Cell Host-Microbe Interactions: Turning Pathogen Mechanisms Into Cell's Advantages

Host-Pathogen Interaction is a very vast field of biological sciences, indeed every year many un- known pathogens are uncovered leading to an exponential growth of this field. The present work lyes between its boundaries, touching different aspects of host-pathogen interaction: We have evaluate the permissiveness of Mesenchimal Stem cell (FM-MSC from now on) to all known human affecting herpesvirus. Our study demonstrate that FM-MSC are full permissive to HSV1, HSV2, HCMV and VZV. On the other hand HHV6, HHV7, EBV and HHV8 are susceptible, but failed to activate a lytic infection program. FM-MSC are pluripotent stem cell and have been studied intensely in last decade. FM-MSC are employed in some clinical applications. For this reason it is important to known the degree of susceptibility to transmittable pathogens. Our atten- tion has then moved to bacterial pathogens: we have performed a proteome-wide in silico analy- sis of Chlamydiaceae family, searching for putative Nuclear localization Signal (NLS). Chlamy- diaceae are a family of obligate intracellular parasites. It’s reasonably to think that its members could delivered to nucleus effector proteins via NLS sequences: if that were the case the identifi- cation of NLS carrying proteins could open the way to therapeutic approaches. Our results strengthen this hypothesis: we have identified 72 protein bearing NLS, and verified their func- tionality with in vivo assays. Finally we have conceived a molecular scissor, creating a fusion protein between HIV-1 IN protein and FokI catalytic domain (a deoxyexonuclease domain). Our aim is to obtain chimeric enzyme (trojIN) which selectively identify IN naturally occurring target (HIV LTR sites) and cleaves subsequently LTR carrying DNA (for example integrated HIV1 DNA). Our preliminary results are promising since we have identified trojIN mutated version capable to selectively recognize LTR carrying DNA in an in vitro experiments.

Drosophila melanogaster as a model to study host-parasitoid interactions: the case of the polydnaviral protein TnBVANK1

Parasitic wasps attack a number of insect species on which they feed, either externally or internally. This requires very effective strategies for suppressing the immune response and a finely tuned interference with the host physiology that is co-opted for the developing parasitoid progeny. The wealth of physiological host alterations is mediated by virulence factors encoded by the wasp or, in some cases, by polydnaviruses (PDVs), unique viral symbionts injected into the host at oviposition along with the egg, venom and ovarian secretions. PDVs are among the most powerful immunosuppressors in nature, targeting insect defense barriers at different levels. During my PhD research program I have used Drosophila melanogaster as a model to expand the functional analysis of virulence factors encoded by PDV focusing on the molecular processes underlying the disruption of the host endocrine system. I focused my research on a member of the ankyrin (ank) gene family, an immunosuppressant found in bracovirus, which associates with the parasitic wasp Toxoneuron nigriceps. I found that ankyrin disrupts ecdysone biosynthesis by impairing the vesicular traffic of ecdysteroid precursors in the cells of the prothoracic gland and results in developmental arrest.

Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.

Changes in plant metabolism induced by dioxygenase inhibitors and their effect on the epiphytic microbial community and fire blight (Erwinia amylovora) control

Fire blight, caused by the gram negative bacterium Erwinia amylovora, is one of the most destructive bacterial diseases of Pomaceous plants. Therefore, the development of reliable methods to control this disease is desperately needed. This research investigated the possibility to interfere, by altering plant metabolism, on the interactions occurring between Erwinia amylovora, the host plant and the epiphytic microbial community in order to obtain a more effective control of fire blight. Prohexadione-calcium and trinexapac-ethyl, two dioxygenase inhibitors, were chosen as a chemical tool to influence plant metabolism. These compounds inhibit the 2-oxoglutarate-dependent dioxygenases and, therefore, they greatly influence plant metabolism. Moreover, dioxygenase inhibitors were found to enhance plant resistance to a wide range of pathogens. In particular, dioxygenase inhibitors application seems a promising method to control fire blight. From cited literature, it is assumed that these compounds increase plant defence mainly by a transient alteration of flavonoids metabolism. We tried to demonstrate, that the reduction of susceptibility to disease could be partially due to an indirect influence on the microbial community established on plant surface. The possibility to influence the interactions occurring in the epiphytic microbial community is particularly interesting, in fact, the relationships among different bacterial populations on plant surface is a key factor for a more effective biological control of plant diseases. Furthermore, we evaluated the possibility to combine the application of dioxygenase inhibitors with biological control in order to develop an integrate strategy for control of fire blight. The first step for this study was the isolation of a pathogenic strain of E. amylovora. In addition, we isolated different epiphytic bacteria, which respond to general requirements for biological control agents. Successively, the effect of dioxygenase inhibitors treatment on microbial community was investigated on different plant organs (stigmas, nectaries and leaves). An increase in epiphytic microbial population was found. Further experiments were performed with aim to explain this effect. In particular, changes in sugar content of nectar were observed. These changes, decreasing the osmotic potential of nectar, might allow a more consistent growth of epiphytic bacteria on blossoms. On leaves were found similar differences as well. As far as the interactions between E. amylovora and host plant, they were deeply investigated by advanced microscopical analysis. The influence of dioxygenase inhibitors and SAR inducers application on the infection process and migration of pathogen inside different plant tissues was studied. These microscopical techniques, combined with the use of gpf-labelled E. amylovora, allowed the development of a bioassay method for resistance inducers efficacy screening. The final part of the work demonstrated that the reduction of disease susceptibility observed in plants treated with prohexadione-calcium is mainly due to the accumulation of a novel phytoalexins: luteoforol. This 3-deoxyflavonoid was proven to have a strong antimicrobial activity.

