S431F mutation on AChE1 and overexpression of P450 genes confer high pirimicarb resistance in Sitobion miscanthi.

Sitobion miscanthi is a destructive wheat pest responsible for significant wheat yield losses. Pirimicarb, one of the most important representatives of N, N-dimethylcarbamate insecticides, is widely used to control wheat aphids. In present work, heterozygous S431F mutation of acetylcholinesterase 1 (AChE1) was identified and verified in three pirimicarb-resistant S. miscanthi populations (two field populations (HA and HS, >955.8-fold) and one lab-selected population (PirR, 486.1-fold)), which has not been reported in S. miscanthi yet. The molecular docking results revealed that AChE1 containing the S431F mutation of S. miscanthi (SmAChE1S431F) showed higher free binding energy to three insecticides (pirimicarb, omethoate, and methomyl) than wild-type AChE1 of S. miscanthi (SmAChE1). Enzyme kinetic and inhibition experiments showed that the recombinant SmAChE1S431F was more insensitive to pirimicarb and omethoate than the recombinant SmAChE1. Furthermore, two overexpression P450 genes (CYP6K1 and CYP6A14) associated with pirimicarb resistance of S. miscanthi were verified by RNAi. These results suggested both target alteration and enhanced metabolism contributed to high pirimicarb resistance of S. miscanthi in the field and laboratory. These findings lay a foundation for further elucidating the mechanism of pirimicarb resistance in S. miscanthi, and have important implications for the resistance management of S. miscanthi control.

Effects of dimpropyridaz on feeding behavior, locomotivity and biological parameters of Aphis gossypii.

Aphis gossypii is a worldwide agricultural pest insect that has developed resistance to multiple pesticides. Dimpropyridaz is a new chordotonal organ regulator and has been registered for control of sap-sucking insects including A. gossypii. For the aim to effectively apply dimpropyridaz for A. gossypii control, it is necessary to clarify the toxic effects of dimpropyridaz on cotton aphids. In the present study, the effects of dimpropyridaz on feeding behavior, locomotivity and biological parameters of A. gossypii were investigated. The bioassay results showed that dimpropyridaz had good insecticidal activity against A. gossypii, with LC50 as 1.91 mg/L at 72 h post exposure. Moreover, the dimpropyridaz treated A. gossypii showed obvious poisoning symptoms of dehydration and shrivel. Through the gentle-touch experiment and feeding experiment, it was found that dimpropyridaz treatment had significant adverse impacts on the locomotivity and feeding behavior of A. gossypii. Compared with the control group, the coordinated movement ability of the treated A. gossypii attenuated, moreover the feeding behavior of A. gossypii was inhibited. The feeding rate decreased by 62.00%, 64.00% and 71.67% after treatment with 50.33 mg/L dimpropyridaz for 24 h, 48 h and 72 h, respectively. Especially, EPG recordings showed that the number of intracellular stylet puncture and the total duration of phloem sap ingestion and concurrent salivation decreased substantially, while the total duration of non-probing increased after exposure to dimpropyridaz. Furthermore, the treatments with LC10 and LC30 of dimpropyridaz significantly reduced the longevity and fecundity of F0, and led to a decrease of the relative fitness of F0 to 0.48 and 0.32, respectively. The net reproductive rate (R0) and mean generation time (T) of F1 generation were also significantly reduced, moreover the duration of reproduction was significantly shortened. In addition, at 72 h post treatment with LC30 dimpropyridaz, the gene expression levels of JHEH and USP of cotton aphids significantly increased, while the expression of FOXO, INR, EcR and INRS decreased. These results provide basis for clarifying the toxicology of dimpropyridaz to cotton aphids, and also are beneficial for effective control of cotton aphid using dimpropyridaz.

Cross-Resistance and Fitness Costs of the cis-Nitromethylene Neonicotinoid Cycloxaprid Resistance in Melon Aphid, Aphis gossypii (Hemiptera: Aphididae).

