV: CACGGCCTTGCTCTTGTTTGAPDHFW: TGCACCACCAACTGCTTAGC
RV: GGCATGGACTGTGGTCATGAG

































Table 3: Size and Polydispersion from the outer membrane vesicles 


Strain OMVSize (r.nm)Superficial charge C2135114.6-19.0 + 27.6P214382.4-21.6 + 5.92P37475.8-16.9 + 8.14 C2135 
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la�p�������ytf��pilE71.8-26.0 + 13.00C2135 �synA55.4-27.9 + 9.27





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la�p�������ytf�d�r�|�~����h�:�h�n�OJQJ^Jh�;2OJQJ^Jhf�h�;2OJQJ^J<P1�h:p�n���. ��A!��"��#��$��%�������Dp�	D<EndNote><Cite><Author>Caugant</Author><Year>1994</Year><RecNum>9032</RecNum><DisplayText>(Caugant, Hoiby et al. 1994)</DisplayText><record><rec-number>9032</rec-number><foreign-keys><key app="EN" db-id="0swvdszxlr0d9oexsvk5exebtffd29frrpx0">9032</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Caugant, D. A.</author><author>Hoiby, E. A.</author><author>Magnus, P.</author><author>Scheel, O.</author><author>Hoel, T.</author><author>Bjune, G.</author><author>Wedege, E.</author><author>Eng, J.</author><author>Froholm, L. O.</author></authors></contributors><auth-address>Department of Bacteriology, National Institute of Public Health, Oslo, Norway.</auth-address><titles><title>Asymptomatic carriage of Neisseria meningitidis in a randomly sampled population</title><secondary-title>J Clin Microbiol</secondary-title><alt-title>Journal of clinical microbiology</alt-title></titles><periodical><full-title>J Clin Microbiol</full-title><abbr-1>Journal of clinical microbiology</abbr-1></periodical><alt-periodical><full-title>J Clin Microbiol</full-title><abbr-1>Journal of clinical microbiology</abbr-1></alt-periodical><pages>323-30</pages><volume>32</volume><number>2</number><edition>1994/02/01</edition><keywords><keyword>Adolescent</keyword><keyword>Adult</keyword><keyword>Aged</keyword><keyword>Aged, 80 and over</keyword><keyword>Carrier State/*epidemiology/microbiology</keyword><keyword>Child</keyword><keyword>Child, Preschool</keyword><keyword>Drug Resistance, Microbial</keyword><keyword>Female</keyword><keyword>Humans</keyword><keyword>Infant</keyword><keyword>Male</keyword><keyword>Meningococcal Infections/*epidemiology/microbiology</keyword><keyword>Middle Aged</keyword><keyword>Multivariate Analysis</keyword><keyword>Neisseria meningitidis/classification/drug effects/*isolation &amp; purification</keyword><keyword>Norway/epidemiology</keyword><keyword>Risk Factors</keyword><keyword>Sampling Studies</keyword><keyword>Serotyping</keyword></keywords><dates><year>1994</year><pub-dates><date>Feb</date></pub-dates></dates><isbn>0095-1137 (Print)&#xD;0095-1137 (Linking)</isbn><accession-num>8150942</accession-num><work-type>Research Support, Non-U.S. Gov&apos;t</work-type><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/8150942</url></related-urls></urls><custom2>263032</custom2><language>eng</language></record></Cite></EndNote>�	D<EndNote><Cite><Author>Caugant</Author><Year>1994</Year><RecNum>9032</RecNum><DisplayText>(Caugant, Hoiby et al. 1994)</DisplayText><record><rec-number>9032</rec-number><foreign-keys><key app="EN" db-id="0swvdszxlr0d9oexsvk5exebtffd29frrpx0">9032</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Caugant, D. A.</author><author>Hoiby, E. A.</author><author>Magnus, P.</author><author>Scheel, O.</author><author>Hoel, T.</author><author>Bjune, G.</author><author>Wedege, E.</author><author>Eng, J.</author><author>Froholm, L. O.</author></authors></contributors><auth-address>Department of Bacteriology, National Institute of Public Health, Oslo, Norway.