Determination of the signaling proteins responsible for the antioxidant potential of the Yishenhuoxue formula for treating asthenospermia in rats.

Asthenospermia is a predominant cause of male infertility, and antioxidant supplements can be effective in treating asthenospermia. We demonstrate the antioxidant potential of traditional Chinese medicine, the Yishenhuoxue (YSHX) formula, in treating polyglycosides of Tripterygium wilfordii (GTW)-induced asthenospermia in rats. Fifty male rats were randomly divided into the normal, model, and treatment groups. HE staining was used to evaluate the improvement of spermatogenic function of rats, and TBA reaction, qRT-PCR, Western Blot and other methods were used to determine the changes of oxidative stress indicators and to evaluate the improvement of antioxidant capacity of rats by YSHX. Comparison with the model group showed significant improvement in pathological damage caused by GTW to seminiferous tubules. MDA and NO content in rat testes decreased, especially in middle- and high-dosage groups. No significant changes were observed in SOD and CAT activity or mRNA expression. GSH-Px activity and GSH mRNA expression were significantly higher in the low-dosage group than in the model group. Compared to the model group, GR activity was significantly lower in the middle and high dosage groups, while the mRNA expression was higher. The PKC-beta level increased, while p-ERK1/2, NF-κB, and the ratio of p-ERK1/2*(ERK1/2)-1 decreased significantly in the treatment groups. Therefore, YSHX can alleviate GTW-induced testicular damage, enhance GSH-Px activity, regulate GSH redox cycling, and mitigate oxidative stress injury. Furthermore, YSHX can promote PKC-beta expression and inhibit the phosphorylation of ERK1/2 and NF-κB. Using YSHX may be an effective way to increase sperm motility via the PKC-ERK1/2-NF-ĸB axis.

Incorporation of PD-1 blockade into induction chemotherapy improved tumor response in patients with locoregionally advanced nasopharyngeal carcinoma in a retrospective patient cohort.

OBJECTIVE: To investigate the short-term efficacy and safety of induction chemotherapy (IC) combined with anti-PD-1 immunotherapy in locoregionally advanced nasopharyngeal carcinoma (LA-NPC). METHODS: A total of 217 patients diagnosed with LA-NPC at the First Affiliated Hospital of Nanchang University, including 67 who received IC combined with anti-PD-1 and 150 who received IC, were retrospectively enrolled. Efficacy was evaluated at the end of the IC cycles and one month after radiotherapy based on RECIST v1.1 criteria. Acute toxicities were graded based on the CTCAE v5.0 criteria. Quantitative variables were compared by unpaired t-tests, and categorical variables were evaluated by Fisher Freeman-Halton test or Pearson Chi-square test. RESULTS: At the end of all induction therapy cycles, the objective response rate (ORR) of the IC + anti-PD-1 group was 88.1 % (59/67) as opposed to 70.0 % (105/150) in the IC group. Subgroup analysis showed that patients in both stage Ⅲ and ⅣA achieved a significant improvement in ORR with the inclusion of anti-PD-1 therapy. Patients with T3-4 or N2-3 category appeared to benefit more from anti-PD-1 compared to patients with T1-2 or N0-1 category. However, neither ORR nor the complete response (CR) rate was significantly different between the two treatment groups one month after the end of radiotherapy. In addition, the frequency of Grade 3-4 adverse events were also similar in both groups. CONCLUSIONS: IC combined with anti-PD-1 immunotherapy significantly improved the ORR of LA-NPC patients after induction therapy compared to IC alone.

Relationship between Rumen Microbial Differences and Phenotype Traits among Hu Sheep and Crossbred Offspring Sheep.