The melon aphid, Aphis gossypii Glover, is an important pest on various vegetables around the world and has developed resistance to neonicotinoids in fields. Cycloxaprid is a novel cis-nitromethylene configuration neonicotinoid insecticide that is different from trans-configuration neonicotinoids like imidacloprid and thiamethoxam. Herein, the cross-resistance to the other seven insecticides and fitness costs were investigated in the cycloxaprid-resistant A. gossypii strain (Cpd-R), which has developed 69.5-fold resistance to cycloxaprid. The results showed that the Cpd-R strain had very low levels of cross-resistance to imidacloprid (4.3-fold), acetamiprid (2.9-fold), thiamethoxam (3.7-fold), nitenpyram (6.1-fold), flupyradifurone (2.2-fold), and sulfoxaflor (4.5-fold), while it exhibited a cross-resistance to dinotefuran (10.6-fold). The fitness of the Cpd-R strain by life table analysis was only 0.799 compared to the susceptible strain (Cpd-S). This Cpd-R strain exhibited significantly reduction in fecundity, oviposition days, and developmental time of nymph stage compared to the Cpd-S strain. Moreover, the expression levels of some genes related to the development and reproduction, including EcR, USP, JHAMT, and JHEH were significantly up-regulated, while Vg was down-regulated in the Cpd-R strain. This study indicates that the Cpd-R strain possessed a certain fitness cost. The above research results are useful for rational application of cycloxaprid and implementing the appropriate resistance management strategy for A. gossypii.

CYP4CJ6-mediated resistance to two neonicotinoid insecticides in Sitobion miscanthi (Takahashi).

The wheat aphid Sitobion miscanthi (CWA) is an important harmful pest in wheat fields. Insecticide application is the main method to effectively control wheat aphids. However, CWA has developed resistance to some insecticides due to its extensive application, and understanding resistance mechanisms is crucial for the management of CWA. In our study, a new P450 gene, CYP4CJ6, was identified from CWA and showed a positive response to imidacloprid and thiamethoxam. Transcription of CYP4CJ6 was significantly induced by both imidacloprid and thiamethoxam, and overexpression of CYP4CJ6 in the imidacloprid-resistant strain was also observed. The sensitivity of CWA to these two insecticides was increased after the knockdown of CYP4CJ6. These results indicated that CYP4CJ6 could be associated with CWA resistance to imidacloprid and thiamethoxam. Subsequently, the posttranscriptional regulatory mechanism was assessed, and miR-316 was confirmed to participate in the posttranscriptional regulation of CYP4CJ6. These results are crucial for clarifying the roles of P450 in the resistance of CWA to insecticides.

Mutations in the nAChR ß1 subunit and overexpression of P450 genes are associated with high resistance to thiamethoxam in melon aphid, Aphis gossypii Glover.

The TMXR is a strain of melon aphids (Aphis gossypii Glover) that has extremely high resistance (resistance ratio > 2300 fold) to thiamethoxam. We explored the basis of this resistance by examining differences in nicotinic acetylcholine receptors (nAChRs) and cytochrome P450 monooxygenase (CYP450s) between the TMXR and the susceptible strain. The results showed that two mutation sites of nAChR ß1 subunit, V62I and R81T, were found in TMXR, with the mutation frequencies of the two mutation sites as 93.75%. Meanwhile, compared with the susceptible strain, the expression level of nAChR ß1 subunit gene in the TMXR decreased by 38%. In addition, piperonyl butoxide (PBO) showed a synergistic ratio of 17.78-fold on TMX toxicity against the TMXR, which suggested the involvement of CYP450s in the TMX resistance of melon aphid. Moreover, the expression levels of 4 P450s genes were significantly higher in the TMXR than the susceptible strain. Through RNAi, we verified that down-regulating CYP6DA1 increased the sensitivity of TMXR to TMX toxicity, demonstrating that a decrease in CYP6DA1 expression may reduce resistance in vivo. These results suggest that A. gossypii has the capacity to develop extremely high resistance to TMX through aggregated resistance mechanisms including enhancement of detoxification by upregulation of CYP450s, and target insensitivity caused by alteration of nAChR ß1 subunit with mutation and low expression. These findings provide basic information for further clarifying the molecular mechanism of insecticide resistance in A. gossypii.

