The Knockout of Enterobactin-Related Gene in Pectobacterium atrosepticum Results in Reduced Stress Resistance and Virulence towards the Primed Plants

Siderophores produced by microorganisms to scavenge iron from the environment have been shown to contribute to virulence and/or stress resistance of some plant pathogenic bacteria. Phytopathogenic bacteria of Pectobacterium genus possess genes for the synthesis of siderophore enterobactin, which role in plant-pathogen interactions has not been elucidated. In the present study we characterized the phenotype of the mutant strain of Pba deficient for the enterobactin-biosynthetic gene entA. We showed that enterobactin may be considered as a conditionally beneficial virulence factor of Pba. The entA knockout did not reduce Pba virulence on non-primed plants; however, salicylic acid-primed plants were more resistant to ΔentA mutant than to the wild type Pba. The reduced virulence of ΔentA mutant towards the primed plants is likely explained by its compromised resistance to oxidative stress.     

Complementary Regulation of BfmRS Two-Component and AbaIR Quorum Sensing Systems to Express Virulence-Associated Genes in Acinetobacter baumannii

Acinetobacter baumannii expresses various virulence factors to adapt to hostile environments and infect susceptible hosts. This study investigated the regulatory network of the BfmRS two-component and AbaIR quorum sensing (QS) systems in the expression of virulence-associated genes in A. baumannii ATCC 17978. The ΔbfmS mutant exhibited a significant decrease in surface motility, which presumably resulted from the low expression of pilT and A1S_0112-A1S_0119 gene cluster. The ΔbfmR mutant displayed a significant reduction in biofilm and pellicle formation due to the low expression of csu operon. The deletion of abaR did not affect the expression of bfmR or bfmS. However, the expression of abaR and abaI was upregulated in the ΔbfmR mutant. The ΔbfmR mutant also produced more autoinducers than did the wild-type strain, suggesting that BfmR negatively regulates the AbaIR QS system. The ΔbfmS mutant exhibited no autoinducer production in the bioassay system. The expression of the A1S_0112-A1S_0119 gene cluster was downregulated in the ΔabaR mutant, whereas the expression of csu operon was upregulated in this mutant with a high cell density. In conclusion, for the first time, we demonstrated that the BfmRS-AbaIR QS system axis regulated the expression of virulence-associated genes in A. baumannii. This study provides new insights into the complex network system involved in the regulation of virulence-associated genes underlying the pathogenicity of A. baumannii.     

Aryl Hydrocarbon Receptor Repressor and TiPARP (ARTD14) Use Similar,but also Distinct Mechanisms to Repress Aryl Hydrocarbon Receptor Signaling

The aryl hydrocarbon receptor (AHR) regulates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AHR repressor (AHRR) is an AHR target gene and functions as a ligand-induced repressor of AHR; however, its mechanism of inhibition is controversial. Recently, we reported that TCDD-inducible poly (ADP-ribose) polymerase (TiPARP; ARTD14) also acts as a repressor of AHR, representing a new player in the mechanism of AHR action. Here we compared the ability of AHRR- and TiPARP-mediated inhibition of AHR activity. TCDD increased AHRR mRNA levels and recruitment of AHRR to cytochrome P450 1A1 (CYP1A1) in MCF7 cells. Knockdown of TiPARP, but not AHRR, increased TCDD-induced CYP1A1 mRNA and AHR protein levels. Similarly, immortalized TiPARP^−/− mouse embryonic fibroblasts (MEFs) and AHRR^−/− MEFs exhibited enhanced AHR transactivation. However, unlike TiPARP^−/− MEFs, AHRR^−/− MEFs did not exhibit increased AHR protein levels. Overexpression of TiPARP in AHRR^−/− MEFs or AHRRΔ8, the active isoform of AHRR, in TiPARP^−/− MEFs reduced TCDD-induced CYP1A1 mRNA levels, suggesting that they independently repress AHR. GFP-AHRRΔ8 and GFP-TiPARP expressed as small diffuse nuclear foci in MCF7 and HuH7 cells. GFP-AHRRΔ8_Δ1-49, which lacks its putative nuclear localization signal, localized to both the nucleus and the cytoplasm, while the GFP-AHRRΔ8_Δ1-100 mutant localized predominantly in large cytoplasmic foci. Neither GFP-AHRRΔ8_Δ1-49 nor GFP-AHRRΔ8_Δ1-100 repressed AHR. Taken together, AHRR and TiPARP repress AHR transactivation by similar, but also different mechanisms.     

