New Insights into the Biological Functions of Essential TsaB/YeaZ Protein in Staphylococcus aureus.

TsaB/YeaZ represents a promising target for novel antibacterial agents due to its indispensable role in bacterial survival, high conservation within bacterial species, and absence of eukaryotic homologs. Previous studies have elucidated the role of the essential staphylococcal protein, TsaB/YeaZ, in binding DNA to mediate the transcription of the ilv-leu operon, responsible for encoding key enzymes involved in the biosynthesis of branched-chain amino acids-namely isoleucine, leucine, and valine (ILV). However, the regulation of ILV biosynthesis does not account for the essentiality of TsaB/YeaZ for bacterial growth. In this study, we investigated the impact of TsaB/YeaZ depletion on bacterial morphology and gene expression profiles using electron microscopy and deep transcriptomic analysis, respectively. Our results revealed significant alterations in bacterial size and surface smoothness upon TsaB/YeaZ depletion. Furthermore, we pinpointed specific genes and enriched biological pathways significantly affected by TsaB/YeaZ during the early and middle exponential phases and early stationary phases of growth. Crucially, our research uncovered a regulatory role for TsaB/YeaZ in bacterial autolysis. These discoveries offer fresh insights into the multifaceted biological functions of TsaB/YeaZ within S. aureus.

Enhanced Photocatalytic Properties of All-Organic IDT-COOH/O-CN S-Scheme Heterojunctions Through π-π Interaction and Internal Electric Field.

Herein, we present a distinct methodology for the in situ electrostatic assembly method for synthesizing a conjugated (IDT-COOH)/oxygen-doped g-C3N4 (O-CN) S-scheme heterojunction. The electron delocalization effect due to π-π interactions between O-CN and self-assembled IDT-COOH favors interfacial charge separation. The self-assembled IDT-COOH/O-CN exhibits a broadened visible absorption to generate more charge carriers. The internal electric field between the IDT-COOH and the O-CN interface provides a directional charge-transfer channel to increase the utilization of photoinduced charge carriers. Moreover, the active species (•O2-, h+, and 1O2) produced by IDT-COOH/O-CN under visible light play important roles in photocatalytic disinfection. The optimum 40% IDT-COOH/O-CN can kill 7-log of methicillin-resistant Staphylococcus aureus (MRSA) cells in 2 h and remove 88% tetracycline (TC) in 5 h, while O-CN only inactivates 1-log of MRSA cells and degrades 40% TC. This work contributes to a promising method to fabricate all-organic g-C3N4-based S-scheme heterojunction photocatalysts with a wide range of optical responses and enhanced exciton dissociation.

Self-assembled A-D-A type indacenodithiophene-based small conjugated molecule/TiO2 for enhancing the photocatalytic activity.

Herein, A-D-A type indacenodithiophene-based small conjugated molecule IDT-COOH and IDT-COOH/TiO2 photocatalysts with stable non-covalent bonding have been successfully synthesized via in situ electrostatic assembly. The self-assembled three-dimensional π-conjugation structure IDT-COOH with high crystallinity not only broadens the visible absorption to produce more photogenerated carriers but also provides directional charge-transfer channels to accelerate the charge mobility. Thus, 7 log inactivation of S. aureus in 2 h and 92.5% decomposition of TC in 4 h under visible light exposure are achieved for optimized 30% IDT-COOH/TiO2. The dynamic constants (k) of S. aureus disinfection and TC degradation for 30% IDT-COOH/TiO2 are 3.69 and 2.45 times compared to those of self-assembled IDT-COOH, respectively. The notable inactivation performance is among the best reported for conjugated semiconductor/TiO2 photocatalysts for photocatalytic sterilization. ˙O2-, e- and ˙OH are the primary reactive species in the photocatalytic process. The strong interfacial interaction between TiO2 and IDT-COOH is in favour of rapid charge transfer, which leads to enhanced photocatalytic performance. This work offers a feasible method to fabricate TiO2-based photocatalytic agents with a wide visible light response and improved exciton dissociation.