This experiment was conducted to investigate the effect of three-way hybrid sheep and Hu sheep on serum indicators, rumen fermentation, rumen enzyme activity, and microorganisms in sheep. Healthy and similar birth weights from three groups (Hu, n = 11; Charolais × Australian White × Hu, CAH, n = 11; Charolais × Dorper × Hu, CDH, n = 11) were selected to be fed by the ewes until 45 days of age. Subsequently, they were weaned intensively and underwent short-term fattening for 3 months along with selected male lambs fed intensively. During this period, they were fed and watered ad libitum. Blood and rumen fluid were collected and analyzed for serum indicators and rumen fluid microorganisms, enzyme activity, and VFA, respectively, at the end of the fattening period. Compared with Hu lamb, the offspring of the three-way hybrid lamb showed significant improvements in body weight, serum lactate dehydrogenase, and creatinine content. However, there was no significant effect on serum immunity and antioxidant indices. In addition, the rumen fluid volatile fatty acid (VFA) molar concentration and microcrystalline cellulose and lipase content were significantly lower in the three-way hybrid lamb compared to Hu lamb, but ß-glucosidase, amylase, pepsin, and VFA molar ratio were not significantly affected. Subsequently, 16S rRNA sequencing diversity analysis revealed that three-way hybrid lamb significantly increased rumen microbial ACE and Chao1 indices compared to Hu lamb. Meanwhile, the abundance of Verrucomicrobiota and Synergistota significantly increased at the phylum level. Correlation analysis showed that Prevotella had the highest proportion, while Rikenellaceae_RC9_gut_group correlated most closely with others genus. The microbial communities isovaleric acid molar concentration and proportion were strongly correlated. In addition, there were significant differences in correlations between microbial communities and isobutyric acid, butyric acid and valeric acid content, and their molar proportion, but they were not significantly correlated with digestive enzymes. From the functional enrichment analysis, it was found that hybrid progeny were mainly enriched in the pyruvate metabolism, microbial metabolism in diverse environments, carbon metabolism, and quorum sensing pathways. In contrast, the Hu sheep were primarily enriched in the cysteine and methionine, amino sugar and nucleotide sugar, and biosynthesis of secondary metabolite pathways. These results suggest that hybridization can play a role in regulating organismal metabolism and improve animal production performance by influencing the structure and characteristics of microbial communities.

High-Throughput Sequencing Reveals a Dynamic Bacterial Linkage between the Captive White Rhinoceros and Its Environment.

Soil is an essential part of the animal habitat and has a large diversity of microbiota, while the animal body was colonized by a complex bacterial community; so far, the relationship between the animal host microbial community and the soil microbial ecosystem remains largely unknown. In this study, 15 white rhinoceros from three different captive grounds were selected and the bacterial community of the gut, skin, and environment of these rhinoceros were analyzed by 16S rRNA sequencing technology. Our results showed that Firmicutes and Bacteroidota were the predominant phyla in the gut microbiome, whereas skin and environment samples share similar microbiome profiles and are dominated by the phyla of Actinobacteriota, Chloroflexi, and Proteobacteria. Although the bacterial composition of the gut differs from that of the skin and environment, the Venn diagrams showed that there were 22 phyla and 186 genera shared by all the gut, skin, and environmental microbes in white rhinoceroses. Further cooccurrence network analysis indicated a bacterial linkage based on a complex interaction was established by the bacterial communities from the three different niches. In addition, beta diversity and bacterial composition analysis showed that both the captive ground and host ages induced shifts in the microbial composition of white rhinoceroses, which suggested that the bacterial linkage between the captive white rhinoceros and its environment is dynamic. Overall, our data contribute to a better understanding of the bacterial community of the captive white rhinoceros, especially for the relationship between the environment and animal bacterial communities. IMPORTANCE The white rhinoceros is one of the world's most endangered mammals. The microbial population plays a key role in animal health and welfare; however, studies regarding the microbial communities of the white rhinoceros are relatively limited. As the white rhinoceros has a common behavior of mud baths and thus is in direct contact with the environment, a relationship between the animal microbial community and the soil microbial ecosystem appears possible, but it remains unclear. Here, we described the characteristics and interaction of bacterial communities of the white rhinoceros in three different niches, including gut, skin, and environment. We also analyzed the effects of captive ground and age on the composition of the bacterial community. Our findings highlighted the relationship among the three niches and may have important implications for the conservation and management of this threatened species.

