Phylogeographic evidence of cognate recognition site patterns and transformation efficiency differences in H. pylori: theory of strain dominance

BACKGROUND: Helicobacter pylori has diverged in parallel to its human host, leading to distinct phylogeographic populations. Recent evidence suggests that in the current human mixing in Latin America, European H. pylori (hpEurope) are increasingly dominant at the expense of Amerindian haplotypes (hspAmerind). This phenomenon might occur via DNA recombination, modulated by restriction-modification systems (RMS), in which differences in cognate recognition sites (CRS) and in active methylases will determine direction and frequency of gene flow. We hypothesized that genomes from hspAmerind strains that evolved from a small founder population have lost CRS for RMS and active methylases, promoting hpEurope’s DNA invasion. We determined the observed and expected frequencies of CRS for RMS in DNA from 7 H. pylori whole genomes and 110 multilocus sequences. We also measured the number of active methylases by resistance to in vitro digestion by 16 restriction enzymes of genomic DNA from 9 hpEurope and 9 hspAmerind strains, and determined the direction of DNA uptake in co-culture experiments of hspAmerind and hpEurope strains. RESULTS: Most of the CRS were underrepresented with consistency between whole genomes and multilocus sequences. Although neither the frequency of CRS nor the number of active methylases differ among the bacterial populations (average 8.6 ± 2.6), hspAmerind strains had a restriction profile distinct from that in hpEurope strains, with 15 recognition sites accounting for the differences. Amerindians strains also exhibited higher transformation rates than European strains, and were more susceptible to be subverted by larger DNA hpEurope-fragments than vice versa. CONCLUSIONS: The geographical variation in the pattern of CRS provides evidence for ancestral differences in RMS representation and function, and the transformation findings support the hypothesis of Europeanization of the Amerindian strains in Latin America via DNA recombination

Interactions between Bacteria and Bile Salts in the Gastrointestinal and Hepatobiliary Tracts

Bile salts and bacteria have intricate relationships. The composition of the intestinal pool of bile salts is shaped by bacterial metabolism. In turn, bile salts play a role in intestinal homeostasis by controlling the size and the composition of the intestinal microbiota. As a consequence, alteration of the microbiome–bile salt homeostasis can play a role in hepatic and gastrointestinal pathological conditions. Intestinal bacteria use bile salts as environmental signals and in certain cases as nutrients and electron acceptors. However, bile salts are antibacterial compounds that disrupt bacterial membranes, denature proteins, chelate iron and calcium, cause oxidative damage to DNA, and control the expression of eukaryotic genes involved in host defense and immunity. Bacterial species adapted to the mammalian gut are able to endure the antibacterial activities of bile salts by multiple physiological adjustments that include remodeling of the cell envelope and activation of efflux systems and stress responses. Resistance to bile salts permits that certain bile-resistant pathogens can colonize the hepatobiliary tract, and an outstanding example is the chronic infection of the gall bladder by Salmonella enterica. A better understanding of the interactions between bacteria and bile salts may inspire novel therapeutic strategies for gastrointestinal and hepatobiliary diseases that involve microbiome alteration, as well as novel schemes against bacterial infectionsEspaña, MINECO BIO2013-44220-

Structural studies of potential new-generation antibiotic targets and the use of one of them, TonB as a model protein in protein engineering

