The adenoviral protein E3-14.7K (14.7K) is an inhibitor of TNF-induced apoptosis, but the molecular mechanism underlying this protective effect has not yet been explained exhaustively. TNF-mediated apoptosis is initiated by ligand-induced recruitment of TNF receptor–associated death domain (TRADD), Fas-associated death domain (FADD), and caspase-8 to the death domain of TNF receptor 1 (TNFR1), thereby establishing the death-inducing signaling complex (DISC). Here we report that adenovirus 14.7K protein inhibits ligand-induced TNFR1 internalization. Analysis of purified magnetically labeled TNFR1 complexes from murine and human cells stably transduced with 14.7K revealed that prevention of TNFR1 internalization resulted in inhibition of DISC formation. In contrast, 14.7K did not affect TNF-induced NF-κB activation via recruitment of receptor-interacting protein 1 (RIP-1) and TNF receptor–associated factor 2 (TRAF-2). Inhibition of endocytosis by 14.7K was effected by failure of coordinated temporal and spatial assembly of essential components of the endocytic machinery such as Rab5 and dynamin 2 at the site of the activated TNFR1. Furthermore, we found that the same TNF defense mechanisms were instrumental in protecting wild-type adenovirus–infected human cells expressing 14.7K. This study describes a new molecular mechanism implemented by a virus to escape immunosurveillance by selectively targeting TNFR1 endocytosis to prevent TNF-induced DISC formation.
Wulf Schneider-Brachert, Vladimir Tchikov, Oliver Merkel, Marten Jakob, Cora Hallas, Marie-Luise Kruse, Peter Groitl, Alexander Lehn, Eberhard Hildt, Janka Held-Feindt, Thomas Dobner, Dieter Kabelitz, Martin Krönke, Stefan Schütze
Topical microbicides represent a promising new approach to preventing HIV and other sexually transmitted infections. TLR agonists are ideal candidates for microbicides, as they trigger a multitude of antiviral genes effective against a broad range of viruses. Although vaginal application of CpG oligodeoxynucleotides (ODNs) and poly I:C has been shown to protect mice from genital herpes infection, the mechanism by which these agents provide protection remains unclear. Here, we show that plasmacytoid DCs (pDCs) are required for CpG ODN–mediated protection against lethal vaginal challenge with herpes simplex virus type 2 (HSV-2). Moreover, we demonstrate that cells of both the hematopoietic and stromal compartments must respond to CpG ODN via TLR9 and to type I IFNs through IFN-αβ receptor (IFN-αβR) for protection. Thus, crosstalk between pDCs and vaginal stromal cells provides for optimal microbicide efficacy. Our results imply that temporally and spatially controlled targeting of CpG ODN to pDCs and epithelial cells can potentially maximize their effectiveness as microbicides while minimizing the associated inflammatory responses.
Hong Shen, Akiko Iwasaki
To develop an animal model of Kaposi sarcoma–associated herpesvirus (KSHV) infection uniquely suited to evaluate longitudinal patterns of viral gene expression, cell tropism, and immune responses, we injected NOD/SCID mice intravenously with purified virus and measured latent and lytic viral transcripts in distal organs over the subsequent 4 months. We observed sequential escalation of first latent and then lytic KSHV gene expression coupled with electron micrographic evidence of virion production within the murine spleen. Using novel technology that integrates flow cytometry with immunofluorescence microscopy, we found that the virus establishes infection in murine B cells, macrophages, NK cells, and, to a lesser extent, dendritic cells. To investigate the potential for human KSHV–specific immune responses within this immunocompromised host, we implanted NOD/SCID mice with functional human hematopoietic tissue grafts (NOD/SCID-hu mice) and observed that a subset of animals produced human KSHV–specific antibodies. Furthermore, treatment of these chimeric mice with ganciclovir at the time of inoculation led to prolonged but reversible suppression of KSHV DNA and RNA levels, suggesting that KSHV can establish latent infection in vivo despite ongoing suppression of lytic replication.
