Recombinase polymerase amplification combined with lateral flow biosensor for rapid visual detection of Clostridium perfringens in chicken meat and milk.

Aims: Clostridium perfringens is one of the major anaerobic pathogen causing food poisoning and animal enteritis. With the rise of antibiotic resistance and the restrictions of the use of antibiotic growth promoting agents (AGPs) in farming, Clostridium enteritis and food contamination have become more common. It is time-consuming and labor-intensive to confirm the detection by standard culture methods, and it is necessary to develop on-site rapid detection tools. In this study, a combination of recombinase polymerase amplification (RPA) and lateral flow biosensor (LFB) was used to visually detect C. perfringens in chicken meat and milk. Methods and results: Two sets of primers were designed for the plc gene of C. perfringens, and the amplification efficiency and specificity of the primers. Selection of primers produces an amplified fragment on which the probe is designed. The probe was combined with the lateral flow biosensor (LFB). The reaction time and temperature of RPA-LFB assay were optimized, and the sensitivity of the assay was assessed. Several common foodborne pathogens were selected to test the specificity of the established method. Chicken and milk samples were artificially inoculated with different concentrations (1 × 102 CFU/mL to 1 × 106 CFU/mL) of C. perfringens, and the detection efficiency of RPA-LFB method and PCR method was compared. RPA-LFB can be completed in 20 min and the results can be read visually by the LFB test strips. The RPA-LFB has acceptable specificity and the lowest detection limit of 100 pg./µL for nucleic acid samples. It was able to stably detect C. perfringens contamination in chicken and milk at the lowest concentration of 1 × 104 CFU/mL and 1 × 103 CFU/mL, respectively. Conclusion: In conclusion, RPA-LFB is specific and sensitive. It is a rapid, simple and easy-to-visualize method for the detection of C. perfringens in food and is suitable for use in field testing work.

Combined Immunoinformatics to Design and Evaluate a Multi-Epitope Vaccine Candidate against Streptococcus suis Infection.

Streptococcus suis (S. suis) is a zoonotic pathogen with multiple serotypes, and thus, multivalent vaccines generating cross-protection against S. suis infections are urgently needed to improve animal welfare and reduce antibiotic abuse. In this study, we established a systematic and comprehensive epitope prediction pipeline based on immunoinformatics. Ten candidate epitopes were ultimately selected for building the multi-epitope vaccine (MVSS) against S. suis infections. The ten epitopes of MVSS were all derived from highly conserved, immunogenic, and virulence-associated surface proteins in S. suis. In silico analyses revealed that MVSS was structurally stable and affixed with immune receptors, indicating that it would likely trigger strong immunological reactions in the host. Furthermore, mice models demonstrated that MVSS elicited high titer antibodies and diminished damages in S. suis serotype 2 and Chz infection, significantly reduced sequelae, induced cytokine transcription, and decreased organ bacterial burdens after triple vaccination. Meanwhile, anti-rMVSS serum inhibited five important S. suis serotypes in vitro, exerted beneficial protective effects against S. suis infections and significantly reduced histopathological damage in mice. Given the above, it is possible to develop MVSS as a universal subunit vaccine against multiple serotypes of S. suis infections.

Novel Flavonol Alkaloids in Green Tea: Synthesis, Detection, and Anti-Alzheimer's Disease Effect in a Transgenic Caenorhabditis elegans CL4176 Model.

Novel N-ethy-2-pyrrolidinone-substituted flavonols, myricetin alkaloids A-C (1-3), quercetin alkaloids A-C (4a, 4b, and 5), and kaempferol alkaloids A and B (6 and 7), were prepared from thermal reaction products of myricetin, quercetin, kaempferol─l-theanine, respectively. We used HPLC-ESI-HRMS/MS to detect 1-7 in 14 cultivars of green tea and found that they were all present in "Shuchazao," "Longjing 43", "Fudingdabai", and "Zhongcha 108" green teas. The structures of 1-4 and 6 were determined by extensive 1D and 2D NMR spectroscopies. These flavonol alkaloids along with their skeletal flavonols were assessed for anti-Alzheimer's disease effect based on molecular docking, acetylcholinesterase inhibition, and the transgenic Caenorhabditis elegans CL4176 model. Compound 7 strongly binds to the protein amyloid ß (Aß1-42) through hydrogen bonds (BE: -9.5 kcal/mol, Ki: 114.3 nM). Compound 3 (100 µM) is the strongest one in significantly extending the mean lifespan (13.4 ± 0.5 d, 43.0% promotion), delaying the Aß1-42-induced paralysis (PT50: 40.7 ± 1.9 h, 17.1% promotion), enhancing the locomotion (140.0% promotion at 48 h), and alleviating glutamic acid (Glu)-induced neurotoxicity (153.5% promotion at 48 h) of CL4176 worms (p < 0.0001).

