Genomic insights of Leclercia adecarboxylata strains linked to an outbreak in public hospitals in Mexico.

BACKGROUND: Leclercia adecarboxylata is a bacteria closely related to Escherichia coli according to its biochemical characteristics and is commonly considered non-pathogenic although a growing number of publications classify it as an emerging pathogen. Fosfomycin resistance is a common trait for L. adecarboxylata encoded by fosALA gene. OBJECTIVE: To analyze genomic traits of sixteen L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition. METHODS: Twenty-eight L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition were identified biochemically with a Vitek ® automated system. The strains were phenotyped by their growth on Eosin Methylene Blue agar or MacConkey agar plates. Additionally, Pulsed field gel electrophoresis (PFGE) was performed to establish the clonal relationship. The genomic DNA of sixteen strains was obtained using a Qubit ® dsDNA HS Assay Kit and sequenced on an Illumina ® MiSeq instrument. Draft genomes were assembled using PROKKA and Rast. Assemblies were submitted to Resfinder and PathogenFinder from the Center for Genomic Epidemiology in order to find resistance genes and pathogenic potential. IslandViewer4 was also used to find Pathogenicity and Phage Islands. For identification of the fosA gene, manual curation and Clustal analysis was performed. A novel FosA variant was identified. Finally, phylogenetic analysis was performed using VAMPhyRE software and Mega X. RESULTS: In this paper, we report the genomes of sixteen strains of Leclercia adecarboxylata causing an outbreak associated with parenteral nutrition in public hospitals in Mexico. The genomes were analyzed for genetic determinants of virulence and resistance. A high pathogenic potential (pathogenicity index 0.82) as well as multiple resistance genes including carbapenemics, colistin and efflux pumps were determined. Based on sequence analysis, a new variant of the fosALA gene was described. Finally, the outbreak was confirmed by establishing the clonal relationship among the sixteen genomes obtained. CONCLUSIONS: Commensal strains of L. adecarboxylata may acquire genetic determinants that provide mechanisms of host damage and go unnoticed in clinical diagnosis. L. adecarboxylata can evolve in a variety of ways including the acquisition of resistance and virulence genes representing a therapeutic challenge in patient care.

Combinatorial interactions between viral proteins expand the potential functional landscape of the tomato yellow leaf curl virus proteome.

Viruses manipulate the cells they infect in order to replicate and spread. Due to strict size restrictions, viral genomes have reduced genetic space; how the action of the limited number of viral proteins results in the cell reprogramming observed during the infection is a long-standing question. Here, we explore the hypothesis that combinatorial interactions may expand the functional landscape of the viral proteome. We show that the proteins encoded by a plant-infecting DNA virus, the geminivirus tomato yellow leaf curl virus (TYLCV), physically associate with one another in an intricate network, as detected by a number of protein-protein interaction techniques. Importantly, our results indicate that intra-viral protein-protein interactions can modify the subcellular localization of the proteins involved. Using one particular pairwise interaction, that between the virus-encoded C2 and CP proteins, as proof-of-concept, we demonstrate that the combination of viral proteins leads to novel transcriptional effects on the host cell. Taken together, our results underscore the importance of studying viral protein function in the context of the infection. We propose a model in which viral proteins might have evolved to extensively interact with other elements within the viral proteome, enlarging the potential functional landscape available to the pathogen.

In planta vs viral expression of HCPro affects its binding of nonplant 21-22 nucleotide small RNAs, but not its preference for 5'-terminal adenines, or its effects on small RNA methylation.

Previous studies have found a correlation between the abilities of PVX vector-expressed HCPro variants to bind small RNAs (sRNAs), and to suppress silencing. Moreover, HCPro preferred to bind viral sRNAs of 21-22 nucleotides (nt) containing 5'-terminal adenines. This would require such viral sRNAs to have either different access to the suppressor than those of plant sequences, or different molecular properties. To investigate this preference further, we have used suppressor-competent or suppressor-deficient HCPro variants, expressed from either T-DNAs or potyvirus constructs. Then, the sRNAs generated in plants and associated with the purified HCPro variants were characterized. Marked differences were observed in the ratios of sRNAs of plant vs nonplant origin that bound to suppressor-competent HCPro, depending on the mode of its expression. Regardless of the means of expression, HCPro retained the same preference among the nonplant sRNAs of 21-22 nt for those with 5'-terminal adenines. Relative methylation levels of individual sRNAs were assessed, and the nonplant sRNAs were found to be significantly less methylated in the presence of the suppressor. Targeted binding of sRNAs based on size, 5'-terminal sequence and origin, together with affecting their methylation, could explain how HCPro counteracts silencing.

