High-dose Infection Research Articles

DifferentialRequirements for Soluble and Transmembrane Tumor Necrosis Factor in theImmunological Control of Primary and SecondaryListeria monocytogenesInfection

The relative contributions of transmembrane tumor necrosis factor (memTNF) and soluble tumor necrosis factor (solTNF) in innate and adaptive immunity are poorly defined. We examined the capacities of wild-type (WT) mice, TNF-/- mice, and memTNF mice, which express only transmembrane TNF, to control primary and secondary Listeria monocytogenes infections. Soluble TNF was not required for induction or maintenance of protective immunity against a low-dose (200-CFU) Listeria infection. In contrast to TNF-/- mice, both WT and memTNF mice cleared the bacilli within 10 days and were fully protected against rechallenge with a lethal infective dose. Furthermore, T cells transferred from immune mice, but not from naïve, WT, and memTNF mice, protected TNF-/- recipients against an otherwise lethal infection. By contrast, infection with a higher dose of Listeria (2,000 CFU) clearly demonstrated that solTNF is required to coordinate an optimal protective inflammatory response. memTNF mice were more susceptible to a high-dose infection, and they exhibited delayed bacterial clearance, increased inflammation, and necrosis in the liver that resulted in 55% mortality. The dysregulated inflammation was accompanied by prolonged elevated expression of mRNAs for several chemokines as well as the macrophage effector molecules inducible nitric oxide synthase and LRG-47 in the livers of memTNF mice but not in the livers of WT mice. These data demonstrated that memTNF is sufficient for establishing protective immunity against a primary low-dose Listeria infection but that solTNF is required for optimal control of cellular inflammation and resistance to a primary high-dose infection. By contrast, memTNF alone is sufficient for resolution of a secondary, high-dose infection and for the transfer of protective immunity with memory T cells.

Dynamic observation of polypide in semen and blood of rabbits infected with Toxoplasma tachyzoites

Toxoplasma gondii (T。gondii ) 是细胞内部的寄生于的原生动物之一并且可以在损害的胎儿或免疫引起严重医药复杂并发症个人。当时， T.gondii 在尖锐舞台期间作为 tachyzoite 存在作为 bradyzoite 在在人的房间的长期的阶段期间。改进疾病的预防，诊断，和治疗的理解，探索分发和变化和 T 的另外的生物特征是重要的。在主人的 gondii。trophozoite 在主人的口水，血或尿被发现了。一些研究在血或在尿建议了传播抗体和 toxoplasma 传播抗原(TCA ) 的动态变化。T。因为它仅仅是在尺寸的几测微计，因此，大多数研究被执行，在织物或血的 gondii 不能完全在显微镜下面被数由老鼠接种或免疫学方法的品质上。量的荧光灯的聚合酶链反应(QF-PCR ) 和它的申请在主人为 polypide 的动态观察提起了可能性。在这研究，血和精液从感染 toxoplasma tachyzoites 和 T 的雄的兔子模型被收集。gondii 被检测由 QF-PCRquantitatively。

Immune responses in Eimeria acervulina infected one-day-old broilers compared to amount of Eimeria in the duodenum, measured by real-time PCR

