Type II and IV toxin-antitoxin systems coordinately stabilize the integrative and conjugative element of the ICESa2603 family conferring multiple drug resistance in Streptococcus suis.

Integrative and conjugative elements (ICEs) play a vital role in bacterial evolution by carrying essential genes that confer adaptive functions to the host. Despite their importance, the mechanism underlying the stable inheritance of ICEs, which is necessary for the acquisition of new traits in bacteria, remains poorly understood. Here, we identified SezAT, a type II toxin-antitoxin (TA) system, and AbiE, a type IV TA system encoded within the ICESsuHN105, coordinately promote ICE stabilization and mediate multidrug resistance in Streptococcus suis. Deletion of SezAT or AbiE did not affect the strain's antibiotic susceptibility, but their duple deletion increased susceptibility, mainly mediated by the antitoxins SezA and AbiEi. Further studies have revealed that SezA and AbiEi affect the genetic stability of ICESsuHN105 by moderating the excision and extrachromosomal copy number, consequently affecting the antibiotic resistance conferred by ICE. The DNA-binding proteins AbiEi and SezA, which bind palindromic sequences in the promoter, coordinately modulate ICE excision and extracellular copy number by binding to sequences in the origin-of-transfer (oriT) and the attL sites, respectively. Furthermore, AbiEi negatively regulates the transcription of SezAT by binding directly to its promoter, optimizing the coordinate network of SezAT and AbiE in maintaining ICESsuHN105 stability. Importantly, SezAT and AbiE are widespread and conserved in ICEs harbouring diverse drug-resistance genes, and their coordinated effects in promoting ICE stability and mediating drug resistance may be broadly applicable to other ICEs. Altogether, our study uncovers the TA system's role in maintaining the genetic stability of ICE and offers potential targets for overcoming the dissemination and evolution of drug resistance.

SssP1, a Fimbria-like component of Streptococcus suis, binds to the vimentin of host cells and contributes to bacterial meningitis.

Streptococcus suis (S. suis) is one of the important pathogens that cause bacterial meningitis in pigs and humans. Evading host immune defences and penetrating the blood-brain barrier (BBB) are the preconditions for S. suis to cause meningitis, while the underlying mechanisms during these pathogenic processes are not fully understood. By detecting the red blood and white blood cells counts, IL-8 expression, and the pathological injury of brain in a mouse infection model, a serine-rich repeat (SRR) glycoprotein, designated as SssP1, was identified as a critical facilitator in the process of causing meningitis in this study. SssP1 was exported to assemble a fimbria-like component, thus contributed to the bacterial adhesion to and invasion into human brain microvascular endothelial cells (HBMECs), and activates the host inflammatory response during meningitis but is not involved in the actin cytoskeleton rearrangement and the disruption of tight junctions. Furthermore, the deletion of sssP1 significantly attenuates the ability of S. suis to traverse the BBB in vivo and in vitro. A pull-down analysis identified vimentin as the potential receptors of SssP1 during meningitis and following Far-Western blot results confirmed this ligand-receptor binding mediated by the NR2 (the second nonrepeat region) region of SssP1. The co-localisation of vimentin and S. suis observed by laser scanning confocal microscopy with multiplex fluorescence indicated that vimentin significantly enhances the interaction between SssP1 and BBB. Further study identified that the NR216-781 and NR1711-2214 fragments of SssP1 play critical roles to bind to the BBB depending on the sialylation of vimentin, and this binding is significantly attenuated when the antiserum of NR216-781 or NR1711-2214 blocked the bacterial cells, or the vimentin antibody blocked the BBB. Similar binding attenuations are observed when the bacterial cells were preincubated with the vimentin, or the BBB was preincubated with the recombinant protein NR216-781, NR1711-2214 or sialidase. In conclusion, these results reveal a novel receptor-ligand interaction that enhances adhesion to and penetration of the BBB to cause bacterial meningitis in the S. suis infection and highlight the importance of vimentin in host-pathogen interactions.

Insight into the role of Streptococcus suis zinc metalloprotease C from the new serotype causing meningitis in piglets.