Streptococcal arginine deiminase system defences macrophage bactericidal effect mediated by XRE family protein XtrSs.

The arginine deiminase system (ADS) has been identified in various bacteria and functions to supplement energy production and enhance biological adaptability. The current understanding of the regulatory mechanism of ADS and its effect on bacterial pathogenesis is still limited. Here, we found that the XRE family transcriptional regulator XtrSs negatively affected Streptococcus suis virulence and significantly repressed ADS transcription when the bacteria were incubated in blood. Electrophoretic mobility shift (EMSA) and lacZ fusion assays further showed that XtrSs directly bind to the promoter of ArgR, an acknowledged positive regulator of bacterial ADS, to repress ArgR transcription. Moreover, we provided compelling evidence that S. suis could utilize arginine via ADS to adapt to acid stress, while ΔxtrSs enhanced this acid resistance by upregulating the ADS operon. Moreover, whole ADS-knockout S. suis increased arginine and antimicrobial NO in the infected macrophage cells, decreased intracellular survival, and even caused significant attenuation of bacterial virulence in a mouse infection model, while ΔxtrSs consistently presented the opposite results. Our experiments identified a novel ADS regulatory mechanism in S. suis, whereby XtrSs regulated ADS to modulate NO content in macrophages, promoting S. suis intracellular survival. Meanwhile, our findings provide a new perspective on how Streptococci evade the host's innate immune system.

Discovery of novel phenanthridone derivatives with anti-streptococcal activity.

To address the growing health threat posed by drug-resistant pathogenic microorganisms, the development of novel antimicrobial medications with multiple mechanisms of action is in urgent demand. With traditional antibacterial drug resources challenging to push forward, developing new antibacterial drugs has become a hot spot in biomedical research. In this study, we tested the antibacterial activity of 119 phenanthridine derivatives via the antibacterial assay and obtained 5 candidates. The cytotoxicity assay showed one phenanthridine derivative, HCK20, was safe for mammalian cells below 125 µM. HCK20 was verified to possess significant antibacterial activity to Streptococcus spp., such as Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis, Streptococcus dysgalactiae, and Streptococcus equi with MICs ranging from 15 to 60 µM. Furthermore, we found that HCK20 probably achieved its bacterial inhibition by influencing the permeability of bacterial cell walls via interacting with Streptococcal penicillin-binding proteins (PBPs). Our results suggest that this phenanthridine derivative, HCK20, has great potential to become a novel antibacterial agent that can be a potent treatment for streptococcal infections.

luxS contributes to intramacrophage survival of Streptococcus agalactiae by positively affecting the expression of fruRKI operon.

The LuxS quorum sensing system is a widespread system employed by many bacteria for cell-to-cell communication. The luxS gene has been demonstrated to play a crucial role in intramacrophage survival of piscine Streptococcus agalactiae, but the underlying mechanism remains largely unknown. In this study, transcriptome analysis, followed by the luxS gene deletion and subsequent functional studies, confirmed that impaired bacterial survival inside macrophages due to the inactivation of luxS was associated with reduced transcription of the fruRKI operon, encoding the fructose-specific phosphotransferase system. Further, luxS was determined not to enhance the transcription of fruRKI operon by binding its promoter, but to upregulate the expression of this operon via affecting the binding ability of catabolite control protein A (CcpA) to the catabolite responsive element (cre) in the promoter of fruRKI. Collectively, our study identifies a novel and previously unappreciated role for luxS in bacterial intracellular survival, which may give a more thorough understanding of the immune evasion mechanism in S. agalactiae.

Conjugative transfer of streptococcal prophages harboring antibiotic resistance and virulence genes.

