scholarly journals Innate Immune Response to Streptococcus iniae Infection in Zebrafish Larvae

2012 ◽  
Vol 81 (1) ◽  
pp. 110-121 ◽  
Author(s):  
Elizabeth A. Harvie ◽  
Julie M. Green ◽  
Melody N. Neely ◽  
Anna Huttenlocher
Keyword(s):  
Systemic Infection ◽  
Neutrophil Function ◽  
Otic Vesicle ◽  
Streptococcus Iniae ◽  
Wild Type ◽  
Zebrafish Model ◽  
Content Type ◽  
Larval Zebrafish ◽  
Zebrafish Larvae ◽  
Lethal Infection

Streptococcus iniaecauses systemic infection characterized by meningitis and sepsis. Here, we report a larval zebrafish model ofS. iniaeinfection. Injection of wild-typeS. iniaeinto the otic vesicle induced a lethal infection by 24 h postinfection. In contrast, anS. iniaemutant deficient in polysaccharide capsule (cpsAmutant) was not lethal, with greater than 90% survival at 24 h postinfection. Live imaging demonstrated that both neutrophils and macrophages were recruited to localized otic infection with mutant and wild-typeS. iniaeand were able to phagocytose bacteria. Depletion of neutrophils and macrophages impaired host survival following infection with wild-typeS. iniaeand thecpsAmutant, suggesting that leukocytes are critical for host survival in the presence of both the wild-type and mutant bacteria. However, zebrafish larvae with impaired neutrophil function but normal macrophage function had increased susceptibility to wild-type bacteria but not thecpsAmutant. Taking these findings together, we have developed a larval zebrafish model ofS. iniaeinfection and have found that although neutrophils are important for controlling infection with wild-typeS. iniae, neutrophils are not necessary for host defense against thecpsAmutant.

scholarly journals Aerobactin, but Not Yersiniabactin, Salmochelin, or Enterobactin, Enables the Growth/Survival of Hypervirulent (Hypermucoviscous) Klebsiella pneumoniaeEx VivoandIn Vivo

2015 ◽  
Vol 83 (8) ◽  
pp. 3325-3333 ◽  
Author(s):  
Thomas A. Russo ◽  
Ruth Olson ◽  
Ulrike MacDonald ◽  
Janet Beanan ◽  
Bruce A. Davidson
Keyword(s):  
Virulence Factor ◽  
Human Urine ◽  
Ex Vivo ◽  
Systemic Infection ◽  
Relative Activity ◽  
Ascites Fluid ◽  
Specific Gene ◽  
Wild Type ◽  
Content Type

The siderophore aerobactin is the dominant siderophore produced by hypervirulentKlebsiella pneumoniae(hvKP) and was previously shown to be a major virulence factor in systemic infection. However, strains of hvKP commonly produce the additional siderophores yersiniabactin, salmochelin, and enterobactin. The roles of these siderophores in hvKP infection have not been optimally defined. To that end, site-specific gene disruptions were created in hvKP1 (wild type), resulting in the generation of hvKP1ΔiucA(aerobactin deficient), hvKP1ΔiroB(salmochelin deficient), hvKP1ΔentB(enterobactin and salmochelin deficient), hvKP1Δirp2(yersiniabactin deficient), and hvKP1ΔentBΔirp2(enterobactin, salmochelin, and yersiniabactin deficient). The growth/survival of these constructs was compared to that of their wild-type parent hvKP1ex vivoin human ascites fluid, human serum, and human urine andin vivoin mouse systemic infection and pulmonary challenge models. Interestingly, in contrast to aerobactin, the inability to produce enterobactin, salmochelin, or yersiniabactin individually or in combination did not decrease theex vivogrowth/survival in human ascites or serum or decrease virulence in thein vivoinfection models. Surprisingly, none of the siderophores increased growth in human urine. In human ascites fluid supplemented with exogenous siderophores, siderophores increased the growth of hvKP1ΔiucA, with the relative activity being enterobactin > aerobactin > yersiniabactin > salmochelin, suggesting that the contribution of aerobactin to virulence is dependent on both innate biologic activity and quantity produced. Taken together, these data confirm and extend a role for aerobactin as a critical virulence factor for hvKP. Since it appears that aerobactin production is a defining trait of hvKP strains, this factor is a potential antivirulence target.