Streptococcus suis (S. suis) is an important gram-positive pathogen and an emerging zoonotic pathogen that causes meningitis in swine and humans. Although several virulence factors have been characterized in S. suis, the underlying mechanisms of pathogenesis are not fully understood. In this study, we identified Zinc metalloproteinase C (ZmpC) probably as a critical virulence factor widely distributed in S. suis strains. ZmpC was identified as a critical facilitator in the development of bacterial meningitis, as evidenced by the detection of increased expression of TNF-α, IL-8, and matrix metalloprotease 9 (MMP-9). Subcellular localization analysis further revealed that ZmpC was localized to the cell wall surface and gelatin zymography analysis showed that ZmpC could cleave human MMP-9. Mice challenge demonstrated that ZmpC provided protection against S. suis CZ130302 (serotype Chz) and ZY05719 (serotype 2) infection. In conclusion, these results reveal that ZmpC plays an important role in promoting CZ130302 to cause mouse meningitis and may be a potential candidate for a S. suis CZ130302 vaccine.

Streptococcus suis Deploys Multiple ATP-Dependent Proteases for Heat Stress Adaptation.

Streptococcus suis is an important zoonotic pathogen, causing cytokine storms of Streptococcal toxic shock-like syndrome amongst humans after a wound infection into the bloodstream. To overcome the challenges of fever and leukocyte recruitment, invasive S. suis must deploy multiple stress responses forming a network and utilize proteases to degrade short-lived regulatory and misfolded proteins induced by adverse stresses, thereby adapting and evading host immune responses. In this study, we found that S. suis encodes multiple ATP-dependent proteases, including single-chain FtsH and double-subunit Clp protease complexes ClpAP, ClpBP, ClpCP, and ClpXP, which were activated as the fever of infected mice in vivo. The expression of genes ftsH, clpA/B/C, and clpP, but not clpX, were significantly upregulated in S. suis in response to heat stress, while were not changed notably under the treatments with several other stresses, including oxidative, acidic, and cold stimulation. FtsH and ClpP were required for S. suis survival within host blood under heat stress in vitro and in vivo. Deletion of ftsH or clpP attenuated the tolerance of S. suis to heat, oxidative and acidic stresses, and significantly impaired the bacterial survival within macrophages. Further analysis identified that repressor CtsR directly binds and controls the clpA/B/C and clpP operons and is relieved by heat stress. In summary, the deployments of multiple ATP-dependent proteases form a flexible heat stress response network that appears to allow S. suis to fine-tune the degradation or refolding of the misfolded proteins to maintain cellular homeostasis and optimal survival during infection.

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.

The effectiveness of extended binding affinity of prophage lysin PlyARI against Streptococcus suis infection.

Streptococcus suis is an important zoonotic pathogen. An increase in multi-drug-resistant strains has led to poor performance of traditional antibiotic therapies. Thus, alternative antibacterial agents are urgently needed. In this study, we identified a recombined and expressed lysin PlyARI derived from the novel serotype S. suis (Chz) prophage PhiARI0460-1. The recombinant PlyARI at a concentration of 10 µg/mL showed high bacteriolytic activity against 30 S. suis isolates. The minimum inhibitory concentration (MIC) of PlyARI against S. suis was found to be as low as 2 µg/mL, and the lytic efficiency could be maintained between the range of pH 4 and 12. Additionally, in a mouse infection model, a dose of 0.5 mg of PlyARI protected 10 out of 10 mice that were challenged with highly virulent S. suis strain HA9801. Furthermore, the binding specificity of PlyARI was evaluated by constructing a green fluorescent protein (GFP-ARIb), where GFP was fused with the PlyARI-SH3b (cell wall-binding domain, CBD), revealing a high affinity to S. suis, Staphylococcus aureus, and Streptococcus equi along with exhibiting a medium affinity to Streptococcus pneumoniae as well as Streptococcus agalactiae. Overall, our findings indicated that PlyARI may be an alternative antibacterial agent that was useful in treating and possibly the prevention of Streptococcal infections.

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.

Phenanthridine Derivative Host Heat Shock Cognate 70 Down-Regulators as Porcine Epidemic Diarrhea Virus Inhibitors.

Porcine epidemic diarrhea virus (PEDV) has become increasingly problematic around the world, not only for its hazards to livestock but also due to the possibility that it is a zoonotic disease. Although vaccine therapy has made some progress toward PEDV control, additional effective therapeutic strategies against PEDV are needed, such as the development of chemotherapeutic agents. The aim of this work was to identify novel anti-PEDV agents by designing and synthesizing a series of phenanthridine derivatives. Among them, three compounds (compounds 1, 2, and 4) were identified as potent anti-PEDV agents exhibiting suppression of host cell heat shock cognate 70 (Hsc70) expression. Mechanism studies revealed that host Hsc70 is involved in the replication of PEDV, and its expression can be suppressed by destabilization of the mRNA, resulting in inhibition of PEDV replication. Activity against PEDV in vivo in PEDV-infected piglets suggested that phenanthridine derivatives are the first host-acting potential anti-PEDV agents.