Christopher H. Parsons, Laura A. Adang, Jon Overdevest, Christine M. O’Connor, J. Robert Taylor, David Camerini, Dean H. Kedes
Failure to clear persistent viral infections results from the early loss of T cell activity. A pertinent question is whether the immune response is programmed to fail or if nonresponsive T cells can specifically be fixed to eliminate infection. Although evidence indicates that T cell expansion is permanently programmed during the initial priming events, the mechanisms that determine the acquisition of T cell function are less clear. Herein we show that in contrast to expansion, the functional programming of T cell effector and memory responses in vivo in mice is not hardwired during priming but is alterable and responsive to continuous instruction from the antigenic environment. As a direct consequence, dysfunctional T cells can be functionally reactivated during persistent infection even after an initial program of inactivation has been instituted. We also show that early therapeutic reductions in viral replication facilitate the preservation of antiviral CD4+ T cell activity, enabling the long-term control of viral replication. Thus, dysfunctional antiviral T cells can regain activity, providing a basis for future therapeutic strategies to treat persistent viral infections.
David G. Brooks, Dorian B. McGavern, Michael B.A. Oldstone
HIV infection selectively targets CD4+ effector memory T (TEM) cells, resulting in dramatic depletion of CD4+ T cells in mucosal effector sites in early infection. Regeneration of the TEM cell compartment is slow and incomplete, even when viral replication is controlled by antiretroviral therapy (ART). Here, we demonstrate that IL-15 dramatically increases in vivo proliferation of rhesus macaque (RM) CD4+ and CD8+ TEM cells with little effect on the naive or central memory T (TCM) cell subsets, a response pattern that is quite distinct from that of either IL-2 or IL-7. TEM cells produced in response to IL-15 did not accumulate in blood. Rather, 5-bromo-2′-deoxyuridine (BrdU) labeling studies suggest that many of these cells rapidly disperse to extralymphoid effector sites, where they manifest (slow) decay kinetics indistinguishable from that of untreated controls. In RMs with uncontrolled SIV infection and highly activated immune systems, IL-15 did not significantly increase CD4+ TEM cell proliferation, but with virologic control and concomitant reduction in immune activation by ART, IL-15 responsiveness was again observed. These data suggest that therapeutic use of IL-15 in the setting of ART might facilitate specific restoration of the CD4+ T cell compartment that is the primary target of HIV with less risk of exhausting precursor T cell compartments or generating potentially deleterious regulatory subsets.
Louis J. Picker, Edward F. Reed-Inderbitzin, Shoko I. Hagen, John B. Edgar, Scott G. Hansen, Alfred Legasse, Shannon Planer, Michael Piatak, Jeffrey D. Lifson, Vernon C. Maino, Michael K. Axthelm, Francois Villinger
CD8+ T cells play a key role in clearing primary virus infections and in protecting against subsequent challenge. The potent antiviral effects of these cells make them important components of vaccine-induced immunity and, because of this, peptide vaccines often contain epitopes designed to induce strong CD8+ T cell responses. However, the same effector functions that protect the host also can be harmful if they are not tightly regulated, and virus-specific CD8+ T cells are a frequent cause of immunopathology. Here, we report that the administration of peptide to virus-immune recipient mice can lead to the synchronous activation of preexisting virus-specific CD8+ T cells with serious, and even lethal, consequences. Mice infected with LCMV or vaccinia virus developed rapid and profound hypothermia following injection of cognate synthetic peptides, and LCMV-infected mice frequently died within hours. Detailed analyses of the LCMV infected mice revealed enterocyte apoptosis and implicated TNF produced by peptide-specific CD8+ T cells as the major mediator of disease. The caspase inhibitor zVADfmk had no demonstrable effect on the development of hypothermia, but diminished enterocyte apoptosis and greatly reduced the number of deaths. These findings, if similarly observed in patients, counsel caution when administering powerful immunogens such as peptide vaccines to individuals who may have a large preexisting pool of epitope-specific CD8+ T cells.
Fei Liu, Ralph Feuer, Daniel E. Hassett, J. Lindsay Whitton
Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell–independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modifed DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.