The receptor-like kinases BAM1 and BAM2 are required for root xylem patterning.

Xylem patterning in the root is established through the creation of opposing gradients of miRNAs and their targets, transcripts of the HD-ZIP III family of transcriptions factors, enabled by the cell-to-cell spread of the former. The miRNAs regulating xylem patterning, miR165/6, move through plasmodesmata, but how their trafficking is regulated remains elusive. Here, we describe that simultaneous mutation of the plasma membrane- and plasmodesmata-localized receptor-like kinases (RLKs) BARELY ANY MERISTEM (BAM) 1 and 2 or expression of the geminivirus-encoded BAM1/2-interactor C4 results in higher accumulation and broader distribution of the HD-ZIP III transcripts despite normal total accumulation of miR165/6, and ultimately causes defects in xylem patterning, which depend on the function of the aforementioned miRNA targets. Taken together, our results show that BAM1 and BAM2 are redundantly required for proper xylem patterning in the Arabidopsis root, by ensuring the proper distribution and accumulation of miR165/6-targeted transcripts.

Overexpression of polygalacturonase-inhibiting protein (PGIP) gene from Hypericum perforatum alters expression of multiple defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens in tobacco.

Hypericum perforatum L is a remarkable source of high-value secondary metabolites with increasing applications in pharmaceutical industry. However, improvement in the production of secondary metabolites through genetic engineering is a demanding task, as H. perforatum is not amenable to Agrobacterium tumefaciens-mediated transformation. In this study, we identified a Polygalacturonase-inhibiting protein (PGIP) gene from a subtractive cDNA library of A. tumefaciens-treated H. perforatum suspension cells. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum overexpressing HpPGIP alone or fused at the N-terminus to Phenolic oxidative coupling protein (Hyp-1), a gene that positively modulates resistance to A. tumefaciens. Furthermore, virus-induced gene silencing was employed to knock down the expression of the PGIP homologous in N. benthamiana. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in both HpPGIP and Hyp-1-PGIP transgenic plants, as assessed by GUS staining assays. However, silencing of PGIP in N. benthamiana increased the resistance to A. tumefaciens rather than susceptibility, which correlated with induction of pathogenesis-related proteins (PRs). The expression of core genes involved in several defense pathways was also analyzed in transgenic tobacco plants. Overexpression of HpPGIP led to up-regulation of key genes involved in hormone signaling, microRNA-based gene silencing, homeostasis of reactive oxygen species, and the phenylpropanoid pathway. Overexpression of Hyp-1-PGIP seemed to enhance the effect of PGIP on the expression of most genes analyzed. Moreover, HpPGIP was detected in the cytoplasm, nucleus and the plasma membrane or cell wall by confocal microscopy. Overall, our findings suggest HpPGIP modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Recent advances on the plant manipulation by geminiviruses.

As intracellular parasites, viruses co-opt the molecular machinery of the cells they infect in order to multiply and spread, and the extensiveness and effectiveness of this manipulation ultimately determine the outcome of the interaction between virus and host. Members of the Geminiviridae family, causal agents of devastating diseases in crops, encode only a handful of multifunctional, fast-evolving proteins, which efficiently target host proteins to re-wire plant development and physiology and enable replication and spread of the viral genome. In this review, we offer an overview of the different steps in the geminiviral invasion of the host plant, and explore the knowns and unknowns in geminivirus biology.

Virus infection induces resistance to Pseudomonas syringae and to drought in both compatible and incompatible bacteria-host interactions, which are compromised under conditions of elevated temperature and CO2 levels.