Identification of differentially expressed microRNAs under imidacloprid exposure in Sitobion miscanthi.

Imidacloprid is a neonicotinoid that targets sucking pests, such as aphids and the green leaf bug and has been widely applied in wheat fields to control wheat aphids in China. To investigate the involvement of miRNAs in imidacloprid resistance, we sequenced small RNA libraries of Sitobion miscanthi Fabricius, across two different treatments using Illumina short-read sequencing technology. As a result, 265 microRNAs (miRNAs), of which 242 were known and 23 were novel, were identified. Quantitative analysis of miRNA levels showed that 23 miRNAs were significantly up-regulated, and 54 miRNAs were significantly down-regulated in the nymphs of S. miscanthi treated with imidacloprid in comparison with those of the control. Modulation of the abundances of differentially expressed miRNAs, smi-miR-316, smi-miR-1000, and smi-miR-iab-4 by the addition of the corresponding antagomir/inhibitor to the artificial diet significantly changed the susceptibility of S. miscanthi to imidacloprid. Subsequently, the post-transcriptional regulatory mechanism was conducted, smi-miR-278 and smi-miR-316 were confirmed to be participated in the post-transcriptional regulation of nAChRα1A and CYP4CJ6, respectively. The results suggested that miRNAs differentially expressed in response to imidacloprid could play a critical regulatory role in the metabolism of S. miscanthi to imidacloprid.

Comparative genetic analyses provide clues about capsule switching in Streptococcus suis 2 strains with different virulence levels and genetic backgrounds.

Streptococcus suis (S. suis) is a major bacterial pathogen in the swine industry and an emerging zoonotic agent. S. suis produces an important extracellular component, capsular polysaccharide (CPS), based on which dozens of serotypes have been identified. Through virulence genotyping, we revealed the relatedness between subpopulations of S. suis serotype 2 (SS2), S. suis serotype 3 (SS3) and S. suis serotype 7 (SS7) strains despite their serotype differences. Multilocus sequence typing (MLST) was used to characterize the whole S. suis population and revealed capsule switching between S. suis strains. Importantly, capsule switching occurred in the SS2, SS3 and SS7 strains belonging to CC28 and CC29, which are phylogenetically distinct from the main CC1 SS2 lineage. To further explore capsule switching in S. suis, comparative genomic analyses were performed using available complete S. suis genomes. Phylogenetic analyses suggested that the SS2 strains could be divided into two clades (1 and 2), and those classified into clade 2 colocalized with SS3 and SS7 strains, in accordance with the above virulence genotyping and MLST analyses. Clade 2 SS2 strains presented high genetic similarity to SS3 and SS7 and shared common competence and defensive elements with them but were significantly different from Clade 1 SS2 strains. Notably, although the cps loci shared by Clade 1 and 2 SS2 strains were almost identical, a specific region of the cps locus of strain NSUI002 (Clade 2 SS2) could be found in the SS3 cps locus but not in the Clade 1 SS2 strain. These data indicated that the SS2 strains in CC28 and CC29 might have acquired the cps locus through capsule switching, which could explain the distinct genetic lineages within the SS2 population.

CrfP, a fratricide protein, contributes to natural transformation in Streptococcus suis.