</auth-address><titles><title>Asymptomatic carriage of Neisseria meningitidis in a randomly sampled population</title><secondary-title>J Clin Microbiol</secondary-title><alt-title>Journal of clinical microbiology</alt-title></titles><periodical><full-title>J Clin Microbiol</full-title><abbr-1>Journal of clinical microbiology</abbr-1></periodical><alt-periodical><full-title>J Clin Microbiol</full-title><abbr-1>Journal of clinical microbiology</abbr-1></alt-periodical><pages>323-30</pages><volume>32</volume><number>2</number><edition>1994/02/01</edition><keywords><keyword>Adolescent</keyword><keyword>Adult</keyword><keyword>Aged</keyword><keyword>Aged, 80 and over</keyword><keyword>Carrier State/*epidemiology/microbiology</keyword><keyword>Child</keyword><keyword>Child, Preschool</keyword><keyword>Drug Resistance, Microbial</keyword><keyword>Female</keyword><keyword>Humans</keyword><keyword>Infant</keyword><keyword>Male</keyword><keyword>Meningococcal Infections/*epidemiology/microbiology</keyword><keyword>Middle Aged</keyword><keyword>Multivariate Analysis</keyword><keyword>Neisseria meningitidis/classification/drug effects/*isolation &amp; purification</keyword><keyword>Norway/epidemiology</keyword><keyword>Risk Factors</keyword><keyword>Sampling Studies</keyword><keyword>Serotyping</keyword></keywords><dates><year>1994</year><pub-dates><date>Feb</date></pub-dates></dates><isbn>0095-1137 (Print)&#xD;0095-1137 (Linking)</isbn><accession-num>8150942</accession-num><work-type>Research Support, Non-U.S. Gov&apos;t</work-type><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/8150942</url></related-urls></urls><custom2>263032</custom2><language>eng</language></record></Cite></EndNote>�D<EndNote><Cite><Author>Brandtzaeg</Author><Year>1995</Year><RecNum>8996</RecNum><DisplayText>(Brandtzaeg, Ovstebo et al. 1995)</DisplayText><record><rec-number>8996</rec-number><foreign-keys><key app="EN" db-id="0swvdszxlr0d9oexsvk5exebtffd29frrpx0">8996</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Brandtzaeg, P.</author><author>Ovstebo, R.</author><author>Kierulf, P.</author></authors></contributors><auth-address>Department of Pediatrics, Ulleval University Hospital, Oslo, Norway.</auth-address><titles><title>Bacteremia and compartmentalization of LPS in meningococcal disease</title><secondary-title>Prog Clin Biol Res</secondary-title><alt-title>Progress in clinical and biological research</alt-title></titles><periodical><full-title>Prog Clin Biol Res</full-title><abbr-1>Progress in clinical and biological research</abbr-1></periodical><alt-periodical><full-title>Prog Clin Biol Res</full-title><abbr-1>Progress in clinical and biological research</abbr-1></alt-periodical><pages>219-33</pages><volume>392</volume><edition>1995/01/01</edition><keywords><keyword>Bacteremia/*etiology</keyword><keyword>Carbohydrate Sequence</keyword><keyword>Chemistry, Physical</keyword><keyword>Cytokines/blood/cerebrospinal fluid</keyword><keyword>Female</keyword><keyword>Humans</keyword><keyword>Lipid A/chemistry/pharmacokinetics/toxicity</keyword><keyword>Lipopolysaccharides/chemistry/*pharmacokinetics/toxicity</keyword><keyword>Male</keyword><keyword>Meningitis, Meningococcal/etiology</keyword><keyword>Meningococcal Infections/*etiology</keyword><keyword>Molecular Sequence Data</keyword><keyword>Molecular Structure</keyword><keyword>Neisseria meningitidis/isolation &amp; purification</keyword><keyword>Physicochemical Phenomena</keyword><keyword>Shock, Septic/etiology</keyword></keywords><dates><year>1995</year></dates><isbn>0361-7742 (Print)&#xD;0361-7742 (Linking)</isbn><accession-num>8524927</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/8524927</url></related-urls></urls><language>eng</language></record></Cite></EndNote>�D<EndNote><Cite><Author>Brandtzaeg</Author><Year>1995</Year><RecNum>8996</RecNum><DisplayText>(Brandtzaeg, Ovstebo et al. 1995)</DisplayText><record><rec-number>8996</rec-number><foreign-keys><key app="EN" db-id="0swvdszxlr0d9oexsvk5exebtffd29frrpx0">8996</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Brandtzaeg, P.</author><author>Ovstebo, R.</author><author>Kierulf, P.</author></authors></contributors><auth-address>Department of Pediatrics, Ulleval University Hospital, Oslo, Norway.