HP0953 - hypothetical virulence factor overexpresion and localization during Helicobacter pylori infection of gastric epithelium.

BACKGROUND: The high prevalence and persistence of Helicobacter pylori (H. pylori) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are varied. Moreover, as its proteome contains 340 hypothetical proteins, it is important to investigate them to completely understand the mechanisms of its virulence and survival. We have previously reported that the hypothetical protein HP0953 is overexpressed during the first hours of adhesion to inert surfaces, under stress conditions, suggesting its role in the environmental survival of this bacterium and perhaps as a virulence factor. AIM: To investigate the expression and localization of HP0953 during adhesion to an inert surface and against gastric (AGS) cells. METHODS: Expression analysis was performed for HP0953 during H. pylori adhesion. HP0953 expression at 0, 3, 12, 24, and 48 h was evaluated and compared using the Kruskal-Wallis equality-of-populations rank test. Recombinant protein was produced and used to obtain polyclonal antibodies for immunolocalization. Immunogold technique was performed on bacterial sections during adherence to inert surfaces and AGS cells, which was analyzed by transmission electron microscopy. HP0953 protein sequence was analyzed to predict the presence of a signal peptide and transmembrane helices, both provided by the ExPASy platform, and using the GLYCOPP platform for glycosylation sites. Different programs, via, I-TASSER, RaptorX, and HHalign-Kbest, were used to perform three-dimensional modeling. RESULTS: HP0953 exhibited its maximum expression at 12 h of infection in gastric epithelium cells. Immunogold technique revealed HP0953 localization in the cytoplasm and accumulation in some peripheral areas of the bacterial body, with greater expression when it is close to AGS cells. Bioinformatics analysis revealed the presence of a signal peptide that interacts with the transmembrane region and then allows the release of the protein to the external environment. The programs also showed a similarity with the Tip-alpha protein of H. pylori. Tip-alpha is an exotoxin that penetrates cells and induces tumor necrosis factor alpha production, and HP0953 could have a similar function as posttranslational modification sites were found; modifications in turn require enzymes located in eukaryotic cells. Thus, to be functional, HP0953 may necessarily need to be translocated inside the cell where it can trigger different mechanisms producing cellular damage. CONCLUSION: The location of HP0953 around infected cells, the probable posttranslational modifications, and its similarity to an exotoxin suggest that this protein is a virulence factor.

Antibacterial mechanism of gold nanoparticles on Streptococcus pneumoniae.

Streptococcus pneumoniae is a causal agent of otitis media, pneumonia, meningitis and severe cases of septicemia. This human pathogen infects elderly people and children with a high mortality rate of approximately one million deaths per year worldwide. Antibiotic-resistance of S. pneumoniae strains is an increasingly serious health problem; therefore, new therapies capable of combating pneumococcal infections are indispensable. The application of gold nanoparticles has emerged as an option in the control of bacterial infections; however, the mechanism responsible for bacterial cell lysis remains unclear. Specifically, it has been observed that gold nanoparticles are capable of crossing different structures of the S. pneumoniae cells, reaching the cytosol where inclusion bodies of gold nanoparticles are noticed. In this work, a novel process for the separation of such inclusion bodies that allowed the analysis of the biomolecules such as carbohydrates, lipids and proteins associated with the gold nanoparticles was developed. Then, it was possible to separate and identify proteins associated with the gold nanoparticles, which were suggested as possible candidates that facilitate the interaction and entry of gold nanoparticles into S. pneumoniae cells.

Characterization of Spbhp-37, a Hemoglobin-Binding Protein of Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive microorganism that is the cause of bacterial pneumonia, sinusitis and otitis media. This human pathogen also can cause invasive diseases such as meningitis, bacteremia and septicemia. Hemoglobin (Hb) and haem can support the growth and viability of S. pneumoniae as sole iron sources. Unfortunately, the acquisition mechanism of Hb and haem in this bacterium has been poorly studied. Previously we identified two proteins of 37 and 22 kDa as putative Hb- and haem-binding proteins (Spbhp-37 and Spbhp-22, respectively). The sequence of Spbhp-37 protein was database annotated as lipoprotein without any function or localization. Here it was immunolocalized in the surface cell by transmission electron microscopy using specific antibodies produced against the recombinant protein. The expression of Spbhp-37 was increased when bacteria were grown in media culture supplied with Hb. In addition, the affinity of Sphbp-37 for Hb was determined. Thus, in this work we are presenting new findings that attempt to explain the mechanism involved in iron acquisition of this pathogen. In the future these results could help to develop new therapy targets in order to avoid the secondary effects caused by the traditional therapies.

Inclusion bodies and pH lowering: as an effect of gold nanoparticles in Streptococcus pneumoniae.