scholarly journals Cytoplasmic Copper Detoxification in Salmonella Can Contribute to SodC Metalation but Is Dispensable during Systemic Infection

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
Luke A. Fenlon ◽  
James M. Slauch
Keyword(s):  
Superoxide Dismutase ◽  
Immune Cells ◽  
Gastrointestinal Disease ◽  
Systemic Infection ◽  
Copper Binding ◽  
Wild Type ◽  
Triple Mutant ◽  
Content Type

ABSTRACT Salmonella enterica serovar Typhimurium is a leading cause of foodborne disease worldwide. Severe infections result from the ability of S. Typhimurium to survive within host immune cells, despite being exposed to various host antimicrobial factors. SodCI, a copper-zinc-cofactored superoxide dismutase, is required to defend against phagocytic superoxide. SodCII, an additional periplasmic superoxide dismutase, although produced during infection, does not function in the host. Previous studies suggested that CueP, a periplasmic copper binding protein, facilitates acquisition of copper by SodCII. CopA and GolT, both inner membrane ATPases that pump copper from the cytoplasm to the periplasm, are a source of copper for CueP. Using in vitro SOD assays, we found that SodCI can also utilize CueP to acquire copper. However, both SodCI and SodCII have a significant fraction of activity independent of CueP and cytoplasmic copper export. We utilized a series of mouse competition assays to address the in vivo role of CueP-mediated SodC activation. A copA golT cueP triple mutant was equally as competitive as the wild type, suggesting that sufficient SodCI is active to defend against phagocytic superoxide independent of CueP and cytoplasmic copper export. We also confirmed that a strain containing a modified SodCII, which is capable of complementing a sodCI deletion, was fully virulent in a copA golT cueP background competed against the wild type. These competitions also address the potential impact of cytoplasmic copper toxicity within the phagosome. Our data suggest that Salmonella does not encounter inhibitory concentrations of copper during systemic infection. IMPORTANCE Salmonella is a leading cause of gastrointestinal disease worldwide. In severe cases, Salmonella can cause life-threatening systemic infections, particularly in very young children, the elderly, or people who are immunocompromised. To cause disease, Salmonella must survive the hostile environment inside host immune cells, a location in which most bacteria are killed. Our work examines how one particular metal, copper, is acquired by Salmonella to activate a protein important for survival within immune cells. At high levels, copper itself can inhibit Salmonella. Using a strain of Salmonella that cannot detoxify intracellular copper, we also addressed the in vivo role of copper as an antimicrobial agent.

scholarly journals The Streptococcus iniae Transcriptional Regulator CpsY Is Required for Protection from Neutrophil-Mediated Killing and Proper GrowthInVitro

2011 ◽  
Vol 79 (11) ◽  
pp. 4638-4648 ◽  
Author(s):  
Jonathan P. Allen ◽  
Melody N. Neely
Keyword(s):  
Whole Blood ◽  
Local Environment ◽  
Growth Defect ◽  
Infection Model ◽  
Streptococcus Iniae ◽  
Virulence Determinants ◽  
Wild Type ◽  
Content Type ◽  
Proteose Peptone ◽  
Lysr Family

ABSTRACTThe 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 theStreptococcus iniaeLysR family regulator CpsY have been shown to regulate methionine biosynthesis and uptake pathways but appear to influence expression of several virulence genes as well. AnS. iniaemutant with an in-frame deletion ofcpsY(ΔcpsYmutant) is highly attenuated in a zebrafish infection model. The ΔcpsYmutant displays a methionine-independent growth defect in serum, which differs from the methionine-dependent defect observed for orthologous mutants ofStreptococcus mutansandStreptococcus agalactiae. On the contrary, the ΔcpsYmutant 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 ofS. iniaefrom clearance by neutrophils in whole blood but is dispensable for intracellular survival in macrophages. Susceptibility of the ΔcpsYmutant 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 forS. iniae, integrating metabolism and virulence. Furthermore,S. iniaeprovides a unique model to investigate the paradigm of CpsY-dependent regulation during systemic streptococcal infection.