RpoN Regulates Virulence Factors of Pseudomonas aeruginosa via Modulating the PqsR Quorum Sensing Regulator

The alternative sigma factor RpoN regulates many cell functions, such as motility, quorum sensing, and virulence in the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa often evolves rpoN-negative variants during the chronic infection in cystic fibrosis patients. It is unclear how RpoN interacts with other regulatory mechanisms to control virulence of P. aeruginosa. In this study, we show that RpoN modulates the function of PqsR, a quorum sensing receptor regulating production of virulence factors including the phenazine pyocyanin. The ∆rpoN mutant is able to synthesize 4-quinolone signal molecule HHQ but unable to activate PqsR and Pseudomonas quinolone signal (pqs) quorum sensing. The ∆rpoN mutant produces minimal level of pyocyanin and is unable to produce the anti-staphylococcal agents. Providing pqsR in trans in the ∆rpoN mutant restores its pqs quorum sensing and virulence factor production to the wild-type level. Our study provides evidence that RpoN has a regulatory effect on P. aeruginosa virulence through modulating the function of the PqsR quorum sensing regulator.     

The LAMMER Kinase MoKns1 Regulates Growth,Conidiation and Pathogenicity in Magnaporthe oryzae

Magnaporthe oryzae is an important pathogen that causes a devastating disease in rice. It has been reported that the dual-specificity LAMMER kinase is conserved from yeast to animal species and has a variety of functions. However, the functions of the LAMMER kinase have not been reported in M. oryzae. In this study, we identified the unique LAMMER kinase MoKns1 and analyzed its function in M. oryzae. We found that in a MoKNS1 deletion mutant, growth and conidiation were primarily decreased, and pathogenicity was almost completely lost. Furthermore, our results found that MoKns1 is involved in autophagy. The ΔMokns1 mutant was sensitive to rapamycin, and MoKns1 interacted with the autophagy-related protein MoAtg18. Compared with the wild-type strain 70−15, autophagy was significantly enhanced in the ΔMokns1 mutant. In addition, we also found that MoKns1 regulated DNA damage stress pathways, and the ΔMokns1 mutant was more sensitive to hydroxyurea (HU) and methyl methanesulfonate (MMS) compared to the wild-type strain 70−15. The expression of genes related to DNA damage stress pathways in the ΔMokns1 mutant was significantly different from that in the wild-type strain. Our results demonstrate that MoKns1 is an important pathogenic factor in M. oryzae involved in regulating autophagy and DNA damage response pathways, thus affecting virulence. This research on M. oryzae pathogenesis lays a foundation for the prevention and control of M. oryzae.     

Aspergillus parasiticus crzA,Which Encodes Calcineurin Response Zinc-Finger Protein,Is Required for Aflatoxin Production under Calcium Stress

Two morphologically different Aspergillus parasiticus strains, one producing aflatoxins, abundant conidia but few sclerotia (BN9) and the other producing O-methyl-sterimatocystin (OMST), copious sclerotia but a low number of conidia (RH), were used to assess the role of crzA which encodes a putative calcium-signaling pathway regulatory protein. Under standard culture conditions, BN9ΔcrzA mutants conidiated normally but decreased slightly in radial growth, regardless of illumination conditions. RHΔcrzA mutants produced only conidia under light and showed decreased conidiation and delayed sclerotial formation in the dark. Regulation of conidiation of both A. parasiticus strains by light was independent of crzA. Increased concentrations of lithium, sodium, and potassium impaired conidiation and sclerotial formation of the RHΔcrzA mutants but they did not affect conidiation of the BN9ΔcrzA mutants. Vegetative growth and asexual development of both ΔcrzA mutants were hypersensitive to increased calcium concentrations. Calcium supplementation (10 mM) resulted in 3-fold and 2-fold decreases in the relative expression of the endoplasmic reticulum calcium ATPase 2 gene in the BN9 and RH parental strains, respectively, but changes in both ΔcrzA mutants were less significant. Compared to the parental strains, the ΔcrzA mutants barely produced aflatoxins or OMST after the calcium supplementation. The relative expression levels of aflatoxin biosynthesis genes, nor1, ver1, and omtA, in both ΔcrzA mutants were decreased significantly, but the decreases in the parental strains were at much lower extents. CrzA is required for growth and development and for aflatoxin biosynthesis under calcium stress conditions.     