Streptococcus suis (S. suis) is an important zoonotic pathogen that causes septicaemia, meningitis and streptococcal toxic shock-like syndrome in its host, and recent studies have shown that S. suis could be competent for natural genetic transformation. Transformation is an important mechanism for the horizontal transfer of DNA, but some elements that affect the transformation process need to be further explored. Upon entering the competent state, Streptococcus species stimulate the transcription of competence-related genes that are responsible for exogenous DNA binding, uptake and processing. In this study, we performed conserved promoter motif and qRT-PCR analyses and identified CrfP as a novel murein hydrolase that is widespread in S. suis and stimulated with a peptide pheromone in the competent state through a process controlled by ComX. A bioinformatics analysis revealed that CrfP consists of a CHAP hydrolase domain and two bacterial Src homology 3-binding (SH3b) domains. Further characterization showed that CrfP could be exported to extracellular bacterial cells and lytic S. suis strains of different serotypes, and this finding was verified by TEM and a turbidity assay. To investigate the potential effect of CrfP in vivo, a gene-deletion mutant (ΔcrfP) was constructed. Instead of stopping the natural transformation process, the inactivation of CrfP clearly reduced the effective transformation rate. Overall, these findings provide evidence showing that CrfP is important for S. suis serovar 2 competence.

Cross-resistance and Fitness Cost Analysis of Resistance to Thiamethoxam in Melon and Cotton Aphid (Hemiptera: Aphididae).

The melon/cotton aphid, Aphis gossypii Glover, is a notorious pest in many crops. The neonicotinoid insecticide thiamethoxam is widely used for A. gossypii control. To evaluate thiamethoxam resistance risk, a melon/cotton aphid strain with an extremely high level of resistance to thiamethoxam (>2,325.6-fold) was established after selection with thiamethoxam for 24 generations. Additionally, the cross-resistance pattern to other neonicotinoids and fitness were analyzed. The cross-resistance results showed the thiamethoxam-resistant strain had extremely high levels of cross-resistance against clothianidin (>311.7-fold) and nitenpyram (299.9-fold), high levels of cross-resistance against dinotefuran (142.3-fold) and acetamiprid (76.6-fold), and low cross-resistance against imidacloprid (9.3-fold). Compared with the life table of susceptible strain, the thiamethoxam-resistant strain had a relative fitness of 0.950, with significant decreases in oviposition days and fecundity and prolonged developmental duration. The molecular mechanism for fitness costs was studied by comparing the mRNA expression levels of juvenile hormone acid O-methyltransferase (JHAMT), juvenile hormone-binding protein (JHBP), juvenile hormone epoxide hydrolase (JHEH), ecdysone receptor (EcR), ultraspiracle protein (USP), and Vitellogenin (Vg) in the susceptible and thiamethoxam-resistant strains. Significant overexpression of JHEH and JHBP and downregulation of EcR and Vg expression were found in the thiamethoxam-resistant strain. These results indicate that A. gossypii has the potential to develop extremely high resistance to thiamethoxam after continuous exposure, with a considerable fitness cost and cross-resistance to other neonicotinoids.

Streptococcus pneumoniae Infection Promotes Histone H3 Dephosphorylation by Modulating Host PP1 Phosphatase.

Pathogenic bacteria can alter host gene expression through post-translational modifications of histones. We show that a natural colonizer, Streptococcus pneumoniae, induces specific histone modifications, including robust dephosphorylation of histone H3 on serine 10 (H3S10), during infection of respiratory epithelial cells. The bacterial pore-forming toxin pneumolysin (PLY), along with the pyruvate oxidase SpxB responsible for H2O2 production, play important roles in the induction of this modification. The combined effects of PLY and H2O2 trigger host signaling that culminates in H3S10 dephosphorylation, which is mediated by the host cell phosphatase PP1. Strikingly, S. pneumoniae infection induces dephosphorylation and subsequent activation of PP1 catalytic activity. Colonization of PP1 catalytically deficient cells results in impaired intracellular S. pneumoniae survival and infection. Interestingly, PP1 activation and H3S10 dephosphorylation are not restricted to S. pneumoniae and appear to be general epigenomic mechanisms favoring intracellular survival of pathogenic bacteria.