Plants are simultaneously exposed to a variety of biotic and abiotic stresses, such as infections by viruses and bacteria, or drought. This study aimed to improve our understanding of interactions between viral and bacterial pathogens and the environment in the incompatible host Nicotiana benthamiana and the susceptible host Arabidopsis thaliana, and the contribution of viral virulence proteins to these responses. Infection by the Potato virus X (PVX)/Plum pox virus (PPV) pathosystem induced resistance to Pseudomonas syringae (Pst) and to drought in both compatible and incompatible bacteria-host interactions, once a threshold level of defence responses was triggered by the virulence proteins P25 of PVX and the helper component proteinase of PPV. Virus-induced resistance to Pst was compromised in salicylic acid and jasmonic acid signalling-deficient Arabidopsis but not in N. benthamiana lines. Elevated temperature and CO2 levels, parameters associated with climate change, negatively affected resistance to Pst and to drought induced by virus infection, and this correlated with diminished H2O2 production, decreased expression of defence genes and a drop in virus titres. Thus, diminished virulence should be considered as a potential factor limiting the outcome of beneficial trade-offs in the response of virus-infected plants to drought or bacterial pathogens under a climate change scenario.

cfr(B), cfr(C), and a New cfr-Like Gene, cfr(E), in Clostridium difficile Strains Recovered across Latin America.

Cfr is a radical S-adenosyl-l-methionine (SAM) enzyme that confers cross-resistance to antibiotics targeting the 23S rRNA through hypermethylation of nucleotide A2503. Three cfr-like genes implicated in antibiotic resistance have been described, two of which, cfr(B) and cfr(C), have been sporadically detected in Clostridium difficile However, the methylase activity of Cfr(C) has not been confirmed. We found cfr(B), cfr(C), and a cfr-like gene that shows only 51 to 58% protein sequence identity to Cfr and Cfr-like enzymes in clinical C. difficile isolates recovered across nearly a decade in Mexico, Honduras, Costa Rica, and Chile. This new resistance gene was termed cfr(E). In agreement with the anticipated function of the cfr-like genes detected, all isolates exhibited high MIC values for several ribosome-targeting antibiotics. In addition, in vitro assays confirmed that Cfr(C) and Cfr(E) methylate Escherichia coli and, to a lesser extent, C. difficile 23S rRNA fragments at the expected positions. The analyzed isolates do not have mutations in 23S rRNA genes or genes encoding the ribosomal proteins L3 and L4 and lack poxtA, optrA, and pleuromutilin resistance genes. Moreover, these cfr-like genes were found in Tn6218-like transposons or integrative and conjugative elements (ICE) that could facilitate their transfer. These results indicate selection of potentially mobile cfr-like genes in C. difficile from Latin America and provide the first assessment of the methylation activity of Cfr(C) and Cfr(E), which belong to a cluster of Cfr-like proteins that does not include the functionally characterized enzymes Cfr, Cfr(B), and Cfr(D).

Transgenic expression of Hyp-1 gene from Hypericum perforatum L. alters expression of defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens.

MAIN CONCLUSION: Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Cell death triggered by the P25 protein in Potato virus X-associated synergisms results from endoplasmic reticulum stress in Nicotiana benthamiana.

The synergistic interaction of Potato virus X (PVX) with a number of potyviruses results in systemic necrosis in Nicotiana spp. Previous investigations have indicated that the viral suppressor of RNA silencing (VSR) protein P25 of PVX triggers systemic necrosis in PVX-associated synergisms in a threshold-dependent manner. However, little is still known about the cellular processes that lead to this necrosis, and whether the VSR activity of P25 is involved in its elicitation. Here, we show that transient expression of P25 in the presence of VSRs from different viruses, including the helper component-proteinase (HC-Pro) of potyviruses, induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which ultimately lead to ER collapse. However, the host RNA silencing pathway was dispensable for the elicitation of cell death by P25. Confocal microscopy studies in leaf patches co-expressing P25 and HC-Pro showed dramatic alterations in ER membrane structures, which correlated with the up-regulation of bZIP60 and several ER-resident chaperones, including the ER luminal binding protein (BiP). Overexpression of BiP alleviated the cell death induced by the potexviral P25 protein when expressed together with VSRs derived from different viruses. Conversely, silencing of the UPR master regulator, bZIP60, led to an increase in cell death elicited by the P25/HC-Pro combination as well as by PVX-associated synergism. In addition to its role as a negative regulator of P25-induced cell death, UPR partially restricted PVX infection. Thus, systemic necrosis caused by PVX-associated synergistic infections is probably the effect of an unmitigated ER stress following the overaccumulation of a viral protein, P25, with ER remodelling activity.

HCPro-mediated transmission by aphids of purified virions does not require its silencing suppression function and correlates with its ability to coat cell microtubules in loss-of-function mutant studies.