T-cell responses are supposed to be the major immune reactions in broilers infected with Eimeria. The nature of such T-cell responses is influenced by the species of Eimeria involved, age of the host, amount of parasites and the preceding infection history. In young chicks the intestine is still developing in length while the lymphocyte populations in the gut develop and differentiate. In chicks infected at young age the immune response may be different in quality as compared to responses in adults. We investigated the (T-cell) immune responses of young broilers to a primary Eimeria acervulina infection in relation to the number of parasites used for infection. In our experiment we infected one-day-old broilers with a low (5 × 10 2 oocysts) and a high (5 × 10 4 oocysts) dose of E. acervulina. We used a newly developed species specific real-time PCR to quantify total amount of parasites in the duodenum as the number of oocysts in faeces may not be representative for the exposure of the gut immune system. We characterized T-cell subsets in the duodenum by means of FACS-analyses, lymphocyte proliferation assays with spleen lymphocytes and the mRNA profiles of different cytokines (TGF-β2, -4, IFN-γ, IL-2, -6, -8 and -18) in the duodenum by means of real-time PCR. From day 5 p.i. broilers with a high dose of E. acervulina had a significantly lower body weight than the control group. No increase in CD4 + cells, but a strong increase in CD8 + cells was observed at days 7 and 9 p.i. in the duodenum of broilers infected with a high dose E. acervulina. IL-8 mRNA responses were observed after infection with low and with high infection doses, but no IFN-γ and TGF-β mRNA responses were found in the duodenum. The specific proliferative T-cell responses to a low infectious dose were not significantly different as compared to the control group. In conclusion, based on the kinetics of observed responses a primary infection with a high dose of E. acervulina in one-day-old broilers seems to generate an immune response that shows a peak at the time of oocyst excretion, whereas the immune response to a low dose is less explicit.

Diptericin expression in bacteria infected Drosophila mbn‐2 cells – effect of infection dose and phagocytosis

Drosophila haemocytes play a key role in defence against microbial aggression. Their capacity to sense and dispose of bacteria and also to signal to other immune tissues is probably vital to overcome an infection. In this work we used the haemocyte-like mbn-2 cell line to investigate how expression of the antimicrobial peptide diptericin is affected after a high dose bacterial challenge with diaminopimelic acid (DAP)-peptidoglycan Gram-positive and Gram-negative bacteria. We report that diptericin expression is negatively affected by high infection dose and rapid bacterial growth regardless of the type of infection and bacterial virulence and occurs in the absence of mbn-2 cell death. Furthermore we show that the mbn-2 cell population is heterogeneous, containing both phagocytic and nonphagocytic cells and that contact with large numbers of bacteria decreases diptericin expression in the phagocytic cell population.

Lymph Node Dendritic Cells Control CD8 + T Cell Responses through Regulated FasL Expression

The lethal outcome of high-dose pulmonary virus infection is thought to reflect high-level, sustained virus replication and associated lung inflammation prior to development of an adaptive immune response. Herein, we demonstrate that the outcome of lethal/sublethal influenza infection instead correlates with the initial virus replication tempo. Furthermore, the magnitude of early lung antiviral CD8+ T cell responses varies inversely with inoculum dose and is controlled by lymph-node-resident dendritic cells (LNDC) through IL-12p40-regulated FasL-dependent T cell apoptosis. These results suggest that the inoculum dose and replication rate of a pathogen entering the respiratory tract may regulate the strength of the adaptive immune response, and the subsequent outcome of infection and that LNDC may serve as regulators (gatekeepers) in the development of CD8+ T cell responses.

Requirement for Neutralizing Antibodies to Control Bone Marrow Transplantation-Associated Persistent Viral Infection and to Reduce Immunopathology

Bone marrow transplantation (BMT) is commonly used in the treatment of leukemia, however its therapeutic application is partly limited by the high incidence of associated opportunistic infections. We modeled this clinical situation by infecting mice that underwent BMT with lymphocytic choriomeningitis virus (LCMV) and investigated the potential of immunotherapeutic strategies to counter such infections. All mice that received BMT survived LCMV infection and developed a virus carrier status. Immunotherapy by adoptive transfer of naive splenocytes protected against low (200 PFU), but not high (2 x 10(6) PFU), doses of LCMV. Attempts to control infection of high viral titers using strongly elevated frequencies of activated LCMV-specific T cells failed to control virus and resulted in immunopathology and death. In contrast, virus neutralizing Abs combined with naive splenocytes were able to efficiently control high-dose LCMV infection without associated side effects. Thus, cell transfer combined with neutralizing Abs represented the most effective means of controlling BMT-associated opportunistic viral infection in our in vivo model. These data underscore the in vivo efficacy and immunopathological "safety" of neutralizing antibodies.

