Pathogenic plant-microbe interactions. What we know and how we benefit

Plants, like humans and other animals, also get sick, exhibit disease symptoms, and die. Plant diseases are caused by environmental stress, genetic or physiological disorders and infectious agents including viroids, viruses, bacteria and fungi. Plant pathology originated from the convergence of microbiology, botany and agronomy; its ultimate goal is the control of plant disease. Microbiologists have been attracted to this field of research because of the need for identification of the agents causing infectious diseases in economically important crops. In 1878—only two years after Pasteur and Koch had shown for the first time that anthrax in animals was caused by a bacteria—Burril, in the USA, discovered that the fire blight disease of apple and pear was also caused by a bacterium (nowadays known as Erwinia amylovora). In 1898, Beijerinck concluded that tobacco mosaic was caused by a “contagium vivum fluidum” which he called a virus. In 1971, Diener proved that a potato disease named potato spindle tuber was caused by infectious RNA which he called viroid

A pathogenic mechanism in Huntington"s disease involves small CAG-repeated RNAs with neurotoxic activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches

Unexpected genomic variability in clinical and environmental strains of the pathogenic yeast Candida parapsilosis

Invasive candidiasis is the most commonly reported invasive fungal infection worldwide. Although Candida albicans remains the main cause, the incidence of emerging Candida species, such as C. parapsilosis is increasing. It has been postulated that C. parapsilosis clinical isolates result from a recent global expansion of a virulent clone. However, the availability of a single genome for this species has so far prevented testing this hypothesis at genomic scales. We present here the sequence of three additional strains from clinical and environmental samples. Our analyses reveal unexpected patterns of genomic variation, shared among distant strains, that argue against the clonal expansion hypothesis. All strains carry independent expansions involving an arsenite transporter homolog, pointing to the existence of directional selection in the environment, and independent origins of the two clinical isolates. Furthermore, we report the first evidence for the existence of recombination in this species. Altogether, our results shed new light onto the dynamics of genome evolution in C. parapsilosis.

Transfer of penicillin resistance between Neisseriae in microcosm

Horizontal gene transfer between commensal and pathogenic Neisseriae is the mechanism proposed to explain how pathogenic species acquire altered portions of the penA gene, which encodes penicillin binding protein 2. These changes resulted in a moderately penicillin-resistant phenotype in the meningococci, whose frequency of isolation in Spain increased at the end of the 1980s. Little has been published about the possibility of this gene transfer in nature or about its simulation in the laboratory. We designed a simple microcosm, formed by solid and liquid media, that partially mimics the upper human respiratory tract. In this microcosm, penicillin-resistant commensal strains and the fully susceptible meningococcus were co-cultivated. The efficiency of gene transfer between the strains depended on the phase of bacterial growth and the conditions of culture. Resistance of penicillin was acquired in different steps irrespective of the source of the DNA. The presence of DNase in the medium had no effect on gene transfer, but it was near zero when nicked DNA was used. Cell-to-cell contact or membrane blebs could explain these results. The analysis of sequences of the transpeptidase domain of PBP2 from transformants, and from donor and recipient strains demonstrated that the emergence of moderately resistant transformants was due to genetic exchange between the co-cultivated strains. Finally, mechanisms other than penA modification could be invoked to explain decreased susceptibility

Transfer of penicillin resistance between Neisseriae in microcosm

Horizontal gene transfer between commensal and pathogenic Neisseriae is the mechanism proposed to explain how pathogenic species acquire altered portions of the penA gene, which encodes penicillin binding protein 2. These changes resulted in a moderately penicillin-resistant phenotype in the meningococci, whose frequency of isolation in Spain increased at the end of the 1980s. Little has been published about the possibility of this gene transfer in nature or about its simulation in the laboratory. We designed a simple microcosm, formed by solid and liquid media, that partially mimics the upper human respiratory tract. In this microcosm, penicillin-resistant commensal strains and the fully susceptible meningococcus were co-cultivated. The efficiency of gene transfer between the strains depended on the phase of bacterial growth and the conditions of culture. Resistance of penicillin was acquired in different steps irrespective of the source of the DNA. The presence of DNase in the medium had no effect on gene transfer, but it was near zero when nicked DNA was used. Cell-to-cell contact or membrane blebs could explain these results. The analysis of sequences of the transpeptidase domain of PBP2 from transformants, and from donor and recipient strains demonstrated that the emergence of moderately resistant transformants was due to genetic exchange between the co-cultivated strains. Finally, mechanisms other than penA modification could be invoked to explain decreased susceptibility

