SummaryMany 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 H ± H 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 H ± H lacZ in S-30 extracts and bound to the 5 H 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.

The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA-lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development.

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 ϳ24 h failed to repress and generally activated biofilm formation.In the natural environment, bacteria predominantly exist in matrix-enclosed, sessile communities referred to as biofilms (4). Biofilms protect cells from deleterious conditions, such as attack by the mammalian immune system (5). Biofilms are complex assemblages of cells which exhibit channels and pillars that are thought to permit the exchange of nutrients and wastes. A recent model for biofilm development proposes that it is initiated by the attachment of individual cells to a surface, followed by their migration and replication to form microcolonies that eventually produce the mature biofilm (20,22). A variety of extracellular molecules and surface organelles participate in E. coli biofilm formation (6,7,23,33).Central carbon flux and its regulation may represent key features of bacterial biofilm development. We recently reported that the RNA binding protein CsrA of Escherichia coli represses biofilm formation and activates biofilm dispersal (13). The effect of CsrA on biofilm formation is mediated largely through its regulatory role in central carbon flux and intracellular glycogen synthesis and catabolism (17,18,24,25,28,34). The influence of CsrA is substantially greater than that of other regulators of E. coli biofilm formation, OmpR, RpoS, or the Cpx two-component system (1,8,33). Studies with other species have revealed that the global regulator Crc (catabolite repression control) of Pseudomonas aeruginosa activates biofilm formation (21), and the expression of the staphylococcal biofilm polysaccharide PIA (polysaccharide intracellular adhesin) requires a functional glucose phosphoenolpyruvate:sugar phosphotransferase system (15).During studies of biofilm formation, we noted that the addition of glucose to media was inhibitory. To substantiate this observation, E. coli K-12 parental strains MG1655, MC4100, and W3110 and their isogenic csrA mutants (Table 1) were grown in microtiter wells in colony-forming antigen (CFA) medium (9) with or without glucose (0.2% wt/vol), and biofilm was quantitated after 24 h of growth using crystal violet staining (A 630 ), as described previously (13) (Fig. 1A). Essentially identical results were observed in Luria-Bertani (LB) medium (19) (data not shown). These and other biofilm experiments described in this article were performed at least in triplicate with three samples per experiment, and data were analyzed by Tukey multigroup analysis (Stat View; SAS Institute Inc., Cary, N.C.). Glucose caused a statistically significant decrease in biofilm formation in every case, which varied from ϳ30 to 95% reduction, depending primarily on the strain background but also on the medium. Biofilm formation by related clinical i...

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.

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.

One major concern about using adenoviral vectors for repetitive gene delivery to lung epithelial cells is the induction of an immune response to the vector, thus, impeding effective gene transduction. To assess the immune response to the adenoviral vector, repetitive intratracheal (i.t.) gene dosing was performed in CD-1 mice using the replication-deficient adenovirus 5 (Ade5) vector carrying the lacZ gene, and compared to the antibody responses induced by conventional intranasal (i.n.) and intraperitoneal (i.p.) routes of immunization. Kinetics of serum IgG, IgA, and IgM antibody responses to the adenoviral vector and to beta-galactosidase (beta-Gal) were evaluated. Two or three adenoviral vector doses given by i.t., i.n., or i.p. routes resulted in serum IgG titers in excess of 1:200,000, whereas serum IgM and IgA were moderately induced. Analysis of the predominant murine IgG subclass was determined to be IgG2b and IgG2a. To determine the localization of this antibody response, the ELISPOT assay was employed. Lymphocytes were isolated from the lung, the lower respiratory lymph nodes (LRLN), the nasal passages (NP), and the spleen. For i.t- and i.n.-administered mice, the highest IgA spot-forming cell (SFC) response to Ade5 and beta-Gal was located in the NP and in the lung. Both the lung and the LRLN showed elevated numbers of IgG SFCs (4- to 12-fold greater than splenic IgG SFC response) for Ade5 and beta-Gal. This evidence suggests that the lung and associated lymphoid tissues were the source for serum antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between mycoplasmas and B cells consist primarily of the development of specific antibody and of nonspecific interactions with B lymphocytes or antibody. Antibody responses are important in the resistance to mycoplasmal disease in both humans and animals. However, the ability of mycoplasmas to survive in their host despite vigorous responses suggests that these play a limited role in the host's recovery from infection. Antibody also may prevent dissemination of mycoplasmal infections from mucosal sites and may account for the appearance of systemic mycoplasmal infections in immunocompromised patients. In some cases, antibody responses may contribute to disease pathogenesis through the development of hypersensitivity responses or the deposition of immune complexes. In addition, nonspecific interactions between mycoplasmas and B lymphocytes have been implicated in disease pathogenesis, possibly leading to autoimmune reactions, modulation of immunity, and/or promotion of lesion development. For example, several mycoplasmas, including Mycoplasma pneumoniae and Mycoplasma pulmonis, are able to activate B cells polyclonally in vitro and in vivo, but the mechanisms and consequences of these responses have yet to be defined. In addition to activating B lymphocytes, mycoplasmas are capable of producing chemotactic factors, Fc receptors, and immunoglobulin proteases that may also be involved in lesion development and/or survival of the organisms. Thus, both specific and nonspecific interactions of mycoplasmas with B cells can have important effects on disease progression, especially since many mycoplasmal infections are chronic and the cumulative effect of these interactions may be substantial.

LPT significantly increased both thoracic duct lymph flow and leukocyte count, so lymph leukocyte flux was markedly enhanced. Increased mobilization of immune cells is likely and important mechanism responsible for the enhanced immunity and recovery from infection of patients treated with LPT.