Computational methods for the analysis of protein structure and function

The vast majority of known proteins have not yet been experimentally characterized and little is known about their function. The design and implementation of computational tools can provide insight into the function of proteins based on their sequence, their structure, their evolutionary history and their association with other proteins. Knowledge of the three-dimensional (3D) structure of a protein can lead to a deep understanding of its mode of action and interaction, but currently the structures of <1% of sequences have been experimentally solved. For this reason, it became urgent to develop new methods that are able to computationally extract relevant information from protein sequence and structure. The starting point of my work has been the study of the properties of contacts between protein residues, since they constrain protein folding and characterize different protein structures. Prediction of residue contacts in proteins is an interesting problem whose solution may be useful in protein folding recognition and de novo design. The prediction of these contacts requires the study of the protein inter-residue distances related to the specific type of amino acid pair that are encoded in the so-called contact map. An interesting new way of analyzing those structures came out when network studies were introduced, with pivotal papers demonstrating that protein contact networks also exhibit small-world behavior. In order to highlight constraints for the prediction of protein contact maps and for applications in the field of protein structure prediction and/or reconstruction from experimentally determined contact maps, I studied to which extent the characteristic path length and clustering coefficient of the protein contacts network are values that reveal characteristic features of protein contact maps. Provided that residue contacts are known for a protein sequence, the major features of its 3D structure could be deduced by combining this knowledge with correctly predicted motifs of secondary structure. In the second part of my work I focused on a particular protein structural motif, the coiled-coil, known to mediate a variety of fundamental biological interactions. Coiled-coils are found in a variety of structural forms and in a wide range of proteins including, for example, small units such as leucine zippers that drive the dimerization of many transcription factors or more complex structures such as the family of viral proteins responsible for virus-host membrane fusion. The coiled-coil structural motif is estimated to account for 5-10% of the protein sequences in the various genomes. Given their biological importance, in my work I introduced a Hidden Markov Model (HMM) that exploits the evolutionary information derived from multiple sequence alignments, to predict coiled-coil regions and to discriminate coiled-coil sequences. The results indicate that the new HMM outperforms all the existing programs and can be adopted for the coiled-coil prediction and for large-scale genome annotation. Genome annotation is a key issue in modern computational biology, being the starting point towards the understanding of the complex processes involved in biological networks. The rapid growth in the number of protein sequences and structures available poses new fundamental problems that still deserve an interpretation. Nevertheless, these data are at the basis of the design of new strategies for tackling problems such as the prediction of protein structure and function. Experimental determination of the functions of all these proteins would be a hugely time-consuming and costly task and, in most instances, has not been carried out. As an example, currently, approximately only 20% of annotated proteins in the Homo sapiens genome have been experimentally characterized. A commonly adopted procedure for annotating protein sequences relies on the "inheritance through homology" based on the notion that similar sequences share similar functions and structures. This procedure consists in the assignment of sequences to a specific group of functionally related sequences which had been grouped through clustering techniques. The clustering procedure is based on suitable similarity rules, since predicting protein structure and function from sequence largely depends on the value of sequence identity. However, additional levels of complexity are due to multi-domain proteins, to proteins that share common domains but that do not necessarily share the same function, to the finding that different combinations of shared domains can lead to different biological roles. In the last part of this study I developed and validate a system that contributes to sequence annotation by taking advantage of a validated transfer through inheritance procedure of the molecular functions and of the structural templates. After a cross-genome comparison with the BLAST program, clusters were built on the basis of two stringent constraints on sequence identity and coverage of the alignment. The adopted measure explicity answers to the problem of multi-domain proteins annotation and allows a fine grain division of the whole set of proteomes used, that ensures cluster homogeneity in terms of sequence length. A high level of coverage of structure templates on the length of protein sequences within clusters ensures that multi-domain proteins when present can be templates for sequences of similar length. This annotation procedure includes the possibility of reliably transferring statistically validated functions and structures to sequences considering information available in the present data bases of molecular functions and structures.

