Melody N. Neely

Streptococcus-Zebrafish Model of Bacterial Pathogenesis

ABSTRACT Due to its small size, rapid generation time, powerful genetic systems, and genomic resources, the zebrafish has emerged as an important model of vertebrate development and human disease. Its well-developed adaptive and innate cellular immune systems make the zebrafish an ideal model for the study of infectious diseases. With a natural and important pathogen of fish, Streptococcus iniae, we have established a streptococcus- zebrafish model of bacterial pathogenesis. Following injection into the dorsal muscle, zebrafish developed a lethal infection, with a 50% lethal dose of 103 CFU, and died within 2 to 3 days. The pathogenesis of infection resembled that of S. iniae in farmed fish populations and that of several important human streptococcal diseases and was characterized by an initial focal necrotic lesion that rapidly progressed to invasion of the pathogen into all major organ systems, including the brain. Zebrafish were also susceptible to infection by the human pathogen Streptococcus pyogenes. However, disease was characterized by a marked absence of inflammation, large numbers of extracellular streptococci in the dorsal muscle, and extensive myonecrosis that occurred far in advance of any systemic invasion. The genetic systems available for streptococci, including a novel method of mutagenesis which targets genes whose products are exported, were used to identify several mutants attenuated for virulence in zebrafish. This combination of a genetically amenable pathogen with a well-defined vertebrate host makes the streptococcus-zebrafish model of bacterial pathogenesis a powerful model for analysis of infectious disease.

Functional and genetic analysis of regulatory regions of coliphage H-19B: location of shiga-like toxin and lysis genes suggest a role for phage functions in toxin release

Analysis of the DNA sequence of a 17 kb region of the coli lambdoid phage H‐19B genome located the genes encoding shiga‐like toxin I (Stx‐I) downstream of the gene encoding the analogue of the phage λ Q transcription activator with its site of action, qut at the associated pR′ late promoter, and upstream of the analogues of λ genes encoding lysis functions. Functional studies, including measurement of the effect of H‐19B Q action on levels of Stx expressed from an H‐19B prophage, show that the H‐19B Q acts as a transcription activator with its associated pR′(qut) by promoting readthrough of transcription terminators. Another toxin‐producing phage, 933W, has the identical Q gene and pR′(qut) upstream of the stx‐II genes. The H‐19B Q also activates Stx‐II expression from a 933W prophage. An ORF in H‐19B corresponding to the holin lysis genes of other lambdoid phages differs by having only one instead of the usual two closely spaced translation initiation signals that are thought to contribute to the time of lysis. These observations suggest that stx‐I expression can be enhanced by transcription from pR′ as well as a model for toxin release through cell lysis mediated by action of phage‐encoded lysis functions. Functional studies show that open reading frames (ORFs) and sites in H‐19B that resemble components of the N transcription antitermination systems controlling early operons of other lambdoid phages similarly promote antitermination. However, this N‐like system differs significantly from those of other lambdoid phages.

Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli

The stx genes of many Shiga toxin‐encoding Escherichia coli (STEC) strains are encoded by prophages of the λ bacteriophage family. In the genome of the Stx1‐encoding phage H‐19B, the stx1AB genes are found ≈ 1 kb downstream of the late phage promoter, pR′, but are known to be regulated by the associated iron‐regulated promoter, pStx1. Growth of H‐19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H‐19B prophage. We found that replication of the phage genome, with the associated increase in stx1AB copy number, is the most quantitatively important mechanism by which H‐19B induction increases Stx1 production. Three promoters are shown to be involved in stx1AB transcription after phage induction, the iron‐regulated pStx1 and the phage‐regulated pR and pR′ promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the pR′ promoter, contrary to previous findings in the related Stx2‐encoding phage φ361, was found to be unnecessary for high‐level Stx1 production after phage induction. Finally, we present evidence that phage‐mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx1AB copy number, regulating stx1AB transcription and allowing for Stx1 release, the biology of the Stx‐encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.