Chemokines induced by PEDV infection and chemotactic effects on monocyte, T and B cells.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging pathogen that causes severe economic loss in the pig industry. The host's innate immune system is the first line of defense on virus invasion of the small intestinal epithelial cells. Chemokines, as a part of the innate immune system, play an important role in host immunity against infection, however, and their expression and chemotactic effect on key immune cells in PEDV infection remains unclear. In this study, cDNA microarray was firstly performed to analyzed ileum tissue of piglets on the third day after PEDV infection. The differentially expressed genes mainly involved in multiple biological processes, chemokine signaling pathway and cytokine receptor interaction signaling pathway had the highest enrichment according to GO and KEGG enrichment analysis. The expression levels of chemokines MCP-1, MIP-1ß, IL-8, CXCL9, CXCL10 and CXCL13 in ileum of PEDV- infected piglets were significantly higher than those in the control group. The expression of chemokines in vivo experiment was further verified by RT-qPCR and ELISA using PEDV-infected IPEC-J2 cells. The results showed that the PEDV-infected IPEC-J2 cells had significantly induced protein expression of MCP-1, MIP-1ß, IL-8, CXCL9, CXCL-10 and CXCL13. These results indicated that the changes of chemokines expressed in the ileum of piglets (in vivo) were consistent with those in IPEC-J2 cells (in vitro) after PEDV infection. Finally, the role of chemokines in immune cell migration during PEDV infection was illustrated by siRNA-mediated knock down method and the co-culture model of IPEC-J2 cells with peripheral blood leukocyte cells (PBLCs). The FACS analysis showed that MCP-1 induced by PEDV infection played a chemotactic effect on CD14+ cells, CXCL9 on CD3+CD4-CD8-γδ T, CD3+CD4-CD8+ Tc, CD3+CD4+CD8- Th and CD3+CD4+CD8+ Tm subsets, and CXCL13 on CD19+ B cells. Collectively, our findings first indicate that PEDV-induced chemokines MCP-1, CXCL-9 and CXCL-13 attracted CD14+ cells, T cells and B cells, respectively. These results provide a theoretical basis for studying the mechanism of anti-PEDV infection in piglets.

Differences in cytokines expression between Vero cells and IPEC-J2 cells infected with porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) primarily infects suckling piglets and causes severe economic losses to the swine industry. Cytokines, as part of the innate immune response, are important in PEDV infection. The cytokines secreted by cell infection models in vitro might reflect true response to viral infection of target cells in vivo. Vero cells and IPEC-J2 are commonly used as an in vitro model to investigate PEDV infection. However, it is not clear which type of cells is more beneficial to the study of PEDV. In our study, firstly, Vero cells and IPEC-J2 were successfully infected with PEDV virulent strains (HBQY2016) and attenuated vaccine strains (CV777) and were capable of supporting virus replication and progeny release. Moreover, cytokine differences expression by Vero cells and IPEC-J2 cells infected with two PEDV strains were analyzed. Compared with IPEC-J2 cells, only the mRNA levels of TGF-ß, MIP-1ß and MCP-1 were detected in Vero cells. ELISA assay indicated that compared to the control group, the PEDV-infected group had significantly induced expression levels of IL-1ß, MIP-1ß, MCP-1, IL-8, and CXCL10 in IPEC-J2 cells, while only secretion level of IL-1ß, MIP-1ß and IL-8 in Vero cells were higher in PEDV infected group. Finally, cytokines change of piglets infected PEDV-HBQY2016 strains were detected by cDNA microarray, and similar to those of IPEC-J2 cells infected PEDV. Collectively, these data determined that the IPEC-J2 could be more suitable used as a cell model for studying PEDV infection in vitro compared with Vero cells, based on the close approximation of cytokine expression profile to in vivo target cells.

Visible-light-induced bactericidal properties of a novel thiophene-based linear conjugated polymer/TiO2 heterojunction.