Revealing eukaryotic histone-modifying mechanisms through bacterial infection.

In the long co-evolution of host-pathogen interaction, bacteria have developed sophisticated strategies to manipulate host cell mechanisms and reprogram host transcription. Targeting chromatin, mainly through post-translational modification (PTM) of histone proteins, is one strategy that has been revealed over the last decade. Indeed, histone modifications play a crucial role in regulating transcription during cell type and stimulus specific responses, making them good targets during infection. Therefore, the study of host-pathogen interactions provides breakthroughs in understanding virulence mechanisms, but also in host cell mechanisms. Although chromatin is regulated by DNA methylation, noncoding RNAs, and post-translational modifications of histones, most studies have concentrated on bacteria-induced histone modifications, which will be the focus of this review. We will discuss the different mechanisms used by bacteria to induce histone PTMs, whether it is through direct targeting of pathogen effector enzymes, or indirectly through modulation of cellular signaling cascade. We will summarize the concepts we learned in cell biology from exploring bacteria-triggered histone modifications, by focusing on the signaling cascades modified by bacteria, bacterial mimics of eukaryotic enzymes, and the novel histone marks imposed upon infection.

Identification of an Autorepressing Two-Component Signaling System That Modulates Virulence in Streptococcus suis Serotype 2.

Streptococcus suis is one of the most important pathogens affecting the swine industry and is also an emerging zoonotic agent for humans. Two-component signaling systems (TCSs) play important roles in the adaptation of pathogenic bacteria to host environments. In this study, we identified a novel TCS, named TCS09HKRR, which facilitated Streptococcus suis serotype 2 (SS2) resistance to clearance by the host immune system and contributed to bacterial pathogenicity. Furthermore, RNA-sequencing analyses identified 79 genes that were differentially expressed between the wild-type (WT) and ΔTCS09HKRR strains, among which half of the 39 downregulated genes belonged to the capsular biosynthesis clusters. Transmission electron microscopy confirmed that the capsule of the ΔTCS09HKRR strain was thinner than that of the WT strain. Electrophoretic mobility shift assays (EMSA) showed that the regulator of TCS09HKRR (TCS09RR) could not bind the promoter regions of cps and neu clusters, which suggested that TCS09HKRR regulates capsule biosynthesis by indirect pathways. Unexpectedly, the TCS09HKRR operon was upregulated when TCS09HKRR was deleted. A specific region, ATGACATTTGTCAC, which extends from positions -193 to -206 upstream of the TCS09HKRR operon, was further identified as the TCS09RR-binding site using EMSA. These results suggested the involvement of a negative feedback loop in this regulation. In addition, TCS09RR was significantly upregulated by more than 18-fold when coincubated with RAW264.7 macrophages. Our data suggested that autorepression renders TCS09HKRR more sensitive to host stimuli, which optimizes the regulatory network of capsular biosynthesis in SS2.

A novel autolysin AtlASS mediates bacterial cell separation during cell division and contributes to full virulence in Streptococcus suis.

Streptococcus suis (SS) is a major pathogen in the swine industry, and also an important zoonotic agent for humans. The novel SS cell surface protein, AtlASS, comprising the special GW module and N-acetylmuramidases domain, was designated as a putative autolysin. Indeed, the atlASS deletion mutant almost completely lost its activity in Triton X-100 induced bacterial autolysis, while the wild-type and CΔatlASS strains showed significant decrease, to less than 20% of the initial OD600 values. Unexpectedly, both immunofluorescence and immunogold electron microscopy confirmed that AtlASS is mainly located in the cell division septum, suggesting autolytic activity in peptidoglycan hydrolysis may be required for cell separation, thus modulating and truncating bacterial chain length. The biofilm capacity of the AtlASS mutation was reduced ˜ 40%, as compared to the wild-type strain. The ΔatlASS strain also attenuated bacterial adherence in human brain microvessel endothelial cells (HBMECs). Furthermore, we confirmed that AtlASS has fibrinogen/fibronectin binding capacities. In mouse infection model, the AtlASS inactivation also significantly attenuated bacterial virulence and proliferation in vivo. In conclusion, these results indicate that AtlASS autolysin modulates bacterial chain length, and contributes to the full virulence of SS during infection.