Prophages play important roles in the transduction of various functional traits, including virulence factors, but remain debatable in harboring and transmitting antimicrobial resistance genes (ARGs). Herein we characterize a prevalent family of prophages in Streptococcus, designated SMphages, which harbor twenty-five ARGs that collectively confer resistance to ten antimicrobial classes, including vanG-type vancomycin resistance locus and oxazolidinone resistance gene optrA. SMphages integrate into four chromosome attachment sites by utilizing three types of integration modules and undergo excision in response to phage induction. Moreover, we characterize four subtypes of Alp-related surface proteins within SMphages, the lethal effects of which are extensively validated in cell and animal models. SMphages transfer via high-frequency conjugation that is facilitated by integrative and conjugative elements from either donors or recipients. Our findings explain the widespread of SMphages and the rapid dissemination of ARGs observed in members of the Streptococcus genus.

Characterization of G-type Clostridium perfringens bacteriophages and their disinfection effect on chicken meat.

OBJECTIVE: Clostridium perfringens is one of most important bacterial pathogens in the poultry industry and mainly causes necrotizing enteritis (NE). This pathogen and its toxins can cause foodborne diseases in humans through the food chain. In China, with the rise of antibiotic resistance and the banning of antibiotic growth promoters (AGPs) in poultry farming, food contamination and NE are becoming more prevalent. Bacteriophages are a viable technique to control C. perfringens as an alternative to antibiotics. We isolated Clostridium phage from the environment, providing a new method for the prevention of NE and C. perfringens contamination in meat. METHODS: In this study, we selected C. perfringens strains from various regions and animal sources in China for phage isolation. The biological characteristics of Clostridium phage were studied in terms of host range, MOI, one-step curve, temperature and pH stability. We sequenced and annotated the genome of the Clostridium phage and performed phylogenetic and pangenomic analyses. Finally, we studied its antibacterial activity against bacterial culture and its disinfection effect against C. perfringens in meat. RESULTS: A Clostridium phage, named ZWPH-P21 (P21), was isolated from chicken farm sewage in Jiangsu, China. P21 has been shown to specifically lyse C. perfringens type G. Further analysis of basic biological characteristics showed that P21 was stable under the conditions of pH 4-11 and temperature 4-60 °C, and the optimal multiple severity of infection (MOI) was 0.1. In addition, P21 could form a "halo" on agar plates, suggesting that the phage may encode depolymerase. Genome sequence analysis showed that P21 was the most closely related to Clostridium phage CPAS-15 belonging to the Myoviridae family, with a recognition rate of 97.24% and a query coverage rate of 98%. No virulence factors or drug resistance genes were found in P21. P21 showed promising antibacterial activity in vitro and in chicken disinfection experiments. In conclusion, P21 has the potential to be used for preventing and controlling C. perfringens in chicken food production.

Contributions of local emissions and regional background to summertime ozone in central China.

Source contributions and regional transport of maximum daily average 8-h (MDA8) O3 during a high O3 month (June 2019) in Henan province in central China are explored using a source-oriented Community Multiscale Air Quality (CMAQ) model. The monthly average MDA8 O3 exceeds ∼70 ppb in more than half of the areas and shows a clear spatial gradient, with lower O3 concentrations in the southwest and higher in the northeast. Significant contributions of anthropogenic emissions to monthly average MDA8 O3 concentrations of more than 20 ppb are predicted in the provincial capital Zhengzhou, mostly due to emissions from the transportation sector (∼50%) and in the areas in the north and northeast regions where industrial and power generation-related emissions are high. Biogenic emissions in the region only contribute to approximately 1-3 ppb of monthly average MDA8 O3. In industrial areas north of the province, their contributions reach 5-7 ppb. Two CMAQ-based O3-NOx-VOCs sensitivity assessments (the local O3 sensitivity ratios based on the direct decoupled method and the production ratio of H2O2 to HNO3) and the satellite HCHO to NO2 column density ratio consistently show that most of the areas in Henan are in NOx-limited regime. In contrast, the high O3 concentration areas in the north and at the city centers are in the VOC-limited or transition regimes. The results from this study suggest that although reducing NOx emissions to reduce O3 pollution in the region is desired in most areas, VOC reductions must be applied to urban and industrial regions. Source apportionment simulations with and without Henan anthropogenic emissions show that the benefit of local anthropogenic NOx reduction might be lower than expected from the source apportionment results because the contributions of Henan background O3 increase in response to the reduced local anthropogenic emissions due to less NO titration. Thus, collaborative O3 controls in neighboring provinces are needed to reduce O3 pollution problems in Henan effectively.

