Small Molecule Inhibitors of Staphylococcus aureus RnpA Alter Cellular mRNA Turnover, Exhibit Antimicrobial Activity, and Attenuate Pathogenesis

Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy

Data from: Regulatory CD4+CD25+ T cells dampen inflammatory disease in murine mycoplasma pneumonia and promote IL-17 AND IFN-? responses

Mycoplasmas cause respiratory diseases characterized by persistent infection and chronic airway inflammation. Mycoplasma lung disease is immunopathologic, with CD4+ Th cells determining both disease severity and resistance to infection. Th2 cell responses promote immunopathology, while Th1 cells confer resistance to infection. However, regulatory CD4+ T cells may also have a role in the pathogenesis of mycoplasma respiratory diseases. We hypothesized Treg cells control the severity of the inflammatory lesions and may also promote persistence of infection. To examine this, BALB/c mice were depleted of CD25+ cells, and had increased disease severity due to Mycoplasma pulmonis infection. Increases in mycoplasma antibody responses and lymphocyte infiltration into lungs also occurred after CD25+ cell depletion. CD4+CD25+ regulatory T cells promoted IFN-? and IL-17 mycoplasma-specific CD4+ T cell responses in vitro and in vivo, while dampening IL-13+ Th responses. Neither IL-10 and TGF-? expression was detected in CD4+CD25+ T cells from lymph nodes. Thus, a regulatory T cell population plays an important role in controlling damaging immune responses in mycoplasma respiratory disease but does not contribute to persistence of infection. It appears that a regulatory T cell population preferentially dampens Th2 cell-mediated inflammatory responses to mycoplasma through a mechanism independent of IL-10 or TGF-? characteristic of "classic" Treg cells

Regulatory CD4+CD25+ T Cells Dampen Inflammatory Disease in Murine Mycoplasma Pneumonia and Promote IL-17 and IFN-γ Responses.

Mycoplasmas cause respiratory diseases characterized by persistent infection and chronic airway inflammation. Mycoplasma lung disease is immunopathologic, with CD4+ Th cells determining both disease severity and resistance to infection. Th2 cell responses promote immunopathology, while Th1 cells confer resistance to infection. However, regulatory CD4+ T cells may also have a role in the pathogenesis of mycoplasma respiratory diseases. We hypothesized Treg cells control the severity of the inflammatory lesions and may also promote persistence of infection. To examine this, BALB/c mice were depleted of CD25+ cells, and had increased disease severity due to Mycoplasma pulmonis infection. Increases in mycoplasma antibody responses and lymphocyte infiltration into lungs also occurred after CD25+ cell depletion. CD4+CD25+ regulatory T cells promoted IFN-γ and IL-17 mycoplasma-specific CD4+ T cell responses in vitro and in vivo, while dampening IL-13+ Th responses. Neither IL-10 nor TGF-ß expression was detected in CD4+CD25+ T cells from lymph nodes. Thus, a regulatory T cell population plays an important role in controlling damaging immune responses in mycoplasma respiratory disease but does not contribute to persistence of infection. It appears that a regulatory T cell population preferentially dampens Th2 cell-mediated inflammatory responses to mycoplasma through a mechanism independent of IL-10 or TGF-ß characteristic of "classic" Treg cells

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 ∼24 h failed to repress and generally activated biofilm formation

Regulatory CD4⁺CD25⁺ T Cells Dampen Inflammatory Disease in Murine Mycoplasma Pneumonia and Promote IL-17 and IFN-γ Responses

<div><p>Mycoplasmas cause respiratory diseases characterized by persistent infection and chronic airway inflammation. Mycoplasma lung disease is immunopathologic, with CD4⁺ Th cells determining both disease severity and resistance to infection. Th2 cell responses promote immunopathology, while Th1 cells confer resistance to infection. However, regulatory CD4⁺ T cells may also have a role in the pathogenesis of mycoplasma respiratory diseases. We hypothesized Treg cells control the severity of the inflammatory lesions and may also promote persistence of infection. To examine this, BALB/c mice were depleted of CD25⁺ cells, and had increased disease severity due to <i>Mycoplasma pulmonis</i> infection. Increases in mycoplasma antibody responses and lymphocyte infiltration into lungs also occurred after CD25⁺ cell depletion. CD4⁺CD25⁺ regulatory T cells promoted IFN-γ and IL-17 mycoplasma-specific CD4⁺ T cell responses <i>in vitro</i> and <i>in vivo</i>, while dampening IL-13⁺ Th responses. Neither IL-10 nor TGF-ß expression was detected in CD4⁺CD25⁺ T cells from lymph nodes. Thus, a regulatory T cell population plays an important role in controlling damaging immune responses in mycoplasma respiratory disease but does not contribute to persistence of infection. It appears that a regulatory T cell population preferentially dampens Th2 cell-mediated inflammatory responses to mycoplasma through a mechanism independent of IL-10 or TGF-ß characteristic of “classic” Treg cells.</p></div

CD25 antibody-mediated depletion did not significantly affect non-Treg cells.

<p>CD25 antibody-mediated depletion did not significantly affect non-Treg cells.</p

CD4⁺CD25⁺ T cells stimulate the <i>in vitro</i> production of IFN-γ and IL-17 by CD4⁺ T cells.

<p>CD4⁺CD25⁺ T cells and CD4⁺CD25^- Th cells from LRN of infected mice were isolated. Th cells and Ag-presenting cells (CD3-depleted naïve splenocytes) were cultured <i>in vitro</i> with or without CD4⁺CD25⁺ T cells. The cells were cultured in the presence or absence of mycoplasma Ag as indicated. Four days later, supernatants were assayed for IL-4, IL-10, IL-13, IL-17, TGF-β, and IFN-γ. TGF-β, IL-4 and IL-13 levels were low and did not increase in cultures stimulated with mycoplasma Ag. An asterisk (*) represents a significant (<i>P</i> ≤ 0.05) difference (Ag + CD4⁺CD25⁺ T cells versus Ag only). Individual data points and horizontal lines representing the means are shown (n = 4). A graph for TGF-β levels was not included, as all samples were below the level of detection (< 62.5 pg/ml) using an ELISA.</p

Depletion of CD25⁺ cells decreased the percentage of IL-17 and IFN-γ CD4⁺ Th cells in the LRN of infected mice.

<p>Mice were given anti-CD25 depleting antibody (CD25⁺ cell depleted) or PBS (Control) one day prior to infection with <i>M</i>. <i>pulmonis</i>, followed by antibody or PBS at day 6 post-infection. Mice were sacrificed at day 7 after infection, and cells harvested from LRNs. Data are expressed as a percentage of CD4⁺CD25^-FoxP3^- lymphocytes (Th cells). An asterisk (*) represents a significant (<i>P</i> ≤ 0.05) difference between the percentages of cells found in lymph nodes of control and Treg depleted mice. A double asterisk (**) represents a significant (<i>P</i> ≤ 0.05) difference infected mice that were depleted of CD25⁺ cells and control (PBS-treated) mice. Vertical bars and error bars represent means +/- SE (n = 6).</p