ICESsuHN105, a Novel Multiple Antibiotic Resistant ICE in Streptococcus suis Serotype 5 Strain HN105.

Streptococcus suis serotype 5, an emerging zoonosis bacterial pathogen, has been isolated from infections in both pigs and humans. In this study, we sequenced the first complete genome of a virulent, multidrug-resistant SS5 strain HN105. The strain HN105 displayed enhanced pathogenicity in zebrafish and BABL/c mouse infection models. Comparative genome analysis identified a novel 80K integrative conjugative element (ICE), ICESsuHN105, as required for the multidrug resistance phenotype. Six corresponding antibiotic resistance genes in this ICE were identified, namely tet (O), tet (M), erm (two copies), aph, and spc. Phylogenetic analysis classified the element as a homolog of the ICESa2603 family, containing the typical family backbone and insertion DNA. DNA hybrids mediated by natural transformation between HN105 and ZY05719 verified the antibiotic resistant genes of ICESsuHN105 that could be transferred successfully, while they were dispersedly inserted with a single gene in different genomic locations of ZY05719(HN105) transformants. To further identify the horizontal transfer of ICESsuHN105 as a whole mobile genetic element, a circular intermediate form of ICESsuHN105 was detected by PCR. However, the effective conjugation using serotype 2 S. suis as recipients was not observed in current assays in vitro. Further studies confirmed the presence of the complete lantibiotic locus encoded in ICESsuHN105 that effectively inhibits the growth of other streptococci. In summary, this study demonstrated the presence of antibiotic resistance genes in ICE that are able to transfer between different clinical isolates and adapt to a broader range of Streptococcus serotype or species.

Utilization of the ComRS system for the rapid markerless deletion of chromosomal genes in Streptococcus suis.

AIM: To develop a markerless gene deletion strategy in Streptococcus suis to solve the problem that several serotypes against electrotransformation of foreign DNA. MATERIALS & METHODS: Bioinformatics retrieval was performed to identified ComRS systems functioning for natural transformation. A sacB-spc cassette with the upper and lower homologous fragments was amplification by fusion-PCR for spectinomycin-positive and sucrose-negative selection during gene deletion. RESULTS & CONCLUSION: Three phylogenetic clusters of ComR were identified to function for natural transformation by specific recognition to competence pheromone in S. suis. Thus, they were employed to establish gene deletion method. Its efficiency for genetic replacement was dependent on the length of homologs fragment and the concentration of donor DNA. This rapid gene-editing technique may greatly facilitate molecular studies on S. suis.

A novel integrative conjugative element mediates transfer of multi-drug resistance between Streptococcus suis strains of different serotypes.

Streptococcus suis represents a key antibiotic resistance gene reservoir and an important pathogen for humans and animals. Resistance can be spread through horizontal gene transfer of chromosome-borne mobile genetic elements; however, the exact mechanism by which this occurs remains poorly understood. In the present study, we identified and characterized a novel 82-kb integrative conjugative element (ICE) named ICESsuCZ130302 from the virulent S. suis strain CZ130302. It carries genes that provide resistance to multiple antibiotics, such as tetracycline, doxycycline, erythromycin, lincomycin, neomycin, and kanamycin. It also contains a nisin biosynthesis gene cluster, a toxin-antitoxin system, a type IV secretion system, and an integrase and excisase system. The mobile element can be excised from the chromosome, circulized, and transferred via conjugation from serotype Chz strain CZ130302 to serotype 2 strain P1/7, where it confers resistance to the aforementioned antimicrobial agents. The full length ICE, where multiple antimicrobial resistance genes accumulated, was further identified to be naturally transferred between different serotypes strains of S. suis. This finding illustrates how such elements represent a potential means by which antimicrobial resistance is introduced to a wide range of bacteria of veterinary and medical significance.

