Jerry W. Simecka

Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli.

Many species of bacteria devote considerable metabolic resources and genetic information to the ability to sense the environment and move towards or away from specific stimuli using flagella. In Escherichia coli and related species, motility is regulated by several global regulatory circuits, which converge to modulate the overall expression of the master operon for flagellum biosynthesis, flhDC. We now show that the global regulator CsrA of E. coli K‐12 is necessary for motility under a variety of growth conditions, as a result of its role as an activator of flhDC expression. A chromosomally encoded flhDC′–′lacZ translational fusion was expressed at three‐ to fourfold higher levels in csrA wild‐type strains than in isogenic csrA mutants. Purified recombinant CsrA protein stimulated the coupled transcription‐translation of flhDC′–′ lacZ in S‐30 extracts and bound to the 5′ segment of flhDC mRNA in RNA mobility shift assays. The steady‐state level of flhDC mRNA was higher and its half‐life was ≈ threefold greater in a csrA wild‐type versus a csrA mutant strain. Thus, CsrA stimulates flhDC gene expression by a post‐transcriptional mechanism reminiscent of its function in the repression of glycogen biosynthesis.

Catabolite repression of Escherichia coli biofilm formation.

Biofilm formation was repressed by glucose in several species of Enterobacteriaceae. In Escherichia coli, this effect was mediated at least in part by cyclic AMP (cAMP)-cAMP receptor protein. A temporal role for cAMP in biofilm development was indicated by the finding that glucose addition after approximately 24 h failed to repress and generally activated biofilm formation.

Depletion of CD8+ T Cells Exacerbates CD4+ Th Cell-Associated Inflammatory Lesions During Murine Mycoplasma Respiratory Disease

Mycoplasma infection is a leading cause of pneumonia worldwide and can lead to other respiratory complications. A component of mycoplasma respiratory diseases is immunopathologic, suggesting that lymphocyte activation is a key event in the progression of these chronic inflammatory diseases. The present study delineates the changes in T cell populations and their activation after mycoplasma infection and determines their association with the pathogenesis of murine Mycoplasma respiratory disease, due to Mycoplasma pulmonis infection. Increases in T cell population numbers in lungs and lower respiratory lymph nodes were associated with the development of mycoplasma respiratory disease. Although both pulmonary Th and CD8+ T cells increased after mycoplasma infection, there was a preferential expansion of Th cells. Mycoplasma-specific Th2 responses were dominant in lower respiratory lymph nodes, while Th1 responses predominated in spleen. However, both mycoplasma-specific Th1 and Th2 cytokine (IL-4 and IFN-γ) responses were present in the lungs, with Th1 cell activation as a major component of the pulmonary Th cell response. Although a smaller component of the T cell response, mycoplasma-specific CD8+ T cells were also a significant component of pulmonary lymphoid responses. In vivo depletion of CD8+ T cells resulted in dramatically more severe pulmonary disease, while depletion of CD4+ T cells reduced its severity, but there was no change in mycoplasma numbers in lungs after cell depletion. Thus, mycoplasma-specific Th1 and CD8+ T cell activation in the lung plays a critical regulatory role in development of immunopathologic reactions in Mycoplasma respiratory disease.

The Pulmonary Environment Promotes Th2 Cell Responses After Nasal-Pulmonary Immunization with Antigen Alone, but Th1 Responses Are Induced During Instances of Intense Immune Stimulation

