Large-scale screen highlights the importance of capsule for virulence in the zoonotic pathogen Streptococcus iniae

Zoonotic pathogens have the unique ability to cross the species barrier, causing disease in both humans and specific animal hosts. Streptococcus iniae is a zoonotic pathogen of both fish and humans, and the clinical presentations of S. iniae infections in fish and humans are very similar to those caused by various human-specific streptococcal pathogens. Virulence mechanisms required for infection by this pathogen of either host have yet to be determined. Using the previously reported zebrafish infectious disease model, we performed a large-scale screening to determine genes required for systemic infection. Screening 1,128 signature-tagged transposon mutants through the zebrafish model allowed identification of 41 potential mutants that were unable to survive within the host environment. Greater than 50% of the mutants that could be identified through homology searches were highly homologous to genes found in other human-specific streptococcal pathogens, while 32% were found to have no homology to any sequences found in the databases, suggesting as yet unknown gram-positive bacterial virulence factors. A large percentage of the insertions were found to be located in several putative capsule synthesis genes, an important virulence component for other systemic pathogens. Density gradient assays demonstrated that several of these putative capsule mutants have dissimilar buoyant densities, suggesting different levels of capsule synthesis. Putative capsule mutants were also less resistant to phagocytosis in whole-blood assays than wild-type S. iniae. Our initial large-scale characterization of S. iniae virulence highlights the importance of the capsule for successful infection.     

Zebrafish and Streptococcal Infections

Streptococcal bacteria are a versatile group of gram‐positive bacteria capable of infecting several host organisms, including humans and fish. Streptococcal species are common colonizers of the human respiratory and gastrointestinal tract, but they also cause some of the most common life‐threatening, invasive infections in humans and aquaculture. With its unique characteristics and efficient tools for genetic and imaging applications, the zebrafish (Danio rerio) has emerged as a powerful vertebrate model for infectious diseases. Several zebrafish models introduced so far have shown that zebrafish are suitable models for both zoonotic and human‐specific infections. Recently, several zebrafish models mimicking human streptococcal infections have also been developed. These models show great potential in providing novel information about the pathogenic mechanisms and host responses associated with human streptococcal infections. Here, we review the zebrafish infection models for the most relevant streptococcal species: the human‐specific Streptococcus pneumoniae and Streptococcus pyogenes, and the zoonotic Streptococcus iniae and Streptococcus agalactiae. The recent success and the future potential of these models for the study of host–pathogen interactions in streptococcal infections are also discussed.     

Streptococcus iniae virulence is associated with a distinct genetic profile

Streptococcus iniae causes meningoencephalitis and death in commercial fish species and has recently been identified as an emerging human pathogen producing fulminant soft tissue infection. As identified by pulsed-field gel electrophoresis (PFGE), strains causing disease in either fish or humans belong to a single clone, whereas isolates from nondiseased fish are genetically diverse. In this study, we used in vivo and in vitro models to examine the pathogenicity of disease-associated isolates. Strains with the clonal (disease-associated) PFGE profile were found to cause significant weight loss and bacteremia in a mouse model of subcutaneous infection. As little as 10(2) CFU of a disease-associated strain was sufficient to establish bacteremia, with higher inocula (10(7)) resulting in increased mortality. In contrast, non-disease-associated (commensal) strains failed to cause bacteremia and weight loss, even at inocula of 10(8) CFU. In addition, disease-associated strains were more resistant to phagocytic clearance in a human whole blood killing assay compared to commensal strains, which were almost entirely eradicated. Disease-associated strains were also cytotoxic to human endothelial cells as measured by lactate dehydrogenase release from host cells. However, both disease-associated and commensal strains adhered to and invaded cultured human epithelial and endothelial cells equally well. While cellular invasion may still contribute to the pathogenesis of invasive S. iniae disease, resistance to phagocytic clearance and direct cytotoxicity appear to be discriminating virulence attributes of the disease-associated clone.     

The Streptococcus iniae transcriptional regulator CpsY is required for protection from neutrophil-mediated killing and proper growth in vitro

The ability of a pathogen to metabolically adapt to the local environment for optimal expression of virulence determinants is a continued area of research. Orthologs of the Streptococcus iniae LysR family regulator CpsY have been shown to regulate methionine biosynthesis and uptake pathways but appear to influence expression of several virulence genes as well. An S. iniae mutant with an in-frame deletion of cpsY (ΔcpsY mutant) is highly attenuated in a zebrafish infection model. The ΔcpsY mutant displays a methionine-independent growth defect in serum, which differs from the methionine-dependent defect observed for orthologous mutants of Streptococcus mutans and Streptococcus agalactiae. On the contrary, the ΔcpsY mutant can grow in excess of the wild type (WT) when supplemented with proteose peptone, suggesting an inability to properly regulate growth. CpsY is critical for protection of S. iniae from clearance by neutrophils in whole blood but is dispensable for intracellular survival in macrophages. Susceptibility of the ΔcpsY mutant to killing in whole blood is not due to a growth defect, because inhibition of neutrophil phagocytosis rescues the mutant to WT levels. Thus, CpsY appears to have a pleiotropic regulatory role for S. iniae, integrating metabolism and virulence. Furthermore, S. iniae provides a unique model to investigate the paradigm of CpsY-dependent regulation during systemic streptococcal infection.     

