Regulation of Biofilm Aging and Dispersal in Bacillus subtilis by the Alternative Sigma Factor SigB.

Bacterial biofilms are important in natural settings, biotechnology, and medicine. However, regulation of biofilm development and its persistence in different niches is complex and only partially understood. One key step during the biofilm life cycle is dispersal, when motile cells abandon the mature biofilm to spread out and colonize new niches. Here, we show that in the model bacterium Bacillus subtilis the general stress transcription factor SigB is essential for halting detrimental overgrowth of mature biofilm and for triggering dispersal when nutrients become limited. Specifically, SigB-deficient biofilms were larger than wild-type biofilms but exhibited accelerated cell death, significantly greater sensitivity to different stresses, and reduced dispersal. Interestingly, the signal detected by SigB to limit biofilm growth was transduced through the RsbP-dependent metabolic arm of the SigB regulatory cascade, which in turn positively controlled expression of SinR, the master regulator of biofilm formation and cell motility. This novel SigB-SinR regulatory circuit might be important in controlling the fitness of biofilms (either beneficial or harmful) in diverse environments.IMPORTANCE Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical systems. Sessile cells embedded in the self-produced extracellular matrix of the biofilm benefit from a division of labor and are protected from environmental insults. However, as the biofilm ages, cells become stressed because of overcrowding, starvation, and accumulation of waste products. How does the sessile biofilm community sense and respond to stressful conditions? Here, we show that in Bacillus subtilis, the transcription factors SigB and SinR control whether cells remain in or leave a biofilm when metabolic conditions become unfavorable. This novel SigB-SinR regulatory circuit might be important for controlling the fitness of biofilms (either beneficial or harmful) in diverse environments.

Influence of disease severity on nitrite and cytokine production by peripheral blood mononuclear cells (PBMC) from patients with pulmonary tuberculosis (TB).

Earlier studies in patients with pulmonary TB have revealed a higher production of Th1 cell type cytokines in moderate TB, with predominant Th2-like responses in advanced disease. Given the influence of IL-12 in T cell differentiation, as well as the roles of transforming growth factor-beta (TGF-beta), nitric oxide and tumour necrosis factor-alpha (TNF-alpha) in the immune response against intracellular pathogens, we decided to analyse the interferon-gamma (IFN-gamma), IL-4, IL-12, TGF-beta, TNF-alpha and nitrite concentrations in culture supernatants of PBMC from TB patients showing different degrees of lung involvement. The sample population comprised 18 untreated TB patients with either moderate (n = 9) or advanced (n = 9) disease and 12 age- and sex-matched healthy controls (total population (patients and controls) 12 women, 18 men, aged 37 +/- 13 years (mean +/- s.d.)). PBMC were stimulated with whole sonicate from Mycobacterium tuberculosis and the supernatants were collected on day 4 for measurement of cytokine and nitrite levels. Antigen-stimulated IFN-gamma, TGF-beta and TNF-alpha production was found to be significantly increased in TB patients, both moderate and advanced, compared with the controls. Levels of IFN-gamma were significantly higher in moderate disease than advanced cases, whereas advanced cases showed significantly higher IL-12, TGF-beta and TNF-alpha concentrations when compared with cases of moderate TB. Nitrite levels were also increased in TB patients and the increase was statistically significant when advanced cases were compared with controls. These findings may contribute to a clearer picture of the net effect of cytokine interactions in TB, essential for a better understanding of the immunopathological mechanisms underlying the distinct clinical forms of the disease.

In vitro synthesis of interferon-gamma, interleukin-4, transforming growth factor-beta and interleukin-1 beta by peripheral blood mononuclear cells from tuberculosis patients: relationship with the severity of pulmonary involvement.

