Analysis of the biofilm proteome of Xylella fastidiosa

<p>Abstract</p> <p>Background</p> <p><it>Xylella fastidiosa </it>is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters). The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp). The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density) presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of <it>X. fastidiosa </it>strain 9a5c, in comparison to planktonic growth condition.</p> <p>Results</p> <p>We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins) different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of <it>X. fastidiosa</it>. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments.</p> <p>Conclusions</p> <p>We observed overexpression of proteins related to quorum sensing, proving the existence of communication between cells, and thus the development of structuring the biofilm (mature biofilm) leading to obstruction of vessels and development of disease. This paper reports a first proteomic analysis of mature biofilm of <it>X. fastidiosa</it>, opening new perspectives for understanding the biochemistry of mature biofilm growth in a plant pathogen.</p

Microsatellite markers for urochloa Humidicola (poaceae) and their transferability to other urochloa species

Urochloa humidicola is a warm-season grass commonly used as forage in the tropics and is recognized for its tolerance to seasonal flooding. This grass is an important forage species for the Cerrado and Amazon regions of Brazil. U. humidicola is a polyploid species with variable ploidy (6X-9X) and facultative apomixis with high phenotypic plasticity. However, this apomixis and ploidy, as well as the limited knowledge of the genetic basis of the germplasm collection, have constrained genetic breeding activities, yet microsatellite markers may enable a better understanding of the species' genetic composition. This study aimed to develop and characterize new polymorphic microsatellite molecular markers in U. humidicola and to evaluate their transferability to other Urochloa species. Findings: A set of microsatellite markers for U. humidicola was identified from two new enriched genomic DNA libraries: the first library was constructed from a single sexual genotype and the second from a pool of eight apomictic genotypes selected on the basis of previous results. Of the 114 loci developed, 72 primer pairs presented a good amplification product, and 64 were polymorphic among the 34 genotypes tested. The number of bands per simple sequence repeat (SSR) locus ranged from 1 to 29, with a mean of 9.6 bands per locus. The mean polymorphism information content (PIC) of all loci was 0.77, and the mean discrimination power (DP) was 0.87. STRUCTURE analysis revealed differences among U. humidicola accessions, hybrids, and other Urochloa accessions. The transferability of these microsatellites was evaluated in four species of the genus, U. brizantha, U. decumbens, U. ruziziensis, and U. dictyoneura, and the percentage of transferability ranged from 58.33% to 69.44% depending on the species. Conclusions: This work reports new polymorphic microsatellite markers for U. humidicola that can be used for breeding programs of this and other Urochloa species, including genetic linkage mapping, quantitative trait loci identification, and marker-assisted selection8

Effect of oral biofilms on the biotribocorrosion of titanium

The oral cavity is a complex environment where corrosive substances from dietary, human saliva and oral biofilms may accumulate in retentive areas of implant-supported prostheses promoting a corrosion of their surfaces. On the other side, during mastication, micro-movements may occur in artificial joints causing a relative contact motion between surfaces. Both processes result in a tribocorrosion system. The objective of this work is to investigate the in-vitro tribocorrosion behavior of titanium covered by biofilms and immersed in artificial saliva. Reciprocating sliding wear tests coupled with electrochemical techniques were performed at 25 o C on titanium samples covered or not with biofilms. Mixed biofilms of Candida albicans and Streptococcus mutans were grown at 37 oC on titanium surfaces for 9 days in Tryptic Soy Broth medium, containing mucin, yeast extract, peptone and sucrose. The open-circuit potential (OCP) was measured until stabilization in artificial saliva. After that, electrochemical impedance spectroscopy (EIS) measurements were carried out at open circuit potential. Subsequently, sliding tests were performed at normal loads of 0.1, 0.2 and 3 N, at a sliding frequency of 1 Hz, and linear displacement amplitudes of 0.5 and 2 mm using a tribometer equipped with a test viewer software. During the sliding tests, the OCP was constantly monitored. At the end of the sliding test, the OCP was further monitored until stabilization, and another series of EIS measurements was made. Worn and unworn surfaces were inspected by SEM. The detachment of the biofilms was not noticed when low loads were applied, and biofilms appear to have some lubricating effect. However, it was found that acidic substances released from Streptococcus mutans and external substances accumulated into the biofilm might corrode surfaces located below and around the biofilms. At a normal load of 3 N, the biofilms were removed from the contact region by the sliding action, and it was observed that the repassivation rate of titanium covered with biofilms was lower than without biofilms.Fundação para a Ciência e a Tecnologia (FCT)ALBANFlemish Science Foundation. Scientific Research Community on Surface Modification of MaterialsPrograma Alban - Bolsas de Alto Nível da União Europeia para a América Latin