Mingquan Zheng, Alistair J. Ramsay, Myles B. Robichaux, Karen A. Norris, Corrine Kliment, Christopher Crowe, Rekha R. Rapaka, Chad Steele, Florencia McAllister, Judd E. Shellito, Luis Marrero, Paul Schwarzenberger, Qiu Zhong, Jay K. Kolls
Reovirus induces apoptosis in cultured cells and in vivo. In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-κB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-κB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirus-induced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-β mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-β substantially diminished reovirus replication and apoptosis, which suggests that IFN-β induction by NF-κB protects against reovirus-induced myocarditis. These findings reveal an organ-specific role for NF-κB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.
Sean M. O’Donnell, Mark W. Hansberger, Jodi L. Connolly, James D. Chappell, Melissa J. Watson, Janene M. Pierce, J. Denise Wetzel, Wei Han, Erik S. Barton, J. Craig Forrest, Tibor Valyi-Nagy, Fiona E. Yull, Timothy S. Blackwell, Jeffrey N. Rottman, Barbara Sherry, Terence S. Dermody
Difficulties in understanding the mechanisms of HIV neuropathogenesis include the inability to study dynamic processes of infection, cumulative effects of the virus, and contributing host immune responses. We used 1H magnetic resonance spectroscopy and studied monocyte activation and progression of CNS neuronal injury in a CD8 lymphocyte depletion model of neuroAIDS in SIV-infected rhesus macaque monkeys. We found early, consistent neuronal injury coincident with viremia and SIV infection/activation of monocyte subsets and sought to define the role of plasma virus and monocytes in contributing to CNS disease. Antiretroviral therapy with essentially non–CNS-penetrating agents resulted in slightly decreased levels of plasma virus, a significant reduction in the number of activated and infected monocytes, and rapid, near-complete reversal of neuronal injury. Robust macrophage accumulation and productive virus replication were found in brains of infected and CD8 lymphocyte–depleted animals, but no detectable virus and few scattered infiltrating macrophages were observed in CD8 lymphocyte–depleted animals compared with animals not receiving antiretroviruses that were sacrificed at the same time after infection. These results underscore the role of activated monocytes and monocyte infection outside of the brain in driving CNS disease.
Kenneth Williams, Susan Westmoreland, Jane Greco, Eva Ratai, Margaret Lentz, Woong-Ki Kim, Robert A. Fuller, John P. Kim, Patrick Autissier, Prahbat K. Sehgal, Raymond F. Schinazi, Norbert Bischofberger, Michael Piatak Jr., Jeffrey D. Lifson, Eliezer Masliah, R. Gilberto González
Patients with infectious mononucleosis (IM) undergoing primary EBV infection show large expansions of EBV-specific CD8+ T cells in the blood. While latent infection of the B cell pool is quickly controlled, virus shedding from lytically infected cells in the oropharynx remains high for several months. We therefore studied how responses localize to the tonsil, a major target site for EBV, during primary infection and persistence. In acute IM, EBV-specific effectors were poorly represented among CD8+ T cells in tonsil compared with blood, coincident with absence of the CCR7 lymphoid homing marker on these highly activated cells. In patients who had recently recovered from IM, latent epitope reactivities were quicker than lytic reactivities both to acquire CCR7 and to accumulate in the tonsil, with some of these cells now expressing the CD103 integrin, which mediates retention at mucosal sites. By contrast, in long-term virus carriers in whom both lytic and latent infections had been controlled, there was 2- to 5-fold enrichment of lytic epitope reactivities and 10- to 20-fold enrichment of latent epitope reactivities in tonsil compared with blood; up to 20% of tonsillar CD8+ T cells were EBV specific, and many now expressed CD103. We suggest that efficient control of EBV infection requires appropriate CD8+ T cell homing to oropharyngeal sites.