Understanding the two-component sensing System of virulent bacteria

Report for the scientific sojourn carried out at the l’ Institute for Computational Molecular Science of the Temple University, United States, from 2010 to 2012. Two-component systems (TCS) are used by pathogenic bacteria to sense the environment within a host and activate mechanisms related to virulence and antimicrobial resistance. A prototypical example is the PhoQ/PhoP system, which is the major regulator of virulence in Salmonella. Hence, PhoQ is an attractive target for the design of new antibiotics against foodborne diseases. Inhibition of the PhoQ-mediated bacterial virulence does not result in growth inhibition, presenting less selective pressure for the generation of antibiotic resistance. Moreover, PhoQ is a histidine kinase (HK) and it is absent in animals. Nevertheless, the design of satisfactory HK inhibitors has been proven to be a challenge. To compete with the intracellular ATP concentrations, the affinity of a HK inhibidor must be in the micromolar-nanomolar range, whereas the current lead compounds have at best millimolar affinities. Moreover, the drug selectivity depends on the conformation of a highly variable loop, referred to as the “ATP-lid, which is difficult to study by X-Ray crystallography due to its flexibility. I have investigated the binding of different HK inhibitors to PhoQ. In particular, all-atom molecular dynamics simulations have been combined with enhanced sampling techniques in order to provide structural and dynamic information of the conformation of the ATP-lid. Transient interactions between these drugs and the ATP-lid have been identified and the free energy of the different binding modes has been estimated. The results obtained pinpoint the importance of protein flexibility in the HK-inhibitor binding, and constitute a first step in developing more potent and selective drugs. The computational resources of the hosting institution as well as the experience of the members of the group in drug binding and free energy methods have been crucial to carry out this work.

Evidence for classification of c.1852_1853AA>GC in MLH1 as a neutral variant for Lynch syndrome

Background: Lynch syndrome (LS) is an autosomal dominant inherited cancer syndrome characterized by early onset cancers of the colorectum, endometrium and other tumours. A significant proportion of DNA variants in LS patients are unclassified. Reports on the pathogenicity of the c.1852_1853AA>GC (p.Lys618Ala) variant of the MLH1 gene are conflicting. In this study, we provide new evidence indicating that this variant has no significant implications for LS.Methods: The following approach was used to assess the clinical significance of the p.Lys618Ala variant: frequency in a control population, case-control comparison, co-occurrence of the p.Lys618Ala variant with a pathogenic mutation, co-segregation with the disease and microsatellite instability in tumours from carriers of the variant. We genotyped p.Lys618Ala in 1034 individuals (373 sporadic colorectal cancer [CRC] patients, 250 index subjects from families suspected of having LS [revised Bethesda guidelines] and 411 controls). Three well-characterized LS families that fulfilled the Amsterdam II Criteria and consisted of members with the p.Lys618Ala variant were included to assess co-occurrence and co-segregation. A subset of colorectal tumour DNA samples from 17 patients carrying the p.Lys618Ala variant was screened for microsatellite instability using five mononucleotide markers.Results: Twenty-seven individuals were heterozygous for the p.Lys618Ala variant; nine had sporadic CRC (2.41%), seven were suspected of having hereditary CRC (2.8%) and 11 were controls (2.68%). There were no significant associations in the case-control and case-case studies. The p.Lys618Ala variant was co-existent with pathogenic mutations in two unrelated LS families. In one family, the allele distribution of the pathogenic and unclassified variant was in trans, in the other family the pathogenic variant was detected in the MSH6 gene and only the deleterious variant co-segregated with the disease in both families. Only two positive cases of microsatellite instability (2/17, 11.8%) were detected in tumours from p.Lys618Ala carriers, indicating that this variant does not play a role in functional inactivation of MLH1 in CRC patients.Conclusions: The p.Lys618Ala variant should be considered a neutral variant for LS. These findings have implications for the clinical management of CRC probands and their relatives.