The low utilization of visible light and the fast recombination of photogenerated electron-hole pairs are the two intrinsic defects that have hindered the antibacterial applications of TiO2. The addition of organic photocatalytic agents to form heterojunctions with TiO2 powder can effectively address these problems. A novel linear conjugated polymer poly[(thiophene-ethylene-thiophene)-thiophene-3-carboxylic acid decyl ester] (PTCD) was successfully synthesized via Stille coupling polymerization. PTCD and TiO2 can form a heterojunction photocatalyst (PTCD/TiO2), and this product was characterized using NMR and XRD. The UV-vis spectra showed that the absorption edge of the PTCD/TiO2 heterojunction extends to approximately 700 nm, which indicates that the visible-light utilization is greatly improved. Staphylococcus aureus (S. aureus) was selected as the target organism to test the photocatalytic antimicrobial activity of the material. Photogenerated electrons can undergo directional transmission of the PTCD polymer to TiO2 on the PTCD/TiO2 heterojunction to realize excellent antibacterial properties. With an optimized PTCD loading ratio of 30%, PTCD/TiO2 showed an antibacterial rate that was 14.5 times greater than that of pure TiO2 in 4 h under visible-light irradiation.

Contribution of Coagulase and Its Regulator SaeRS to Lethality of CA-MRSA 923 Bacteremia.

Coagulase is a critical factor for distinguishing Staphylococcus aureus and coagulase-negative Staphylococcus. Our previous studies demonstrated that the null mutation of coagulase (coa) or its direct regulator, SaeRS, significantly enhanced the ability of S. aureus (CA-MRSA 923) to survive in human blood in vitro. This led us to further investigate the role of coagulase and its direct regulator, SaeRS, in the pathogenicity of CA-MRSA 923 in bacteremia during infection. In this study, we found that the null mutation of coa significantly decreased the mortality of CA-MRSA 923; moreover, the single null mutation of saeRS and the double deletion of coa/saeRS abolished the virulence of CA-MRSA 923. Moreover, the mice infected with either the saeRS knockout or the coa/saeRS double knockout mutant exhibited fewer histological lesions and less neutrophils infiltration in the infected kidneys compared to those infected with the coa knockout mutant or their parental control. Furthermore, we examined the impact of coa and saeRS on bacterial survival in vitro. The null mutation of coa had no impact on bacterial survival in mice blood, whereas the deletion mutation of saeRS or coa/saeRS significantly enhanced bacterial survival in mice blood. These data indicate that SaeRS plays a key role in the lethality of CA-MRSA 923 bacteremia, and that coagulase is one of the important virulence factors that is regulated by SaeRS and contributes to the pathogenicity of CA-MRSA 923.

Visible Light-Driven D-A Conjugated Linear Polymer and Its Coating for Dual Highly Efficient Photocatalytic Degradation and Disinfection.

Herein, a novel donor-acceptor (D-A) conjugated linear polymeric system, poly[(2,6-(4,8-bis(5-(2-ethylhexyl)-4-fluorothiophen-2-yl)-benzo[1,2-b:4,5-b']dithiophene))-alt-2,5-(3-carboxyl)-thiophene] (PBDT-F-COOH), with outstanding processing ability and its all-organic PBDT-F-COOH coating featuring chemical bonding for combination with polyurethane were prepared. Wide visible spectrum-driven PBDT-F-COOH and PBDT-F-COOH-PU showed dual efficient photocatalytic activities toward degradation and disinfection, mainly attributing to efficient dissociation of excitons and transfer of charge carriers, resulting from the large dipole moment of D-A PBDT-F-COOH. PBDT-F-COOH demonstrated >99.2% inactivation of Staphylococcus aureus (S. aureus) within 1 h and a 7-log decrease in 4 h under visible light irradiation. Additionally, the coating showed the 7-log inactivation of S. aureus in 7 h. These inactivation efficiency results are among those of the best reported D-A conjugated linear polymers. Importantly, PBDT-F-COOH and the PBDT-F-COOH-PU coating both presented satisfactory stability with high photocatalytic activity after recycling runs. This work provides a feasible approach for fabricating nontoxic and highly active organic photocatalysts with wide visible spectra and a large dipole moment via a D-A linear structure design protocol.