Precisely Controlling Csr sRNA Levels by MshH Enhances Vibrio cholerae Colonization in Adult Mice.

Vibrio cholerae is the causative agent of cholera. Effective intestinal colonization is a key step for V. cholerae pathogenicity and transmission. In this study, we found that deleting mshH, a homolog of the Escherichia coli CsrD protein, caused a V. cholerae colonization defect in the intestine of adult mice. By analyzing the RNA levels of CsrB, CsrC, and CsrD, we found that deleting mshH increased the levels of CsrB and CsrD but decreased the level of CsrC. However, deleting CsrB and -D not only recovered the mshH deletion mutant colonization defect but also recovered CsrC to wild-type levels. These results indicated that controlling the RNA levels of CsrB, -C, and -D is crucial for V. cholerae colonization of adult mice. We further demonstrated that the RNA levels of CsrB and CsrD were mainly controlled by MshH-dependent degradation, yet the level of CsrC was mainly determined by the CsrA-dependent stabilization. Our data show that V. cholerae differentially controls CsrB, -C, and -D abundance through the MshH-CsrB/C/D-CsrA regulatory pathway to finely regulate the activity of CsrA targets such as ToxR, so as to better survive in adult mouse intestine. IMPORTANCE The ability of V. cholerae to colonize the intestine is a key factor for its fitness and transmissibility between hosts. Here, we investigated the mechanism of V. cholerae colonization of adult mammal intestine and found that precisely controlling the CsrB, -C, and -D contents by MshH and CsrA plays an essential role for V. cholerae colonization in the adult mouse intestine. These data expand our knowledge on the mechanism of V. cholerae controlling the RNA level of CsrB, -C, and -D and highlight the importance that the different strategies used by V. cholerae to regulate the RNA level of CsrB, -C, and -D confer the bacterium with a survival advantage.

Quorum sensing signal synthases enhance Vibrio parahaemolyticus swarming motility.

Vibrio parahaemolyticus is a significant food-borne pathogen that is found in diverse aquatic habitats. Quorum sensing (QS), a signaling system for cell-cell communication, plays an important role in V. parahaemolyticus persistence. We characterized the function of three V. parahaemolyticus QS signal synthases, CqsAvp , LuxMvp , and LuxSvp , and show that they are essential to activate QS and regulate swarming. We found that CqsAvp , LuxMvp , and LuxSvp activate a QS bioluminescence reporter through OpaR. However, V. parahaemolyticus exhibits swarming defects in the absence of CqsAvp , LuxMvp , and LuxSvp , but not OpaR. The swarming defect of this synthase mutant (termed Δ3AI) was recovered by overexpressing either LuxOvp D47A , a mimic of dephosphorylated LuxOvp mutant, or the scrABC operon. CqsAvp , LuxMvp , and LuxSvp inhibit lateral flagellar (laf) gene expression by inhibiting the phosphorylation of LuxOvp and the expression of scrABC. Phosphorylated LuxOvp enhances laf gene expression in a mechanism that involves modulating c-di-GMP levels. However, enhancing swarming requires phosphorylated and dephosphorylated LuxOvp which is regulated by the QS signals that are synthesized by CqsAvp , LuxMvp , and LuxSvp . The data presented here suggest an important strategy of swarming regulation by the integration of QS and c-di-GMP signaling pathways in V. parahaemolyticus.

Cancer of unknown primary site in the mandibular region: A case report.