Inactivation of the viral interleukin 1 receptor improves CD8+ T-cell memory responses elicited upon immunization with modified vaccinia virus Ankara

Interleukin 1 (IL1) is an important regulator of inflammatory responses and contributes to host immune defence against infection. Vaccinia virus encodes a viral soluble IL1beta receptor (IL1betaR), which modulates the acute-phase host response to infection and might influence the induction of immune responses against virus-associated antigens. Here, modified vaccinia virus Ankara (MVA) mutants defective in IL1betaR production were produced by insertion of selectable marker gene sequences that precisely deleted the IL1betaR coding sequences from the MVA genome (MVA-DeltaIL1betaR). Analysis of MVA mutants indicated that deletion of the IL1betaR gene did not abrogate the formation of MVA progeny upon tissue culture propagation. After high-dose intranasal infection with MVA-DeltaIL1betaR, mice showed no signs of fever or other illness, suggesting that the avirulent phenotype remained preserved for MVA-DeltaIL1betaR. Following vaccination of mice, MVA-DeltaIL1betaR or non-mutated MVA induced similar acute-phase immune responses. Importantly, when monitored at the memory phase, significantly higher vaccinia virus-specific total CD8(+) and HLA-A*0201-binding peptide epitope-specific T-cell responses were found after vaccination of HLA-A*0201-transgenic and non-transgenic mice with MVA-DeltaIL1betaR. Moreover, 4-6 months after vaccination, MVA-DeltaIL1betaR provided higher levels of protection against lethal respiratory challenge infection with virulent vaccinia virus strain Western Reserve compared with wild-type MVA. These data suggest that deletion of the viral IL1betaR gene may be considered a relevant approach to amplify the virus-specific CD8+ memory T-cell response and duration of protective immunity obtained after MVA vaccination.

Aptamers directed to HIV-1 reverse transcriptase display greater efficacy over small hairpin RNAs targeted to viral RNA in blocking HIV-1 replication

RNA molecules can be powerful inhibitors of HIV-1 replication. To determine the relative efficacy of siRNAs and RNA aptamers, a direct comparison of three anti-HIV reverse transcriptase aptamers and three shRNAs targeted to HIV-1(R3b) was made. U6 promoter-driven anti-HIV genes were delivered into CEMx174 cells via a retroviral vector, and transduced cells were sorted out via green fluorescent protein function and challenged with HIV. The results show that, at low virus input, shRNAs can block HIV as efficiently as aptamers. When expressed in target cells, both classes of inhibitors blocked early events of reverse transcription, suggesting they are both able to access intracellular reverse transcription complexes. However, at higher multiplicities of infection (m.o.i. of 50), while the aptamers could efficiently inhibit HIV replication, shRNAs did not. RNase protection assays indicated similar steady-state levels or nucleocytoplasmic distribution showing that the differential efficacy was not a reflection of intracellular concentration. The higher potency of anti-RT aptamers could be due to their ability to inhibit two successive rounds of reverse transcription owing to their unique ability to be encapsidated into virion particles. Furthermore, anti-RT aptamers expressed in T cells afforded protection against high-dose infection by chimeric RT-SHIV viruses.

Systemic and Local Antibody Responses after Experimental Infection withActinobacillus pleuropneumoniaein Piglets with Passive or Active Immunity

The objectives of the present study was to describe different dynamics of humoral immune responses to experimental infection in piglets of different stages of infection and immunity. Two groups of piglets originating from non-immune (group 1) and immune (group 2) sows at the age of 3 weeks were subdivided as follows: a half of each group of piglets was exposed to a low-dose infection with Actinobacillus pleuropneumoniae (APP) strain 9. At the age of 8 weeks, all four groups of piglets were challenged with a high infection dose of APP of the same strain. Isotype characterization of the specific antibodies in sera and in bronchoalveolar lavage fluids (BALF) to a lipopolysaccharide was carried out, besides monitoring clinical signs and post-mortem examinations. A typical primary immune response was observed in specific antibody-free piglets infected with a challenge infection. Colostrum-derived immunoglobulin-G (IgG) antibodies persisted in sera and BALF of piglets up to the age of 8 weeks. However, they did not prevent induction of specific-primary antibody response, either in 8 or 4 weeks of age, when levels of specific colostrum-derived antibodies were still high. It was demonstrated by the increase of specific IgM antibodies in sera. The infection induced an increase in the levels of IgA antibodies in BALF regardless the severity of infection and presence of specific colostrum-derived antibodies. The specific antibodies of IgG isotype increased only in BALF from piglets without colostrum-derived antibodies.