Humoral immune responses in piglets experimentally infected with a field strain of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) causes severe clinical diarrhea in neonatal piglets, with reported mortality rates between 70-100%. The humoral immunity, especially the local intestinal IgA responses, plays an important role in the immune protection against PEDV infection. In this study, we evaluated the isotype antibody responses against the PEDV nucleocapsid (N) protein and the spike (S) protein subunits 1 (S1) and 2 (S2) in the serum and intestine of piglets. We also determined its serum neutralizing activity against the PEDV field strain HBMC2012 in 21-day-old piglets. Enzyme-linked immunosorbent assays (ELISA) revealed that the production of IgM against the N protein and S1 subunit was higher compared to the S2 subunit. The anti-S2 IgA antibodies were higher than the anti-N protein and anti-S1 IgA at 3 days post-infection (dpi). The specific IgA responses to the S2 subunit were higher than the responses observed in S1. The specific IgG responses against S1 and S2 subunits exceeded those of N protein. The serum neutralizing activities against PEDV were relatively low with a tendency to decline over time. No isotype-specific antibodies were found in the intestinal contents from infected pigs, except the one with weak IgA responses against N protein at 28 dpi. Immunohistochemical staining showed that a few IgM, IgA, and IgG antibody-secreting cells were mainly located in the mucosa of the duodenum and ileum of PEDV-infected pigs at 3 dpi. This study suggests poor systemic and intestinal isotype-specific antibody responses, especially those of IgA, and weak serum neutralizing activities against the field PEDV strain in piglets.

Isolation and characteristics of multi-drug resistant Streptococcus porcinus from the vaginal secretions of sow with endometritis.

BACKGROUND: Sow endometritis is a common disease in pig breeding farms after artificial insemination, which leads to gray-green vaginal secretions and decreased conception rates. It is important to perform an etiologic diagnosis for effective treatments and control of diseases. The aim of this study was to carry out a pathogenic detection in five specimens of vaginal secretions collected from sick pigs with endometritis, implement identification of the pathogens by phenotypic detection and 16 s rDNA sequence and phylogeny analysis, and determinate antibiotic susceptibility of the isolates. RESULTS: A Streptococcus strain was isolated and identified from all of the five specimens. The isolate was positive for Voges-Proskauer (V-P) and for the hydrolysis of arginine, esculin and myelin-associated glycoprotein (MAG). Acid formation was observed for sorbitol, mushroom sugar, sucrose, and glucose. The 16S rDNA sequence of the isolate possessed 99.93% similarity to that of Streptococcus porcinus. The phylogenetic analysis of 16S rDNA sequence showed that the isolate belonged to the same clade as the S. porcinus strains from humans, pigs, and other animals. The isolate exhibited multi-drug resistance to aminoglycosides, quinolones, macrolides and tetracyclines except being sensitive to some ß- lactams such as penicillin G, cephalothin, cefazolin, cephradine and cefuroxime. CONCLUSIONS: A S. porcinus isolate with multi-drug resistance was identified from vaginal secretions of sows with endometritis in one pig breeding farm, which suggests that the sow endometritis was caused by S. porcinus infection during artificial insemination. This study indicates that sensitive antibiotics such as penicillin G or some cephalosporins could be used for treatment of the diseases. In addition, the study hints that bacterial multi-drug resistance is a tough problem for disease treatment in pig farms.

Interaction between two essential, conserved bacterial proteins YeaZ and glycoprotease as a potential antibacterial target in multi-drug-resistant Staphylococcus aureus.

Protein-protein interactions among highly conserved and essential proteins can serve as new targets for antibacterial therapies. One protein-protein interaction between two widely conserved and essential bacterial proteins, YeaZ and its paralog, a putative glycoprotease, is being looked into for its antimicrobial drug potential. These two proteins possess tandem functions, including repression of the branched-chain amino acids biosynthesis and induction of a tRNA modification important in enhancing translation fidelity through anticodon-codon base pairing. Heterodimer formation between these two proteins is essential for Staphylococcus aureus, and other bacterial species including Escherichia coli and Salmonella typhimurium. Such YeaZ-glycoprotease interaction could thus be a target for antimicrobial drugs designed for multi-drug-resistant S. aureus. In this review, we discuss the function, structure, and interaction between these two proteins and their orthologs in other bacteria.