The diagnosis and treatment of cancer of unknown primary site (CUP) present with difficulties and produce a poor prognosis. The current study presents the case of a patient with CUP in the mandibular region was treated with docetaxel and lobaplatin chemotherapy, and vascular embolization of the tumor. The tumor size was markedly reduced and the patient's quality of life improved following radiotherapy. The present case report is accompanied by a discussion of the literature to contextualize the treatment regimen for patients with CUP. These findings will support current treatment practices, inform oncologists and benefit patients with cancer.

Immunomodulatory role of estrogen in ischemic stroke: neuroinflammation and effect of sex.

Although estrogen is predominantly related to the maintenance of reproductive functioning in females, it mediates various physiological effects in nearly all tissues, especially the central nervous system. Clinical trials have revealed that estrogen, especially 17ß-estradiol, can attenuate cerebral damage caused by an ischemic stroke. One mechanism underlying this effect of 17ß-estradiol is by modulating the responses of immune cells, indicating its utility as a novel therapeutic strategy for ischemic stroke. The present review summarizes the effect of sex on ischemic stroke progression, the role of estrogen as an immunomodulator in immune reactions, and the potential clinical value of estrogen replacement therapy. The data presented here will help better understand the immunomodulatory function of estrogen and may provide a basis for its novel therapeutic use in ischemic stroke.

Developmental stage variation in the gut microbiome of South China tigers.

South China tigers (Panthera tigris amoyensis, SC) are the most threatened tiger subspecies in the world. All the living SCs are captive in zoos or reserves and depend on artificial feeding. The composition of the gut microbiome plays an important role in sustaining the health of the host. A comprehensive understanding of the composition and development of the microbial community of SC is helpful to improve the feeding of captive SC. In this study, we collected 47 fecal samples, 37 of which were from SC of three developmental stages, 5 from adult Amur tigers (Am), and 5 from adult Bengal tigers (Bg), which were all housed in the same zoo. We investigated the diversity, richness, and composition of the bacterial microbiomes and we found that the gut microbiome of SC is strongly affected by host aging. The composition of the gut microbiome of juvenile SC experienced dramatic changes from 5 months old to 1 year old, and it showed much less difference when compared to the samples of 1 year old and the subadult. No significant differences were observed between the samples of subadult and the adult groups. The predominant phylum of 5-month-old SC is Fusobacteriota (33.99%) when the juvenile tigers were older than 5 months, and Firmicutes, but not Fusobacteriota, became the predominant phylum of bacteria in their gut. The gut microbiome of SC, Am, and Bg is possibly affected by their genetic variation; however, the core microbiome of these three subspecies is the same. Our data suggest that the gut microbiome of SC undergoes a developmental progression: a developmental phase (cub), a transitional phase (subadult), and a stable phase (adult). These results expand our understanding of the role of age in the development of the gut microbiome of SC.

Characterization and epidemiological analysis of Vibrio parahaemolyticus isolated from different marine products in East China.

Vibrio parahaemolyticus is a major foodborne pathogen with a wide distribution in the world that causes economic and public health problems. Here, we isolated 152 V. parahaemolyticus strains from shellfish, shrimp, crab, and snails from 5 provinces in East China, and analyzed the genetic diversity, population structure, and virulence profiles of these isolates. Our results showed that the 152 V. parahaemolyticus strains belong to 84 different sequence types (STs), of which 69 (82.14 %) STs and 60 alleles were newly identified. The thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH) were present in 4 V. parahaemolyticus isolates (2.63 %) respectively, while toxRS/new, a distinctive toxRS sequence that was associated with pandemic V. parahaemolyticus strains, is present in 52 isolates (34.21 %). Thereinto, both the ZJ11 and ZJ12 strains measure up to the standard of toxRS/new+, tdh+, and trh-, which was widely used marker for the rapid screening of pandemic strains, and thus these strains may have the risk of infectious outbreaks. In addition, we observed that all the 152 V. parahaemolyticus isolates encode type III secretion systems 1 and type VI secretion system (T6SS) 2, while 119 isolates (78.29 %) of them also contain T6SS1. The genetic relatedness of our isolates to the human V. parahaemolyticus collection was explored, which showed that ST6, ST79, ST162, ST1060, and ST1061 were all identified in both human isolates (7 isolates) uploaded in the PubMLST database and our marine products isolates (7 isolates). Our findings expand the views of the genetic diversity of V. parahaemolyticus and will contribute to understanding the potential risk of the transboundary spread of this bacterium.