The purpose of this study was to determine the nature of the CD4+ Th cell responses induced after nasal-pulmonary immunization, especially those coinciding with previously described pulmonary inflammation associated with the use of the mucosal adjuvant, cholera toxin (CT). The major T cell population in the lungs of naive mice was CD4+, and these cells were shown to be predominantly of Th2 type as in vitro polyclonal stimulation resulted in IL-4, but not IFN-γ, production. After nasal immunization with influenza Ag alone, Th2 cytokine mRNA (IL-4 and IL-5) levels were increased, whereas there was no change in Th1 cytokine (IL-2 and IFN-γ) mRNA expression. The use of the mucosal adjuvant, CT, markedly enhanced pulmonary Th2-type responses; however, there was also a Th1 component to the T cell response. Using in vitro Ag stimulation of pulmonary lymphocytes, influenza virus-specific cytokine production correlated with the mRNA cytokine results. Furthermore, there was a large increase in CD4+ Th cell numbers in lungs after nasal immunization using CT, correlating with the pulmonary inflammatory infiltrate previously described. Coincidentally, both macrophage-inflammatory protein-1α (MIP-1α) and MIP-1β mRNA expression increased in the lungs after immunization with Ag plus CT, while only MIP-1β expression increased when mice were given influenza Ag alone. Our study suggests a mechanism to foster Th1 cell recruitment into the lung, which may impact on pulmonary immune responses. Thus, while Th2 cell responses may be prevalent in modulating mucosal immunity in the lungs, Th1 cell responses contribute to pulmonary defenses during instances of intense immune stimulation.

Co-expression of granulocyte-macrophage colony-stimulating factor with antigen enhances humoral and tumor immunity after DNA vaccination

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance humoral and tumor immunity resulting from DNA immunization. The genes encoding GM-CSF and antigen were cloned onto the same plasmid backbone, but separate promoters drove expression of each gene. beta-Galactosidase was used as the model antigen to generate antibody responses while the human tumor antigen, MAGE-1, was used to monitor tumor resistance. Immunization with a DNA vaccine co-expressing GM-CSF and beta-gal resulted in higher antigen-specific IgG responses than immunization with antigen encoding plasmid alone or co-inoculated with GM-CSF expressing plasmid. Similarly, DNA vaccines expressing both MAGE-1 antigen and GM-CSF were more effective in protecting against B16-MAGE-1 melanoma. However, both GM-CSF co-expressing DNA vaccines and co-inoculation with plasmids encoding the cytokine or antigen enhanced the generation antigen-specific IFN-gamma and IL-6 responses. These results demonstrate that co-expressing both GM-CSF and antigen on a DNA vaccine enhances humoral and tumor immune responses.

Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats

Restriction and modification (R–M) systems are generally thought to protect bacteria from invasion by foreign DNA. This paper proposes the existence of an alternative role for the phase‐variable R–M systems encoded by the hsd loci of Mycoplasma pulmonis. Populations of M. pulmonis cells that arose during growth in different environments were compared with respect to R–M activity and surface antigen production. When M. pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony‐forming units (cfu) lacked R–M activity and produced the variable surface protein VsaA. Mycoplasmas isolated from the nose of experimentally infected rats also lacked R–M activity and produced VsaA. In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex. The most dramatic results were obtained for mycoplasmas isolated from the trachea. At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems. These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R–M activity of the mycoplasmal cell population. We propose that variations in the production of R–M activity and cell surface proteins are important for the survival of the mycoplasma within the host.

A Novel IL-17 Dependent Mechanism of Cross Protection: Respiratory Infection with Mycoplasma Protects Against a Secondary Listeria Infection

Immune responses to pathogens occur within the context of current and previous infections. Cross protection refers to the phenomena where infection with a particular pathogen provides enhanced resistance to a subsequent unrelated pathogen in an antigen‐independent manner. Proposed mechanisms of antigen‐independent cross protection have involved the secretion of IFN‐γ, which activates macrophages, thus providing enhanced innate immunity against the secondary viral or bacterial pathogen. Here we provide evidence that a primary infection with the chronic respiratory pathogen, Mycoplasma pulmonis, provides a novel form of cross protection against a secondary infection with Listeria monocytogenes that is not mediated by IFN‐γ, but instead relies upon IL‐17 and mobilization of neutrophils. Mice infected with M. pulmonis have enhanced clearance of L. monocytogenes from the spleen and liver, which is associated with increased numbers of Gr‐1+CD11b+ cells and higher levels of IL‐17. This enhanced clearance of L. monocytogenes was absent in mice depleted of Gr‐1+ cells or in mice deficient in the IL‐17 receptor. Additionally, both the IL‐17 receptor and neutrophils were essential for optimal clearance of M. pulmonis. Thus, a natural component of the immune response directed against M. pulmonis was able to enhance clearance of L. monocytogenes.