scholarly journals Distinct Innate Immune Phagocyte Responses to Aspergillus fumigatus Conidia and Hyphae in Zebrafish Larvae

2014 ◽  
Vol 13 (10) ◽  
pp. 1266-1277 ◽  
Author(s):  
Benjamin P. Knox ◽  
Qing Deng ◽  
Mary Rood ◽  
Jens C. Eickhoff ◽  
Nancy P. Keller ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Host Defense ◽  
Leukocyte Adhesion ◽  
Human Leukocyte ◽  
Invasive Disease ◽  
Neutrophil Function ◽  
Innate Immune ◽  
Infection Model ◽  
Zebrafish Model ◽  
Content Type

ABSTRACTAspergillus fumigatusis the most common filamentous fungal pathogen of immunocompromised hosts, resulting in invasive aspergillosis (IA) and high mortality rates. Innate immunity is known to be the predominant host defense againstA. fumigatus; however, innate phagocyte responses toA. fumigatusin an intact host and their contributions to host survival remain unclear. Here, we describe a larval zebrafishA. fumigatusinfection model amenable to real-time imaging of host-fungal interactions in live animals. Following infection withA. fumigatus, innate phagocyte populations exhibit clear preferences for different fungal morphologies: macrophages rapidly phagocytose conidia and form aggregates around hyphae, while the neutrophil response is dependent upon the presence of hyphae. Depletion of macrophages rendered host larvae susceptible to invasive disease. Moreover, a zebrafish model of human leukocyte adhesion deficiency with impaired neutrophil function also resulted in invasive disease and impaired host survival. In contrast, macrophage-deficient but not neutrophil-deficient larvae exhibited attenuated disease following challenge with a less virulent (ΔlaeA) strain ofA. fumigatus, which has defects in secondary metabolite production. Taking these results together, we have established a new vertebrate model for studying innate immune responses toA. fumigatusthat reveals distinct roles for neutrophils and macrophages in mediating host defense against IA.

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

2005 ◽  
Vol 73 (2) ◽  
pp. 921-934 ◽  
Author(s):  
Jesse D. Miller ◽  
Melody N. Neely
Keyword(s):  
Large Scale ◽  
Disease Model ◽  
Systemic Infection ◽  
Streptococcus Iniae ◽  
Species Barrier ◽  
Zebrafish Model ◽  
Zoonotic Pathogen ◽  
Infectious Disease Model ◽  
Successful Infection ◽  
Human Specific

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.

scholarly journals CpsY Influences Streptococcus iniae Cell Wall Adaptations Important for Neutrophil Intracellular Survival

2012 ◽  
Vol 80 (5) ◽  
pp. 1707-1715 ◽  
Author(s):  
Jonathan P. Allen ◽  
Melody N. Neely
Keyword(s):  
Cell Wall ◽  
Streptococcal Infection ◽  
Systemic Infection ◽  
Streptococcus Iniae ◽  
Methionine Metabolism ◽  
Content Type ◽  
Phagosomal Maturation ◽  
Mature Neutrophil ◽  
Invasive Infections ◽  
Phagocytic Killing

ABSTRACTThe ability of a pathogen to evade neutrophil phagocytic killing mechanisms is critically important for dissemination and establishment of a systemic infection. Understanding how pathogens overcome these innate defenses is essential for the development of optimal therapeutic strategies for invasive infections. CpsY is a conserved transcriptional regulator previously identified as an important virulence determinant for systemic infection ofStreptococcus iniae. While orthologs of CpsY have been associated with the regulation of methionine metabolism and uptake pathways, CpsY additionally functions in protection from neutrophil-mediated killing.S. iniaedoes not alter neutrophil phagosomal maturation but instead is able to adapt to the extreme bactericidal environment of a mature neutrophil phagosome, a property dependent upon CpsY. This CpsY-dependent adaptation appears to involve stabilization of the cell wall through peptidoglycan O-acetylation and repression of cellular autolysins. Furthermore,S. iniaecontinues to be a powerful model for investigation of bacterial adaptations during systemic streptococcal infection.