Given the role of cell-mediated immune responses in resistance to mycobacteria, we sought to analyse whether there was a relationship between the severity of pulmonary tuberculosis (TB) and lymphocyte proliferation as well as in vitro cytokine production. To achieve this, 25 untreated TB patients showing mild (n = 5), moderate (n = 9) or advanced (n = 11) pulmonary disease, and 12 age-matched healthy controls (mean+/-SD, 37+/-14.5 years) were studied. Peripheral blood mononuclear cells were cultured for 5 days with 10 microg/ml whole, sonicated Mycobacterium tuberculosis (WSA) or 2.5 microg/ml Concanavalin A (Con A). Supernatants were collected on day 4, from cultures grown with or without WSA, for measurement of interferon-gamma (IFN-gamma), interleukin (IL)-4, IL-1beta and transforming growth factor-beta (TGF-beta). Antigen-specific proliferation was found to be reduced among patients and more profound in those with advanced disease who also displayed a depressed response to Con A. Patients with mild TB showed a preferential production of IFN-gamma over IL-4, gave the highest level of IFN-gamma synthesis upon specific antigen stimulation and showed increased levels of IL-1beta production. Findings in patients with moderate TB appeared compatible with a mixed production of IFN-gamma and IL-4 coexisting with a higher synthesis of TGF-beta, by comparison to patients with mild TB. Advanced disease showed the highest IL-4 and TGF-beta production, with IFN-gamma synthesis readily noticeable, yet decreased in comparison with the other patient groups. Differences in cytokine response according to the amount of lung involvement suggest a role for such mediators in the immunopathogenesis underlying the distinct clinical forms of pulmonary TB, that is a predominant T helper Th)1-like or Th2-like activity in mild or in progressive TB, respectively.

Circulating profile of Th1 and Th2 cytokines in tuberculosis patients with different degrees of pulmonary involvement.

To investigate whether differences in the degree of pulmonary tuberculosis lesions could be accompanied by changes in the pattern of circulating cytokines, 29 untreated tuberculosis patients showing mild (n = 10), moderate (n = 5) or advanced (n = 14) pulmonary disease, and 12 age-matched healthy controls (mean +/- S.D., 36 +/- 15 years) were studied. ELISA methods for the evaluation of interferon-gamma, interleukin-2, interleukin-4, and interleukin-10 indicated that all patients had increased serum levels of the four cytokines in relation to controls. Mean titers of interferon-gamma and interleukin-2 in mild and moderate patients appeared higher than in those with advanced disease, whereas moderate and advanced patients showed the higher levels of IL-4 in comparison to mild cases. Raised levels of interleukin-10 were more prevalent in advanced disease, and statistically different from those in mild patients. This cytokine pattern may help to explain findings wherein mild tuberculosis is characterized by preserved cellular immune responses while advanced disease is accompanied by an impairment of such parameters.

Levels of interleukin-8 in tuberculous pleurisy and the profile of immunocompetent cells in pleural and peripheral compartments.

Our study investigated the presence of IL-8 in pleural exudates from tuberculosis patients (TBP) (n = 13), and evaluated whether it was related with the profile of major immunocompetent cells present in their pleural and peripheral compartments. To allow comparisons, an additional group of patients with parapneumonic pleural effusions (PNE) (n = 7) was included. Blood peripheral immunophenotypic studies were also carried out in 12 age-matched healthy controls (Co), and 39 tuberculosis patients classified, according to the extent of pulmonary involvement, into mild (n = 9), and advanced (n = 30) cases. Patients were recruited before starting therapy, had HIV negative serology, and showed no age differences among groups (mean +/- SD., 40.7 +/- 14.7 years). IL-8 concentrations were measured by an ELISA method while immunophenotypic analysis was performed by using FITC-conjugated monoclonal antibodies reacting against the following cell surface molecules: CD3, CD4, CD8, CD25 (IL-2R+ cells), CD19, and CD68. IL-8 was detected in all pleural exudates though levels in the TB patients, 384 +/- 110 pg/ml, appeared significantly higher than the PNE group, 185 +/- 110 pg/mg, (P < 0.015, mean +/- S.D.). In turn, the former group presented values of pleural CD3+, CD4+, and CD25, which were found increased in comparison with PNE patients (P < 0.01). Unlike the pleural compartment, patients with TBP showed a marked and significant decrease in their circulating levels of cells bearing the CD3, CD4, CD19, CD25, and CD68 phenotypes not only when comparing with Co but also with PNE and mild patients. Differences between the levels of pleural and peripheral T-cells from TBP patients may be the reflection of an important influx of T-lymphocytes from the circulatory system to the pleural cavity, probably linked to the presence of chemotactic factors within the pleural fluid like IL-8.