Sanguinarine, Isolated From Macleaya cordata, Exhibits Potent Antifungal Efficacy Against Candida albicans Through Inhibiting Ergosterol Synthesis.

In recent decades, infections caused by the opportunistic fungus Candida albicans have increased, especially in patients with immunodeficiency. In this study, we investigated the mechanism of action of sanguinarine (SAN) against C. albicans both in vitro and in vivo. SAN exhibited antifungal activity against C. albicans clinical isolates, with MICs in the range of 112.8-150.5 µM. Furthermore, scanning electron and transmission electron microscopy showed that SAN induced morphological changes as well as structure disruption in C. albicans cells, including masses of cellular debris, ruptured cell walls, and membrane deformation. Flow cytometry revealed that SAN could lead to cell membrane damage, and ergosterol content analysis indicated that SAN could cause ergosterol content reduction exceeding 90%. Further, we validated the efficacy of SAN against candidiasis caused by C. albicans in a murine model, and SAN significantly improved survival and reduced weight loss compared to vehicle. The treatment of 1.5 and 2.5 mg/kg/d SAN obviously reduced the fungal burden in the kidney. In addition, histopathological examination indicated that no fungal cells were observed in lung and kidney tissues after SAN treatment. Hence, this study suggests that SAN is a promising plant-derived compound for the development of an effective anticandidal agent.

The Effects of Different Induction Chemotherapy Cycles and Adjuvant Chemotherapy on the Survival Outcomes of Patients With Locally Advanced Nasopharyngeal Carcinoma.

Objective: This study investigated whether differences in the induction chemotherapy (IC) cycle number and adjuvant chemotherapy (AC) affect survival outcomes in patients with locally advanced nasopharyngeal carcinoma (LA-NPC). Methods: The survival outcomes of 386 consecutive LA-NPC patients treated between January 2015 and March 2018 were retrospectively analyzed. Univariate and multivariate analyses were used to compare treatment groups defined by IC< 3 or ≥3 IC cycles followed by radiotherapy with or without AC (i.e., IC<3+AC, IC<3+non-AC, IC≥3+AC, and IC≥3+non-AC groups). Results: The median follow-up time was 53 months (range: 2-74 months) and the median number of IC cycles was 2 (range: 1-6 cycles). The 3-year overall survival (OS) rate was significantly higher in patients with IC≥3 cycles compared to IC<3 cycles (95.7% vs. 90.3%, P=0.020). Multivariate analysis indicated that the IC cycle number is an independent factor for OS (hazard ratio=0.326, P=0.007). Furthermore, patients in the IC<3+AC group had a better OS rate than those in the IC<3+non-AC group (91.6% vs. 79.1%, P=0.030), indicating that AC positively affected OS in patients with IC<3. However, no significant difference in the OS rate was found between IC≥3+non-AC and IC≥3+AC groups (92.1% vs. 94.6%, P =0.550). Conclusion: The IC cycle number appears to be an independent prognostic factor for higher OS in LA-NPC patients who received ≥3 cycles. Sequential AC after IC plus radiotherapy may improve OS in patients with IC<3 cycles.

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.

Characterization of GefA, a GGEEF Domain-Containing Protein That Modulates Vibrio parahaemolyticus Motility, Biofilm Formation, and Virulence.