Trojan horse effect: phagocyte-mediated Streptococcus iniae infection of fish

The salmonid macrophage-like cell line RTS-11 and purified trout pronephros phagocytes were used to analyze in vitro entry and survival of two Streptococcus iniae serotypes. Efficient invasion by S. iniae occurred in both cells, but only the type II strain persisted in pronephros phagocytes for at least 48 h. Ex vivo models of opsonin-dependent phagocytosis by pronephros phagocytes demonstrated increased phagocytosis efficacy. Analysis of phagocytes collected from diseased fish demonstrated that approximately 70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system.     

Invasive Streptococcus iniae infections outside North America

Streptococcus iniae, a fish pathogen causing infections in aquaculture farms worldwide, has only been reported to cause human infections in North America. In this article, we report the first two cases of invasive S. iniae infections in two Chinese patients outside North America. While the first patient presented with bacteremic cellulitis, which is the most common presentation in previous cases, the second patient represents the first recognized case of S. iniae osteomyelitis. Both S. iniae strains isolated from the two patients were either misidentified or unidentified by three commercial systems and were only identified by 16S rRNA gene sequencing. Since no currently available commercial system for bacterial identification includes S. iniae in its database, 16S rRNA gene sequencing is the most practical and reliable method to identify the bacterium at the moment. In contrast to the distinct genetic profile described previously in clinical isolates from Canada, the present two isolates and a clinical isolate from a Canadian patient were found to be genetically unrelated, as demonstrated by pulsed-field gel electrophoresis. Morphologically, colonies of both isolates were also larger, more beta-hemolytic and mucoid, which differ from the usual morphotype described for S. iniae. Owing to their habit of cooking and eating fresh fish, the Asian population is strongly associated with S. iniae infections. As a result of the difficulty in making microbiological diagnosis in patients with cellulitis and the problem of identification in most clinical microbiology laboratories, the prevalence of S. iniae infections, especially in the Asian population, may have been under-estimated.     

Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development

Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Tn917 mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniae mutants. The Tn917 insertion in one mutant disrupted an S. iniae homologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with DeltaPGM mutant cells reaching sizes approximately 3-fold larger than those of the wild type (WT). The DeltaPGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the DeltaPGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the DeltaPGM mutant. In analysis of the aborted infectious process, we found that DeltaPGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the DeltaPGM mutant and later challenged with a lethal dose of WT S. iniae survived. We conclude that the pgm gene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniae DeltaPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.     

Development of loop-mediated isothermal amplification method for rapid detection of Streptococcus iniae, the causative agent of streptococcicosis in fish

Streptococcus iniae is a major pathogen that causes sever economic losses in tilapia aquaculture. A set of four specific primers was designed by targeting lctO gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64 °C in a simple water bath. The sensitivity of the LAMP assay for the detection of S. iniae is about 12.4 cells per reaction in both of pure cultures and added fish tissues cultures. LAMP products could be judged with agar gel or naked eye after addition of SYBR Green I. There were no cross-reactions with other bacterial strains indicating high specificity of the LAMP. The LAMP method was also applied to detect S. iniae-infected tilapia tissues effectively. The LAMP assay reported here indicates the potential usefulness of the technique as a valuable simple, rapid alternative procedure for the detection of S. iniae during streptococcicosis monitoring of cultured fish.     

Identification of a streptolysin S-associated gene cluster and its role in the pathogenesis of Streptococcus iniae disease

Streptococcus iniae causes meningoencephalitis and death in cultured fish species and soft-tissue infection in humans. We recently reported that S. iniae is responsible for local tissue necrosis and bacteremia in a murine subcutaneous infection model. The ability to cause bacteremia in this model is associated with a genetic profile unique to strains responsible for disease in fish and humans (J. D. Fuller, D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo, Infect. Immun. 69:1994-2000, 2001). S. iniae produces a cytolysin that confers a hemolytic phenotype on blood agar media. In this study, we characterized the genomic region responsible for S. iniae cytolysin production and assessed its contribution to virulence. Transposon (Tn917) mutant libraries of commensal and disease-associated S. iniae strains were generated and screened for loss of hemolytic activity. Analysis of two nonhemolytic mutants identified a chromosomal locus comprising 9 genes with 73% homology to the group A streptococcus (GAS) sag operon for streptolysin S (SLS) biosynthesis. Confirmation that the S. iniae cytolysin is a functional homologue of SLS was achieved by PCR ligation mutagenesis, complementation of an SLS-negative GAS mutant, and use of the SLS inhibitor trypan blue. SLS-negative sagB mutants were compared to their wild-type S. iniae parent strains in the murine model and in human whole-blood killing assays. These studies demonstrated that S. iniae SLS expression is required for local tissue necrosis but does not contribute to the establishment of bacteremia or to resistance to phagocytic clearance.     