Streptococcus pneumoniae is a human pathogen whose principal virulence factor is its capsule. This structure allows the bacterium to evade the human immune system. Treatment of infections caused by this bacterium is based on antibiotics; however, the emergence of antibiotic-resistant strains makes this task increasingly difficult. Therefore, it is necessary to investigate new therapies, such as those based on gold nanoparticles, for which unfortunately the mechanisms involved have not yet been investigated. As far as we know, this study is the first that attempts to explain how gold nanoparticles destroy the bacterium Streptococcus pneumoniae. We found that the mean particle size was an important issue, and that the effect on the bacterium was dose-dependent. Cellular growth was inhibited by the presence of the nanoparticles, as was cell viability. The pH of the bacterial growth media was acidified, but interestingly the reactive species were not affected. A transmission electron microscopy analysis revealed the presence of inclusion bodies of gold nanoparticles within the bacterium. We present the first findings that attempt to explain how gold nanoparticles lyse Gram-positive bacteria.

Vancomycin modifies the expression of the agr system in multidrug-resistant Staphylococcus aureus clinical isolates.

UNLABELLED: Staphylococcus aureus is an opportunistic pathogen that colonizes human hosts and causes a wide variety of diseases. Two interacting regulatory systems called agr (accessory gene regulator) and sar (staphylococcal accessory regulator) are involved in the regulation of virulence factors. The aim of this study was to evaluate the effect of vancomycin on hld and spa gene expression during the exponential and post-exponential growth phases in multidrug-resistant (MDR) S. aureus. METHODS: Antibiotic susceptibility was evaluated by the standard microdilution method. The phylogenetic profile was obtained by pulsed-field gel electrophoresis (PFGE). Polymorphisms of agr and SCCmec (staphylococcal cassette chromosome mec) were analyzed by multiplex polymerase chain reaction (PCR). The expression levels of hld and spa were analyzed by reverse transcription-PCR. An enzyme-linked immunosorbent assay (ELISA) was performed to detect protein A, and biofilm formation was analyzed via crystal violet staining. RESULTS: In total, 60.60% (20/33) of S. aureus clinical isolates were MDR. Half (10/20) of the MDR S. aureus isolates were distributed in subcluster 10, with >90% similarity among them. In the isolates of this subcluster, a high prevalence (100%) for the agrII and the cassette SCCmec II polymorphisms was found. Our data showed significant increases in hld expression during the post-exponential phase in the presence and absence of vancomycin. Significant increases in spa expression, protein A production and biofilm formation were observed during the post-exponential phase when the MDR S. aureus isolates were challenged with vancomycin. CONCLUSION: The polymorphism agrII, which is associated with nosocomial isolates, was the most prevalent polymorphism in MDR S. aureus. Additionally, under our study conditions, vancomycin modified hld and spa expression in these clinical isolates. Therefore, vancomycin may regulate alternative systems that jointly participate in the regulation of these virulence factors.

Streptococcus pneumoniae secretes a glyceraldehyde-3-phosphate dehydrogenase, which binds haemoglobin and haem.

Streptococcus pneumoniae is a gram positive encapsulated bacterium responsible of septicaemia and upper respiratory infections in children. This pathogen requires iron to survive in the host, which it can obtain of haemoglobin (Hb) or haem. Only two Hb-binding membrane proteins have been identified up to now. However it is unknown whether this pathogen secretes proteins in order to scavenge iron from the Hb or haem. Therefore, in order to explore these possibilities, cellular growth of S. pneumoniae was tested with several alternative iron supplies. The bacterial growth was supported with iron, Hb and haem. Additionally, S. pneumoniae expressed and secreted a protein of 38 kDa which was purified and characterized as Hb and haem-binding protein. This protein was also identified by mass spectrometry as glyceraldehyde-3-phosphate dehydrogenase. Our overall results suggest that S. pneumoniae secretes a protein capable of binding two usefull iron sources for this bacterium (Hb and haem). This protein could be playing a dynamic role in the success of the invasive and infective processes of this pathogen.

Helicobacter pylori secretes the chaperonin GroEL (HSP60), which binds iron.

Helicobacter pylori is a bacterium that can use multiple iron sources. However, it is unknown whether this bacterium secretes molecules such as siderophores or haemophores to scavenge iron. Here, we report the first secreted iron-binding protein of H. pylori, which we purified by haem-affinity chromatography. Mass spectrometry analysis revealed its identity as chaperonin (HpGroEL). When we compared HpGroEL with EcGroEL from Escherichia coli, they were homologous, showing 60% similarity. Additionally, purified cytoplasmic HpGroEL could also bind iron. Perhaps H. pylori secretes HpGroEL to maintain the appropriate folding of extracellular proteins and to bind iron.

