Francisco Pablo Chavez Espinosa | Universidad de Chile (original) (raw)

Papers by Francisco Pablo Chavez Espinosa

The outcome of a host-pathogen interaction is determined by the conditions of the host, the patho... more The outcome of a host-pathogen interaction is determined by the conditions of the host, the pathogen, and the environment. Although numerous proteomic studies of in vitro-grown microbial pathogens have been performed, in vivo proteomic approaches are still rare. In addition, increasing evidence supports that in vitro studies inadequately reflect in vivo conditions. Choosing the proper host is essential to detect the expression of proteins from the pathogen in vivo. Numerous studies have demonstrated the suitability of zebrafish (Danio rerio) embryos as a model to in vivo studies of Pseudomonas aeruginosa infection. In most zebrafish-pathogen studies, infection is achieved by microinjection of bacteria into the larvae. However, few reports using static immersion of bacterial pathogens have been published. In this study we infected 3 days post-fertilization (DPF) zebrafish larvae with P. aeruginosa PAO1 by immersion and injection and tracked the in vivo immune response by the zebrafish. Additionally, by using non-isotopic (Q-exactive) metaproteomics we simultaneously evaluated the proteomic response of the pathogen (P. aeruginosa PAO1) and the host (zebrafish). We found some zebrafish metabolic pathways, such as hypoxia response via HIF activation pathway, were exclusively enriched in the larvae exposed by static immersion. In contrast, we found that inflammation mediated by chemokine and cytokine signaling pathways was exclusively enriched in the larvae exposed by injection, while the integrin signaling pathway and angiogenesis were solely enriched in the larvae exposed by immersion. We also found important virulence factors from P. aeruginosa that were enriched only after exposure by injection, such as the Type-III secretion system and flagella-associated proteins. On the other hand, P. aeruginosa proteins involved in processes like biofilm formation, and cellular responses to antibiotic and starvation were enriched exclusively after exposure by immersion. We demonstrated the suitability of zebrafish embryos as a model for in vivo host-pathogen based proteomic studies in P. aeruginosa. Our global proteomic profiling identifies novel molecular signatures that give systematic insight into zebrafish-Pseudomonas interaction.

The dynamic response of organisms exposed to environmental pathogens determines their survival or... more The dynamic response of organisms exposed to environmental pathogens determines their survival or demise, and the outcome of this interaction depends on the host's susceptibility and pathogen-dependent virulence factors. The transmission of acquired information about the nature of a pathogen to progeny may ensure effective defensive strategies for the progeny's survival in adverse environments. Environmental RNA interference (RNAi) is a systemic and heritable mechanism and has recently been linked to antibacterial and antifungal defenses in both plants and animals. Here, we report that the second generation of Caenorhabditis el-egans living on pathogenic bacteria can avoid bacterial infection by entering dia-pause in an RNAi pathway-dependent mechanism. Furthermore, we demonstrate that the information encoding this survival strategy is transgenerationally transmitted to the progeny via the maternal germ line. IMPORTANCE Bacteria vastly influence physiology and behavior, and yet, the specific mechanisms by which they cause behavioral changes in hosts are not known. We use C. elegans as a host and the bacteria they eat to understand how microbes trigger a behavioral change that helps animals to survive. We found that animals faced with an infection for two generations could enter a hibernationlike state, arresting development by forming dauer larvae. Dauers have closed mouths and effectively avoid infection. Animals accumulate information that is transgenerationally transmitted to the next generations to form dauers. This work gives insight on how bacteria communicate in noncanonical ways with their hosts, resulting in long-lasting effects providing survival strategies to the community.

A periplasmic invertase from the yeast Candida utilis was purified to homogeneity from cells full... more A periplasmic invertase from the yeast Candida utilis was purified to homogeneity from cells fully derepressed for invertase synthesis. The enzyme was purified by successive Sephacryl S-300, and affinity chromatography and shown to be a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa. After EndoH treatment, the deglycosylated protein showed an apparent molecular weight of 60 kDa. The apparent Km values for sucrose and raffinose were 11 and 150 mM, respectively, similar to those reported in Saccharomyces cerevisiae. The range of optimum temperature was 60–75°C. The optimum pH was 5.5 and the enzyme was stable over pH range 3–6.