Risk of Occupationally Acquired Illnesses from Biological Threat Agents in Unvaccinated Laboratory Workers

Many vaccines for bioterrorism agents are investigational and therefore not available (outside of research protocol use) to all at-risk laboratory workers who have begun working with these agents as a result of increased interest in biodefense research. Illness surveillance data archived from the U.S. offensive biological warfare program (from 1943 to 1969) were reviewed to assess the impact of safety measures on disease prevention (including biosafety cabinets [BSCs]) before and after vaccine availability. Most laboratory-acquired infections from agents with higher infective doses (e.g., anthrax, glanders, and plague) were prevented with personal protective measures and safety training alone. Safety measures (including BSCs) without vaccination failed to sufficiently prevent illness from agents with lower infective doses in this high-risk research setting. Infections continued with tularemia (average 15/year), Venezuelan equine encephalitis (1.9/year), and Q fever (3.4/year) but decreased dramatically once vaccinations became available (average of 1, 0.6, and 0 infections per year, respectively). While laboratory-acquired infections are not expected to occur frequently in the current lower-risk biodefense research setting because of further improvements in biosafety equipment and changes in biosafety policies, the data help to define the inherent risks of working with the specific agents of bioterrorism. The data support the idea that research with these agents should be restricted to laboratories with experience in handling highly hazardous agents and where appropriate safety training and precautions can be implemented.

Release kinetics and cell trafficking in relation to bacterial growth explain the time course of blood neutrophils and monocytes during primary Salmonella infection

Granulocytes and neutrophils are predominantly responding cells during the early phase of infection of rats with Salmonella. We propose mathematical and experimental models of the kinetics of neutrophil and monocyte responses in Salmonella infection via the oral route. Using the models, we estimate that approximately 1 in 500 inoculated Salmonella cells actually infected the rat and multiplied with a doubling time of 5 h in Peyer's patches, reaching a maximum of approximately 10(6) c.f.u./g. In low-dose infection, neutrophil and monocyte responses are delayed, but further resemble the responses in high-dose infection. Important processes influencing neutrophil and monocyte recruitment are: massive migration into the infected tissue, and non-linear release kinetics of neutrophils and monocytes from the bone marrow. In conclusion, we can predict time series of neutrophil and monocyte responses in low-dose and high-dose experimental infection via the oral route.

Low-dose Salmonella infection evades activation of flagellin-specific CD4 T cells.

Many pathogens can establish a lethal infection from relatively small inocula, yet the effect of infectious dose upon CD4 T cell activation is not clearly understood. This issue was examined by tracking Salmonella flagellin-specific SM1 T cells in vivo, after i.v. and oral challenge of mice with virulent Salmonella typhimurium. SM1 T cells rapidly expressed activation markers and expanded in response to high-dose infection but remained completely unresponsive in mice challenged with low doses of Salmonella. SM1 T cells, in these mice, remained unresponsive, despite massive bacterial replication in vivo. Naive SM1 T cells in low-dose Salmonella-infected mice were activated rapidly after the injection of flagellin peptide, demonstrating that these T cells were fully capable of responding, ruling out the possibility of a bacterial-induced suppressive environment. The inability of flagellin-specific SM1 T cells to respond to low-dose infection was not due to Ag down-regulation, because flagellin expression was detected using a functional assay. Together, these data suggest that low-dose Salmonella infection can evade flagellin-specific CD4 T cell activation in vivo.