At the present time resistance to every main class of antibiotic has been observed. Therefore, the continuous development of new-generation antibiotics is crucial to combat the rise of antibiotic resistant strains. Identification of potential antibiotic targets and investigation of their structure and function represent a rational approach to developing a better understanding of the essential processes in which they are involved, and may lead to finding a mechanism to inhibit these processes. The first part of this thesis covers structural characterization and functional studies of the potential antibiotic targets TonB protein and the cell shape determining protein MreC. TonB is needed for TonB-dependent uptake of scarce nutrients, such as iron and vitamin B 12. The cell shape determining protein MreC is involved in cell wall synthesis, which is the target of penicillin and its derivatives. In this study, the three dimensional structures of the C-terminal domain of Helicobacter pylori TonB of different lengths and the C-terminal domain of Bacillus subtilis MreC were determined using nuclear magnetic resonance (NMR) spectroscopy. Additionally, interaction studies of MreC with penicillin-binding proteins were done using NMR spectroscopy and a bacterial two-hybrid system. NMR spectroscopy is a versatile tool to investigate protein structure, dynamics and interactions. One of the advantages of NMR is that proteins can be studied in solution under nearly physiological conditions. However, with increasing molecular size (> 25 – 30 kDa), structural investigation by NMR becomes more complex and often requires specific labelling techniques and alternative methodologies for NMR measurements. Segmental isotopic labelling, where only a part of the protein is stable isotopic labelled, is an attractive method to overcome the challenges of studying large proteins by NMR. Segmental isotopic labelling allows, e.g. investigation of individual protein domains in a full-length context. Furthermore, NMR is a powerful tool to study the integrity of a protein. Certain requirements, however, have to be fulfilled: the protein has to be soluble at high concentrations and stable over the whole measurement time. Therefore it is important to optimize protein production in order to obtain soluble, properly folded proteins at high concentrations. In the second part of this study, TonB has been used as a model protein to show traceless intein-mediated segmental isotopic labelling by salt induced protein trans splicing using a halophilic intein. This approach facilitates structural investigations of TonB by NMR. TonB consists of a well-structured C-terminal domain and a flexible proline-rich region, which would severely interfere with spectral quality in a uniformly labelled sample. Furthermore TonB was used as a model protein to show the benefit in protein expression of using tandem SUMO fusion vectors as tools for the expression of more soluble proteins, which tend to be expressed in an insoluble form. Both of these applications are beneficial for structural investigation of proteins by NMR and can be applied to other proteins.Kaikille antibiooteille resistenttien mikrobikantojen yleistyminen on maailmanlaajuinen terveysuhka. Tällaisten multiresistenttien kantojen hoitamiseksi tarvitaan uusia antibiootteja, joiden vaikutus perustuu uusiin kohdemolekyyleihin tai –rakenteisiin bakteerien pinnoilla. Uusien molekyylien rakenteellinen ja toiminnallinen tutkimus on tärkeää selvitettäessä niiden soveltuvuutta antibioottien kohdemolekyyleiksi. Tämän väitöskirjatyön ensimmäisessä osassa on tutkittu mahdollista antibioottien kohdeproteiinia TonB:tä sekä solun muotoa säätelevää proteiinia MreC:tä. Bakteereilla TonB-proteiini osallistuu eräiden bakteereille niukasti saatavilla olevien ravintoaineiden, kuten raudan ja B 12–vitamiinin, ottamiseen ympäristöstä. MreC toimii soluseinän synteesissä, ja soluseinässä ovat mm. penisilliinin ja sen johdannaisten kohderakenteet. Tässä tutkimuksessa selvitettiin ydinmagneettisen resonanssispektroskopian eli NMR-spektroskopian avulla Helicobacter pylori-bakteerin TonB-proteiinin erimittaisten C-terminaalisten osien sekä Bacillus subtilis-bakteerin MreC-proteiinin kolmiulotteinen rakenne. Tämän lisäksi tutkittiin MreC-proteiinin ja penisilliiniä sitovien proteiinien vuorovaikutusta käyttäen NMR-spektroskopiaa sekä bakteerin kaksihybridi-menetelmää. NMR-spektroskopia on monipuolinen menetelmä, jonka avulla tutkitaan proteiinien rakennetta, dynamiikkaa ja vuorovaikutuksia esimerkiksi toisten proteiinien kanssa. Menetelmän merkittävä etu on se, että proteiineja voidaan tutkia liuostilassa fysiologisen kaltaisissa olosuhteissa. Haittapuolena menetelmässä on, että molekyylin koon kasvaessa (> 25 – 30 kDa) NMR:n käyttö on haasteellisempaa ja vaatii usein erityisiä leimausmenetelmiä. Osittainen isotooppinen leimaaminen, jossa vain osa proteiinista leimataan pysyvästi, voi auttaa suurten proteiinien NMR-mittauksissa. Tutkimuksen toisessa osassa TonB–proteiinia on käytetty mallina tutkittaessa suolasta riippuvaisen inteiinin soveltuvuutta osittaiseen isotooppiseen leimaamiseen. Suolan läsnäollessa halofiilinen inteiini tekee ns. trans splicing reaktion ja yhdistää kaksi proteiinidomeenia. Osittainen isotooppinen leimaus mahdollistaa esimerkiksi yksittäisten domeenien tutkimisen monidomeenisessa proteiinissa. TonB-proteiinissa on rakenteellinen C-terminaalinen domeeni sekä joustava proliinirikas alue, joka vaikuttaisi haitallisesti NMR-spektrien laatuun täysin leimatussa näytteessä. Sen lisäksi TonB-proteiinia on käytetty mallina tutkittaessa tandem SUMO-fuusiovektoreita, joiden avulla saatiin yleensä liukenemattomana tuottuvasta proteiinista liukoinen. NMR-tutkimusta varten proteiinien on pysyttävä liukoisina korkeissa pitoisuuksissa. Tämän vuoksi on tärkeä optimoida proteiinin tuotto siten, että saadaan liukoinen, oikeassa kolmiulotteisessa muodossa oleva proteiini, jonka pitoisuus on riittävän korkea. Molempia optimoituja menetelmiä voidaan käyttää myös muiden proteiinien tuottamiseen ja leimaamiseen, mikä edesauttaa proteiinien rakennetutkimuksia NMR-spektroskopian avulla