Novel Camellia sinensis O-Methyltransferase Regulated by CsMADSL1 Specifically Methylates EGCG in Cultivar "GZMe4".

Epigallocatechin-3-O-(4-O-methyl)gallate (EGCG4″Me) in Camellia sinensis possesses numerous beneficial biological activities. However, the germplasm rich in EGCG4″Me and the O-methyltransferase responsible for EGCG4″Me biosynthesis are poorly understood. Herein, the content of EGCG3″Me and EGCG4″Me in the shoots of 13 cultivars was analyzed to demonstrate that EGCG4″Me is characteristically accumulated in the "GZMe4" cultivar but not in the other 12 cultivars. A novel O-methyltransferase (CsOMTL1) was identified from "GZMe4" using RNA-Seq and correlation analysis. Using the recombinant enzyme, EGCG4″Me was synthesized in vitro. Overexpression of CsOMTL1 via Agrobacterium-mediated genetic transformation caused constitutive accumulation of EGCG4″Me in C. sinensis callus. Moreover, the transcription factor CsMADSL1 localized in the nucleus activated the transcription of CsOMTL1 and specifically interacted with its promoter. Hence, our study identified a novel O-methyltransferase that characteristically catalyzes the synthesis of EGCG4″Me and a positive regulator of EGCG4″Me synthesis in "GZMe4", which might provide a strategy for the breeding of a tea cultivar rich in EGCG4″Me.

Development and evaluation of a multi-epitope subunit vaccine against group B Streptococcus infection.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a multi-host pathogen, even causing life-threatening infections in newborns. Vaccination with GBS crossed serotypes vaccine is one of the best options for long-term infection control. Here we built a comprehensive in silico epitope-prediction workflow pipeline to design a multivalent multiepitope-based subunit vaccine containing 11 epitopes against Streptococcus agalactiae (MVSA). All epitopes in MVSA came from the proteins which were antigenic-confirmed, virulent-associated, surface-exposed and conserved in ten GBS serotypes. The in-silico analysis showed MVSA had potential to evoke strong immune responses and enable worldwide population coverage. To validate MVSA protection efficacy against GBS infection, immune protection experiments were performed in a mouse model. Importantly, MVSA induced a high titre of antibodies, significant proliferation of mice splenocytes and elicited strong protection against lethal-dose challenge with a survival rate of 100% in mice after three vaccinations. Meanwhile, the polyclonal antibody against MVSA did not only inhibit for growth of GBS from six crucial serotypes in vitro, but also protect 100% naive mice from GBS lethal challenge. These active and passive immunity assay results suggested that MVSA could therefore be an efficacious multi-epitope vaccine against GBS infection.

[One-year Simulation of Air Pollution in Central China, Characteristics, Distribution, Inner Region Cross-transmission, and Pathway Research in 18 Cities].

Air pollution in Henan province is serious and is significantly impacted by pollution transmission and interactions with surrounding areas. The emission sources in 18 cities in Henan province were labeled and applied to the WRF-CMAQ traceability model for simulation in January, April, July, and October of 2017. The pollutant distribution results showed that due to the combined influence of emissions and meteorology, the concentrations of PM2.5, NO2, and SO2 in Henan province were the highest in winter and the lowest in summer. The seasonal variation in O3-8h concentration was the highest in summer, followed by spring, and the lowest in winter. There was a large difference in pollutant concentrations between different seasons. The average concentrations of PM2.5, NO2, and SO2 in winter in Henan province were 4.17, 4.12, and 6.24 times those in summer, respectively, whereas the concentration of O3-8h in summer was 2.24 times that in winter. Since PM2.5, NO2, and SO2 are closely related to primary emissions and have a certain homology, the distributions of high values of these three pollutants were higher in the north and lower in the south, and the seasonal trends were more consistent. The seasonal distribution of O3-8h varied widely, with high O3-8h values mainly distributed in the northeastern region of Henan province in summer, when meteorological conditions contributed to O3 production; in winter, spring, and autumn, high O3-8h values were mainly distributed in the southern part of Henan province due to the suppression of meteorological conditions and NOx consumption. The results of the study on the transport of pollutants showed that extra-provincial transport and natural sources contributed the most to the concentrations of PM2.5, O3-8h, NO2, and SO2 in winter, with 36.20%-72.32%, 77.96%-96.08%, 49.45%-78.80%, and 59.05%-88.85%, respectively. When considering only local emissions and intra-provincial transmission, the contributions of emissions to local concentrations of the four pollutants in summer were the highest in all cities of Henan province. The contributions of intra-provincial transmission to PM2.5 and O3-8h in spring were the largest, with 25.63%-74.69% and 30.21%-80.01%, respectively, and the contributions of intra-provincial transmission to NO2 and SO2 in winter were larger, with 26.02%-76.96% and 20.30%-82.34%. The transmission paths of PM2.5, NO2, and SO2 were more similar in Henan province, with more transmission from north to south in winter, from west to east and southwest to northeast in spring, from southwest to northeast in summer, and from north to south in autumn; however, the transmission of PM2.5 was more complicated. The O3-8h transport path was more different from the others, especially in autumn when pollutants were mostly transported from north to south, but the O3-8h transport path from southwest to northeast was obvious.