Analysis of the polysaccharide capsule of the systemic pathogen Streptococcus iniae and its implications in virulence

Systemic pathogens have developed numerous strategies for evading the defenses of the host, permitting dissemination and multiplication in various tissues. One means of survival in the host, particularly in the bloodstream, has been attributed to the ability to avoid phagocytosis via capsular polysaccharide. To further define the virulence capacity of Streptococcus iniae, a zoonotic pathogen with the ability to cause severe systemic disease in both fish and humans, we performed an analysis of the capsule locus. The initial analysis included cloning and sequencing of the capsule synthesis operon, which revealed an approximately 21-kb region that is highly homologous to capsule operons of other streptococci. A genetic comparison of S. iniae virulent strain 9117 and commensal strain 9066 revealed that the commensal strain does not have the central region of the capsule operon composed of several important capsule synthesis genes. Four 9117 insertion or deletion mutants with mutations in the beginning, middle, or end of the capsule locus were analyzed to determine their capsule production and virulence. Virulence profiles were analyzed for each mutant using three separate criteria, which demonstrated the attenuation of each mutant in several tissue environments. These analyses also provided insight into the different responses of the host to each mutant strain compared to a wild-type infection. Our results demonstrate that capsule is not required for all host environments, while excess capsule is also not optimal, suggesting that for an "ideal" systemic infection, capsule production is most likely regulated while the bacterium is in different environments of the host.     

The Genus Streptococcus – Part II: New Species and Pathogens in the “Miscellaneous” Streptococci and “Viridans” Streptococci Groups

In the second part of this two-part series on an update on the streptococci, new species and emergent human pathogens in the “viridans streptococci” and the “miscellaneous streptococci” groups are discussed. Among the “miscellaneous streptococci,” the most important organism in human infectious diseases is Streptococcus suis. This organism is primarily an agent of disease in swine and other animals and has now emerged as a significant human pathogen, causing bacteremia and meningitis, particularly among populations in Asia and the Far East. While S. suis capsular serotype 2 is the most frequently isolated agent, infections caused by other serotypes are increasingly being reported. In addition, disease caused by S. suis is now being described more frequently in North America in individuals with significant occupational exposure to swine. Among the viridans streptococci, several new species in the mitis-sanguinis group have been described as human pathogens, including Streptococcus pseudopneumoniae, Streptococcus oligofermentans, and Streptococcus tigurinus. Other newly reported viridans group species either comprise part of the human oral microbiome or have been isolated from animals. This article presents information on these bacterial agents, including characteristics for the recognition and identification of the more important clinical isolates in these streptococcal groups.     

Zebrafish as a model for zoonotic aquatic pathogens

Aquatic habitats harbor a multitude of bacterial species. Many of these bacteria can act as pathogens to aquatic species and/or non-aquatic organisms, including humans, that come into contact with contaminated water sources or colonized aquatic organisms. In many instances, the bacteria are not pathogenic to the aquatic species they colonize and are only considered pathogens when they come into contact with humans. There is a general lack of knowledge about how the environmental lifestyle of these pathogens allows them to persist, replicate and produce the necessary pathogenic mechanisms to successfully transmit to the human host and cause disease. Recently, the zebrafish infectious disease model has emerged as an ideal system for examining aquatic pathogens, both in the aquatic environment and during infection of the human host. This review will focus on how the zebrafish has been used successfully to analyze the pathogenesis of aquatic bacterial pathogens.     

Host Immune Response and Acute Disease in a Zebrafish Model of Francisella Pathogenesis

Members of the bacterial genus Francisella are highly virulent and infectious pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species are needed to provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. We took advantage of the recent discovery of a novel species of Francisella to establish a zebrafish/Francisella comparative model of pathogenesis and host immune response. Adult zebrafish were susceptible to acute Francisella-induced disease and suffered mortality in a dose-dependent manner. Using immunohistochemical analysis, we localized bacterial antigens primarily to lymphoid tissues and livers of zebrafish following infection by intraperitoneal injection, which corresponded to regions of local cellular necrosis. Francisella sp. bacteria replicated rapidly in these tissues beginning 12 h postinfection, and bacterial titers rose steadily, leveled off, and then decreased by 7 days postinfection. Zebrafish mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of interleukin-1β (IL-1β), gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h postinfection and persisting for up to 7 days postinfection. In addition, exposure of zebrafish to heat-killed bacteria demonstrated that the significant induction of IL-1β was highly specific to live bacteria. Taken together, the pathology and immune response to acute Francisella infection in zebrafish share many features with those in mammals, highlighting the usefulness of this new model system for addressing both general and specific questions about Francisella host-pathogen interactions via an evolutionary approach.     

Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish

The zebrafish (Danio rerio) is a widely used model for developmental biology, neurobiology, toxicology, and genetic disease. Recently, the zebrafish has been recognized as a valuable model for infectious disease and immunity. In this study the pathogenesis and inflammatory cytokine response of zebrafish to experimental Edwardsiella tarda infection was characterized. In challenge experiments, zebrafish embryos were susceptible to infection by immersion. Adult fish were susceptible to challenge by intraperitoneal (ip) injection but not static immersion unless the epithelial layer was perturbed by scraping prior to exposure. To determine if E. tarda infection induces a typical acute inflammatory response, mRNA expression levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) were assessed by quantitative real-time PCR. The expression levels of IL-1beta and TNFalpha were significantly upregulated in infected zebrafish embryos and adults. The methods developed in this study will be particularly valuable for targeted gene disruption studies of host immune components and in zebrafish genetic screens.     

Transgenic zebrafish models of neurodegenerative diseases

Since the introduction of the zebrafish as a model for the study of vertebrate developmental biology, an extensive array of techniques for its experimental manipulation and analysis has been developed. Recently it has become apparent that these powerful methodologies might be deployed in order to elucidate the pathogenesis of human neurodegenerative diseases and to identify candidate therapeutic approaches. In this article, we consider evidence that the zebrafish central nervous system provides an appropriate setting in which to model human neurological disease and we review techniques and resources available for generating transgenic models. We then examine recent publications showing that appropriate phenotypes can be provoked in the zebrafish through transgenic manipulations analogous to genetic abnormalities known to cause human tauopathies, polyglutamine diseases or motor neuron degenerations. These studies show proof of concept that findings in zebrafish models can be applicable to the pathogenic mechanisms underlying human diseases. Consequently, the prospects for providing novel insights into neurodegenerative diseases by exploiting transgenic zebrafish models and discovery-driven approaches seem favorable.     

Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity

The zebrafish, a genetically tractable model vertebrate, is naturally susceptible to tuberculosis caused by Mycobacterium marinum, a close genetic relative of the causative agent of human tuberculosis, Mycobacterium tuberculosis. We previously developed a zebrafish embryo-M. marinum infection model to study host-pathogen interactions in the context of innate immunity. Here, we have constructed a flowthrough fish facility for the large-scale longitudinal study of M. marinum-induced tuberculosis in adult zebrafish where both innate and adaptive immunity are operant. We find that zebrafish are exquisitely susceptible to M. marinum strain M. Intraperitoneal injection of five organisms produces persistent granulomatous tuberculosis, while the injection of approximately 9,000 organisms leads to acute, fulminant disease. Bacterial burden, extent of disease, pathology, and host mortality progress in a time- and dose-dependent fashion. Zebrafish tuberculous granulomas undergo caseous necrosis, similar to human tuberculous granulomas. In contrast to mammalian tuberculous granulomas, zebrafish lesions contain few lymphocytes, calling into question the role of adaptive immunity in fish tuberculosis. However, like rag1 mutant mice infected with M. tuberculosis, we find that rag1 mutant zebrafish are hypersusceptible to M. marinum infection, demonstrating that the control of fish tuberculosis is dependent on adaptive immunity. We confirm the previous finding that M. marinum DeltaRD1 mutants are attenuated in adult zebrafish and extend this finding to show that DeltaRD1 predominantly produces nonnecrotizing, loose macrophage aggregates. This observation suggests that the macrophage aggregation defect associated with DeltaRD1 attenuation in zebrafish embryos is ongoing during adult infection.     

Expanding Francisella models: Pairing up the soil amoeba Dictyostelium with aquatic Francisella

The bacterial genus Francisella comprises highly pathogenic species that infect mammals, arthropods, fish and protists. Understanding virulence and host defense mechanisms of Francisella infection relies on multiple animal and cellular model systems. In this review, we want to summarize the most commonly used Francisella host model platforms and highlight novel, alternative model systems using aquatic Francisella species. Established mouse and macrophage models contributed extensively to our understanding of Francisella infection. However, murine and human cells display significant differences in their response to Francisella infection. The zebrafish and the amoeba Dictyostelium are well-established model systems for host-pathogen interactions and open up opportunities to investigate bacterial virulence and host defense. Comparisons between model systems using human and fish pathogenic Francisella species revealed shared virulence strategies and pathology between them. Hence, zebrafish and Dictyostelium might complement current model systems to find new vaccine candidates and contribute to our understanding of Francisella infection.