scholarly journals Host and Pathogen Copper-Transporting P-Type ATPases Function Antagonistically during Salmonella Infection

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Erik Ladomersky ◽  
Aslam Khan ◽  
Vinit Shanbhag ◽  
Jennifer S. Cavet ◽  
Jefferson Chan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Systemic Infection ◽  
Copper Transport ◽  
Wild Type ◽  
Macrophage Cell ◽  
Content Type ◽  
Serovar Typhimurium ◽  
P Type ◽  
Toxic Trace Element

ABSTRACT Copper is an essential yet potentially toxic trace element that is required by all aerobic organisms. A key regulator of copper homeostasis in mammalian cells is the copper-transporting P-type ATPase ATP7A, which mediates copper transport from the cytoplasm into the secretory pathway, as well as copper export across the plasma membrane. Previous studies have shown that ATP7A-dependent copper transport is required for killing phagocytosed Escherichia coli in a cultured macrophage cell line. In this investigation, we expanded on these studies by generating Atp7a LysMcre mice, in which the Atp7a gene was specifically deleted in cells of the myeloid lineage, including macrophages. Primary macrophages isolated from Atp7a LysMcre mice exhibit decreased copper transport into phagosomal compartments and a reduced ability to kill Salmonella enterica serovar Typhimurium compared to that of macrophages isolated from wild-type mice. The Atp7a LysMcre mice were also more susceptible to systemic infection by S. Typhimurium than wild-type mice. Deletion of the S. Typhimurium copper exporters, CopA and GolT, was found to decrease infection in wild-type mice but not in the Atp7a LysMcre mice. These studies suggest that ATP7A-dependent copper transport into the phagosome mediates host defense against S. Typhimurium, which is counteracted by copper export from the bacteria via CopA and GolT. These findings reveal unique and opposing functions for copper transporters of the host and pathogen during infection.

scholarly journals Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus

2016 ◽  
Vol 84 (12) ◽  
pp. 3379-3387 ◽  
Author(s):  
Mohammad Gharaibeh ◽  
Monica Hagedorn ◽  
Stefanie Lilla ◽  
Matthias Hauptmann ◽  
Holger Heine ◽  
...  
Keyword(s):  
Scrub Typhus ◽  
Orientia Tsutsugamushi ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Content Type ◽  
Lethal Infection ◽  
Tnf Α ◽  
Toll Like Receptor 2 ◽  
Deficient Mice ◽  
Increased Susceptibility

Scrub typhus is a potentially lethal infection that is caused by the obligate intracellular bacteriumOrientia tsutsugamushi. The roles of Toll-like receptor 2 (TLR2) and TLR4 in innate recognition ofO. tsutsugamushihave not been elucidated. By overexpression of TLR2 or TLR4 in HEK293 cells, we demonstrated that TLR2, but not TLR4, recognizes heat-stable compounds ofO. tsutsugamushithat were sensitive to treatment with sodium hydroxide, hydrogen peroxide, and proteinase K. TLR2 was required for the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) by dendritic cells. In an intradermal mouse infection model, TLR2-deficient mice did not show impaired control of bacterial growth or reduced survival. Moreover, after intraperitoneal infection, TLR2-deficient mice were even more resistant to lethal infection than C57BL/6 wild-type mice, which showed stronger symptoms and lower survival rates during the convalescent phase. Compared to the time of reduction of bacterial loads in TLR2-deficient mice, the reduction of bacterial loads in infected organs was accelerated in wild-type mice. The higher mortality of wild-type mice was associated with increased concentrations of serum alkaline phosphatase but not aspartate aminotransferase. The transcription of mRNA for TNF-α and IL-6 decreased more rapidly in peritoneum samples from wild-type mice than in those from TLR2-deficient mice and was therefore not a correlate of increased susceptibility. Thus, although TLR2 is an important mediator of the early inflammatory response, it is dispensable for protective immunity againstO. tsutsugamushi. Increased susceptibility toO. tsutsugamushiinfection in TLR2-competent mice rather suggests a TLR2-related immunopathologic effect.