Wear and corrosion of titanium in oral simulating environments

The oral cavity can be considered as an aggressive environment to restorative materials considering that several acidic substances can be retained in the saliva and biofilms as well as loads from mastication and abrasive particles take place in the wear of oral surfaces. The objective of this work is to investigate the in-vitro corrosion and wear in fluoridated artificial saliva and in presence of biofilms. Electrochemical tests of titanium surfaces were performed in artificial saliva containing different fluoride concentrations at 37 oC. Another group of titanium surfaces were prepared to the growth of oral mixed biofilms for 9 days in a specific culture medium. Then, electrochemical tests were carried out with titanium surfaces covered with biofilms in artificial saliva without fluorides. After that, wear sliding tests were performed in the same mediums using a tribometer equipped with a test viewer software. Also, electrochemical measurements were carried during the wear sliding tests. After corrosion-wear tests, worn and unworn surfaces were analyzed by AFM and SEM. A localized corrosion of titanium was only noticed in high fluoride concentration. Therefore, there was a decrease of the corrosion resistance of titanium when the fluoride concentration was increased. Moreover, the presence of biofilms affected harmfully the corrosion resistance of titanium probably due to acids release from the biofilms. However, the wear was decreased when low loads were applied on the biofilms.Fundação para a Ciência e a Tecnologia (FCT) - PTDC/CTM/67500/2006Programa Alban - Bolsas de Alto Nível da União Europeia para a América Latina - E06D103407BRFlemish Science Foundation. Scientific Research Community on Surface Modification of Material

Tribocorrosion of commercially pure titanium in oral simulating environments

According to several studies, commercially pure (cp) titanium presents the required properties for dental applications such as mechanical resistance, low density, corrosion resistance and biocompatibility [1-3]. Also, it is widely accepted that the surface properties of the metallic material will play an essential role in the interaction between implanted materials and tissue. Whatever Ti is used in crowns or implants, a relative displacement between the implant and the adjacent material (gum, bone, porcelain…) might occur, always in the presence of an environment constituted by inorganic and organic species. Therefore, a tribocorrosion system may be formed, and the synergistic effect of wear and corrosion may lead to accelerated failure of the system. Nevertheless, few studies have focused on the tribocorrosion behavior of titanium in oral simulating environments, considering the presence of fluoride solutions and/or bacterial biofilms [4-8]. The aim of this work was to study the effect of sliding wear of cp titanium in the presence of artificial saliva containing different amounts of fluoride. Also, the behavior of the tribocorrosion system when a biofilm is present at the surface of the metallic materials was evaluated. Samples of grade 2 Ti were prepared by grinding using SiC sandpapers till 1200 mesh. Tribocorrosion tests were accomplished in Fusayama’s artificial saliva (AS) containing two different fluoride concentrations: 30 and 227 ppmF-. Open-circuit potential (OCP) was monitored during the entire experiment and electrochemical impedance spectroscopy tests (EIS) were carried out at 50 mV above corrosion potential (Ecorr) at frequencies from 100 KHz to 15.8 mHz, before and after sliding. The sliding tests were carried out using an alumina sphere as counterbody under a normal load of 3 N, an amplitude of 2 mm for 20 min. Results were statistically analyzed at significance level of p < 0.05. Samples were inspected by SEM-EDS and AFM. As shown in fig. 1, the addition of 227 ppmF- to the AS resulted in a higher Ecorr during sliding. However, in the same solution, Ti presents a significantly higher passive current density in the absence of mechanical action. During sliding, third-body particles were produced, and chemical analysis of the solution revealed a significant release of Ti ions. The influence of the presence of bacterial cultures of S.mutans on the corrosion and tribocorrosion behavior of titanium is also discussed.Fundação para a Ciência e a Tecnologia (FCT)Programa Alban - Bolsas de Alto Nível da União Europeia para a América LatinaFlemish Science Foundation.Scientific Research Community on Surface Modification of Material

Corrosion behaviour of titanium in the presence of Streptococcus mutans

Objective The main aim of this in vitro study was to evaluate the influence of Streptococcus mutans on the corrosion of titanium. Methods S. mutans biofilms were formed on commercially pure titanium (CP-Ti) square samples (10 mm × 10 mm × 1 mm) using a culture medium enriched with sucrose. Open circuit potential (OCP) and electrochemical impedance spectroscopy (EIS) measurements were used to evaluate the corrosion behaviour of CP-Ti in the presence of S. mutans in Fusayama's artificial saliva. The corrosion of biofilm-free CP-Ti samples was also evaluated in artificial saliva. Biofilms biomass was measured by spectrophotometry, using crystal violet staining, after 1, 2 and 7 days. Results The OCP values recorded on CP-Ti in the presence of S. mutans (−0.3 ± 0.02 V vs. SCE) was lower than those on biofilm-free CP-Ti (−0.1 ± 0.01 V vs. SCE) after 2 h of immersion in artificial saliva (p < 0.05). That reveals a high reactivity of titanium in presence of S. mutans. Impedance spectra revealed the formation of a compact passive film on titanium in artificial saliva or in the presence of a 2 days old S. mutans biofilm even though the corrosion resistance of CP-Ti has decreased in presence of a S. mutans biofilm. Conclusion The presence of bacterial colonies, such as S. mutans, negatively affected the corrosion resistance of the titanium.The authors acknowledge the financial support provided by Alban Programme (cod. E06D103407BR), the Erasmus Student Exchange Programme of the CEC, FCT (PTDC/CTM/67500/2006) and the Scientific Research Community on Surface Modification of Materials funded by the Flemish Science Foundation (WOG-FWO-Vlaanderen)