</auth-address><titles><title>Bacteremia and compartmentalization of LPS in meningococcal disease</title><secondary-title>Prog Clin Biol Res</secondary-title><alt-title>Progress in clinical and biological research</alt-title></titles><periodical><full-title>Prog Clin Biol Res</full-title><abbr-1>Progress in clinical and biological research</abbr-1></periodical><alt-periodical><full-title>Prog Clin Biol Res</full-title><abbr-1>Progress in clinical and biological research</abbr-1></alt-periodical><pages>219-33</pages><volume>392</volume><edition>1995/01/01</edition><keywords><keyword>Bacteremia/*etiology</keyword><keyword>Carbohydrate Sequence</keyword><keyword>Chemistry, Physical</keyword><keyword>Cytokines/blood/cerebrospinal fluid</keyword><keyword>Female</keyword><keyword>Humans</keyword><keyword>Lipid A/chemistry/pharmacokinetics/toxicity</keyword><keyword>Lipopolysaccharides/chemistry/*pharmacokinetics/toxicity</keyword><keyword>Male</keyword><keyword>Meningitis, Meningococcal/etiology</keyword><keyword>Meningococcal Infections/*etiology</keyword><keyword>Molecular Sequence Data</keyword><keyword>Molecular Structure</keyword><keyword>Neisseria meningitidis/isolation &amp; purification</keyword><keyword>Physicochemical Phenomena</keyword><keyword>Shock, Septic/etiology</keyword></keywords><dates><year>1995</year></dates><isbn>0361-7742 (Print)&#xD;0361-7742 (Linking)</isbn><accession-num>8524927</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/8524927</url></related-urls></urls><language>eng</language></record></Cite></EndNote>�D<EndNote><Cite><Author>Zarantonelli</Author><Year>2008</Year><RecNum>8363</RecNum><record><rec-number>8363</rec-number><foreign-keys><key app="EN" db-id="v5epp0v5vpwtaxe0xv0xtzfees5pwpdazr5e">8363</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Zarantonelli, M. L.</author><author>Lancellotti, M.</author><author>Deghmane, A. E.</author><author>Giorgini, D.</author><author>Hong, E.</author><author>Ruckly, C.</author><author>Alonso, J. M.</author><author>Taha, M. 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P.</author><author>Robert, V.</author><author>Tommassen, J.</author></authors></contributors><auth-address>Department of Molecular Microbiology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.</auth-address><titles><title>Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain</title><secondary-title>EMBO Rep</secondary-title><alt-title>EMBO reports</alt-title></titles><periodical><full-title>EMBO Rep</full-title><abbr-1>EMBO reports</abbr-1></periodical><alt-periodical><full-title>EMBO Rep</full-title><abbr-1>EMBO reports</abbr-1></alt-periodical><pages>1149-54</pages><volume>8</volume><number>12</number><edition>2007/11/17</edition><keywords><keyword>Bacterial Outer Membrane Proteins/classification/genetics/*metabolism</keyword><keyword>Binding Sites/genetics</keyword><keyword>Cell Membrane/metabolism</keyword><keyword>Electrophoresis, Polyacrylamide Gel</keyword><keyword>Immunoblotting</keyword><keyword>Mutation</keyword><keyword>Neisseria meningitidis/genetics/*metabolism</keyword><keyword>Phylogeny</keyword></keywords><dates><year>2007</year><pub-dates><date>Dec</date></pub-dates></dates><isbn>1469-221X (Print)&#xD;1469-221X (Linking)</isbn><accession-num>18007659</accession-num><work-type>Research Support, Non-U.S. Gov&apos;t</work-type><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/18007659</url></related-urls></urls><custom2>2267228</custom2><electronic-resource-num>10.1038/sj.embor.7401092</electronic-resource-num><language>eng</language></record></Cite><Cite><Author>Bos</Author><Year>2007</Year><RecNum>8935</RecNum><record><rec-number>8935</rec-number><foreign-keys><key app="EN" db-id="0swvdszxlr0d9oexsvk5exebtffd29frrpx0">8935</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Bos, M. 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Germany.</auth-address><titles><title>Interaction of Neisseria meningitidis with human dendritic cells</title><secondary-title>Infect Immun</secondary-title><alt-title>Infection and immunity</alt-title></titles><periodical><full-title>Infect Immun</full-title><abbr-1>Infection and immunity</abbr-1></periodical><alt-periodical><full-title>Infect Immun</full-title><abbr-1>Infection and immunity</abbr-1></alt-periodical><pages>6912-22</pages><volume>69</volume><number>11</number><edition>2001/10/13</edition><keywords><keyword>Adult</keyword><keyword>Bacterial Adhesion/physiology</keyword><keyword>Cell Differentiation</keyword><keyword>Cells, Cultured</keyword><keyword>Cytokines/biosynthesis</keyword><keyword>Dendritic Cells/cytology/immunology/*microbiology</keyword><keyword>Humans</keyword><keyword>Neisseria 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