We report here the development of an auxotrophic transformation system for the food yeast Candida... more We report here the development of an auxotrophic transformation system for the food yeast Candida utilis. To facilitate molecular studies in Candida utilis, we isolated auxotrophic strains for uracil biosynthesis by the combination of NTGmutagenesis and 5-fluorotic acid (FOA) selection. The ura 3 mutation could be functionally complemented by the homologous URA3 gene. We used both, LiAc and electroporation methods to direct insertions at the ura3 locus through homologous recombination. z

The Mig1p repressor from the food yeast Candida utilis has been isolated using a homologous PCR h... more The Mig1p repressor from the food yeast Candida utilis has been isolated using a homologous PCR hybridization probe. This probe was amplified with two sets of degenerate primers designed on the basis of highly conserved motifs in the DNA-binding region (zinc-finger domain) from yeast Mig1p and fungi CreA repressors. The cloned gene was sequenced and found to encode a polypeptide of 345 amino acids which shows significant identity with other yeast and fungus repressors in the DNA-binding domain and also with the yeast Mig1 proteins in the C-terminal region (effector domain). The MIG1 repressor gene from C. utilis was able to complement functionally the mig1 mutation of S. cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ277830.

A DNA fragment, carrying the Candida utilis HIS3 gene, has been isolated from a genomic DNA libra... more A DNA fragment, carrying the Candida utilis HIS3 gene, has been isolated from a genomic DNA library by complementation of the E. coli hisB mutant. Its nucleotide sequence was determined and it predicts a single open reading frame of 675 bp (224 aa). The deduced amino acid sequence is highly homologous to other yeast and fungi HIS3 genes.

Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Althou... more Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Although the presence of polyP in members of the domain Archaea has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain. We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (ppx) from thermophilic Sulfolobus solfataricus. The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa). The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60°C. The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs. In contrast, informatic analysis revealed that the deduced amino acid sequence of S. solfataricus PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs. To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the Archaea.

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance ... more Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors. Polyphosphate (polyP) is a ubiquitous linear polymer consisting of hundreds of orthophosphate residues (P i) linked by high-energy phosphoanhydride bonds. The best-known enzymes involved in the metabolism of polyP in bacteria are the polyphosphate kinase (PPK) that catalyzes the reversible conversion of the terminal phosphate of ATP into polyP and the exopolyphosphatase that processively hydrolyzes the terminal residues of polyP to liberate P i (15). The involvement of polyP in the regulation of both enzyme activities and expression of large group of genes is the basis of survival for different bacteria, including pathogens, under stress conditions and of adaptation to the stationary growth phase (reviewed in reference 16). Mutant bacterial cells that lack polyP survive poorly during growth in the stationary phase and are less resistant to heat, oxidants, osmotic challenge, antibiotics, and UV radiation (6, 13, 24, 25, 35). polyP accumulation in response to nutrient deprivation has also been reported in the genus Pseudomonas, and recent studies have demonstrated that PPK is essential in Pseudomonas aeruginosa not only for various forms of motility (26, 27) but also for biofilm development, quorum sensing, production of virulence factors, and virulence in the burned-mouse patho-genesis model (28). Chlorinated biphenyls (CBs) and polychlorinated biphenyls (PCBs) belong to one of the most widely distributed classes of chlorinated chemicals in the environment (33, 34). The toxic-ities and carcinogenicities of some PCB congeners make them a serious environmental and health problem (14). For cleanup of large areas of PCB-contaminated soils and aquatic environments bioremediation seems to be a promising approach (22). Although many genetic, enzymological, and biochemical analyses of PCB-degradative pathways have provided the basis for the engineering of specific enzymes and genetically modified microorganisms in order to improve performance in bioreme-diation of PCBs, little is known about the physiological adjustments of PCB-degrading bacteria during growth with these kinds of organochlorine compounds. Here we demonstrate that the PCB-degrading bacterium Pseudomonas sp. strain B4 accumulates much higher levels of polyP during exponential growth with biphenyl than when glucose is the sole carbon source. Following a shift from a defined medium with glucose to a medium with biphenyl or CBs as the single carbon source, numerous polyP granules accumulated in the cytoplasm. Additionally, induction of the general stress protein GroEl and oxygen reactive species (ROS) was observed , probably as a physiological adjustment to growth in the presence of these contaminating compounds, which appear to stress the cells.