The Novel Streptococcal Transcriptional Regulator XtgS Negatively Regulates Bacterial Virulence and Directly Represses PseP Transcription.

Streptococcus agalactiae (group B streptococcus [GBS]) has received continuous attention for its involvement in invasive infections and its broad host range. Transcriptional regulators have an important impact on bacterial adaptation to various environments. Research on transcriptional regulators will shed new light on GBS pathogenesis. In this study, we identified a novel XRE-family transcriptional regulator encoded on the GBS genome, designated XtgS. Our data demonstrate that XtgS inactivation significantly increases bacterial survival in host blood and animal challenge test, suggesting that it is a negative regulator of GBS pathogenicity. Further transcriptomic analysis and quantitative reverse transcription-PCR (qRT-PCR) mainly indicated that XtgS significantly repressed transcription of its upstream gene pseP Based on electrophoretic mobility shift and lacZ fusion assays, we found that an XtgS homodimer directly binds a palindromic sequence in the pseP promoter region. Meanwhile, the PseP and XtgS combination naturally coexists in diverse Streptococcus genomes and has a strong association with sequence type, serotype diversification and host adaptation of GBS. Therefore, this study reveals that XtgS functions as a transcriptional regulator that negatively affects GBS virulence and directly represses PseP expression, and it provides new insights into the relationships between transcriptional regulator and genome evolution.

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

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

Extraintestinal pathogenic Escherichia coli increase extracytoplasmic polysaccharide biosynthesis for serum resistance in response to bloodstream signals.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the leading causes of bloodstream infections. Characteristically, these organisms exhibit strong resistance to the bactericidal action of host serum. Although numerous serum resistance factors in ExPEC have been identified, their regulatory mechanisms during in vivo infection remain largely unknown. Here, RNA sequencing analyses together with quantitative reverse-transcription PCR revealed that ExPEC genes involved in the biosynthesis of extracytoplasmic polysaccharides (ECPs) including K-capsule, lipopolysaccharide (LPS), colanic acid, peptidoglycan and Yjb exopolysaccharides were significantly upregulated in response to serum under low oxygen conditions and during bloodstream infection. The oxygen sensor FNR directly activated the expression of K-capsule and colanic acid and also indirectly modulated the expression of colanic acid, Yjb exopolysaccharides and peptidoglycan via the known Rcs regulatory system. The global regulator Fur directly or indirectly repressed the expression ofECP biosynthesis genes in iron replete media, whereas the low iron conditions in the bloodstream could relieve Fur repression. Using in vitro and animal models, FNR, Fur and the Rcs system were confirmed as contributing to ExPEC ECP production, serum resistance and virulence. Altogether, these findings indicated that the global regulators FNR andFur and the signaling transduction system Rcs coordinately regulated the expression of ECP biosynthesis genes leading to increased ExPEC serum resistance in response to low oxygen and low iron levels in the bloodstream.

Bacitracin resistance and enhanced virulence of Streptococcus suis via a novel efflux pump.

BACKGROUND: Streptococcus suis is a prominent pathogen causing septicemia and meningitis in swine and humans. Bacitracin is used widely as a growth promoter in animal feed and to control the spread of necrotic enteritis in most developing countries. This study aimed to characterize a novel membrane transporter module Sst comprising SstE, SstF, and SstG for bacitracin resistance. RESULTS: Comparative genomics and protein homology analysis found a potential efflux pump SstFEG encoded upstream of well-known bacitracin-resistance genes bceAB and bceRS. A four-fold decrease in bacitracin susceptibility was observed in sstFEG deletion mutant comparing with S. suis wildtype strain CZ130302. Further studies indicated that the bacitracin tolerance mediated by SstFEG is not only independent of the BceAB transporter, but also regulated by the two-component system BceSR. Given that SstFEG are harbored by almost all virulent strains, but not in the avirulent strains, we managed to explore its potential role in bacterial pathogencity. Indeed, our results showed that SstFEG is involved in S. suis colonization and virulence in animal infection model by its potential competitive survival advantage against host bactericidal effect. CONCLUSION: To our knowledge, this is the first study to functionally characterize the bacitracin efflux pump in S. suis to provide evidence regarding the important roles of the novel ABC transporter system SstFEG with respect to drug resistance and virulence.

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

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

Uropathogenic Escherichia coli preferentially utilize metabolites in urine for nucleotide biosynthesis through salvage pathways.

Growth in urinary tract depends on the ability of uropathogenic E. coli to adjust metabolism in response to available nutrients, especially to synthesize metabolites that are present in urinary tract with limited concentrations. In this study, a genome-wide assay was applied and identified five nucleotide biosynthetic genes purA, guaAB and carAB that are required for optimal growth of UPEC in human urine and colonization in vivo. Subsequent functional analyses revealed that either interruption of de novo nucleotide biosynthesis or blocking of salvage pathways alone could not decrease UPEC's growth, while only simultaneous interruption of both two pathways significantly reduced UPEC's growth in urine. Evidences showed that uracil, xanthine, and hypoxanthine in human urine could support nucleotide biosynthesis through salvage pathways when the de novo pathways were interrupted. Moreover, the expression of genes involved in salvage pathways of nucleotide biosynthesis were significantly upregulated when UPEC are cultured in human urine and artificial urine medium with uracil, xanthine or hypoxanthine. Finally, animal tests showed that further deletion of genes involved in salvage nucleotide biosynthesis from mutants with defects in de novo pathways significantly reduced UPEC's colonization in host bladders and kidneys. These results indicated that UPEC preferentially utilize abundant metabolites in urine for nucleotide biosynthesis through salvage pathways, which is not like in serum, where the limiting amounts of substrates for salvage biosynthesis force invading pathogens to rely on de novo nucleotide biosynthesis. Taken together, our study implied the importance of salvage pathways of nucleotides biosynthesis for UPEC's fitness during urinary tract infection.

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

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

SBP1 is an adhesion-associated factor without the involvement of virulence in Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that infects swine and humans with high mortality and morbidity. Although a number of virulence-associated factors have been reported, the understanding of the molecular mechanism underlying SS2 pathogenicity remains limited. Our previous studies revealed that srtBCD-associated protein 2' (SBP2') contributed to the pathogenesis of SS2, but the function of another member in the srtBCD cluster, srtBCD-associated protein 1 (SBP1) was still unknown. Here, we found that sbp1 was widely distributed among high virulent SS2 strains, suggesting that sbp1 may be involved in the pathogenesis of SS2. To investigate the function of SBP1, we firstly conducted Western blotting analyses to confirm that SBP1 was expressed in the high virulent SS2 strain ZY05719 both in vivo and in vitro, then constructed the deletion mutant of sbp1 by homologous recombination. Bacterial adhesion assay, indirect immunofluorescence assay and protein binding assay all demonstrated that SBP1 was associated with adhesion of SS2 to HEp-2 cells. However, SBP1 did not influence the invasion, phagocytosis or intracellular survival of SS2. Furthermore, infection assays in vivo showed that inactivation of sbp1 failed to impair the ability of SS2 to cause zebrafish and mouse mortality. Overall, these results indicate that SBP1 is an adhesion-associated factor without the involvement of virulence in Streptococcus suis serotype 2.

Diverse toxic effectors are harbored by vgrG islands for interbacterial antagonism in type VI secretion system.

The type VI secretion system (T6SS) is considered as one of the key competition strategies by injecting toxic effectors for intestinal pathogens to acquire optimal colonization in host gut, a microenviroment with high-density polymicrobial community where bacteria compete for niches and resources. Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea in human and animals, widely encode T6SS clusters in their genomes. In this report, we first identified VT1, a novel amidase effector in ETEC, significantly hydrolyzed D-lactyl-L-Ala crosslinks between N-acetylmuramoyl and L-Ala in peptidoglycan. Further study showed that the VT1/VTI1 effector/immunity pair is encoded within a typical vgrG island, and plays a critical role for the successful establishment of ETEC in host gut. Numerous putative effectors with diverse toxin domains were found by retrieving vgrG islands in pathogenic E. coli, and designated as VT modules. Therein, VT5, a lysozyme-like effector widely encoded in ETEC, was confirmed to effectively kill adjacent cells, suggesting that VT toxin modules may be critical for pathogenic E. coli to seize a significantly competitive advantage for optimal intestinal colonization. To expand our analyses for large-scale search of VT antibacterial effectors based on vgrG island, >200 predicted effectors from 20 bacterial species were found and classified into 11 predicted toxins. This work reports a new retrieval strategy for screening T6SS effectors, and provides an example how pathogenic bacteria antagonize and displace commensal microbiome to successfully colonize in the host niches through a T6SS-dependent manner.

PAAR-Rhs proteins harbor various C-terminal toxins to diversify the antibacterial pathways of type VI secretion systems.

The type VI secretion system (T6SS) of bacteria plays a key role in competing for specific niches by the contact-dependent killing of competitors. Recently, Rhs proteins with polymorphic C-terminal toxin-domains that inhibit or kill neighboring cells were identified. In this report, we identified a novel Rhs with an MPTase4 (Metallopeptidase-4) domain (designated as Rhs-CT1) that showed an antibacterial effect via T6SS in Escherichia coli. We managed to develop a specific strategy by matching the diagnostic domain-architecture of Rhs-CT1 (Rhs with an N-terminal PAAR-motif and a C-terminal toxin domain) for effector retrieval and discovered a series of Rhs-CTs in E. coli. Indeed, the screened Rhs-CT3 with a REase-3 (Restriction endonuclease-3) domain also mediated interbacterial antagonism. Further analysis revealed that vgrGO1 and eagR/DUF1795 (upstream of rhs-ct) were required for the delivery of Rhs-CTs, suggesting eagR as a potential T6SS chaperone. In addition to chaperoned Rhs-CTs, neighborless Rhs-CTs could be classified into a distinct family (Rhs-Nb) sharing close evolutionary relationship with T6SS2-Rhs (encoded in the T6SS2 cluster of E. coli). Notably, the Rhs-Nb-CT5 was confirmed bioinformatically and experimentally to mediate interbacterial antagonism via Hcp2B-VgrG2 module. In a further retrieval analysis, we discovered various toxin/immunity pairs in extensive bacterial species that could be systematically classified into eight referential clans, suggesting that Rhs-CTs greatly diversify the antibacterial pathways of T6SS.

Alterations in gp37 Expand the Host Range of a T4-Like Phage.

The use of phages as antibacterial agents is limited by their generally narrow host ranges. The aim of this study was to make a T4-like phage, WG01, obtain the host range of another T4-like phage, QL01, by replacing its host-determinant gene region with that of QL01. This process triggered a direct expansion of the WG01 host range. The offspring of WG01 obtained the host ranges of both QL01 and WG01, as well as the ability to infect eight additional host bacteria in comparison to the wild-type strains. WQD had the widest host range; therefore, the corresponding fragments, named QD, could be used for constructing a homologous sequence library. Moreover, after a sequencing analysis of gene 37, we identified two different mechanisms responsible for the expanded host range: (i) the first generation of WG01 formed chimeras without mutations, and (ii) the second generation of WG01 mutants formed from the chimeras. The expansion of the host range indicated that regions other than the C-terminal region may indirectly change the receptor specificity by altering the supportive capacity of the binding site. Additionally, we also found the novel means by which subsequent generations expanded their host ranges, namely, by exchanging gene 37 to acquire a wider temperature range for lysis. The method developed in this work offers a quick way to change or expand the host range of a phage. Future clinical applications for screening phages against a given clinical isolate could be achieved after acquiring more suitable homologous sequences.IMPORTANCE T4-like phages have been established as safe in numerous phage therapy applications. The primary drawbacks to the use of phages as therapeutic agents include their highly specific host ranges. Thus, changing or expanding the host range of T4-like phages is beneficial for selecting phages for phage therapy. In this study, the host range of the T4-like phage WG01 was expanded using genetic manipulation. The WG01 derivatives acquired a novel means of expanding their host ranges by acquiring a wider temperature range for lysis. A region was located that had the potential to be used as a sequence region for homologous sequence recombination.

Acute meningitis of piglets and mice caused by co-infected with Streptococcus suis and Aerococcus viridans.

The two opportunistic pathogens, Streptococcus suis (S. suis) and Aerococcus. viridans (A. viridans) were isolated from the brains of piglets suffered bacterial meningitis in a farm of China. The murine model has been established to evaluate the pathogenicity and symbiotic relationship of S. suis and A. viridans simultaneously infection. Our results demonstrated the ability of new serotype S. suis to cause the classical bacterial meningitis and death were greatly enhanced during co-infection with A. viridans in mice at a proportion. We also examined the distribution and titer of bacteria coinfection in organs, the titer of S. suis appeared a significant trend for an increase in the lung meanwhile the concentration titer of A. viridans maintain a low level. This is the first reported the A. viridans and S. suis coinfection cause the bacterial meningitis outbroke in the piglets and mice. Moreover, further investigation of the pathogenesis of A. viridans and S. suis is urgently needed in swine industry.