Injectable Schiff base polysaccharide hydrogels for intraocular drug loading and release.

A novel voriconazole (VCZ)-loaded injectable hydrogel was in situ synthetized via a Schiff base reaction between polyaldehyde dextran (PAD) and carboxymethyl chitosan (CMCTS) for intraocular drug loading and release. Water-insoluble VCZ, which is an effective agent in clinic treating fungal endophthalmitis, was loaded through the inclusion into the ß-cyclodextrin (ß-CD) cavity based on host-guest interaction on the linear poly ß-CD chain, which was in situ twined in the cross-linked hydrogel structure. The gelation time, degradation, and drug release process were investigated by studying three kinds of hydrogels with different volume ratio of PAD to CMCTS resolving in phosphate buffered saline (PBS) solution (3:7, 6:7, 9:7), respectively. The property of VCZ-loaded injectable hydrogel should be adjusted through changing the cross-linked density of the hydrogel, the molecular weight or concentration of the linear poly ß-CD to meet the treating requirement, and a multistage drug controlled-release mechanism was proposed. In conclusion, the VCZ-loaded in situ injectable hydrogel should be offered as a promising strategy for intraocular drug delivery in vitreous cavity for the treatment of fungal endophthalmitis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1909-1916, 2019.

Staphylococcal Enterotoxin C Is an Important Virulence Factor for Mastitis.

Staphylococcus aureus is an important bacterial pathogen causing bovine mastitis, but little is known about the virulence factor and the inflammatory responses in the mammary infection. Staphylococcal enterotoxin C (SEC) is the most frequent toxin produced by S. aureus, isolated from bovine mastitis. To investigate the pathogenic activity of SEC in the inflammation of the mammary gland and the immune responses in an animal model, mouse mammary glands were injected with SEC, and the clinical signs, inflammatory cell infiltration, and proinflammatory cytokine production in the mammary glands were assessed. SEC induced significant inflammatory reactions in the mammary gland, in a dose-dependent manner. SEC-injected mammary glands showed a severe inflammation with inflammatory cell infiltration and tissue damage. In addition, interleukin (IL)-1ß and IL-6 production in the SEC-injected mammary glands were significantly higher than those in the PBS control glands. Furthermore, the SEC-induced inflammation and tissue damage in the mammary gland were specifically inhibited by anti-SEC antibody. These results indicated, for the first time, that SEC can directly cause inflammation, proinflammatory cytokine production, and tissue damage in mammary glands, suggesting that SEC might play an important role in the development of mastitis associated with S. aureus infection. This finding offers an opportunity to develop novel treatment strategies for reduction of mammary tissue damage in mastitis.

The SaeRS Two-Component System Controls Survival of Staphylococcus aureus in Human Blood through Regulation of Coagulase.

The SaeRS two-component system plays important roles in regulation of key virulence factors and pathogenicity. In this study, however, we found that the deletion mutation of saeRS enhanced bacterial survival in human blood, whereas complementation of the mutant with SaeRS returned survival to wild-type levels. Moreover, these phenomena were observed in different MRSA genetic background isolates, including HA-MRSA WCUH29, CA-MRSA 923, and MW2. To elucidate which gene(s) regulated by SaeRS contribute to the effect, we conducted a series of complementation studies with selected known SaeRS target genes in trans. We found coagulase complementation abolished the enhanced survival of the SaeRS mutant in human blood. The coa and saeRS deletion mutants exhibited a similar survival phenotype in blood. Intriguingly, heterologous expression of coagulase decreased survival of S. epidermidis in human blood. Further, the addition of recombinant coagulase to blood significantly decreased the survival of S. aureus. Further, analysis revealed staphylococcal resistance to killing by hydrogen peroxide was partially dependent on the presence or absence of coagulase. Furthermore, complementation with coagulase, but not SaeRS, returned saeRS/coa double mutant survival in blood to wild-type levels. These data indicate SaeRS modulates bacterial survival in blood in coagulase-dependent manner. Our results provide new insights into the role of staphylococcal SaeRS and coagulase on bacterial survival in human blood.