SssP1, a Streptococcus suis Fimbria-Like Protein Transported by the SecY2/A2 System, Contributes to Bacterial Virulence.

Streptococcus suis is an important Gram-positive pathogen in the swine industry and is an emerging zoonotic pathogen for humans. In our previous work, we found a virulent S. suis strain, CZ130302, belonging to a novel serotype, Chz, to be associated with acute meningitis in piglets. However, its underlying mechanisms of pathogenesis remain poorly understood. In this study, we sequenced and analyzed the complete genomes of three Chz serotype strains, including strain CZ130302 and two avirulent strains, HN136 and AH681. By genome comparison, we found two putative genomic islands (GIs) uniquely encoded in strain CZ130302 and designated them 50K GI and 58K GI. In mouse infection model, the deletion of 50K and 58K GIs caused 270-fold and 3-fold attenuation of virulence, respectively. Notably, we identified a complete SecY2/A2 system, coupled with its secretory protein SssP1 encoded in the 50K GI, which contributed to the pathogenicity of strain CZ130302. Immunogold electron microscopy and immunofluorescence analyses indicated that SssP1 could form fimbria-like structures that extend outward from the bacterial cell surface. The sssP1 mutation also attenuated bacterial adherence in human laryngeal epithelial (HEp-2) cells and human brain microvessel endothelial cells (HBMECs) compared with the wild type. Furthermore, we showed that two analogous Ig-like subdomains of SssP1 have sialic acid binding capacities. In conclusion, our results revealed that the 50K GI and the inside SecY2/A2 system gene cluster are related to the virulence of strain CZ130302, and we clarified a new S. suis pathogenesis mechanism mediated by the secretion protein SssP1.IMPORTANCEStreptococcus suis is an important zoonotic pathogen. Here, we managed to identify key factors to clarify the virulence of S. suis strain CZ130302 from a novel serotype, Chz. Notably, it was shown that a fimbria-like structure was significantly connected to the pathogenicity of the CZ130302 strain by comparative genomics analysis and animal infection assays. The mechanisms of how the CZ130302 strain constructs these fimbria-like structures in the cell surface by genes encoding and production transport were subsequently elucidated. Biosynthesis of the fimbria-like structure was achieved by the production of SssP1 glycoproteins, and its construction was dependent on the SecA2/Y2 secretion system. This study identified a visible fimbria-like protein, SssP1, participating in adhesion to host cells and contributing to the virulence in S. suis These findings will promote a better understanding of the pathogenesis of S. suis.

The Two-Component Signaling System VraSRss Is Critical for Multidrug Resistance and Full Virulence in Streptococcus suis Serotype 2.

Streptococcus suis has received increasing attention for its involvement in severe human infections worldwide as well as in multidrug resistance. Two-component signaling systems (TCSSs) play important roles in bacterial adaptation to various environmental stimuli. In this study, we identified a novel TCSS located in S. suis serotype 2 (SS2), designated VraSRSS, which is involved in bacterial pathogenicity and susceptibility to antimicrobials. Our data demonstrated that the yvqFSS gene, located upstream of vraSRSS , shared the same promoter with the TCSS genes, which was directly regulated by VraSRSS, as shown in electrophoretic mobility shift assays. Notably, YvqFSS and VraSRSS constitute a novel multidrug resistance module of SS2 that participates in resistance to certain groups of antimicrobials. Further analyses showed that VraSRSS inactivation significantly attenuated bacterial virulence in animal models, which, coupled with the significant activation of VraSRSS expression observed in host blood, strongly suggested that VraSRSS is an important regulator of SS2 pathogenicity. Indeed, RNA-sequencing analyses identified 106 genes that were differentially expressed between the wild-type and ΔvraSRSS strains, including genes involved in capsular polysaccharide (CPS) biosynthesis. Subsequent studies confirmed that VraSRSS indirectly regulated the transcription of CPS gene clusters and, thus, controlled the CPS thickness shown by transmission electron microscopy. Decreased CPS biosynthesis caused by vraSRSS deletion subsequently increased bacterial adhesion to epithelial cells and attenuated antiphagocytosis against macrophages, which partially clarified the pathogenic mechanism mediated by VraSRSS Taken together, our data suggest that the novel TCSS, VraSRSS, plays critical roles for multidrug resistance and full virulence in SS2.