Produzione di virus sintetici per lo studio dei meccanismi di interazione coinvolti nell'induzione di resistenza

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) are members of Benyvirus genus. BSBMV has been reported only in the United States while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae, possess similar host ranges, particles number and morphology. Both viruses are not serologically related but have similar genomic organizations. Field isolates consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles on plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed BSBMV and BNYVV are different enough to be classified in two different species. Additionally in BNYVV/BSBMV mixed infections, a competition was previous described in sugar beet, where BNYVV infection reduces BSBMV accumulation in both susceptible and resistant cultivars. Considering all this observations we hypothesized that BNYVV and BSBMV crossed study, exploiting their similarities and divergences, can improve investigation of molecular interactions between sugar beets and Benyviruses. The main achievement of our research is the production of a cDNA biologically active clones collection of BNYVV and BSBMV RNAs, from which synthetic copies of both Benyviruses can be transcribed. Moreover, through recombination experiments we demonstrated, for the first time, the BNYVV RNA 1 and 2 capability to trans-replicate and encapsidate BSBMV RNA 3 and 4, either the BSBMV RNA 1 and 2 capability to replicate BNYVV RNA2 in planta. We also demonstrated that BSBMV RNA3 support long-distance movement of BNYVV RNA 1 and 2 in B. macrocarpa and that 85 foreign sequence as p29HA, GFP and RFP, are successfully expressed, in C. quinoa, by BSBMV RNA3 based replicon (RepIII) also produced by our research. These results confirm the close correlation among the two viruses. Interestingly, the symptoms induced by BSBMV RNA-3 on C. quinoa leaves are more similar to necrotic local lesions caused by BNYVV RNA-5 p26 than to strongly chlorotic local lesions or yellow spot induced by BNYVV RNA- 3 encoded p25. As previous reported BSBMV p29 share 23% of amino acid sequence identity with BNYVV p25 but identity increase to 43% when compared with sequence of BNYVV RNA-5 p26. Based on our results the essential sequence (Core region) for the longdistance movement of BSBMV and BNYVV in B. macrocarpa, is not only carried by RNA3s species but other regions, perhaps located on the RNA 1 and 2, could play a fundamental role in this matter. Finally a chimeric RNA, composed by the 5’ region of RNA4 and 3’ region of RNA3 of BSBMV, has been produced after 21 serial mechanically inoculation of wild type BSBMV on C. quinoa plants. Chimera seems unable to express any protein, but it is replicated and transcript in planta. It could represent an important tool to study the interactions between Benyvirus and plant host. In conclusion different tools, comprising a method to study synthetic viruses under natural conditions of inoculum through P. Betae, have been produced and new knowledge are been acquired that will allow to perform future investigation of the molecular interactions between sugar beets and Benyviruses.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

Virus a RNA e a DNA: importanza dell’ampliamento dello spettro d’ospite nell’evoluzione virale

L’ampliamento dello spettro d’ospite è strettamente connesso al processo evolutivo a cui i virus sono assoggettati e rappresenta una notevole sfida alla loro capacità di adattarsi. L’attitudine a superare le barriere di specie è conseguente alla costante e relativamente rapida evoluzione che caratterizza i virus; allo stesso tempo, la forza selettiva esercitata dal nuovo ospite rappresenterà un ulteriore stimolo per le capacità adattative del virus. Ad oggi, i meccanismi genetici ed evolutivi responsabili del salto di specie virale, cioè la trasmissione di un virus da un ospite tradizionale ad uno precedentemente resistente all’infezione, sono parzialmente sconosciuti. Nel seguente lavoro verranno presentati gli studi effettuati sulle dinamiche evolutive caratterizzanti virus a RNA e a DNA in cui si sono osservate variazioni dello spettro d’ospite. Gli studi hanno riguardato i coronavirus, con particolare riferimento al ruolo svolto dai pipistrelli nell’evoluzione dei coronavirus SARS-correlati, e l’importanza del gatto nell’evoluzione dei parvovirus dei carnivori. Nella prima sezione saranno mostrate le correlazioni genetiche dei coronavirus identificati in Italia nei pipistrelli appartenenti alla specie Rhinolophus ferrumequinum con i ceppi europei e del resto del mondo, allo scopo di chiarire l’origine evolutiva dei coronavirus dei pipistrelli correlati al virus della SARS (Bat-SARS-like CoV) europei, gli eventi migratori che hanno caratterizzato la loro diffusione nel continente e le potenziali ripercussioni sulla salute pubblica. Nella seconda sezione saranno evidenziate le caratteristiche molecolari dei ceppi di parvovirus circolanti nella popolazione felina, valutandone la diversità di sequenza e la complessità genetica, allo scopo di ottenere importanti informazioni in merito all’evoluzione del virus e alle interazioni tra il parvovirus e l’ospite.