Andrew D. Hislop, Michael Kuo, Adrian B. Drake-Lee, Arne N. Akbar, Wolfgang Bergler, Nicolas Hammerschmitt, Naeem Khan, Umaimainthan Palendira, Alison M. Leese, Judith M. Timms, Andrew I. Bell, Christopher D. Buckley, Alan B. Rickinson
Little is known about the role of CD1d-restricted T cells in antiviral immune responses. Here we show that the lytic replication cycle of the Kaposi sarcoma–associated herpesvirus (KSHV) promotes downregulation of cell-surface CD1d. This is caused by expression of the 2 modulator of immune recognition (MIR) proteins of the virus, each of which promotes the loss of surface CD1d expression following transfection into uninfected cells. Inhibition of CD1d surface expression is due to ubiquitination of the CD1d α-chain on a unique lysine residue in its cytoplasmic tail, which triggers endocytosis. Unlike MIR-mediated MHC class I downregulation, however, CD1d downregulation does not appear to include accelerated lysosomal degradation. MIR2-induced downregulation of CD1d results in reduced activation of CD1d-restricted T cells in vitro. KSHV modulation of CD1d expression represents a strategy for viral evasion of innate host immune responses and implicates CD1d-restricted T cells as regulators of this viral infection.
David Jesse Sanchez, Jenny E. Gumperz, Don Ganem
Kaposi sarcoma–associated herpesvirus (KSHV) is linked with all clinical forms of Kaposi sarcoma and several lymphoproliferative disorders. Like other herpesviruses, KSHV becomes latent in the infected cells, expressing only a few genes that are essential for the establishment and maintenance of its latency and for the survival of the infected cells. Inhibiting the expression of these latent genes should lead to eradication of herpesvirus infection. All currently available drugs are ineffective against latent infection. Here we show, for the first time to our knowledge, that latent infection with KSHV in B lymphocytes can be terminated by glycyrrhizic acid (GA), a triterpenoid compound earlier shown to inhibit the lytic replication of other herpesviruses. We demonstrate that GA disrupts latent KSHV infection by downregulating the expression of latency-associated nuclear antigen (LANA) and upregulating the expression of viral cyclin and selectively induces cell death of KSHV-infected cells. We show that reduced levels of LANA lead to p53 reactivation, an increase in ROS, and mitochondrial dysfunction, which result in G1 cell cycle arrest, DNA fragmentation, and oxidative stress–mediated apoptosis. Latent genes are involved in KSHV-induced oncogenesis, and strategies to interfere with their expression might prove useful for eradicating latent KSHV infection and have future therapeutic implications.
Francesca Curreli, Alvin E. Friedman-Kien, Ornella Flore
Poliovirus replication is limited to a few organs, including the brain and spinal cord. This restricted tropism may be a consequence of organ-specific differences in translation initiation by the poliovirus internal ribosome entry site (IRES). A C-to-U mutation at base 472 in the IRES of the Sabin type 3 poliovirus vaccine strain, known to attenuate neurovirulence, may further restrict tropism by eliminating viral replication in the CNS. To determine the relationship between IRES-mediated translation and poliovirus tropism, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The IRESs of poliovirus, the cardiotropic coxsackievirus B3 (CVB3), and the hepatotropic hepatitis C virus (HCV) mediate translation in many organs, including those that do not support viral replication. A translation defect associated with the Sabin type 3 IRES was observed in all organs examined. Poliovirus type 1 and recombinant polioviruses dependent on the IRES of CVB3 or HCV replicate in the CNS of mice and cause paralysis. Although the type 3 Sabin strain is an effective vaccine, polioviruses with a U at base 472 of the IRES cause paralysis in newborn mice. Tropism of wild-type and vaccine strains of poliovirus is therefore determined after internal ribosome entry.