Polychlorinated biphenyls (PCBs) are one of the most widely distributed classes of chlorinated ch... more Polychlorinated biphenyls (PCBs) are one of the most widely distributed classes of chlorinated chemicals in the environment. For cleanup of large areas of PCB-contaminated environments, bioremediation seems to be a promising approach. However, the multitude of PCB congeners, their low bioavailability and high toxicity are important factors that affect the cleanup progression. Elucidating how the PCB-degrading microorganisms involved in the process adapt to and deal with the stressing conditions caused by this class of compounds may help to improve the bioremediation process. Also specific physiological characteristics of biphenyl-utilizing bacteria involved in the degradation of PCBs may enhance their availability to these compounds and therefore contribute to a better microbial mineralization. This review will focus in the stress responses caused in aerobic biphenyl-utilizing bacteria by PCBs and its metabolic intermediates and will also analyze bacterial properties such as motility and chemotaxis, adherence to solid surfaces, biosurfactant production and biofilm development, all properties found to enhance bacteria–pollutant interaction.

Titular: CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA ,domiciliado en Ave. 31 entre 158 y 19... more 73) Titular: CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA ,domiciliado en Ave. 31 entre 158 y 190, Cubanacán, Playa.; (CU)

The polychlorinated biphenyl (PCB)-degrading Pseudomonas sp. B4 was tested for its motility and a... more The polychlorinated biphenyl (PCB)-degrading Pseudomonas sp. B4 was tested for its motility and ability to sense and respond to biphenyl, its chloroderivatives and chlorobenzoates in chemotaxis assays. Pseudomonas sp. B4 was attracted to biphenyl, PCBs and benzoate in swarm plate and capillary assays. Chemotaxis towards these compounds correlated with their use as carbon and energy sources. No chemotactic effect was observed in the presence of 2-and 3-chlorobenzoates. Furthermore, a toxic effect was observed when the microorganism was exposed to 3-chlorobenzoate. A nonmotile Pseudomonas sp. B4 transformant and Burkholder-ia xenovorans LB400, the laboratory model strain for PCB degradation, were both capable of growing in biphenyl as the sole carbon source, but showed a clear disadvantage to access the pollutants to be degraded, compared with the highly motile Pseudomonas sp. B4, stressing the importance of motility and chemotaxis in this environmental biodegradation.

Background: Inorganic polyphosphate (polyP), a polymer of tens or hundreds of phosphate residues ... more Background: Inorganic polyphosphate (polyP), a polymer of tens or hundreds of phosphate residues linked by ATP-like bonds, is found in all organisms and performs a wide variety of functions. PolyP is synthesized in bacterial cells by the actions of polyphosphate kinases (PPK1 and PPK2) and degraded by exopolyphosphatase (PPX). Bacterial cells with polyP deficiencies due to knocking out the ppk1 gene are affected in many structural and important cellular functions such as motility, quorum sensing, biofilm formation and virulence among others. The cause of this pleiotropy is not entirely understood. Results: The overexpression of exopolyphosphatase in bacteria mimicked some pleitropic defects found in ppk1 mutants. By using this approach we found new structural and functional defects in the polyP-accumulating bacteria Pseudomonas sp. B4, which are most likely due to differences in the polyP-removal strategy. Colony morphology phenotype, lipopolysaccharide (LPS) structure changes and cellular division malfunction were observed. Finally, we used comparative proteomics in order to elucidate the cellular adjustments that occurred during polyP deficiency in this bacterium and found some clues that helped to understand the structural and functional defects observed. Conclusions: The results obtained suggest that during polyP deficiency energy metabolism and particularly nucleoside triphosphate (NTP) formation were affected and that bacterial cells overcame this problem by increasing the flux of energy-generating metabolic pathways such as tricarboxilic acid (TCA) cycle, b-oxidation and oxidative phosphorylation and by reducing energy-consuming ones such as active transporters and amino acid biosynthesis. Furthermore, our results suggest that a general stress response also took place in the cell during polyP deficiency.