Vibrio parahaemolyticus is a significant foodborne pathogen that causes economic and public health problems worldwide and has a high capacity to adapt to diverse environments and hosts. The second messenger cyclic diguanylate monophosphate (c-di-GMP) allows bacteria to shift from a planktonic form to a communal multicellular lifestyle and plays an important role in bacterial survival and transmission. Here, we characterized single-domain c-di-GMP synthetases in V. parahaemolyticus and identified a novel GGEEF domain-containing protein designated GefA that modulates bacterial swarming motility, biofilm formation, and virulence. GefA inhibits swarming motility by regulating the expression of lateral flagella, while it enhances biofilm formation by controlling exopolysaccharide biosynthesis. Under high-c-di-GMP conditions caused by scrABC knockout, we found that GefA is bifunctional, as it has no effect on swarming motility, but retains the ability to regulate biofilm formation. Subsequent studies suggested that GefA regulates the expression of type III secretion system 1 (T3SS1), which is an important virulence factor in V. parahaemolyticus. Here, we also revealed that the flagella participate in the infection of V. parahaemolyticus. We found that both the T3SS1 and flagella contribute to the GefA-mediated virulence of V. parahaemolyticus in the zebrafish model. Our results expand the knowledge of the V. parahaemolyticus c-di-GMP synthetases and their roles in social behaviors and pathogenicity. IMPORTANCE The c-di-GMP metabolic enzymes constitute one of the largest clusters of potential orthologues in Vibrio parahaemolyticus. However, the specific roles that these individual c-di-GMP metabolic enzymes play are largely unknown. Here, we identified a GGEEF domain-containing protein designated GefA that regulates bacterial behaviors and virulence. We also demonstrated that flagella participate in the infection of this bacterium, through which GefA regulates bacterial virulence. To our knowledge, the roles that c-di-GMP and flagella play in V. parahaemolyticus virulence have never been revealed. Our findings contribute to a better understanding of the function of c-di-GMP and its synthetases in V. parahaemolyticus.

Real-world benefit of combination palbociclib and endocrine therapy for metastatic breast cancer and correlation with neutropenia.

BACKGROUND: Combination CDK4/6 inhibitor and endocrine therapy has been shown to significantly improve progression-free survival (PFS) in patients with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (mBC). The aim of this retrospective study was to evaluate the real-world benefit of first-line combination therapy in this cohort and to correlate treatment efficacy with neutropenia, a common toxicity of CDK4/6 inhibitors. METHODS: This study included HR-positive, HER2-negative advanced or mBC patients who were treated with palbociclib plus endocrine therapy, mainly letrozole, between 1 January 2015 and 1 March 2018. Progression-free survival (PFS) was determined using Kaplan-Meier analysis. The predictive value of absolute neutrophil count (ANC) and neutrophil-to-lymphocyte ratio (NLR) for PFS were explored using Cox regression models. Both ANC and NLR were used as a time-dependent variable. RESULTS: In total, 165 patients were included with median PFS of 24.19 months (95% CI 18.93-NR). Median PFS for patients with bone-only metastases (n = 54) was not reached (95% CI 18.21-NR). Among patients with all other metastases (n = 111), median PFS was 24.19 months (95% CI 16.33-33.82). Lower ANC was correlated with decreased risk of progression (HR 0.84, 95% CI 0.71-0.97, p = 0.008). There was no significant association between NLR and the risk of disease progression (HR 1.07, 95% CI 0.97-1.18, p = 0.203). CONCLUSION: The effectiveness of palbociclib and endocrine therapy in the treatment of HR-positive, HER2-negative mBC in the real-world setting is similar to the efficacy reported in the PALOMA-2 trial. Patients with lower neutrophil count may have a lower risk of early disease progression.