CRISPR Contributes to Adhesion, Invasion, and Biofilm Formation in Streptococcus agalactiae by Repressing Capsular Polysaccharide Production.

The clustered regularly interspaced palindromic repeat (CRISPR)-associated (Cas) system functions classically as a prokaryotic defense system against invading mobile genetic elements, such as phages, plasmids, and viruses. Our previous study revealed that CRISPR deletion caused increased transcription of capsular polysaccharide (CPS) synthesis-related genes and severely attenuated virulence in the hypervirulent piscine Streptococcus agalactiae strain GD201008-001. Here, we found that CRISPR deficiency resulted in reduced adhesion, invasion, and biofilm formation abilities in this strain by upregulating the production of CPS. However, enhanced CPS production was not responsible for the attenuated phenotype of the ΔCRISPR mutant. RNA degradation assays indicated that inhibited transcription of the cps operon by CRISPR RNA (crRNA) was not due to the base pairing of the crRNA with the cps mRNA but to the repression of the promoter activity of cpsA, which is a putative transcriptional regulator of the capsule locus. IMPORTANCE Beyond protection from invading nucleic acids, CRISPR-Cas systems have been shown to have an important role in regulating bacterial endogenous genes. In this study, we demonstrate that crRNA inhibits the transcription of the cps operon by repressing the activity of promoter PcpsA, leading to increases in the abilities of adhesion, invasion, and biofilm formation in S. agalactiae. This study highlights the regulatory role of crRNA in bacterial physiology and provides a new explanation for the mechanism of crRNA-mediated endogenous gene regulation in S. agalactiae.

Identification of the RNA-binding domain-containing protein RbpA that acts as a global regulator of the pathogenicity of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2), an emerging zoonotic pathogen, causes swine diseases and human cases of streptococcal toxic shock syndrome. RNA-binding proteins (RBPs) can modulate gene expression through post-transcriptional regulation. In this study, we identified an RBP harbouring an S1 domain, named RbpA, which facilitated SS2 adhesion to host epithelial cells and contributed to bacterial pathogenicity. Comparative proteomic analysis identified 145 proteins that were expressed differentially between ΔrbpA strain and wild-type strain, including several virulence-associated factors, such as the extracellular protein factor (EF), SrtF pilus, IgA1 protease, SBP2 pilus, and peptidoglycan-binding LysM' proteins. The mechanisms underlying the regulatory effects of RbpA on their encoding genes were explored, and it was found that RbpA regulates gene expression through diverse mechanisms, including post-transcriptional regulation, and thus acts as a global regulator. These results partly reveal the pathogenic mechanism mediated by RbpA, improving our understanding of the regulatory systems of S. suis and providing new insights into bacterial pathogenicity.

XRE family transcriptional regulator XtrSs modulates Streptococcus suis fitness under hydrogen peroxide stress.

Streptococcus suis is an important emerging zoonosis that causes economic losses in the pig industry and severe threats to public health. Transcriptional regulators play essential roles in bacterial adaptation to host environments. In this study, we identified a novel XRE family transcriptional regulator in S. suis CZ130302, XtrSs, involved in the bacterial fitness to hydrogen peroxide stress. Based on electrophoretic mobility shift and ß-galactosidase activity assays, we found that XtrSs auto-regulated its own transcription and repressed the expression of its downstream gene psePs, a surface protein with unknown function in S. suis, by binding to a palindromic sequence from the promoter region. Furthermore, we proved that the deletion of the psePs gene attenuated bacterial antioxidant response. Phylogenetic analysis revealed that XtrSs and PsePs naturally co-existed as a combination in most S. suis genomes. Collectively, we demonstrated the binding characteristics of XtrSs in S. suis and provided a new insight that XtrSs played a critical role in modulating psePs to the hydrogen peroxide resistance of S. suis.