Transmission and infectious dose of Escherichia coli O157:H7 in swine.

Escherichia coli O157:H7 is only occasionally isolated from healthy swine, but some experimentally infected animals will shed the organism in their feces for at least 2 months. Potential explanations for the paucity of naturally occurring infections in swine, as compared to cattle, include a lack of animal-to-animal transmission so that the organism cannot be maintained within a herd, a high infectious dose, or herd management practices that prevent the maintenance of the organism in the gastrointestinal tract. We hypothesized that donor pigs infected with E. coli O157:H7 would transmit the organism to naïve pigs. We also determined the infectious dose and whether housing pigs individually on grated floors would decrease the magnitude or duration of fecal shedding. Infected donor pigs shedding <10(4) CFU of E. coli O157:H7 per g transmitted the organism to 6 of 12 naïve pigs exposed to them. The infectious dose of E. coli O157:H7 for 3-month-old pigs was approximately 6 x 10(3) CFU. There was no difference in the magnitude and duration of fecal shedding by pigs housed individually on grates compared to those housed two per pen on cement floors. These results suggest that swine do not have an innate resistance to colonization by E. coli O157:H7 and that they could serve as a reservoir host under suitable conditions.

Increased early local immune responses and altered worm development in high-dose infections of mice susceptible to the filaria Litomosoides sigmodontis.

The relationship between the number of larvae inoculated and filarial infection outcome is an important fundamental and epidemiological issue. Our study was carried out with BALB/c mice infected with the filaria Litomosoides sigmodontis. For the first time, an immunological analysis of infection with various doses was studied in parallel with parasitological data. Mice were inoculated with 200, 60 or 25 infective larvae (third stage larvae, L3), and monitored over 80 days. At 60 h post-inoculation the immune response was stronger in the 200 L3 group than the 25 L3 group. Cells from lymph nodes draining the site of inoculation proliferated intensely and produced large amounts of IL-5 and IL-4. In the pleural cavity, leukocyte populations accumulated earlier and in larger quantities. IgG1, IL-4 and IL-10 serum concentrations were transiently higher. During the first 10 days the worm recovery rates were identical in all groups, but decreased thereafter in the 200 L3 group. In this group, the development of the worms was altered, with reduced lengths, diminished intra-uterine production of microfilariae and abnormalities of male copulatory organs. Whereas mice inoculated with 25 L3 became microfilaraemic, only one third reached patency in the 200 L3 group. However, detrimental effects of high numbers of worms are not seen in studies using different inoculation protocols. This suggests that the very early events determine subsequent immune response and infection outcome rather than competitive interactions between the worms.

Increased resistance of vaccinated turkeys to experimental infection with an H7N3 low-pathogenicity avian influenza virus

A trial was performed to establish whether turkeys vaccinated against avian influenza with a vaccine containing a strain with a heterologous neuraminidase to the challenge virus required a higher infectious dose to develop infection than naïve birds. Birds were vaccinated with a commercially available, inactivated oil emulsion product containing the strain A/ty/Italy/99/(H7N1) and challenged with different dilutions of a LPAI isolate A/ty/Italy/8000/02(H7N3) obtained during the 2002 to 2003 Italian epidemic. Groups of 10 vaccinated and 10 unvaccinated birds were infected experimentally with a virus suspension containing 10(2), 10(4) and 10(6) median embryo infective dose (EID50)/0.1 ml. Infected birds were observed daily with tracheal and cloacal swabs collected at regular intervals for antigen detection, virus isolation and real-time reverse transcription-polymerase chain reaction. Pre-infection and post-infection serology was also performed. The results of the experiment indicate that infection is achieved in naïve birds with 10(4) EID50, while vaccinated birds are resistant at this challenge dose. Vaccinated and unvaccinated birds were susceptible to infection with 10(6) EID50, although the duration and/or the number of birds shedding was reduced in the vaccinated group. The data presented indicate that heterologous vaccination in the framework of a 'Differentiating Infected from Vaccinated Animals' strategy can be a valid tool to support eradication measures in areas with high densities of susceptible animals. .

