Carlos Medina

NolR Regulates Diverse Symbiotic Signals of Sinorhizobium fredii HH103

We have investigated in Sinorhizobium fredii HH103-1 (=HH103 Str(r)) the influence of the nolR gene on the production of three different bacterial symbiotic signals: Nod factors, signal responsive (SR) proteins, and exopolysaccharide (EPS). The presence of multiple copies of nolR (in plasmid pMUS675) repressed the transcription of all the flavonoid-inducible genes analyzed: nodA, nodD1, nolO, nolX, noeL, rhcJ, hesB, and y4pF. Inactivation of nolR (mutant SVQ517) or its overexpression (presence of pMUS675) altered the amount of Nod factors detected. Mutant SVQ517 produced Nod factors carrying N-methyl residues at the nonreducing N-acetyl-glucosamine, which never have been detected in S. fredii HH103. Plasmid pMUS675 increased the amounts of EPS produced by HH103-1 and SVQ517. The flavonoid genistein repressed EPS production of HH103-1 and SVQ517 but the presence of pMUS675 reduced this repression. The presence of plasmid pMUS675 clearly decreased the secretion of SR proteins. Inactivation, or overexpression, of nolR decreased the capacity of HH103 to nodulate Glycine max. However, HH103-1 and SVQ517 carrying plasmid pMUS675 showed enhanced nodulation capacity with Vigna unguiculata. The nolR gene was positively identified in all S. fredii strains investigated, S. xinjiangense CCBAU110, and S. saheli USDA4102. Apparently, S. teranga USDA4101 does not contain this gene.

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

Identification and Characterization of a nodH Ortholog from the Alfalfa-Nodulating Or191-Like Rhizobia

Nodulation of Medicago sativa (alfalfa) is known to be restricted to Sinorhizobium meliloti and a few other rhizobia that include the poorly characterized isolates related to Rhizobium sp. strain Or191. Distinctive features of the symbiosis between alfalfa and S. meliloti are the marked specificity from the plant to the bacteria and the strict requirement for the presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its reducing end. Here, we present evidence of the presence of a functional nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene, present in single copy, maps to a high molecular weight megaplasmid. As in S. meliloti, a nodF homolog was identified immediately upstream of nodH that was transcribed in the opposite direction (local synteny). This novel nodH ortholog was cloned and shown to restore both NF sulfation and the Nif+Fix+ phenotypes when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH disruption in the Or191-like bacteria did not abolish their ability to nodulate alfalfa, resulting instead in a severely delayed nodulation. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor.

Novel tools to analyze the function of Salmonella effectors show that SvpB ectopic expression induces cell cycle arrest in tumor cells.

In order to further characterize its role in pathogenesis and to establish whether its overproduction can lead to eukaryotic tumor cell death, Salmonella strains able to express its virulence factor SpvB (an ADP-ribosyl transferase enzyme) in a salicylate-inducible way have been constructed and analyzed in different eukaryotic tumor cell lines. To do so, the bacterial strains bearing the expression system have been constructed in a ∆purD background, which allows control of bacterial proliferation inside the eukaryotic cell. In the absence of bacterial proliferation, salicylate-induced SpvB production resulted in activation of caspases 3 and 7 and apoptotic cell death. The results clearly indicated that controlled SpvB production leads to F-actin depolimerization and either G1/S or G2/M phase arrest in all cell lines tested, thus shedding light on the function of SpvB in Salmonella pathogenesis. In the first place, the combined control of protein production by salicylate regulated vectors and bacterial growth by adenine concentration offers the possibility to study the role of Salmonella effectors during eukaryotic cells infection. In the second place, the salicylate-controlled expression of SpvB by the bacterium provides a way to evaluate the potential of other homologous or heterologous proteins as antitumor agents, and, eventually to construct novel potential tools for cancer therapy, given that Salmonella preferentially proliferates in tumors.