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

HP0593 DNA-(N6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5′-GCAG-3′and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori

Flavodoxins as novel therapeutic targets against helicobacter pylori and other gastric pathogens

Flavodoxins are small soluble electron transfer proteins widely present in bacteria and absent in vertebrates. Flavodoxins participate in different metabolic pathways and, in some bacteria, they have been shown to be essential proteins representing promising therapeutic targets to fight bacterial infections. Using purified flavodoxin and chemical libraries, leads can be identified that block flavodoxin function and act as bactericidal molecules, as it has been demonstrated for Helicobacter pylori (Hp), the most prevalent human gastric pathogen. Increasing antimicrobial resistance by this bacterium has led current therapies to lose effectiveness, so alternative treatments are urgently required. Here, we summarize, with a focus on flavodoxin, opportunities for pharmacological intervention offered by the potential protein targets described for this bacterium and provide information on other gastrointestinal pathogens and also on bacteria from the gut microbiota that contain flavodoxin. The process of discovery and development of novel antimicrobials specific for Hp flavodoxin that is being carried out in our group is explained, as it can be extrapolated to the discovery of inhibitors specific for other gastric pathogens. The high specificity for Hp of the antimicrobials developed may be of help to reduce damage to the gut microbiota and to slow down the development of resistant Hp mutants

Expression of Genes for a Flavin Adenine Dinucleotide-Binding Oxidoreductase and a Methyltransferase from Mycobacterium chlorophenolicum Is Necessary for Biosynthesis of 10-Methyl Stearic Acid from Oleic Acid in Escherichia coli

In living organisms, modified fatty acids are crucial for the functions of the cellular membranes and storage lipids where the fatty acids are esterified. Some bacteria produce a typical methyl-branched fatty acid, i.e., 10-methyl stearic acid (19:0Me10). The biosynthetic pathway of 19:0Me10 in vivo has not been demonstrated clearly yet. It had been speculated that 19:0Me10 is synthesized from oleic acid (18:1Δ9) by S-adenosyl-L-methionine-dependent methyltransfer and NADPH-dependent reduction via a methylenated intermediate, 10-methyelene octadecanoic acid. Although the recombinant methyltransferases UmaA and UfaA1 from Mycobacterium tuberculosis H37Rv synthesize 19:0Me10 from 18:1Δ9 and NADPH in vitro, these methyltransferases do not possess any domains functioning in the redox reaction. These findings may contradict the two-step biosynthetic pathway. We focused on novel S-adenosyl-L-methionine-dependent methyltransferases from Mycobacterium chlorophenolicum that are involved in 19:0Me10 synthesis and selected two candidate proteins, WP_048471942 and WP_048472121, by a comparative genomic analysis. However, the heterologous expression of these candidate genes in Escherichia coli cells did not produce 19:0Me10. We found that one of the candidate genes, WP_048472121, was collocated with another gene, WP_048472120, that encodes a protein containing a domain associated with flavin adenine dinucleotide-binding oxidoreductase activity. The co-expression of these proteins (hereafter called BfaA and BfaB, respectively) led to the biosynthesis of 19:0Me10 in E. coli cells via the methylenated intermediate

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylor

Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5′-GTAC and leave a 3′-TA overhang (5′-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure