Zebrafish Infection Model Research Articles

Cold-Shock Domain Family Proteins (Csps) Are Involved in Regulation of Virulence, Cellular Aggregation, and Flagella-Based Motility in Listeria monocytogenes.

Cold shock-domain family proteins (Csps) are highly conserved nucleic acid binding proteins regulating the expression of various genes including those involved in stress resistance and virulence in bacteria. We show here that Csps are involved in virulence, cell aggregation and flagella-based extracellular motility of Listeria monocytogenes. A L. monocytogenes mutant deleted in all three csp genes (ΔcspABD) is attenuated with respect to human macrophage infection as well as virulence in a zebrafish infection model. Moreover, this mutant is incapable of aggregation and fails to express surface flagella or exhibit swarming motility. An evaluation of double csp gene deletion mutant (ΔcspBD, ΔcspAD and ΔcspAB) strains that produce single csp genes showed that there is redundancy as well as functional differences among the three L. monocytogenes Csps in their contributions to virulence, cellular aggregation, flagella production, and swarming motility. Protein and mRNA expression analysis further showed impaired expression of key virulence and motility genes in the csp mutants. Our observations at protein and mRNA level suggest Csp-dependent expression regulation of these genes at transcriptional and post-transcriptional levels. In a mutant lacking all csp genes (ΔcspABD) as well as those possessing single csp genes (ΔcspBD, ΔcspAD, and ΔcspAB) we detected reduced levels of proteins or activity as well as transcripts from the prfA, hly, mpl, and plcA genes suggesting a Csp-dependent transcriptional regulation of these genes. These csp mutants also had reduced or completely lacked ActA proteins and cell surface flagella but contained elevated actA and flaA mRNA levels compared to the parental wild type strain suggesting Csp involvement in post-transcriptional regulation of these genes. Overall, our results suggest that Csps contribute to the expression regulation of virulence and flagella-associated genes thereby promoting host pathogenicity, cell aggregation and flagella-based motility processes in L. monocytogenes.

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

Bacterial size matters: Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis.

Enterococcus faecalis is an opportunistic pathogen frequently isolated in clinical settings. This organism is intrinsically resistant to several clinically relevant antibiotics and can transfer resistance to other pathogens. Although E. faecalis has emerged as a major nosocomial pathogen, the mechanisms underlying the virulence of this organism remain elusive. We studied the regulation of daughter cell separation during growth and explored the impact of this process on pathogenesis. We demonstrate that the activity of the AtlA peptidoglycan hydrolase, an enzyme dedicated to septum cleavage, is controlled by several mechanisms, including glycosylation and recognition of the peptidoglycan substrate. We show that the long cell chains of E. faecalis mutants are more susceptible to phagocytosis and are no longer able to cause lethality in the zebrafish model of infection. Altogether, this work indicates that control of cell separation during division underpins the pathogenesis of E. faecalis infections and represents a novel enterococcal virulence factor. We propose that inhibition of septum cleavage during division represents an attractive therapeutic strategy to control infections.

The Impact of Genome Region of Difference 4 (RD4) on Mycobacterial Virulence and BCG Efficacy.

Comparative genome analyses have revealed a number of regions of difference (RD) among mycobacterial species. The functional consequences of most of these genome variations have not been studied. RD4, which encompasses Rv1506c-Rv1516c of Mycobacterium tuberculosis (M. tb) H37Rv, is absent in the closely related Mycobacterium bovis and M. bovis Bacille Calmette-Guérin (BCG). On the other hand, we previously found that Mycobacterium marinum has an extended RD4 which includes a number of genes involved in the biosynthesis of lipooligosaccharides (LOSs). As such, there appears to be a gradual decay of RD4 in mycobacterial genomes in the order of M. marinum, M. tb, and M. bovis (including BCG). To understand the potential effect of RD4 on mycobacterial virulence, in this study, we cloned the entire (Rv1501–1516c) and partial (Rv1501–1508c) RD4 into an integrating vector. These constructs were introduced to M. bovis BCG and M. marinum and the virulence of the RD4 knock-in strains were evaluated in the SCID mice and zebrafish infection models, respectively. BCG containing the entire RD4 exhibited similar levels of virulence to the parental strain but BCG containing partial RD4 (Rv1501–Rv1508c) was more attenuated. Similarly, zebrafish infection experiments showed that addition of partial RD4 also appeared to attenuate the virulence of M. marinum. However, M. marinum containing entire RD4 was more virulent than the wild type strain. Interestingly, BCG strains containing the entire or partial RD4 exhibited better protection of zebrafish against M. marinum challenge than the parental BCG. Taken together, our data suggest that RD4 plays a role in mycobacterial virulence and that RD4 knock-in BCG strains confer improved protection. Our study has provided new insights into the biological function of RD4 and evolution of mycobacterial genomes.

Membrane vesicles from Piscirickettsia salmonis induce protective immunity and reduce development of salmonid rickettsial septicemia in an adult zebrafish model

Infections caused by the facultative intracellular bacterial pathogen Piscirickettsia salmonis remains an unsolved problem for the aquaculture as no efficient treatments have been developed. As a result, substantial amounts of antibiotic have been used to limit salmonid rickettsial septicemia (SRS) disease outbreaks. The antibiotic usage has not reduced the occurrence, but lead to an increase in resistant strains, underlining the need for new treatment strategies. P. salmonis produce membrane vesicles (MVs); small spherical structures know to contain a variety of bacterial components, including proteins, lipopolysaccharides (LPS), DNA and RNA. MVs mimics' in many aspects their mother cell, and has been reported as alternative vaccine candidates. Here, MVs from P. salmonis was isolated and evaluated as a vaccine candidate against SRS in an adult zebrafish infection model. When zebrafish was immunized with MVs they were protected from subsequent challenge with a lethal dose of P. salmonis. Histological analysis showed a reduced bacterial load upon challenge in the MV immunized group, and the mRNA expression levels of several immune related genes altered, including mpeg1.1, tnfα, il1b, il10 and il6. The MVs induced the secretion of IgM upon immunization, indicating an immunogenic effect of the vesicles. Taken together, the data demonstrate a vaccine potential of MVs against P. salmonis.

Bioactive Molecule from Streptomyces sp. Mitigates MDR Klebsiella pneumoniae in Zebrafish Infection Model.

The emergence and spread of multi-drug resistant (MDR) especially carbapenem-resistant Klebsiella pneumoniae is a major emerging threat to public health, leading to excess in mortality rate as high as 50–86%. MDR K. pneumoniae manifests all broad mechanisms of drug resistance, hence development of new drugs to treat MDR K. pneumoniae infection has become a more relevant question in the scientific community. In the present study a potential Streptomyces sp. ASK2 was isolated from rhizosphere soil of medicinal plant. The multistep HPLC purification identified the active principle exhibiting antagonistic activity against MDR K. pneumoniae. The purified compound was found to be an aromatic compound with aliphatic side chain molecule having a molecular weight of 444.43 Da. FT-IR showed the presence of OH and C=O as functional groups. The bioactive compound was further evaluated for drug induced toxicity and efficacy in adult zebrafish infection model. As this is the first study on K. pneumoniae – zebrafish model, the infectious doses to manifest sub-clinical and clinical infection were optimized. Furthermore, the virulence of K. pneumoniae in planktonic and biofilm state was studied in zebrafish. The MTT assay of ex vivo culture of zebrafish liver reveals non-toxic nature of the proposed ASK2 compound at an effective dose. Moreover, significant increase in survival rate of infected zebrafish suggests that ASK2 compound from a new strain of Streptomyces sp. was potent in mitigating MDR K. pneumoniae infection.

Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion.

SummaryPseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal c-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here we identified an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase WarB. We established that WarA binds to c-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bi-functional Escherichia coli LPS O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.

Streptococcus suis small RNA rss04 contributes to the induction of meningitis by regulating capsule synthesis and by inducing biofilm formation in a mouse infection model

Streptococcus suis (SS) is an important pathogen for pigs, and it is also considered as a zoonotic agent for humans. Meningitis is one of the most common features of the infection caused by SS, but little is known about the mechanisms of SS meningitis. Recent studies have revealed that small RNAs (sRNAs) have emerged as key regulators of the virulence in several bacteria. In the previous study, we reported that SS sRNA rss04 was up-regulated in pig cerebrospinal fluid and contributes to SS virulence in a zebrafish infection model. Here, we show that rss04 facilitates SS invasion of mouse brain and lung in vivo. Label-free quantitation mass spectrometry analysis revealed that rss04 regulates transcriptional regulator CcpA and several virulence factors including LuxS. Transmission electron microscope and Dot-blot analyses indicated that rss04 represses capsular polysaccharide (CPS) production, which in turn facilitates SS adherence and invasion of mouse brain microvascular endothelial cells bEnd.3 in vitro and activates the mRNA expression of TLR2, CCL2, IL-6 and TNF-α in mouse brain in vivo at 12h post-infection. In addition, rss04 positively regulates SS biofilm formation. Survival analysis of infected mice showed that biofilm state in brain contributes to SS virulence by intracranial subarachnoidal route of infection. Together, our data reveal that SS sRNA rss04 contributes to the induction of meningitis by regulating the CPS synthesis and by inducing biofilm formation, thereby increasing the virulence in a mouse infection model. To our knowledge, rss04 represents the first bacterial sRNA that plays definitive roles in bacterial meningitis.

Mac Protein is not an Essential Virulence Factor for the Virulent Reference Strain Streptococcus suis P1/7.

Streptococcus suis is a major pathogen of pigs and also an important zoonotic agent for humans. A S. suis protein containing Mac-1 domain (designated Mac) is a protective antigen, exclusively cleaves porcine IgM, and contributes to complement evasion with the presence of high titers of specific porcine anti-S. suis IgM, but its role in S. suis virulence has not been investigated in natural healthy host without specific IgM. In this study, a mac deletion mutant was constructed by homologous recombination in S. suis serotype 2 virulent reference strain P1/7. Deletion of mac did not significantly influence phagocytosis or intracellular survival within murine macrophages RAW264.7, or the oxidative-burst induction of RAW264.7 and murine neutrophils. Furthermore, the mutant is as virulent as the wild-type strain in pig, mouse, and zebrafish infection models. Our data suggest that Mac is not essential for S. suis virulence in strain P1/7 in natural healthy host without specific IgM, and the immunogenicity of Mac does not appear to correlate with its significance for virulence.

Characteristics of Vibrio parahaemolyticus isolates obtained from crayfish (Procambarus clarkii) in freshwater

Vibrio parahaemolyticus usually occurs in coastal areas and is generally recognized as a marine bacterium. It has become the leading cause of gastroenteritis worldwide. In the present study, 96 V. parahaemolyticus isolates were obtained from freshwater crayfish (Procambarus clarkii) and classified by multilocus sequence typing. Fifty-three sequence types (STs) were identified among the 96 isolates analyzed, 38 of which were novel STs. These isolates fell into six groups and 42 singletons, suggesting a high level of genetic diversity. Screening for 9 virulence and virulence-related genes in the isolates revealed that 40 isolates contained more than two genes with possible roles in pathogenicity. The virulence of the representative isolates VP66 (trh+, ureC+, T3SS1+, T3SS2β+, T6SS2+) and VP80 (T3SS1+, T6SS1+, T6SS2+) were further assessed in zebrafish and mouse infection model in vivo, and the tested isolates were shown to be lethal to both zebrafish and mice. These results suggest that crayfish may serve as a carrier of V. parahaemolyticus in freshwater, and that some isolates may have the potential to cause foodborne disease in humans.

Dithiazole thione derivative as competitive NorA efflux pump inhibitor to curtail multi drug resistant clinical isolate of MRSA in a zebrafish infection model.

Multi drug resistant (MDR) pathogens pose a serious threat to public health since they can easily render most potent drugs ineffective. Efflux pump inhibitors (EPI) can be used to counter the MDR phenotypes arising due to increased efflux. In the present study, a series of dithiazole thione derivatives were synthesized and checked for its antibacterial and efflux pump inhibitory (EPI) activity. Among 10 dithiazole thione derivatives, real-time efflux studies revealed that seven compounds were potent EPIs relative to CCCP. Zebrafish toxicity studies identified four non-toxic putative EPIs. Both DTT3 and DTT9 perturbed membrane potential and DTT6 was haemolytic. Among DTT6 and DTT10, the latter was less toxic as evidenced by histopathology studies. Since DTT10 was non-haemolytic, did not affect the membrane potential, and was least toxic, it was chosen further for in vivo study, wherein DTT10 potentiated effect of ciprofloxacin against clinical strain of MRSA and reduced bacterial burden in muscle and skin tissue of infected zebrafish by ~ 1.7 and 2.5 log fold respectively. Gene expression profiling of major efflux transport proteins by qPCR revealed that clinical isolate of MRSA, in the absence of antibiotic, upregulated NorA, NorB and MepA pump, whereas it downregulates NorC and MgrA relative to wild-type strain of Staphylococcus aureus. In vitro studies with NorA mutant strains and substrate profiling revealed that at higher concentrations DTT10 is likely to function as a competitive inhibitor of NorA efflux protein in S. aureus, whereas at lower concentrations it might inhibit ciprofloxacin efflux through NorB and MepA as implied by docking studies. A novel non-toxic, non-haemolytic dithiazole thione derivative (DTT10) was identified as a potent competitive inhibitor of NorA efflux pump in S. aureus using in silico, in vitro and in vivo studies. This study also underscores the importance of using zebrafish infection model to screen and evaluate putative EPI for mitigating MDR strains of S. aureus.

Mycobacterial WhiB6 Differentially Regulates ESX-1 and the Dos Regulon to Modulate Granuloma Formation and Virulence in Zebrafish

During the course of infection, Mycobacterium tuberculosis (Mtb) is exposed to diverse redox stresses that trigger metabolic and physiological changes. How these stressors are sensed and relayed to the Mtb transcriptional apparatus remains unclear. Here, we provide evidence that WhiB6 differentially regulates the ESX-1 and DosR regulons through its Fe-S cluster. When challenged with NO, WhiB6 continually activates expression of the DosR regulons but regulates ESX-1 expression through initial activation followed by gradual inhibition. Comparative transcriptomic analysis of the holo- and reduced apo-WhiB6 complemented strains confirms these results and also reveals that WhiB6 controls aerobic and anaerobic metabolism, cell division, and virulence. Using the Mycobacterium marinum zebrafish infection model, we find that holo- and apo-WhiB6 modulate levels of mycobacterial infection, granuloma formation, and dissemination. These findings provide fresh insight into the role of WhiB6 in mycobacterial infection, dissemination, and disease development.

Identification and functional characterization of EseH, a new effector of the type III secretion system of Edwardsiella piscicida.

Edwardsiella piscicida, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. However, little is known about the associated effectors and their functional importance. In this study, we identified the ETAE_1757 encoded protein, termed here E. piscicida secretion effector H (EseH) as a novel T3SS effector. We found that upon infection with E. piscicida, EseH is translocated into nucleus of host cells which required the T3SS. Homology modelling analysis suggests that EseH is an enzyme that belongs to the family of phosphothreothine lyases. Consistently, EseH inhibited phosphorylation of ERK1/2, p38α and JNK MAPK pathways in host cells, but had no effect on the NF-kB pathway. Furthermore, mutation of the critical amino acid residues predicted to confer phosphothreonine lyase activity abolished the ability of EseH to inhibit phosphorylation of ERK1/2, p38α and JNK MAPK pathways in host cells. In addition, we found an increase in transcript levels of TNF-α, IL-12, IL-10 and IFN-γ in zebrafish infected with the eseH mutant when compared with the wild type bacterium. Importantly, the virulence of E. piscicida deficient in EseH was highly attenuated in the zebrafish infection model which correlated with decreased loads of the mutant bacterium in both liver and kidney. Complementation of the E. piscicida mutant strain with EseH restored virulence in zebrafish. These results identified EseH as a critical T3SS effector that contributes to virulence by targeting MAPK signalling during E. piscicida infection.

Phenotypic Plasticity Regulates Candida albicans Interactions and Virulence in the Vertebrate Host.

Phenotypic diversity is critical to the lifestyles of many microbial species, enabling rapid responses to changes in environmental conditions. In the human fungal pathogen Candida albicans, cells exhibit heritable switching between two phenotypic states, white and opaque, which yield differences in mating, filamentous growth, and interactions with immune cells in vitro. Here, we address the in vivo virulence properties of the two cell states in a zebrafish model of infection. Multiple attributes were compared including the stability of phenotypic states, filamentation, virulence, dissemination, and phagocytosis by immune cells, and phenotypes equated across three different host temperatures. Importantly, we found that both white and opaque cells could establish a lethal systemic infection. The relative virulence of the two cell types was temperature dependent; virulence was similar at 25°C, but at higher temperatures (30 and 33°C) white cells were significantly more virulent than opaque cells. Despite the difference in virulence, fungal burden, and dissemination were similar between cells in the two states. Additionally, both white and opaque cells exhibited robust filamentation during infection and blocking filamentation resulted in decreased virulence, establishing that this program is critical for pathogenesis in both cell states. Interactions between C. albicans cells and immune cells differed between white and opaque states. Macrophages and neutrophils preferentially phagocytosed white cells over opaque cells in vitro, and neutrophils showed preferential phagocytosis of white cells in vivo. Together, these studies distinguish the properties of white and opaque cells in a vertebrate host, and establish that the two cell types demonstrate both important similarities and key differences during infection.

A prebiotic role of Ecklonia cava improves the mortality of Edwardsiella tarda-infected zebrafish models via regulating the growth of lactic acid bacteria and pathogen bacteria.

In this study, the beneficial prebiotic roles of Ecklonia cava (E.cava, EC) were evaluated on the growth of lactic acid bacteria (LAB) and pathogen bacteria and the mortality of pathogen-bacteria infected zebrafish model. The result showed that the original E.cava (EC) led to the highest growth effects on three LABs (Lactobacillus brevis, L. brevis; Lactobacillus pentosus, L. pentosus; Lactobacillus plantarum; L. plantarum) and it was dose-dependent manners. Also, EC, its Celluclast enzymatic (ECC) and 100% ethanol extracts (ECE) showed the anti-bacterial activities on the fish pathogenic bacteria such as (Edwardsiella tarda; E. tarda, Streptococcus iniae; S. iniae, and Vibrio harveyi; V. harveyi). Interestingly, EC induced the higher production of the secondary metabolites from L.plantarum in MRS medium. The secondary metabolites produced by EC significantly inhibited the growth of pathogen bacteria. In further invivo study, the co-treatment of EC and L.plantarum improved the growth and mortality of E.tarda-infected zebrafish as regulating the expression of inflammatory molecules such as iNOS and COX2. Taken together, our present study suggests that the EC plays an important role as a potential prebiotic and has a protective effect against the infection caused by E.tarda injection in zebrafish. Also, our conclusion from this evidence is that EC can be used and applied as a useful prebiotic.

Proteomic analysis of outer membrane proteins and vesicles of a clinical isolate and a collection strain of Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that displays a high genomic diversity, complicating the study of its pathogenicity, virulence and resistance factors. The interaction of bacterial pathogens with host cells is largely mediated by outer membrane proteins (OMPs). Indeed, several OMPs of Gram-negative bacteria have been recognized as important virulence factors and targets for host immune recognition or to be involved in mechanisms of resistance to antimicrobials. OMPs are also present in outer membrane vesicles (OMVs), which bacteria constitutively secrete to the extracellular milieu and are essential for bacterial survival and pathogenesis. Here, we report the characterization of the OMP and native OMV subproteomes of a clinical isolate (M30) and a collection strain (ATCC13637) of S. maltophilia. We had previously shown that the ATCC13637 strain has an attenuated phenotype in a zebrafish model of infection, as well as a distinct susceptibility profile against a panel of antimicrobials. The protein profiles of the OMP and OMV subproteomes of these two strains and their differences consequently point at pathogenesis, virulence or resistance proteins, such as two variants of the quorum-sensing factor Ax21 that are found to be highly abundant in the OMP fraction and exported to OMVs. Biological significanceStenotrophomonas maltophilia is rapidly climbing positions in the ranking of multidrug-resistant pathogens that are frequently isolated in hospital environments. Being an emerging human pathogen, the knowledge on the factors determining the pathogenicity, virulence and resistance traits of this microorganism is still scarce. Outer membrane proteins (OMPs) and vesicles (OMVs) are key elements for the interaction of Gram-negative bacteria with their environment −including the host–and have fundamental roles in both infection and resistance processes. The present study sets a first basis for a phenotype-dependent characterisation of the OMP subproteome of S. maltophilia and complements very recent work on the OMV subproteome of this species. The variability found among even two strains demonstrates once more that the analysis of genotypically and phenotypically distinct isolates under various conditions will be required before we can draw a significant picture of the OMP and OMV subproteomes of S. maltophilia.

Protective effect of a recombinant VHSV-G vaccine using poly(I:C) loaded nanoparticles as an adjuvant in zebrafish (Danio rerio) infection model

There is a constant need to increase the efficiency of vaccines in the aquaculture industry. Although several nano-based vaccine formulations have been reported, to the best of our knowledge so far only one of them have been implemented in the industry. Here we report on chitosan-poly(I:C) nanoparticles (NPs) that could be used as a non-specific adjuvant in antiviral vaccines in aquaculture. We have characterized the physical parameters of the NPs, studied the in vivo and in vitro bio-distribution of fluorescent NPs and verified NP uptake by zebrafish leucocytes. We used the zebrafish model to test the protective efficiency of the recombinant glycoprotein G (rgpG) of VHSV compared to inactivated whole virus (iV) against VHSV using NPs as an adjuvant in both formulations. In parallel we tested free poly(I:C) and rgpG (pICrgpG), and free chitosan and rgpG (CSrgpG) vaccine formulations. While the iV group (with NP adjuvant) provided the highest overall survival, all vaccine formulations with poly(I:C) provided a significant protection against VHSV; possibly through an early induction of an anti-viral state. Our results suggest that chitosan-poly(I:C) NPs are a promising adjuvant candidate for future vaccine formulations.

Zebrafish as a model host for studies of Piscirickettsia salmonis and its outer membrane vesicles

The intracellular bacterium Piscirickettsia salmonis is the etiologic agent of salmonid rickettsial septicaemia (SRS), a bacterial disease that over the last decades has had a devastating effect on the Chilean aquaculture. Although P. salmonis has been recognized as a major fish pathogen for several years, there is still a lack in knowledge in regard to the bacterium pathogenesis. As outer membrane vesicles (OMVs) has been reported to be important for the infection of several bacteria species, the present study established a zebrafish infection model for studies of P. salmonis and the role of the bacterium’s OMVs during infection. The P. salmonis derived vesicles were, shown to be internalized by cell cultures and OMVs injected into adult zebrafish revealed an upregulation of several proinflammatory genes, indicating a modulating effect on the immune system. Comparison of OMVs from three strains of P. salmonis, isolated from salmonids in Norway (NVI 5692), Canada (NVI 5892) and Chile (LF-89) did, further, reveal several strain-specific differences related to higher virulence capability for LF-89, and stronger similarities between NVI 5692 and NVI 5892. In summary, the present study is the first in-depth analysis of P. salmonis derived OMVs, highlighting strain-specific factors able to contribute to adaptations and virulence.

Salmonella plasmid virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish infection model.

Salmonella enterica serovar typhimurium (S.typhimurium) is a facultative intracellular pathogen that can cause gastroenteritis and systemic infection in a wide range of hosts. Salmonella plasmid virulence gene spvB is closely related to bacterial virulence in different cells and animal models, and the encoded protein acts as an intracellular toxin required for ADP-ribosyl transferase activity. However, until now there is no report about the pathogenecity of spvB gene on zebrafish. Due to the outstanding advantages of zebrafish in analyzing bacteria-host interactions, a S.typhimurium infected zebrafish model was set up here to study the effect of spvB on autophagy and intestinal pathogenesis invivo. We found that spvB gene could decrease the LD50 of S.typhimurium, and the strain carrying spvB promoted bacterial proliferation and aggravated the intestinal damage manifested by the narrowed intestines, fallen microvilli, blurred epithelium cell structure and infiltration of inflammatory cells. Results demonstrated the enhanced virulence induced by spvB in zebrafish. In spvB-mutant strain infected zebrafish, the levels of Lc3 turnover and Beclin1 expression increased, and the double-membraned autophagosome structures were observed, suggesting that spvB can inhibit autophagy activity. In summary, our results indicate that S.typhimurium strain containing spvB displays more virulence, triggering an increase in bacterial survival and intestine injuries by suppressing autophagy for the first time. This model provides novel insights into the role of Salmonella plasmid virulence gene in bacterial pathogenesis, and can help to further elucidate the relationship between bacteria and host immune response.

The Extraintestinal Pathogenic Escherichia coli Factor RqlI Constrains the Genotoxic Effects of the RecQ-Like Helicase RqlH.

Extraintestinal pathogenic Escherichia coli colonize the human gut and can spread to other body sites to induce diseases such as urinary tract infections, sepsis, and meningitis. A complete understanding of the infection process is hindered by both the inherent genetic diversity of E. coli and the large number of unstudied genes. Here, we focus on the uncharacterized gene rqlI, which our lab recently uncovered in a Tn-seq screen for bacterial genes required within a zebrafish model of infection. We demonstrate that the ΔrqlI mutant experiences a growth defect and increased DNA stress in low oxygen conditions. In a genetic screen for suppressor mutations in the Δrql strain, we found that the shortcomings of the Δrql mutant are attributable to the activity of RqlH, which is known in other bacteria to be a helicase of the RecQ family that contains a phosphoribosyltransferase (PRTase) domain. Disruption of rqlH rescues the ΔrqlI strain in both in vivo and in vitro assays, while the expression of RqlH alone activates the SOS response coincident with bacterial filamentation, heightened sensitivity to DNA damage, and an increased mutation rate. The analysis of truncation mutants indicates that, in the absence of RqlI, RqlH toxicity is due to its PRTase domain. Complementary studies demonstrate that the toxicity of RqlH is modulated in a context-dependent fashion by overlapping domains within RqlI. This regulation is seemingly direct, given that the two proteins physically interact and form an operon. Interestingly, RqlH and RqlI orthologs are encoded by a diverse group of bacteria, but in many of these microbes, and especially in Gram-positive organisms, rqlH is found in the absence of rqlI. In total, this work shows that RqlH and RqlI can act in a strain-specific fashion akin to a toxin-antitoxin system in which toxicity is mediated by an atypical helicase-associated PRTase domain.