An Auto-Regulating Type II Toxin-Antitoxin System Modulates Drug Resistance and Virulence in Streptococcus suis.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements that play an essential role in multidrug tolerance and virulence of bacteria. So far, little is known about the TA systems in Streptococcus suis. In this study, the Xress-MNTss TA system, composed of the MNTss toxin in the periplasmic space and its interacting Xress antitoxin, was identified in S. suis. ß-galactosidase activity and electrophoretic mobility shift assay (EMSA) revealed that Xress and the Xress-MNTss complex could bind directly to the Xress-MNTss promoter as well as downregulate streptomycin adenylyltransferase ZY05719_RS04610. Interestingly, the Xress deletion mutant was less pathogenic in vivo following a challenge in mice. Transmission electron microscopy and adhesion assays pointed to a significantly thinner capsule but greater biofilm-formation capacity in ΔXress than in the wild-type strain. These results indicate that Xress-MNTss, a new type II TA system, plays an important role in antibiotic resistance and pathogenicity in S. suis.

Identification of LuxR Family Regulators That Integrate Into Quorum Sensing Circuit in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is one of the most important food-borne pathogens that cause economic and public health problems worldwide. Quorum sensing (QS) is a way for the cell-cell communication between bacteria that controls a wide spectrum of processes and phenotypic behaviors. In this study, we performed a systematic research of LuxR family regulators in V. parahaemolyticus and found that they influence the bacterial growth and biofilm formation. We then established a QS reporter plasmid based on bioluminescence luxCDABE operon of Vibrio harveyi and demonstrated that several LuxR family regulators integrated into QS circuit in V. parahaemolyticus. Thereinto, a novel LuxR family regulator, named RobA, was identified as a global regulator by RNA-sequencing analyses, which affected the transcription of 515 genes in V. parahaemolyticus. Subsequent studies confirmed that RobA regulated the expression of the exopolysaccharides (EPS) synthesis cluster and thus controlled the biofilm formation. In addition, bioluminescence reporter assays showed that RobA plays a key role in the QS circuit by regulating the expression of opaR, aphA, cpsQ-mfpABC, cpsS, and scrO. We further demonstrated that the regulation of RobA to EPS and MfpABC depended on OpaR and CpsQ, which combined the QS signal with bis-(3'-5')-cyclic dimeric GMP to construct a complex regulatory network of biofilm formation. Our data provided new insights into the bacterial QS mechanisms and biofilm formation in V. parahaemolyticus.

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.

YSIRK-G/S-directed translocation is required for Streptococcus suis to deliver diverse cell wall anchoring effectors contributing to bacterial pathogenicity.

The Streptococcus suis serotype 2 (SS2) is a significant zoonotic pathogen that is responsible for various swine diseases, even causing cytokine storms of Streptococcal toxic shock-like syndromes amongst human. Cell wall anchoring proteins with a C-terminal LPxTG are considered to play vital roles during SS2 infection; however, their exporting mechanism across cytoplasmic membranes has remained vague. This study found that YSIRK-G/S was involved in the exportation of LPxTG-anchoring virulence factors MRP and SspA in virulent SS2 strain ZY05719. The whole-genome analysis indicated that diverse LPxTG proteins fused with an N-terminal YSIRK-G/S motif are encoded in strain ZY05719. Two novel LPxTG proteins SspB and YzpA were verified to be exported via a putative transport system that was dependent on the YSIRK-G/S directed translocation, and portrayed vital functions during the infection of SS2 strain ZY05719. Instead of exhibiting an inactivation of C5a peptidase in SspB, another LPxTG protein with an N-terminal YSIRK-G/S motif from Streptococcus agalactiae was depicted to cleave the C5a component of the host complement. The consequent domain-architecture retrieval determined more than 10,000 SspB/YzpA like proteins that are extensively distributed in the Gram-positive bacteria, and most of them harbor diverse glycosyl hydrolase or peptidase domains within their middle regions, thus presenting their capability to interact with host cells. The said findings provide compelling evidence that LPxTG proteins with an N-terminal YSIRK-G/S motif are polymorphic effectors secreted by Gram-positive bacteria, which can be further proposed to define as cell wall anchoring effectors in a new subset.