Identification of a novel broad-spectrum endolysin, Ply0643, with high antibacterial activity in mouse models of streptococcal bacteriaemia and mastitis.

Streptococcal infections are very common in humans and animals, and they are usually treated with antibiotics. Multidrug-resistant Streptococcus strains have continuously emerged in recent years, prompting the search for alternatives to antibiotics. The use of endolysins encoded by phages has presented a promising alternative approach to treatment. In this study, a novel prophage endolysin, Ply0643, was identified from the prophage S. a 04. At an optimal concentration (30 µg/mL), rPly0643 exhibited broad and strong lysosomal enzyme activity against 66 Streptococcus strains from different sources while also maintaining high lytic activity over a wide pH range (pH 6-10) and a broad range of temperatures (28 °C-45 °C). Two in vivo treatments of rPly0643 (total 0.8 mg/mouse) significantly protected mice (80%) from lethal bacteriaemia with Streptococcus suis, and single treatments of rPly0643 (0.1 mg/gland) significantly reduced Streptococcus agalactiae concentrations and inflammation in murine mammary glands. These findings collectively demonstrate that Ply0643 exhibits good bactericidal activity both in vitro and in vivo, and therefore represents a useful antibacterial agent for combatting streptococcal infections.

CRISPR-dependent endogenous gene regulation is required for virulence in piscine Streptococcus agalactiae.

The clustered regularly interspaced palindromic repeats (CRISPR)-Cas (CRISPR-associated) system is a prokaryotic defence against invading mobile genetic elements, such as bacteriophages or exogenous plasmids. Beyond this, this system has been shown to play an important role in controlling the virulence of some bacterial pathogens. Streptococcus agalactiae strain GD201008-001, a causative agent of septicemia and meningitis in tilapia, contains a single type II CRISPR-Cas system with Cas9 as a signature protein. In this study, we found that the deletion of CRISPR significantly reduced adhesion, invasion, cytotoxicity and haemolysis, and caused severely attenuated virulence in the piscine S. agalactiae strain. RNA-Seq identified 236 endogenous genes regulated by CRISPR, with 159 genes upregulated and 77 genes downregulated. The resulting change in gene transcription by CRISPR was much more pronounced than that by cas9 in this bacterium, indicating CRISPR-mediated endogenous gene regulation was mostly independently of cas9. Subsequent studies showed that CovR/S two-component system was transcriptionally upregulated due to CRISPR deletion, which repressed the expression of the cylE gene coding for a cytolytic toxin, and thus decreased the activity of ß-haemolysin/cytolysin. However, upregulation of CovR/S was not the contributor to the attenuation phenotype of ΔCRISPR. Further, we demonstrated that CRISPR is capable of repressing the expression of Toll-like receptor 2 (TLR2)-activating lipoprotein Sag0671 and thus dampens the innate immune response. This study revealed that the CRISPR system of S. agalactiae exhibited extraordinary potential capability in the regulation of endogenous transcripts, which contributes to bacterial innate immune evasion and virulence.

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.

Transcriptional regulator XtgS is involved in iron transition and attenuates the virulence of Streptococcus agalactiae.

Streptococcus agalactiae (GBS) is an important pathogen that has increasingly received attention for its role in invasive infections and its broad host range. Research on the regulation of gene expression could illuminate GBS pathogenesis. We previously identified a novel transcriptional regulator XtgS, which is a negative regulator of GBS pathogenicity. Here, we demonstrate that XtgS overexpression significantly attenuated GBS virulence in zebrafish infection tests, and XtgS indirectly downregulated the transcription of two iron transport systems based on the results of transcriptomic analysis, electrophoretic mobility shift assays (EMSAs) and lacZ fusion assays. Subsequent studies verified that the inactivation of iron transport system 1 resulted in GBS excessive iron accumulation and attenuated virulence. Thus, we infer that the downregulation of iron transport system 1 caused by XtgS overexpression probably attenuates bacterial virulence, which partially clarifies the mechanism by which XtgS alleviates the pathogenesis. These findings provide new insights into the relationship between exogenous transcriptional regulation and bacterial virulence.

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.