X-chromosome tiling path array detection of copy number variants in patients with chromosome X-linked mental retardation

Background: Aproximately 5–10% of cases of mental retardation in males are due to copy number variations (CNV) on the X chromosome. Novel technologies, such as array comparative genomic hybridization (aCGH), may help to uncover cryptic rearrangements in X-linked mental retardation (XLMR) patients. We have constructed an X-chromosome tiling path array using bacterial artificial chromosomes (BACs) and validated it using samples with cytogenetically defined copy number changes. We have studied 54 patients with idiopathic mental retardation and 20 controls subjects. Results: Known genomic aberrations were reliably detected on the array and eight novel submicroscopic imbalances, likely causative for the mental retardation (MR) phenotype, were detected. Putatively pathogenic rearrangements included three deletions and five duplications (ranging between 82 kb to one Mb), all but two affecting genes previously known to be responsible for XLMR. Additionally, we describe different CNV regions with significant different frequencies in XLMR and control subjects (44% vs. 20%). Conclusion:This tiling path array of the human X chromosome has proven successful for the detection and characterization of known rearrangements and novel CNVs in XLMR patients.

Reassessing the role of mitochondrial DNA mutations in autism spectrum disorder

Background: There is increasing evidence that impairment of mitochondrial energy metabolism plays an important role in the pathophysiology of autism spectrum disorders (ASD; OMIM number: 209850). A significant proportion of ASD cases display biochemical alterations suggestive of mitochondrial dysfunction and several studies have reported that mutations in the mitochondrial DNA (mtDNA) molecule could be involved in the disease phenotype. Methods: We analysed a cohort of 148 patients with idiopathic ASD for a number of mutations proposed in the literature as pathogenic in ASD. We also carried out a case control association study for the most common European haplogroups (hgs) and their diagnostic single nucleotide polymorphisms (SNPs) by comparing cases with 753 healthy and ethnically matched controls.Results: We did not find statistical support for an association between mtDNA mutations or polymorphisms and ASD.Conclusions: Our results are compatible with the idea that mtDNA mutations are not a relevant cause of ASD and the frequent observation of concomitant mitochondrial dysfunction and ASD could be due to nuclear factors influencing mitochondrion functions or to a more complex interplay between the nucleus and the mitochondrion/mtDNA.

Multivalent display of the antimicrobial peptides BP100 and BP143

Carbohydrates are considered as promising templates for the display of multiple copies of antimicrobial peptides. Herein, wedescribe the design and synthesis of chimeric structures containing two or four copies of the antimicrobial peptidesKKLFKKILKYL-NH2 (BP100) and KKLfKKILKYL-NH2 (BP143) attached to the carbohydrate template cyclodithioerythritol(cDTE) or α-D-galactopyranoside (Galp). The synthesis involved the preparation of the corresponding peptide aldehyde followedby coupling to an aminooxy-functionalized carbohydrate template. After purification, the multivalent display systems were obtainedin high purities (90–98%) and in good yields (42–64%). These compounds were tested against plant and human pathogenic bacteriaand screened for their cytotoxicity on eukaryotic cells. They showed lower MIC values than the parent peptides against the bacteriaanalyzed. In particular, the carbopeptides derived from cDTE and Galp, which contained two or four copies of BP100, respectively,were 2- to 8-fold more active than the monomeric peptide against the phytopathogenic bacteria. These results suggest thatpreassembling antimicrobial peptides to multimeric structures is not always associated with a significant improvement of theactivity. In contrast, the carbopeptides synthesized were active against human red blood cells pointing out that peptide preassemblyis critical for the hemolytic activity. Notably, peptide preassembly resulted in an enhanced bactericidal effect