Multilocus sequence typing and virulence genotyping of Streptococcus suis serotype 9 isolates revealed high genetic and virulence diversity.

Streptococcus suis is an important swine pathogen that can cause a variety of diseases. Streptococcus suis serotype 9 (SS9) is a prevalent serotype, but limited information is available. Here, we studied and compared 30 SS9 isolates, including 24 isolates from China between year 2004 and 2013, 5 isolates from Vietnam and a serotype reference isolate from Denmark. A multilocus sequence typing (MLST) analysis was performed to exploit the genetic relationships between those isolates. The phylogenetic tree based on the MLST data divides those isolates into two clades (I and II), revealing different evolutionary paths of collected strains. A virulence genotyping analysis was performed by detecting 23 virulence-related genes; the clustering result also revealed two clusters (I and II), highly in accordance with MLST analysis result, showing that phylogenetic relatedness led to the presence of similar virulence genotypes. Murine model infection experiment was performed to assess the virulence of those strains in cluster I and cluster II, which displayed high virulence diversity even within a same cluster/ST. Notably, ST 243 could be regarded as an ST with high virulence potential in SS9. In conclusion, this study has revealed high genetic and virulence diversity in SS9 isolates.

Characterization and virulence clustering analysis of extraintestinal pathogenic Escherichia coli isolated from swine in China.

BACKGROUND: Swine extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen that leads to economic and welfare costs in the swine industry worldwide, and is occurring with increasing frequency in China. By far, various virulence factors have been recognized in ExPEC. Here, we investigated the virulence genotypes and clonal structure of collected strains to improve the knowledge of phylogenetic traits of porcine ExPECs in China. RESULTS: We isolated 64 Chinese porcine ExPEC strains from 2013 to 14 in China. By multiplex PCR, the distribution of isolates belonging to phylogenetic groups B1, B2, A and D was 9.4%, 10.9%, 57.8% and 21.9%, respectively. Nineteen virulence-related genes were detected by PCR assay; ompA, fimH, vat, traT and iutA were highly prevalent. Virulence-related genes were remarkably more prevalent in group B2 than in groups A, B1 and D; notably, usp, cnf1, hlyD, papA and ibeA were only found in group B2 strains. Genotyping analysis was performed and four clusters of strains (named I to IV) were identified. Cluster IV contained all isolates from group B2 and Cluster IV isolates had the strongest pathogenicity in a mouse infection model. As phylogenetic group B2 and D ExPEC isolates are generally considered virulent, multilocus sequence typing (MLST) analysis was performed for these isolates to further investigate genetic relationships. Two novel sequence types, ST5170 and ST5171, were discovered. Among the nine clonal complexes identified among our group B2 and D isolates, CC12 and CC95 have been indicated to have high zoonotic pathogenicity. The distinction between group B2 and non-B2 isolates in virulence and genotype accorded with MLST analysis. CONCLUSION: This study reveals significant genetic diversity among ExPEC isolates and helps us to better understand their pathogenesis. Importantly, our data suggest group B2 (Cluster IV) strains have the highest risk of causing animal disease and illustrate the correlation between genotype and virulence.