Steven E. Kauder, Vincent R. Racaniello
DCs play a pivotal role in bringing forth innate and adaptive immune responses. Viruses can specifically target DCs, rendering them ineffective in stimulating T cells, which can ultimately lead to immunosuppression. In the present study we have identified several potential mechanisms by which lymphocytic choriomeningitis virus (LCMV) induces immunosuppression in its natural murine host. The immunosuppressive LCMV variant clone 13 (Cl 13) infects DCs and interferes with their maturation and antigen-presenting capacity as evidenced by a significant reduction in the surface expression of MHC class I, MHC class II, CD40, CD80, and CD86 molecules. Additionally, Cl 13 infects hematopoietic progenitor cells both in vivo and in vitro, impairing their development. One mechanism by which hematopoietic progenitors are developmentally impaired is through the Cl 13–induced production of IFN-α and IFN-β (IFN-α/β). Mice deficient in the receptor for IFN-α/β show a normal differentiation of progenitors into DCs despite viral infection. Thus, a virus can evolve a strategy to boost its survival by preventing the maturation of DCs from infected progenitor cells and by reducing the expression of antigen-presenting and costimulatory molecules on developed DCs.
Noemí Sevilla, Dorian B. McGavern, Chao Teng, Stefan Kunz, Michael B.A. Oldstone
Antigenic stimulation of T cells gives rise to short-lived effector cells and long-lived memory cells. We used two stable isotope-labeling techniques to identify kinetically distinct subpopulations of T cells and to determine the effect of advanced infection with HIV-1. Long-term deuterated water (2H2O) incorporation into DNA demonstrated biphasic accrual of total and of memory/effector (m/e)–phenotype but not naive-phenotype T cells, consistent with the presence of short-lived and longer-lived subpopulations within the m/e-phenotype T cell pool. These results were mirrored by biphasic die-away kinetics in m/e- but not naive-phenotype T cells after short-term 2H-glucose labeling. Persistent label retention was observed in a subset of m/e-phenotype T cells (presumably memory T cells), confirming the presence of T cells with very different life spans in humans. In advanced HIV-1 infection, much higher proportions of T cells were short-lived, compared to healthy controls. Effective long-term anti-retroviral therapy restored values to normal. These results provide the first quantitative evidence that long-lived and quiescent T cells do indeed predominate in the T cell pool in humans and determine T cell pool size, as in rodents. The greatest impact of advanced HIV-1 infection is to reduce the generation of long-lived, potential progenitor T cells.
Marc K. Hellerstein, Rebecca A. Hoh, Mary Beth Hanley, Denise Cesar, Daniel Lee, Richard A. Neese, Joseph M. McCune
Hepatitis delta virus (HDV) can dramatically worsen liver disease in patients coinfected with hepatitis B virus (HBV). No effective medical therapy exists for HDV. The HDV envelope requires HBV surface antigen proteins provided by HBV. Once inside a cell, however, HDV can replicate its genome in the absence of any HBV gene products. In vitro, HDV virion assembly is critically dependent on prenyl lipid modification, or prenylation, of its nucleocapsid-like protein large delta antigen. To overcome limitations of current animal models and to test the hypothesis that pharmacologic prenylation inhibition can prevent the production of HDV virions in vivo, we established a convenient mouse-based model of HDV infection capable of yielding viremia. Such mice were then treated with the prenylation inhibitors FTI-277 and FTI-2153. Both agents were highly effective at clearing HDV viremia. As expected, HDV inhibition exhibited duration-of-treatment dependence. These results provide the first preclinical data supporting the in vivo efficacy of prenylation inhibition as a novel antiviral therapy with potential application to HDV and a wide variety of other viruses.