Native and amino acid (aa) substitution mutants of HCPro from potato virus Y (PVY) were transiently expressed in Nicotiana benthamiana leaves. Properties of those HCPro variants with regard to silencing suppression activities, mediation of viral transmission by aphids, and subcellular localization dynamics, were determined. One mutant failed to suppress silencing in agropatch assays, but could efficiently mediate the transmission by aphids of purified virions. This mutant also retained the ability to translocate to microtubules (MTs) in stressed cells. By contrast, another single aa substitution mutant displayed native-like silencing suppression activity in agropatch assays, but could not mediate transmission of PVY virions by aphids, and could not relocate to MTs. Our data show that silencing suppression by HCPro is not required in the aphid-mediated transmission of purified virions. In addition, since the same single aa alteration compromised both, viral transmission and coating of MTs, those two properties could be functionally related.

Effects of simultaneously elevated temperature and CO2 levels on Nicotiana benthamiana and its infection by different positive-sense RNA viruses are cumulative and virus type-specific.

We have studied how simultaneously elevated temperature and CO2 levels [climate change-related conditions (CCC) of 30°C, 970 parts-per-million (ppm) of CO2 vs. standard conditions (SC) of 25°C, ~ 405ppm CO2] affect physiochemical properties of Nicotiana benthamiana leaves, and also its infection by several positive-sense RNA viruses. In previous works we had studied effects of elevated temperature, CO2 levels separately. Under CCC, leaves of healthy plants almost doubled their area relative to SC but contained less protein/unit-of-area, similarly to what we had found under conditions of elevated CO2 alone. CCC also affected the sizes/numbers of different foliar cell types differently. Under CCC, infection outcomes in titers and symptoms were virus type-specific, broadly similar to those observed under elevated temperature alone. Under either condition, infections did not significantly alter the protein content of leaf discs. Therefore, effects of elevated temperature and CO2 combined on properties of the pathosystems studied were overall cumulative.

Virulence determines beneficial trade-offs in the response of virus-infected plants to drought via induction of salicylic acid.

It has been hypothesized that plants can get beneficial trade-offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus-induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought-tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus-infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid-independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non-infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.

Identification of MAPKs as signal transduction components required for the cell death response during compatible infection by the synergistic pair Potato virus X-Potato virus Y.

Systemic necrosis is one of the most severe symptoms caused in compatible plant-virus interactions and shares common features with the hypersensitive response (HR). Mitogen-activated protein kinase (MAPK) cascades are associated with responses to compatible and incompatible host-virus interactions. Here, we show that virus-induced gene silencing of the Nicotiana benthamiana MAPK genes salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), and the MAPK kinase (MAPKK) genes MEK1 and MKK1, partially compromised the HR-like response induced by the synergistic interaction of Potato virus X with Potato virus Y (PVX-PVY). Nevertheless, ameliorated cell death induced by PVX-PVY in the MAPK(K)-silenced plants did not facilitate virus accumulation in systemically infected leaves. Dual silencing of SIPK and of the oxylipin biosynthetic gene 9-Lipoxygenase showed that the latter was epistatic to SIPK in response to PVX-PVY infection. These findings demonstrate that SIPK, WIPK, MEK1 and MKK1 function as positive regulators of PVX-PVY-induced cell death.

Potato Virus Y HCPro Suppression of Antiviral Silencing in Nicotiana benthamiana Plants Correlates with Its Ability To Bind In Vivo to 21- and 22-Nucleotide Small RNAs of Viral Sequence.