The role of CD8+ cells in the establishment and maintenance of a Trichuris muris infection.

Chronic infection by the caecal-dwelling intestinal murine nematode Trichuris muris occurs if given as a high-dose infection to 'susceptible' AKR mice or as a low-dose infection to the normally 'resistant' C57BL/6 mouse strain. Both regimes result in a type 1 cytokine response, i.e. high levels of IFN-gamma and IL-12. Here we show this susceptible response is associated with a large population of CD8(+) IFN-gamma(+) cells within the mesenteric lymph nodes and numerous CD8(+) cells infiltrating the caecal mucosa. Despite this, the in vivo abolition of CD8(+) cells within AKR and C57BL/6 mice, either prior to infection or once infection has become established, failed to affect chronicity, implying that CD8(+) T cells are not essential for the initiation or maintenance of the susceptible response to T. muris. Interestingly, the percentage of IFN-gamma(+) CD4(+) cells increased in treated groups, perhaps in a compensatory role. The majority of antigen-specific cytokine responses were comparable in both treated and control groups, although IL-5 was fivefold higher in animals receiving anti-CD8 mAbs and IFN-gamma was also raised in treated mice. Mastocytosis was unaltered by CD8 depletion, however, paradoxically, eosinophilia within the caecum was reduced in treated mice. Together these data clearly demonstrate that CD8(+) T cells are associated with chronic T. muris infection; however, these cells are dispensable for both the early and late phases of this response, but do appear to play a role in the regulation of certain cytokines and caecal eosinophilia.

Protection from bacterial infection by a single vaccination with replication-deficient mutant herpes simplex virus type 1.

Adaptive immune responses in which CD8(+) T cells recognize pathogen-derived peptides in the context of major histocompatibility complex class I molecules play a major role in the host defense against infection with intracellular pathogens. Cells infected with intracellular bacteria such as Listeria monocytogenes, Salmonella enterica serovar Typhimurium, or Mycobacterium tuberculosis are directly lysed by cytotoxic CD8(+) T cells. For this reason, current vaccines for intracellular pathogens, such as subunit vaccines or viable bacterial vaccines, aim to generate robust cytotoxic T-cell responses. In order to investigate the capacity of a herpes simplex virus type 1 (HSV-1) vector to induce strong cytotoxic effector cell responses and protection from infection with intracellular pathogens, we developed a replication-deficient, recombinant HSV-1 (rHSV-1) vaccine. We demonstrate in side-by-side comparison with DNA vaccination that rHSV-1 vaccination induces very strong CD8(+) effector T-cell responses. While both vaccines provided protection from infection with L. monocytogenes at low, but lethal doses, only rHSV-1 vaccines could protect from higher infectious doses; HSV-1 induced potent memory cytotoxic T lymphocytes that, upon challenge by pathogens, efficiently protected the animals. Despite the stimulation of relatively low humoral and CD4-T-cell responses, rHSV-1 vectors are strong candidates for future vaccine strategies that confer efficient protection from subsequent infection with intracellular bacteria.

Glycoprotein C of herpes simplex virus type 1 is required to cause keratitis at low infectious doses in intact rabbit corneas