Bruno B. Bordier, Junko Ohkanda, Ping Liu, So-Young Lee, F.H. Salazar, Patricia L. Marion, Kazuo Ohashi, Leonard Meuse, Mark A. Kay, John L. Casey, Saïd M. Sebti, Andrew D. Hamilton, Jeffrey S. Glenn
Fibrin deposition and thrombosis within the microvasculature is now appreciated to play a pivotal role in the hepatocellular injury observed in experimental and human viral hepatitis. Importantly, the pathways by which fibrin generation is elicited in viral hepatitis may be mechanistically distinct from the classical pathways of coagulation induced by mechanical trauma or bacterial lipopolysaccharide (LPS). In the setting of murine hepatitis virus strain-3 (MHV-3) infection, a member of the Coronaviridae, activated endothelial cells and macrophages express distinct cell-surface procoagulants, including a novel prothrombinase, Fgl2/fibroleukin, which are important for both the initiation and localization of fibrin deposition. To assess the role of Fgl2/fibroleukin in murine viral hepatitis we generated a Fgl2/fibroleukin–deficient mouse. Peritoneal macrophages isolated from Fgl2/fibroleukin–/– mice did not generate a procoagulant response when infected with MHV-3. Fibrin deposition and liver necrosis were markedly reduced, and survival was increased in mice infected with MHV-3. To address the relevance of Fgl2/fibroleukin in human chronic viral hepatitis we studied patients with minimal and marked chronic hepatitis B. We detected robust expression of Fgl2/fibroleukin mRNA transcripts and protein in liver tissue isolated from patients with marked chronic hepatitis B. Fibrin deposition was strongly associated with Fgl2/fibroleukin expression. Collectively, these data indicate a critical role for Fgl2/fibroleukin in the pathophysiology of experimental and human viral hepatitis.
Philip A. Marsden, Qin Ning, Laisum S. Fung, Xioping Luo, Yue Chen, Michael Mendicino, Anand Ghanekar, Jeremy A. Scott, Teresa Miller, Camie W.Y. Chan, Mathew W.C. Chan, Wei He, Reginald M. Gorczynski, David R. Grant, David A. Clark, M. James Phillips, Gary A. Levy
Infection of neonatal mice with some reovirus strains produces a disease similar to infantile biliary atresia, but previous attempts to correlate reovirus infection with this disease have yielded conflicting results. We used isogenic reovirus strains T3SA– and T3SA+, which differ solely in the capacity to bind sialic acid as a coreceptor, to define the role of sialic acid in reovirus encephalitis and biliary tract infection in mice. Growth in the intestine was equivalent for both strains following peroral inoculation. However, T3SA+ spread more rapidly from the intestine to distant sites and replicated to higher titers in spleen, liver, and brain. Strikingly, mice infected with T3SA+ but not T3SA– developed steatorrhea and bilirubinemia. Liver tissue from mice infected with T3SA+ demonstrated intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in humans, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ bound 100-fold more efficiently than T3SA– to human cholangiocarcinoma cells. These observations suggest that the carbohydrate-binding specificity of a virus can dramatically alter disease in the host and highlight the need for epidemiologic studies focusing on infection by sialic acid–binding reovirus strains as a possible contributor to the pathogenesis of neonatal biliary atresia.
Erik S. Barton, Bryan E. Youree, Daniel H. Ebert, J. Craig Forrest, Jodi L. Connolly, Tibor Valyi-Nagy, Kay Washington, J. Denise Wetzel, Terence S. Dermody
Group B streptococcus (GBS) is an important human pathogen. In this study, we sought to identify mechanisms that may protect GBS from host defenses in addition to its capsular polysaccharide. A gene encoding a cell-surface–associated protein (cspA) was characterized from a highly virulent type III GBS isolate, COH1. Its sequence indicated that it is a subtilisin-like extracellular serine protease homologous to streptococcal C5a peptidases and caseinases of lactic acid bacteria. The wild-type strain cleaved the α chain of human fibrinogen, whereas a cspA mutant, TOH121, was unable to cleave fibrinogen. We observed aggregated material when COH1 was incubated with fibrinogen but not when the mutant strain was treated similarly. This suggested that the product(s) of fibrinogen cleavage have strong adhesive properties and may be similar to fibrin. The cspA gene was present among representative clinical isolates from all nine capsular serotypes, as revealed by Southern blotting. A cspA– mutant was ten times less virulent in a neonatal rat sepsis model of GBS infections, as measured by LD50 analysis. In addition, the cspA– mutant was significantly more sensitive than the wild-type strain to opsonophagocytic killing by human neutrophils in vitro. Taken together, the results suggest that cleavage of fibrinogen by CspA may increase the lethality of GBS infection, potentially by protecting the bacterium from opsonophagocytic killing.
Theresa O. Harris, Daniel W. Shelver, John F. Bohnsack, Craig E. Rubens
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