Background: Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. P... more Background: Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence. Findings: By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin. Conclusions: Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.

Inorganic polyphosphate (polyP) is a biopolymer of tens or hundreds of phosphate (Pi) residues li... more Inorganic polyphosphate (polyP) is a biopolymer of tens or hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. PolyP has been studied mainly in prokaryotes but it is present in all species of the three domains of life. In bacteria, polyP and its processing enzymes play important roles in cellular metabolism as well as in pathogenesis. The genomes of many bacterial species, including pathogens, encode orthologs of the main polyP-synthesizing enzyme, PPK1. This enzyme has been studied in E. coli and its metabolic inhibitors have been reported. The high degree of identity between the PPK1 orthologs in some of the major pathogenic species has prompted the knockout of their ppk1 genes to determine the dependence of virulence on polyP. Although viable, mutants lacking the ppk1 gene have reduced levels of polyP and exhibit multiple structural, functional and virulence defects. The emergence of multi-drug resistant (MDR) bacteria is the result of antibiotic overuse. Therefore, novel approaches are much needed to tackle them. One of these combines the reduction of bacterial virulence while simultaneously increasing susceptibility to host defenses instead of killing the pathogen. Considering that no PPK1 orthologs have been identi ed in higher-order eukaryotes, PPK1 exhibits an enormous potential as a novel target for antimicrobial drug design. In this review we focus on the current state of the art regarding polyP deficiency in pathogenic bacteria and attempts to design inhibitors targeting enzymes responsible for the synthesis of polyP in bacteria.

The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant ... more The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In this work, D. discoideum was used as a model to study the ability of Salmonella Typhimurium to survive in amoebae and to evaluate the contribution of selected genes in this process. To do this, we performed infection assays using axenic cultures of D. discoideum co-cultured with wild-type S. Typhimurium and/or defined mutant strains. Our results confirmed that wild-type S. Typhimurium is able to survive intracellularly in D. discoideum. In contrast, mutants aroA and waaL are defective in intracellular survival in this amoeba. Next, we included in our study a group of mutants in genes directly linked to Salmonella virulence. Of note, mutants invA, ssaD, clpV, and phoPQ also showed an impaired ability to survive intracellularly in D. discoideum. This indicates that S. Typhimurium requires a functional biosynthetic pathway of aromatic compounds, a lipopolysaccharide containing a complete O-antigen, the type III secretion systems (T3SS) encoded in SPI- 1 and SPI-2, the type VI secretion system (T6SS) encoded in SPI-6 and PhoP/PhoQ two-component system to survive in D. discoideum. To our knowledge, this is the first report on the requirement of O-antigen and T6SS in the survival of Salmonella within amoebae. In addition, mutants invA and ssaD were internalized in higher numbers than the wild-type strain during competitive infections, suggesting that S. Typhimurium requires the T3SS encoded in SPI-1 and SPI-2 to evade phagocytosis by D. discoideum. Altogether, these results indicate that S. Typhimurium exploits a common set of genes and molecular mechanisms to survive within amoeba and animal host cells. The use of D. discoideum as a model for host–pathogen interactions will allow us to discover the gene repertoire used by Salmonella to survive inside the amoeba and to study the cellular processes that are affected during infection.

The zebrafish model has been used to determine the role of vertebrate innate immunity during bact... more The zebrafish model has been used to determine the role of vertebrate innate immunity during bacterial infections. Here, we compare the in vivo immune response induced by GFP-tagged Salmonella Typhimurium inoculat-ed by immersion and microinjection in transgenic zebrafish larvae. Our novel infection protocols in zebrafish allow live-cell imaging of Salmonella colonization.

Titular: CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA ,domiciliado en Ave. 31 entre 158 y 19... more 73) Titular: CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA ,domiciliado en Ave. 31 entre 158 y 190, Cubanacán, Playa.; (CU)