scholarly journals Efficacy of Voriconazole against Aspergillus fumigatus Infection Depends on Host Immune Function

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Emily E. Rosowski ◽  
Jiaye He ◽  
Jan Huisken ◽  
Nancy P. Keller ◽  
Anna Huttenlocher
Keyword(s):  
Aspergillus Fumigatus ◽  
Fungal Growth ◽  
Fungal Spore ◽  
Antifungal Drugs ◽  
Zebrafish Model ◽  
Content Type ◽  
Larval Zebrafish ◽  
Number Of Patients

ABSTRACT Antifungal therapy can fail in a remarkable number of patients with invasive fungal disease, resulting in significant morbidity worldwide. A major contributor to this failure is that while these drugs have high potency in vitro, we do not fully understand how they work inside infected hosts. Here, we used a transparent larval zebrafish model of Aspergillus fumigatus infection amenable to real-time imaging of invasive disease as an in vivo intermediate vertebrate model to investigate the efficacy and mechanism of the antifungal drug voriconazole. We found that the ability of voriconazole to protect against A. fumigatus infection depends on host innate immune cells and, specifically, on the presence of macrophages. While voriconazole inhibits fungal spore germination and growth in vitro, it does not do so in larval zebrafish. Instead, live imaging of whole, intact larvae over a multiday course of infection revealed that macrophages slow down initial fungal growth, allowing voriconazole time to target and kill A. fumigatus hyphae postgermination. These findings shed light on how antifungal drugs such as voriconazole may synergize with the immune response in living hosts.

scholarly journals Salmonella Infection Enhances Erythropoietin Production by the Kidney and Liver, Which Correlates with Elevated Bacterial Burdens

2016 ◽  
Vol 84 (10) ◽  
pp. 2833-2841 ◽  
Author(s):  
Lin-Xi Li ◽  
Joseph M. Benoun ◽  
Kipp Weiskopf ◽  
K. Christopher Garcia ◽  
Stephen J. McSorley
Keyword(s):  
Regulatory Protein ◽  
Systemic Infection ◽  
Negative Consequences ◽  
Wild Type ◽  
Content Type ◽  
Erythropoietin Production ◽  
Extramedullary Erythropoiesis ◽  
The Impact ◽  
Erythroid Development ◽  
Epor Expression

Salmonellainfection profoundly affects host erythroid development, but the mechanisms responsible for this effect remain poorly understood. We monitored the impact ofSalmonellainfection on erythroid development and found that systemic infection induced anemia, splenomegaly, elevated erythropoietin (EPO) levels, and extramedullary erythropoiesis in a process independent ofSalmonellapathogenicity island 2 (SPI2) or flagellin. The circulating EPO level was also constitutively higher in mice lacking the expression of signal-regulatory protein α (SIRPα). The expression level of EPO mRNA was elevated in the kidney and liver but not increased in the spleens of infected mice despite the presence of extramedullary erythropoiesis in this tissue. In contrast to data from a previous report, mice lacking EPO receptor (EPOR) expression on nonerythroid cells (EPOR rescued) had bacterial loads similar to those of wild-type mice followingSalmonellainfection. Indeed, treatment to reduce splenic erythroblasts and mature red blood cells correlated with elevated bacterial burdens, implying that extramedullary erythropoiesis benefits the host. Together, these findings emphasize the profound effect ofSalmonellainfection on erythroid development and suggest that the modulation of erythroid development has both positive and negative consequences for host immunity.

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