Combination Strategy of Genetic Dereplication and Manipulation of Epigenetic Regulators Reveals a Novel Compound from Plant Endophytic Fungus

The strategies of genetic dereplication and manipulation of epigenetic regulators to activate the cryptic gene clusters are effective to discover natural products with novel structure in filamentous fungi. In this study, a combination of genetic dereplication (deletion of pesthetic acid biosynthetic gene, PfptaA) and manipulation of epigenetic regulators (deletion of histone methyltransferase gene PfcclA and histone deacetylase gene PfhdaA) was developed in plant endophytic fungus Pestalotiopsis fici. The deletion of PfptaA with PfcclA and/or PfhdaA led to isolation of 1 novel compound, pestaloficiol X (1), as well as another 11 known compounds with obvious yield changes. The proposed biosynthesis pathway of pestaloficiol X was speculated using comparative analysis of homologous biosynthetic gene clusters. Moreover, phenotypic effects on the conidial development and response to oxidative stressors in the mutants were explored. Our results revealed that the new strain with deletion of PfcclA or PfhdaA in ΔPfptaA background host can neutralise the hyperformation of conidia in the PfptaA mutant, and that the ΔPfptaA ΔPfhdaA mutant was generally not sensitive to oxidative stressors as much as the ΔPfptaA ΔcclA mutant in comparison with the single mutant ΔPfptaA or the parental strains. This combinatorial approach can be applied to discover new natural products in filamentous fungi.     

Unbalance between Pyridine Nucleotide Cofactors in The SOD1 Deficient Yeast Saccharomyces cerevisiae Causes Hypersensitivity to Alcohols and Aldehydes

Alcohol and aldehyde dehydrogenases are especially relevant enzymes involved in metabolic and detoxification reactions that occur in living cells. The comparison between the gene expression, protein content, and enzymatic activities of cytosolic alcohol and aldehyde dehydrogenases of the wild-type strain and the Δsod1 mutant lacking superoxide dismutase 1, which is hypersensitive to alcohols and aldehydes, shows that the activity of these enzymes is significantly higher in the Δsod1 mutant, but this is not a mere consequence of differences in the enzymatic protein content nor in the expression levels of genes. The analysis of the NAD(H) and NADP(H) content showed that the higher activity of alcohol and aldehyde dehydrogenases in the Δsod1 mutant could be a result of the increased availability of pyridine nucleotide cofactors. The higher level of NAD⁺ in the Δsod1 mutant is not related to the higher level of tryptophan; in turn, a higher generation of NADPH is associated with the upregulation of the pentose phosphate pathway. It is concluded that the increased sensitivity of the Δsod1 mutant to alcohols and aldehydes is not only a result of the disorder of redox homeostasis caused by the induction of oxidative stress but also a consequence of the unbalance between pyridine nucleotide cofactors.     

Identification of Pathogenicity-Related Genes in Biofilm-Defective Acidovorax citrulli by Transposon Tn5 Mutagenesis

Biofilm formation is important for virulence of a large number of plant pathogenic bacteria. Indeed, some virulence genes have been found to be involved in the formation of biofilm in bacterial fruit blotch pathogen Acidovorax citrulli. However, some virulent strains of A. citrulli were unable to format biofilm, indicating the complexity between biofilm formation and virulence. In this study, virulence-related genes were identified in the biofilm-defective strain A1 of A. citrulli by using Tn5 insertion, pathogenicity test, and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). Results from this study indicated that 22 out of the obtained 301 mutants significantly decreased the virulence of strain A1 compared to the wild-type. Furthermore, sequence analysis indicated that the obtained 22 mutants were due to the insertion of Tn5 into eight genes, including Aave 4244 (cation diffusion facilitator family transporter), Aave 4286 (hypothetical protein), Aave 4189 (alpha/beta hydrolase fold), Aave 1911 (IMP dehydrogenase/GMP reductase domain), Aave 4383 (bacterial export proteins, family 1), Aave 4256 (Hsp70 protein), Aave 0003 (histidine kinase, DNA gyrase B, and HSP90-like ATPase), and Aave 2428 (pyridoxal-phosphate dependent enzyme). Furthermore, the growth of mutant Aave 2428 was unaffected and even increased by the change in incubation temperature, NaCl concentration and the pH of the LB broth, indicating that this gene may be directly involved in the bacterial virulence. Overall, the determination of the eight pathogenicity-related genes in strain A1 will be helpful to elucidate the pathogenesis of biofilm-defective A. citrulli.     

N,N′-alkylated Imidazolium-Derivatives Act as Quorum-Sensing Inhibitors Targeting the Pectobacterium atrosepticum-Induced Symptoms on Potato Tubers

Bacteria belonging to the Pectobacterium genus are the causative agents of the blackleg and soft-rot diseases that affect potato plants and tubers worldwide. In Pectobacterium, the expression of the virulence genes is controlled by quorum-sensing (QS) and N-acylhomoserine lactones (AHLs). In this work, we screened a chemical library of QS-inhibitors (QSIs) and AHL-analogs to find novel QSIs targeting the virulence of Pectobacterium. Four N,N′-bisalkylated imidazolium salts were identified as QSIs; they were active at the μM range. In potato tuber assays, two of them were able to decrease the severity of the symptoms provoked by P. atrosepticum. This work extends the range of the QSIs acting on the Pectobacterium-induced soft-rot disease.