We have investigated short and small RNAs (sRNAs) that were bound to a biologically active hexahistidine-tagged Potato virus Y (PVY) HCPro suppressor of silencing, expressed from a heterologous virus vector in Nicotiana benthamiana plants, and purified under nondenaturing conditions. We found that RNAs in purified preparations were differentially enriched in 21-nucleotide (nt) and, to a much lesser extent, 22-nt sRNAs of viral sequences (viral sRNAs [vsRNAs]) compared to those found in a control plant protein background bound to nickel resin in the absence of HCPro or in a purified HCPro alanine substitution mutant (HCPro mutB) control that lacked suppressor-of-silencing activity. In both controls, sRNAs were composed almost entirely of molecules of plant sequence, indicating that the resin-bound protein background had no affinity for vsRNAs and also that HCPro mutB failed to bind to vsRNAs. Therefore, PVY HCPro suppressor activity correlated with its ability to bind to 21- and 22-nt vsRNAs. HCPro constituted at least 54% of the total protein content in purified preparations, and we were able to calculate its contribution to the 21- and the 22-nt pools of sRNAs present in the purified samples and its binding strength relative to the background. We also found that in the 21-nt vsRNAs of the HCPro preparation, 5'-terminal adenines were overrepresented relative to the controls, but this was not observed in vsRNAs of other sizes or of plant sequences.IMPORTANCE It was previously shown that HCPro can bind to long RNAs and small RNAs (sRNAs) in vitro and, in the case of Turnip mosaic virus HCPro, also in vivo in arabidopsis AGO2-deficient plants. Our data show that PVY HCPro binds in vivo to sRNAs during infection in wild-type Nicotiana benthamiana plants when expressed from a heterologous virus vector. Using a suppression-of-silencing-deficient HCPro mutant that can accumulate in this host when expressed from a virus vector, we also show that sRNA binding correlates with silencing suppression activity. We demonstrate that HCPro binds at least to sRNAs with viral sequences of 21 nucleotides (nt) and, to a much lesser extent, of 22 nt, which were are also differentially enriched in 5'-end adenines relative to the purified controls. Together, our results support the physical binding of HCPro to vsRNAs of 21 and 22 nt as a means to interfere with antiviral silencing.

Effects of Elevated CO2and Temperature on Pathogenicity Determinants and Virulence of Potato virus X/Potyvirus-Associated Synergism.

Infections of plants by multiple viruses are common in nature and may result in synergisms in pathologies. Several environmental factors influence plant-virus interactions and act on virulence and host defense responses. Mixed viral infections may be more frequent under environmental conditions associated with global warming. Here, we address how changes in the two main parameters behind global warming, carbon dioxide concentrations ([CO2]) and temperature, may affect virulence of Potato virus X (PVX)/potyvirus-associated synergism compared with single infections in Nicotiana benthamiana. Elevated [CO2] resulted in attenuated virulence of single infection by PVX, which correlated with a lower accumulation of virus. In contrast, virulence of PVX/potyvirus-associated synergism was maintained at elevated [CO2]. On the other hand, elevated temperature decreased markedly both virulence and virus titers in the synergistic infection. We also show that the HR-like response elicited by transient coexpression of PVX P25 together with the potyviral helper component-proteinase protein was significantly enhanced by elevated temperature, whereas it was reduced by elevated [CO2]. Both proteins are main pathogenicity determinants in PVX-associated synergisms. These findings indicate that, under environmental conditions associated with global warming, virulence of PVX/potyvirus-associated synergisms is expected to vary relative to single infections and, thus, may have pathological consequences in the future.

High Temperature, High Ambient CO2 Affect the Interactions between Three Positive-Sense RNA Viruses and a Compatible Host Differentially, but not Their Silencing Suppression Efficiencies.

We compared infection of Nicotiana benthamiana plants by the positive-sense RNA viruses Cucumber mosaic virus (CMV), Potato virus Y (PVY), and by a Potato virus X (PVX) vector, the latter either unaltered or expressing the CMV 2b protein or the PVY HCPro suppressors of silencing, at 25°C vs. 30°C, or at standard (~401 parts per million, ppm) vs. elevated (970 ppm) CO2 levels. We also assessed the activities of their suppressors of silencing under those conditions. We found that at 30°C, accumulation of the CMV isolate and infection symptoms remained comparable to those at 25°C, whereas accumulation of the PVY isolate and those of the three PVX constructs decreased markedly, even when expressing the heterologous suppressors 2b or HCPro, and plants had either very attenuated or no symptoms. Under elevated CO2 plants grew larger, but contained less total protein/unit of leaf area. In contrast to temperature, infection symptoms remained unaltered for the five viruses at elevated CO2 levels, but viral titers in leaf disks as a proportion of the total protein content increased in all cases, markedly for CMV, and less so for PVY and the PVX constructs. Despite these differences, we found that neither high temperature nor elevated CO2 prevented efficient suppression of silencing by their viral suppressors in agropatch assays. Our results suggest that the strength of antiviral silencing at high temperature or CO2 levels, or those of the viral suppressors that counteract it, may not be the main determinants of the observed infection outcomes.