Análisis in silico de proteínas potencialmente involucradas en la biogénesis de fimbrias en Helicobacter pylori / In silico analysis of proteins potentially involved in fimbrial biogenesis in Helicobacter pylori

Introducción. La colonización e infección crónica por Helicobacter pylori es el factor de mayor contribución al desarrollo de cáncer gástrico. Se ha descrito un gran repertorio de adhesinas que contribuyen a la adaptación específica de la bacteria al nicho gástrico y, para H. pylori , al igual que en otras bacterias patógenas, la formación de biopelícula es fundamental en la supervivencia a ambientes no favorables. Las fimbrias o pili tipo IV son responsables de la adhesión de diversas bacterias patógenas ( Escherichia coli , Pseudomonas aeruginosa y Vibrio cholerae) a distintas superficies. El objetivo de este trabajo fue identificar y analizar genes que pudieran codificar para proteínas involucradas en la biogénesis de fimbrias en H. pylori y caracterizar su expresión durante la formación de biopelícula. Métodos. Se emplearon herramientas bioinformáticas y moleculares, tales como la base de datos del NCBI para la búsqueda de secuencias de proteínas relacionadas con la biogénesis de fimbrias, así como la herramienta de PSI BLAST. Los alineamientos múltiples se realizaron con el programa T-COFFEE y HMMER. La predicción de las estructuras secundarias se realizó con ANTHEPROT y las estructuras terciarias se predijeron con el programa I-TASSER. Resultados. Se identificaron dos homólogos, jhp0257 y HP0272, de la proteína PilN de Campylobacter rectus y Xilella fastidiosa , la cual es parte de la maquinaria del ensamble de la fimbria tipo IV. Asimismo, las proteínas jhp0887 y HP0953 presentaron homología a nivel del péptido señal de PilA de P. aeruginosa , y la proteína HP0953 se sobreexpresó durante la formación de la biopelícula. Conclusiones. H. pylori posee proteínas homólogas a las proteínas de familias fimbriales, específicamente PilN y PilA, que ensamblan fimbria tipo IV en otras bacterias. Esta última tiene un nivel de expresión mayor durante la etapa inicial del proceso de formación de biopelícula.

Background. Colonization and chronic infection with Helicobacter pylori is the major contributing factor to the development of gastric cancer. A large repertoire of adhesins has been described that contribute to the adaptation of bacteria to a specific gastric niche. As in other pathogenic bacteria, H. pylori biofilm formation is central to survival on unfavorable environments. Type IV pili or fimbriae are responsible for the adhesion of many pathogenic bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa and Vibrio cholerae ) to various surfaces. The aim of this study was to identify and analyze genes that might encode proteins involved in the biogenesis of fimbriae on H. pylori and characterize their expression during biofilm formation. Methods. PSI BLAST, bioinformatics and molecular tools were used as well as the NCBI database search for sequences related to protein biogenesis of fimbriae. Multiple alignments were performed using the HMMer and T-COFFEE programs. The secondary structure prediction was performed with ANTHEPROT and the tertiary structures were predicted with the I-Tasser. Results. We identified two counterparts-jhp0257 and HP0272-from protein of Campylobacter rectus and PilN Xilella fastidiosa , which is part of the machinery of assembly type IV fimbria. Similarly, proteins jhp0887 and HP0953 show homology from peptide PilA level of P. aeruginosa , and the HP0953 protein is overexpressed during the formation of the biofilm. Conclusions. H. pylori possesses proteins homologous to fimbrial protein families, specifically PilN and PilA, which join type IV fimbriae in other bacteria. The latter has a higher expression level during the initial stage of the formation of biofilm.

Streptococcus pneumoniae requires iron for its viability and expresses two membrane proteins that bind haemoglobin and haem.

Streptococcus pneumoniae, a human pathogen bacterium, can support its growth using haemoglobin (Hb) and haem as sole iron sources, but not when holo-transferrin or holo-lactoferrin is supplied. For this reason, it is easy to think that the principal iron sources for this pathogen inside humans are Hb and haem. Unfortunately, the mechanism has been poorly studied. The findings presented in this study are the first efforts that attempted to explain the mechanism involved in iron acquisition of this pathogen. This pathogen was capable of supporting its viability when iron sources such as Hb or haem were supplied. Membranes of S. pneumoniae were separated and their respective proteins were solubilized in order to be purified by haem-affinity chromatography. This strategy allowed us to purify seven membrane proteins. An experiment of competence with haem and iron showed two potential haem and Hb-binding proteins. Their Hb-binding function was confirmed by overlay assay using Hb and their respective identities were obtained by mass spectrometry. Then by amino acid alignment analysis, the motif involved in binding of Hb or haem was revealed. These results are the first findings that attempt to explain the mechanisms developed by S. pneumoniae to acquire iron from Hb or haem in the host, which could allow a better understanding of the biology of this bacterium.

The frpB1 gene of Helicobacter pylori is regulated by iron and encodes a membrane protein capable of binding haem and haemoglobin.

FrpB1 is a novel membrane protein of Helicobacter pylori that is capable of binding both haem and haemoglobin but consistently shows more affinity for haem. The mRNA levels of frpB1 were repressed by iron and lightly modulated by haem or haemoglobin. The overexpression of the frpB1 gene supported cellular growth when haem or haemoglobin were supplied as the only iron source. Three-dimensional modelling revealed the presence of motifs necessary to bind either haem or haemoglobin. Our overall results support the idea that FrpB1 is a membrane protein of H. pylori that allows this pathogen to survive in the human stomach.