The Upper and Lower Respiratory Tracts Differ in Their Requirement of IFN-γ and IL-4 in Controlling Respiratory Mycoplasma Infection and Disease

The purpose of this study is to evaluate the significance of IFN-γ and IL-4 production in controlling mycoplasma infection and the pathogenesis of disease in the upper and lower respiratory tract. By using IFN-γ knockout and IL-4 knockout BALB/c mice, we were able to study the contribution of these cytokines in the development of pathogenesis and/or protection in response to mycoplasma respiratory infection, in both the upper and lower respiratory tracts. The loss of either IFN-γ or IL-4 does not affect disease pathogenesis or mycoplasma organism numbers in the upper respiratory tract. However, in the absence of IL-4, the nasal passages developed a compensatory immune response, characterized by higher numbers of macrophages and CD8+ T cells, which may be masking detrimental effects due to IL-4 deficiency. This is in contrast to the lower respiratory tract, where the loss of IFN-γ, but not IL-4, leads to higher mycoplasma numbers and increased disease severity. The loss of IFN-γ impacted the innate immune system’s ability to effectively clear mycoplasma, as the number of organisms was higher by day 3 postinfection. This higher organism burden most likely impacted disease pathogenesis; however, the development of Th2 cell-mediated adaptive immune response most likely contributed to lesion severity at later time points during infection. Our studies demonstrate that the upper and lower respiratory tracts are separate and distinct in their cytokine requirements for generating immunity against mycoplasma infection.

Role of Upper and Lower Respiratory Tract Immunity in Resistance to Mycoplasma Respiratory Disease

This study determined whether respiratory tract immunization protects against mycoplasma infection and compared preferential immunization of the upper respiratory tract (nasal) with upper and lower respiratory tract (nasal-pulmonary) immunization. Small volumes of inoculum preferentially deposited antigen and induced IgA responses in nasal passages. Larger inoculums deposited antigen in both the upper and lower respiratory tracts, generating corresponding IgA responses. Mice were given nasal or nasal-pulmonary immunizations with Mycoplasma pulmonis antigen alone or with cholera toxin (CT), and resistance to infection was determined. Generation of upper respiratory tract immunity reduced mycoplasma infection at this site, but CT was needed to elicit protective responses. In the lower respiratory tract, nasal-pulmonary immunization was most effective, but nasal immunization did confer some protection from pulmonary infection. In contrast, intraperitoneal immunization resulted in little protection. Thus, respiratory tract immunity plays a major role in resisting mycoplasma infection, and it should be considered during vaccine development.

Hyperbaric oxygen treatment attenuates the pro-inflammatory and immune responses in apolipoprotein E knockout mice

Chronic hyperbaric oxygen (HBO) therapy significantly attenuates atherosclerosis in New Zealand white rabbits as well as the apoE knockout (KO) mice, independent of plasma lipid concentrations and lipoprotein profiles. Because atherosclerosis has many features of a chronic inflammatory disease, in which both cell-mediated and humoral immune responses participate, we examined the effect of HBO treatment on various aspects of the immune response. We now demonstrate that in apoE KO mice, HBO treatment significantly reduces the circulating levels of antibodies to (MDA)LDL, both in the IgG and IgM class, as well as the delayed-type hypersensitivity (DTH) response to oxLDL challenge. Furthermore, HBO treatment results in a profound attenuation in the production of pro-inflammatory cytokines in response to an inflammatory stimulus (LPS), which is accompanied by a marked increase in the constitutive production of the anti-inflammatory cytokine IL-10 by spleen cells, independent of antigen specificity, as indicated by polyclonal activation of T cells. Our results demonstrate that HBO treatment results in the dampening of T and B cell-mediated responses to oxLDL or inflammatory stimuli.