A branch in the ToxR regulatory cascade of Vibrio cholerae revealed by characterization of toxT mutant strains

Co‐ordinate expression of genes associated with pathogenicity in Vibrio cholerae requires two transcription activators, ToxR and ToxT. Work carried out to date suggests that ToxR activates transcription of the toxT gene and that ToxT directly activates transcription of several genes whose products play a role in colonization or CT production by V. cholerae. Previous work also suggests that ToxR can directly activate transcription of the CT operon (ctxAB) independently of ToxT, thereby implying a degree of complexity in control of the ctxAB operon not found with other genes of the ToxR regulon. We tested the regulatory cascade model of virulence gene expression by constructing strains of classical and El Tor V. cholerae deleted for the coding sequence for the putative DNA‐binding domain of toxT. Phenotypic analysis of these strains suggests that V. cholerae has ToxT‐dependent and ToxT‐independent branches of its virulence regulon. The results also raise questions about the precise role for ToxR in activation of ctxAB transcription.

Role of RopB in Growth Phase Expression of the SpeB Cysteine Protease of Streptococcus pyogenes

The Rgg family of transcription regulators is widely distributed among gram-positive bacteria; however, how the members of this family control transcription is poorly understood. In the pathogen Streptococcus pyogenes, the Rgg family member RopB is required for transcription of the gene that encodes the secreted SpeB cysteine protease. Expression of the protease follows distinct kinetics that involves control of transcription in response to the growth phase. In this study, the contribution of RopB to growth phase control was examined. The gene encoding the protease (speB) and ropB are transcribed divergently from a 940-bp intergenic region. Primer extension analyses, in conjunction with reporter fusion studies, revealed that the major region controlling the transcription of both speB and ropB is adjacent to ropB and that the promoters for the two genes likely overlap. Furthermore, it was found that RopB is a DNA-binding protein that specifically binds to sequences in this control region. The interrelationship between ropB and speB expression was further reflected in the observation that transcription of ropB itself is subject to growth phase control. However, while expression of ropB from a promoter expressed during the early logarithmic phase of growth could complement a ropB deletion mutant, ectopic expression of ropB did not uncouple the expression of speB from its growth phase signal. These data implicate other factors in growth phase control and suggest that regulation of ropB expression itself is not the central mechanism of control.

Large-Scale Screen Highlights the Importance of Capsule for Virulence in the Zoonotic Pathogen Streptococcus iniae

ABSTRACT 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.

Evolution of the zebrafish model: From development to immunity and infectious disease

The successful zebrafish developmental model has now expanded to being used as a model for the analysis of host-pathogen interactions during infectious disease. Numerous pathogens have been demonstrated to infect zebrafish and new mechanisms of virulence, as well as host defense have been uncovered using this new model. In this review we summarize the literature on how the zebrafish infectious disease model is being used to decipher virulence mechanisms used by various pathogens and the host defense mechanisms initiated to combat infection.

Analysis of the Polysaccharide Capsule of the Systemic Pathogen Streptococcus iniae and Its Implications in Virulence

ABSTRACT 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.

Streptococcus iniae Capsule Impairs Phagocytic Clearance and Contributes to Virulence in Fish

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.

Characterization of MtsR, a New Metal Regulator in Group A Streptococcus, Involved in Iron Acquisition and Virulence

ABSTRACT Group A streptococcus (GAS) is a common pathogen of the human skin and mucosal surfaces capable of producing a variety of diseases. In this study, we investigated regulation of iron uptake in GAS and the role of a putative transcriptional regulator named MtsR (for Mts repressor) with homology to the DtxR family of metal-dependent regulatory proteins. An mtsR mutant was constructed in NZ131 (M49 serotype) and analyzed. Western blot and RNA analysis showed that mtsR inactivation results in constitutive transcription of the sia (streptococcal iron acquisition) operon, which was negatively regulated by iron in the parent strain. A recombinant MtsR with C-terminal His6 tag fusion (rMtsR) was cloned and purified. Electrophoretic mobility gel shift assays demonstrated that rMtsR specifically binds to the sia promoter region in an iron- and manganese-dependent manner. Together, these observations indicate that MtsR directly represses the sia operon during cell growth under conditions of high metal levels. Consistent with deregulation of iron uptake, the mtsR mutant is hypersensitive to streptonigrin and hydrogen peroxide, and 55Fe uptake assays demonstrate that it accumulates 80% ± 22.5% more iron than the wild-type strain during growth in complete medium. Studies with a zebrafish infection model revealed that the mtsR mutant is attenuated for virulence in both the intramuscular and the intraperitoneal routes. In conclusion, MtsR, a new regulatory protein in GAS, controls iron homeostasis and has a role in disease production.