Engineering Salmonella as intracellular factory for effective killing of tumour cells

Salmonella have many desirable properties as antitumour-agent due to its ability to proliferate inside tumours and induce tumour regression. Additionally, this bacterium can be genetically engineered to deliver therapeutic proteins intratumourally. The main limitation of this approach is the efficient release of therapeutic molecules from intratumoural bacteria. Here we have developed an inducible autolysis system based in the lysis operon of the lambda phage that, in response to anhydrotetracycline, lysates Salmonella thus releasing its content. The system was combined with a salicylate cascade system that allows efficient production of therapeutic molecules in response to aspirin and with a sifA mutation that liberates bacteria from the vacuoles to a cytosolic location. The combination of these three elements makes this strain a putative powerful instrument in cancer treatment. We have used this engineered strain for the intracellular production and delivery of Cp53 peptide. The engineered strain is able to sequentially produce and release the cytotoxic peptide while proliferating inside tumour cells, thus inducing host cell death. Our results show that temporal separation of protein production from protein release is essential to efficiently kill tumour cells. The combined system is a further step in the engineering of more efficient bacteria for cancer therapy.

NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103.

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103.

Zebrafish as a model organism to study host-pathogen interactions.

Zebrafish have been extensively used in biomedical research as a model to study vertebrate development but it is only recently that it has also been adopted into varied fields such as immunology and host-pathogen interactions. Zebrafish have a rapid life cycle, small size and the adults exhibit no territorial behavior in relatively dense cages. Under standard conditions each female lays an average of a hundred eggs per clutch, providing a large number of larvae per week. Their transparency during early life stages allows real time visualization of the different organs, which makes them especially suitable for the study of bacterial host-pathogen interactions. Traditionally, these studies have been technically challenging in higher organisms, given the loss of control over the bacteria once the pathogen infects its host. Here we describe an emerging approach to monitor Salmonella typhimurium infection progression using in vivo fluorescence upon parenteral infection. We have engineered Salmonella with the Cascade expression system; an efficient method to voluntarily activate bacterial heterologous gene expression at any point during infection once inside the Zebrafish macrophages, using a non-toxic inducer.

The Sinorhizobium (Ensifer) fredii HH103 Nodulation Outer Protein NopI Is a Determinant for Efficient Nodulation of Soybean and Cowpea Plants

ABSTRACT The type III secretion system (T3SS) is a specialized secretion apparatus that is commonly used by many plant and animal pathogenic bacteria to deliver proteins, termed effectors, to the interior of the host cells. These effectors suppress host defenses and interfere with signal transduction pathways to promote infection. Some rhizobial strains possess a functional T3SS, which is involved in the suppression of host defense responses, host range determination, and symbiotic efficiency. The analysis of the genome of the broad-host-range rhizobial strain Sinorhizobium fredii HH103 identified eight genes that code for putative T3SS effectors. Three of these effectors, NopL, NopP, and NopI, are Rhizobium specific. In this work, we demonstrate that NopI, whose amino acid sequence shows a certain similarity with NopP, is secreted through the S. fredii HH103 T3SS in response to flavonoids. We also determined that NopL can be considered an effector since it is directly secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, the symbiotic phenotype of single, double, and triple nopI, nopL, and nopP mutants in soybean and cowpea was assayed, showing that NopI plays an important role in determining the number of nodules formed in both legumes and that the absence of both NopL and NopP is highly detrimental for symbiosis. IMPORTANCE The paper is focused on three Rhizobium-specific T3SS effectors of Sinorhizobium fredii HH103, NopL, NopP, and NopI. We demonstrate that S. fredii HH103 is able to secrete through the T3SS in response to flavonoids the nodulation outer protein NopI. Additionally, we determined that NopL can be considered an effector since it is secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, nodulation assays of soybean and cowpea indicated that NopI is important for the determination of the number of nodules formed and that the absence of both NopL and NopP negatively affected nodulation.

Improved cytotoxic effects of Salmonella-producing cytosine deaminase in tumour cells

In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate‐inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coliu2005codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5‐fluorocytosine, a 5‐fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures.