Yingxue Qin

flrA, flrB and flrC regulate adhesion by controlling the expression of critical virulence genes in Vibrio alginolyticus

Adhesion is an important virulence trait of Vibrio alginolyticus. Bacterial adhesion is influenced by environmental conditions; however, the molecular mechanism underlying this effect remains unknown. The expression levels of flrA, flrB and flrC were significantly downregulated in adhesion-deficient V. alginolyticus strains cultured under Cu2+, Pb2+, Hg2+ and low-pH stresses. Silencing these genes led to deficiencies in adhesion, motility, flagellar assembly, biofilm formation and exopolysaccharide (EPS) production. The expression levels of fliA, flgH, fliS, fliD, cheR, cheV and V12G01_22158 (Gene ID) were significantly downregulated in all of the RNAi groups, whereas the expression levels of toxT, ctxB, acfA, hlyA and tlh were upregulated in flrA- and flrC-silenced groups. These genes play a key role in the virulence mechanisms of most pathogenic Vibrio species. Furthermore, the expression of flrA, flrB and flrC was significantly influenced by temperature, salinity, starvation and pH. These results indicate that (1) flrA, flrB and flrC are important for V. alginolyticus adhesion; (2) flrA, flrB and flrC significantly influence bacterial adhesion, motility, biofilm formation and EPS production by controlling expression of key genes involved in those phenotypes; and (3) flrA, flrB and flrC regulate adhesion in the natural environment with different temperatures, pH levels, salinities and starvation time. Emerging Microbes & Infections (2016) 5, e85; doi:10.1038/emi.2016.82; published online 3 August 2016

Involvement of the flagellar assembly pathway in Vibrio alginolyticus adhesion under environmental stresses.

Adhesion is an important virulence factor of Vibrio alginolyticus. This factor may be affected by environmental conditions; however, its molecular mechanism remains unclear. In our previous research, adhesion deficient strains were obtained by culturing V. alginolyticus under stresses including Cu, Pb, Hg, and low pH. With RNA-seq and bioinformatics analysis, we found that all of these stress treatments significantly affected the flagellar assembly pathway, which may play an important role in V. alginolyticus adhesion. Therefore, we hypothesized that the environmental stresses of the flagellar assembly pathway may be one way in which environmental conditions affect adhesion. To verify our hypothesis, a bioinformatics analysis, QPCR, RNAi, in vitro adhesion assay and motility assay were performed. Our results indicated that (1) the flagellar assembly pathway was sensitive to environmental stresses, (2) the flagellar assembly pathway played an important role in V. alginolyticus adhesion, and (3) motility is not the only way in which the flagellar assembly pathway affects adhesion.

MinD plays an important role in Aeromonas hydrophila adherence to Anguilla japonica mucus.

For a better understanding of the genitic regulation of the adhesion of Aeromonas hydrophila, a mini-Tn10 transposon mutagenesis system was introduced to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOF/Km) and the recipient A. hydrophila strain W. Out of 332 individual colonies, 7 mutants with significantly attenuated adhesion ability were selected. The DNA sequence flanking the mini-Tn10 transposon inserted in the mutant strain M45 was amplified by TAIL-PCR. The results showed that an ORF of approximately 870 bp (GenBank accession number KP271930) of the mutant strain M45 was inserted by mini-Tn10. This ORF putatively encodes a deduced 289 amino acid protein that displays the highest identity (100%) with the CobQ/CobB/MinD/ParA family protein of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type W, the mutant strain M45 and the complemented strain C45 were investigated. The results indicated that mutation of MinD gene in A. hydrophila could lead to abnormal cell division and reduce the ability of adhesion, biofilm formation and bacterial motility.

Identification and characterization of three Vibrio alginolyticus non-coding RNAs involved in adhesion, chemotaxis, and motility processes

The capability of Vibrio alginolyticus to adhere to fish mucus is a key virulence factor of the bacteria. Our previous research showed that stress conditions, such as Cu2+, Pb2+, Hg2+, and low pH, can reduce this adhesion ability. Non-coding (nc) RNAs play a crucial role in regulating bacterial gene expression, affecting the bacterias pathogenicity. To investigate the mechanism(s) underlying the decline in adhesion ability caused by stressors, we combined high-throughput sequencing with computational techniques to detect stressed ncRNA dynamics. These approaches yielded three commonly altered ncRNAs that are predicted to regulate the bacterial chemotaxis pathway, which plays a key role in the adhesion process of bacteria. We hypothesized they play a key role in the adhesion process of V. alginolyticus. In this study, we validated the effects of these three ncRNAs on their predicted target genes and their role in the V. alginolyticus adhesion process with RNA interference (i), quantitative real-time polymerase chain reaction (qPCR), northern blot, capillary assay, and in vitro adhesion assays. The expression of these ncRNAs and their predicted target genes were confirmed by qPCR and northern blot, which reinforced the reliability of the sequencing data and the target prediction. Overexpression of these ncRNAs was capable of reducing the chemotactic and adhesion ability of V. alginolyticus, and the expression levels of their target genes were also significantly reduced. Our results indicated that these three ncRNAs: (1) are able to regulate the bacterial chemotaxis pathway, and (2) play a key role in the adhesion process of V. alginolyticus.

The TCA Pathway is an Important Player in the Regulatory Network Governing Vibrio alginolyticus Adhesion Under Adversity

Adhesion is a critical step in the initial stage of Vibrio alginolyticus infection; therefore, it is important to understand the underlying mechanisms governing the adhesion of V. alginolyticus and determine if environmental factors have any effect. A greater understanding of this process may assist in developing preventive measures for reducing infection. In our previous research, we presented the first RNA-seq data from V. alginolyticus cultured under stress conditions that resulted in reduced adhesion. Based on the RNA-seq data, we found that the Tricarboxylic acid cycle (TCA pathway) might be closely related to adhesion. Environmental interactions with the TCA pathway might alter adhesion. To validate this, bioinformatics analysis, quantitative Real-Time PCR (qPCR), RNAi, and in vitro adhesion assays were performed, while V. alginolyticus was treated with various stresses including temperature, pH, salinity, and starvation. The expression of genes involved in the TCA pathway was confirmed by qPCR, which reinforced the reliability of the sequencing data. Silencing of these genes was capable of reducing the adhesion ability of V. alginolyticus. Adhesion of V. alginolyticus is influenced substantially by environmental factors and the TCA pathway is sensitive to some environmental stresses, especially changes in pH and starvation. Our results indicated that (1) the TCA pathway plays a key role in V. alginolyticus adhesion: (2) the TCA pathway is sensitive to environmental stresses.

Genome-Wide Detection of Predicted Non-coding RNAs Related to the Adhesion Process in Vibrio alginolyticus Using High-Throughput Sequencing

The ability of bacteria to adhere to fish mucus can be affected by environmental conditions and is considered to be a key virulence factor of Vibrio alginolyticus. However, the molecular mechanism underlying this ability remains unclear. Our previous study showed that stress conditions such as exposure to Cu, Pb, Hg, and low pH are capable of reducing the adhesion ability of V. alginolyticus. Non-coding RNAs (ncRNAs) play a crucial role in the intricate regulation of bacterial gene expression, thereby affecting bacterial pathogenicity. Thus, we hypothesized that ncRNAs play a key role in the V. alginolyticus adhesion process. To validate this, we combined high-throughput sequencing with computational techniques to detect ncRNA dynamics in samples after stress treatments. The expression of randomly selected novel ncRNAs was confirmed by QPCR. Among the significantly altered ncRNAs, 30 were up-regulated and 2 down-regulated by all stress treatments. The QPCR results reinforced the reliability of the sequencing data. Target prediction and KEGG pathway analysis indicated that these ncRNAs are closely related to pathways associated with in vitro adhesion, and our results indicated that chemical stress-induced reductions in the adhesion ability of V. alginolyticus might be due to the perturbation of ncRNA expression. Our findings provide important information for further functional characterization of ncRNAs during the adhesion process of V. alginolyticus.

Incidence of antimicrobial-resistance genes and integrons in antibiotic-resistant bacteria isolated from eels and aquaculture ponds

The overuse of antimicrobials in aquaculture has promoted the selection of antimicrobial-resistant bacteria. Here we investigated the abundance of antimicrobial-resistance genes and integrons in 108 strains of antibiotic-resistant bacteria isolated from eels and aquaculture ponds in China. Conventional PCR was implemented to examine common antibiotic-resistance genes, integrons, and their gene cassette arrays. The results showed that the antibiotic-resistance genes blaTEM, tetC, sulI, aadA, floR, and qnrB were detected at high percentages, as were a number of other resistance genes. Class I integrons were present in 79.63% of the strains, and 10 out of 108 isolates carried class II integrons. Class III integrons were not detected. Three strains carried both class I and class II integrons, and 73.26% of the class I integron-positive isolates contained the qacEΔ1/sul1 gene. Fourteen types of integron cassette arrays were found among class I integron-positive isolates. A new array, dfrB4-catB3-blaOXA-10-aadA1, was discovered in this study. The gene cassette array dfrA12-orfF-aadA2 was the most widely distributed. In summary, 23 different gene cassettes encoding resistance to 8 classes of antibiotics were identified in the class I integrons, and the main cassettes contained genes encoding resistance to aminoglycosides (aad) and trimethoprim (dfr). All class II integron-positive strains had only a single gene cassette array, viz. dfrA1-catB2-sat2-aadA1. High levels of antimicrobial-resistance genes and integrons in eels and auqauculture ponds suggest that the overuse of antimicrobials should be strictly controlled and that the levels of bacterial antimicrobial-resistance genes in aquaculture should be monitored.

Role of MshQ in MSHA pili biosynthesis and biofilm formation of Aeromonas hydrophila.

Biofilm formation of pathogen bacterium is currently one of the most widely studied topics; however, little is known regarding pathogen bacteria biofilms in aquaculture. Aeromonas hydrophila is a representative species of the genus Aeromonas, which has been recognized as a common pathogen, is associated with many diseases in aquatic animals, and causes significant mortality. The objectives of this study are i) to confirm that A. hydrophila can form biofilms on abiotic substrates and construct a biofilm growth curve for this bacterium; ii) to identify the genes that play crucial roles in A. hydrophila biofilm formation. The biofilm growth curve of A. hydrophila was constructed using a crystal violet assay, which showed that biofilm formation for this bacterium is a dynamic process. Next, a mutant library of pathogenic A. hydrophila B11 was constructed using the mini-Tn10 transposon mutagenesis system. A total of 861 mutants were screened, and 5 mutants were stably deficient in biofilm formation. Molecular analysis of the mutant B112 revealed that the open reading frame that encodes the protein MshQ was disrupted. Comparison of biological characteristics including growth, motility, and adhesion between the mutant B112 and the wild-type strain B11 suggested that MshQ is necessary for mannose-sensitive hemagglutinin pilus biosynthesis of A. hydrophila, and that these pili play crucial roles in A.hydrophila adherence to a solid surface during the early stages of biofilm formation.

Disruption of chemotaxis-related genes affects multiple cellular processes and the virulence of pathogenic Vibrio harveyi

Chemotactic motility is involved in the virulence of many pathogenic bacteria. In order to understand the role of chemotactic motility of Vibrio harveyi in cellular processes and virulence, mini-Tn10/Kan transposon-induced mutants with deficient chemotactic motility were constructed, screened, and identified. Sequence analysis revealed that the 465-bp fragment (Gen Bank accession number HM630274) flanking the transposon insertion site in mutant TS-CM1 had the highest identity (96.9%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (91.8%) with the pgk gene of V. parahaemolyticus RIMD 2210633. In another mutant, TS-CM2, 356 bp of transposon-flanking sequence (Gen Bank accession number HM630275) also showed the highest identity (94.6%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (92.4%) with the flaB gene of V. alginolyticus HY9901. Studies on virulence-related biological characteristics such as growth, motility, adhesion, and infectivity of themutants showed that disruption of either the flagellin gene or energy metabolism gene led to subsequent loss of chemotactic motility and changes in growth, motility, adhesion, and virulence of the pathogenic V. harveyi. Hence, the flagellin gene and crucial energy metabolism gene played an important role in the chemotactic motility of V. harveyi.

Contributions of the oligopeptide permeases in multistep of Vibrio alginolyticus pathogenesis

Vibrio alginolyticus has been associated with several diseases of cultivated marine animals, and has led to considerable economic losses. The oligopeptide permease (Opp) has been proven to play a variety of important roles in nutrition and virulence in several bacteria. In our previous research, the opp gene cluster was identified in Vibrio alginolyticus with transcriptome sequence, which also indicated that the Opp system might play roles in the regulation of adhesion. In this study, the relationship between V. alginolyticus virulence and the opp gene cluster was determined using gene silencing followed by RT‐qPCR, in vitro adhesion assay, growth curves detection in the presence of glutathione (GSH) as a toxic substrate, hemolysis assay, biofilm assay, and artificial infection. Silencing these genes led to deficiencies in adhesion, peptide internalization, biofilm production, hemolytic activity, and virulence. The expression levels of hapr, hapa, tlh, and hlya, which are important genes closely related to the hemolytic activity of Vibrio, were significantly downregulated in all of the RNAi groups. Furthermore, the expression of oppA, oppB, oppC, oppD, and oppF was significantly influenced by temperature, starvation, and pH. These results indicate that (1) oppABCDF contributed in multistep of V. alginolyticus pathogenesis, including adhesion, biofilm production, and hemolytic activity; (2) oppABCDF was sensitive to different temperatures, changes in pH, and increased starvation time.