The Staphylococcus aureus AirSR Two-Component System Mediates Reactive Oxygen Species Resistance via Transcriptional Regulation of Staphyloxanthin Production.

Staphylococcus aureus is an important opportunistic pathogen and is the etiological agent of many hospital- and community-acquired infections. The golden pigment, staphyloxanthin, of S. aureus colonies distinguishes it from other staphylococci and related Gram-positive cocci. Staphyloxanthin is the product of a series of biosynthetic steps that produce a unique membrane-embedded C30 golden carotenoid and is an important antioxidant. We observed that a strain with an inducible airR overexpression cassette had noticeably increased staphyloxanthin production compared to the wild-type strain under aerobic culturing conditions. Further analysis revealed that depletion or overproduction of the AirR response regulator resulted in a corresponding decrease or increase in staphyloxanthin production and susceptibility to killing by hydrogen peroxide, respectively. Furthermore, the genetic elimination of staphyloxanthin during AirR overproduction abolished the protective phenotype of increased staphyloxanthin production in a whole-blood survival assay. Promoter reporter and gel shift assays determined that the AirR response regulator is a direct positive regulator of the staphyloxanthin-biosynthetic operon, crtOPQMN, but is epistatic to alternative sigma factor B. Taken together, these data indicate that AirSR positively regulates the staphyloxanthin-biosynthetic operon crtOPQMN, promoting survival of S. aureus in the presence of oxidants.

Prevalence and genetic variation of porcine circovirus type 2 in Hebei, China from 2004 to 2014.

Porcine circovirus type 2 (PCV2), the primary causative agent of porcine circovirus-associated disease (PCVAD), causes severe economic losses to the pig industry in China since 2002. To investigate the molecular epidemic characteristics and genetic evolution of PCV2, 12 PCV2 isolates obtained from different pig farms with various clinical symptoms of PCVAD in Hebei, China from 2004 to 2014 were sequenced and analyzed. The phylogenetic analysis showed that the 12 isolates were divided into two distinct genotypes, PCV2b (7/12) and PCV2d (5/12), based on the sequences of either viral complete genome or open reading frame 2 (ORF2). Of the 7 PCV2b strains, 5 were isolated from 2004 to 2008 while all PCV2d were isolated from 2009 to 2014. This exhibited that PCV2b isolates were the most common before 2009 and then PCV2d isolates became predominant and widely distributed in pig farms. Sequence comparisons among total isolates indicated that the nucleotide identity ranged from 95.5% to 100% for complete genome and 93.1%-100% for ORF2. Compared with seven PCV2b isolates, there were thirteen amino-acid substitutions in the ORF2 region and one additional amino-acid K at this region terminal for five PCV2d isolates. The results suggest that a higher genetic variation and a distinct genotype shift occurred among the PCV2 isolates collected from 2004 to 2014 in Hebei.

The AirSR two-component system contributes to Staphylococcus aureus survival in human blood and transcriptionally regulates sspABC operon.

To date, genes identified and transcriptionally regulated by the AirSR TCS have been involved in energy production and cellular homeostasis of the staphylococcal cell. It is well accepted that the state of cellular metabolism impacts the expression of virulence factors in Staphylococcus aureus. For this reason, we conducted experiments to determine if the AirSR TCS contributes to the pathogenesis of S. aureus using an antisense RNA interference technology, an inducible overexpression system, and gene deletions. Depletion of AirSR by antisense RNA expression or deletion of the genes, results in significant decrease in bacterial survival in human blood. Conversely, overexpression of AirR significantly promotes survival of S. aureus in blood. AirR promotes the secretion of virulence factors that inhibits opsonin-based phagocytosis. This enhanced survival is partially linked to the transcriptional regulation of the sspABC operon, encoding V8 protease (SspA), staphopain B (SspB) and staphostatin B (SspC). SspA and SspB are known virulence factors which proteolytically digest opsonins and inhibit killing of S. aureus by professional phagocytes. This is the first evidence linking the AirSR TCS to pathogenesis of S. aureus.