Purpose. To determine whether the herpes simplex virus type 1 (HSV-1) viral glycoprotein C (gC) plays a role in induction of keratitis in unscarified and scarified rabbit eyes.Materials and methods. A gC deletion mutant (ΔgC) was constructed and then rescued back to wild type (wt) for use as a control. Following ocular infection with each virus in rabbit eyes, with or without prior corneal scarification, keratitis was compared.Results. At low infection doses of 2 × 103 and 2 × 104 plaque-forming units (PFU)/eye, in unscarified cornea, ΔgC produced significantly less keratitis than did wt virus (p = 0.007 and 0.03, respectively). In contrast, the keratitis induced by ΔgC was similar to that induced by the wt virus (p > 0.60) in scarified cornea. At high infection dose (2 × 105 PFU/eye), keratitis induced by ΔgC was similar in scarified and unscarified cornea, and the severity of disease was similar to that seen in scarified eyes at the low-dose ΔgC infections. Interestingly, although ΔgC induced keratitis with or without corneal scarification at high infection doses, the severity of disease was significantly less than that induced by wt infection. At all infection doses, keratitis induced by wt infection was similar in scarified and unscarified eyes.Conclusions. These results suggest that (1) at low infection doses, in unscarified corneas, gC is required for HSV-1 induced keratitis; (2) corneal scarification prior to infection can circumvent the need for gC at low doses, but (3) at higher doses, gC is required for wild-type levels of keratitis even in scarified cornea.

Apparently normal tumor necrosis factor receptor 1 signaling in the absence of the silencer of death domains.

The silencer of death domains (SODD) has been proposed to prevent constitutive signaling of tumor necrosis factor receptor 1 (TNFR1) in the absence of ligand. Besides TNFR1, death receptor 3 (DR3), Hsp70/Hsc70, and Bcl-2 have been characterized as binding partners of SODD. In order to investigate the in vivo role of SODD, we generated mice congenitally deficient in expression of the sodd gene. No spontaneous inflammatory infiltrations were observed in any organ of these mice. Consistent with this finding, in the absence of SODD no alteration in the activation patterns of nuclear factor kappaB (NF-kappaB), stress kinases, or ERK1 or -2 was observed after stimulation with tumor necrosis factor (TNF). Activation of NF-kappaB by DR3 was also unchanged. The extents of DR3- and TNF-induced apoptosis were comparable in gene-deficient and wild-type cells. Protection of cells against heat shock as mediated by the Hsp70 system and against staurosporine-induced apoptosis was independent of SODD. Furthermore, resistance to high-dose lipopolysaccharide (LPS) injections, LPS-D-GalN injections, and infection with listeriae was similar in wild-type and gene-deficient mice. In conclusion, our data do not support the concept of a unique, nonredundant role of SODD for the functions of TNFR1, Hsp70, and DR3.

Combined contributions of streptolysin O and streptolysin S to virulence of serotype M5 Streptococcus pyogenes strain Manfredo.

Streptolysin O (SLO) and streptolysin S (SLS) are potent cytolytic toxins produced by almost all clinical isolates of group A streptococci (GAS). Allele-replacement mutagenesis was used to construct nonpolar (in-frame) deletion mutations in the slo and sagB genes of the serotype M5 GAS strain Manfredo, producing isogenic single and double SLO- and SLS-defective mutants. In contrast to recent reports on SLS-defective insertion mutants (I. Biswas, P. Germon, K. McDade, and J. Scott, Infect. Immun. 69:7029-7038, 2001; Z. Li, D. Sledjeski, B. Kreikemeyer, A.Podbielski, and M. Boyle, J. Bacteriol. 181:6019-6027, 1999), none of the mutants described here had notable pleiotropic effects on the expression of other virulence factors examined. Comparison of isogenic parent and mutant strains in various virulence models revealed no differences in their abilities to multiply in human blood or in their 50% lethal doses (LD(50)s) upon intraperitoneal infection of BALB/c mice. A single log unit difference in the LD(50)s of the parent and SLS-defective mutant strains was observed upon infection by the subcutaneous (s.c.) route. Comparisons over a range of infective doses showed that both SLO and SLS contributed to the early stages of infection and to the induction of necrotic lesions in the murine s.c. model. Individually, each toxin made an incremental contribution to virulence that was not apparent at higher infective doses, although the absence of both toxins reduced virulence over the entire dose range examined. Interestingly, in some cases, the contribution of SLO to virulence was clear only from an analysis of the double-mutant strain, highlighting the value of not confining virulence studies to mutant strains defective in the expression of only single virulence factors.