Reciprocal sharing of extracellular proteases and extracellular matrix molecules facilitates Bacillus subtilis biofilm formation.

Extracellular proteases are a class of public good that support growth of Bacillus subtilis when nutrients are in a polymeric form. Bacillus subtilis biofilm matrix molecules are another class of public good that are needed for biofilm formation and are prone to exploitation. In this study, we investigated the role of extracellular proteases in B. subtilis biofilm formation and explored interactions between different public good producer strains across various conditions. We confirmed that extracellular proteases support biofilm formation even when glutamic acid provides a freely available nitrogen source. Removal of AprE from the NCIB 3610 secretome adversely affects colony biofilm architecture, while sole induction of WprA activity into an otherwise extracellular protease-free strain is sufficient to promote wrinkle development within the colony biofilm. We found that changing the nutrient source used to support growth affected B. subtilis biofilm structure, hydrophobicity and architecture. We propose that the different phenotypes observed may be due to increased protease dependency for growth when a polymorphic protein presents the sole nitrogen source. We however cannot exclude that the phenotypic changes are due to alternative matrix molecules being made. Co-culture of biofilm matrix and extracellular protease mutants can rescue biofilm structure, yet reliance on extracellular proteases for growth influences population coexistence dynamics. Our findings highlight the intricate interplay between these two classes of public goods, providing insights into microbial social dynamics during biofilm formation across different ecological niches.

Bacillus subtilis extracellular protease production incurs a context-dependent cost.

Microbes encounter a wide range of polymeric nutrient sources in various environmental settings, which require processing to facilitate growth. Bacillus subtilis, a bacterium found in the rhizosphere and broader soil environment, is highly adaptable and resilient due to its ability to utilise diverse sources of carbon and nitrogen. Here, we explore the role of extracellular proteases in supporting growth and assess the cost associated with their production. We provide evidence of the essentiality of extracellular proteases when B. subtilis is provided with an abundant, but polymeric nutrient source and demonstrate the extracellular proteases as a shared public good that can operate over a distance. We show that B. subtilis is subjected to a public good dilemma, specifically in the context of growth sustained by the digestion of a polymeric food source. Furthermore, using mathematical simulations, we uncover that this selectively enforced dilemma is driven by the relative cost of producing the public good. Collectively, our findings reveal how bacteria can survive in environments that vary in terms of immediate nutrient accessibility and the consequent impact on the population composition. These findings enhance our fundamental understanding of how bacteria respond to diverse environments, which has importance to contexts ranging from survival in the soil to infection and pathogenesis scenarios.

The majority of the matrix protein TapA is dispensable for Bacillus subtilis colony biofilm architecture.

Biofilm formation is a co-operative behaviour, where microbial cells become embedded in an extracellular matrix. This biomolecular matrix helps manifest the beneficial or detrimental outcome mediated by the collective of cells. Bacillus subtilis is an important bacterium for understanding the principles of biofilm formation. The protein components of the B. subtilis matrix include the secreted proteins BslA, which forms a hydrophobic coat over the biofilm, and TasA, which forms protease-resistant fibres needed for structuring. TapA is a secreted protein also needed for biofilm formation and helps in vivo TasA-fibre formation but is dispensable for in vitro TasA-fibre assembly. We show that TapA is subjected to proteolytic cleavage in the colony biofilm and that only the first 57 amino acids of the 253-amino acid protein are required for colony biofilm architecture. Through the construction of a strain which lacks all eight extracellular proteases, we show that proteolytic cleavage by these enzymes is not a prerequisite for TapA function. It remains unknown why TapA is synthesised at 253 amino acids when the first 57 are sufficient for colony biofilm structuring; the findings do not exclude the core conserved region of TapA having a second role beyond structuring the B. subtilis colony biofilm.

Formation of functional, non-amyloidogenic fibres by recombinant Bacillus subtilis TasA.

Bacterial biofilms are communities of microbial cells encased within a self-produced polymeric matrix. In the Bacillus subtilis biofilm matrix, the extracellular fibres of TasA are essential. Here, a recombinant expression system allows interrogation of TasA, revealing that monomeric and fibre forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibres form spontaneously, and share the biological activity of TasA fibres extracted from B. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophysical properties of both native and recombinant TasA fibres indicate that they are not functional amyloid-like fibres. A gel formed by TasA fibres can recover after physical shear force, suggesting that the biofilm matrix is not static and that these properties may enable B. subtilis to remodel its local environment in response to external cues. Using recombinant fibres formed by TasA orthologues we uncover species variability in the ability of heterologous fibres to cross-complement the B. subtilis tasA deletion. These findings are indicative of specificity in the biophysical requirements of the TasA fibres across different species and/or reflect the precise molecular interactions needed for biofilm matrix assembly.

Natural variations in the biofilm-associated protein BslA from the genus Bacillus.

BslA is a protein secreted by Bacillus subtilis which forms a hydrophobic film that coats the biofilm surface and renders it water-repellent. We have characterised three orthologues of BslA from Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus as well as a paralogue from B. subtilis called YweA. We find that the three orthologous proteins can substitute for BslA in B. subtilis and confer a degree of protection, whereas YweA cannot. The degree to which the proteins functionally substitute for native BslA correlates with their in vitro biophysical properties. Our results demonstrate the use of naturally-evolved variants to provide a framework for teasing out the molecular basis of interfacial self-assembly.

Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance.

Glucocorticosteroids are used as a main treatment to reduce airway inflammation in people with asthma who suffer from neutrophilic airway inflammation, a condition frequently associated with Haemophilus influenzae colonization. Here we show that glucocorticosteroids have a direct influence on the behavior of H. influenzae that may account for associated difficulties with therapy. Using a mouse model of infection, we show that corticosteroid treatment promotes H. influenzae persistence. Transcriptomic analysis of bacteria either isolated from infected mouse airway or grown in laboratory medium identified a number of genes encoding regulatory factors whose expression responded to the presence of glucocorticosteroids. Importantly, a number of these corticosteroid-responsive genes also showed elevated expression in H. influenzae within sputum from asthma patients undergoing steroid treatment. Addition of corticosteroid to H. influenzae led to alteration in biofilm formation and enhanced resistance to azithromycin, and promoted azithromycin resistance in an animal model of respiratory infection. Taken together, these data strongly suggest that H. influenzae can respond directly to corticosteroid treatment in the airway potentially influencing biofilm formation, persistence and the efficacy of antibiotic treatment.

The changing face of asthma and its relation with microbes.

During the past 50 years, the prevalence of asthma has increased and this has coincided with our changing relation with microorganisms. Asthma is a complex disease associated with local tissue inflammation of the airway that is determined by environmental, immunological, and host genetic factors. In a subgroup of sufferers, respiratory infections are associated with the development of chronic disease and more frequent inflammatory exacerbations. Recent studies suggest that these infections are polymicrobial in nature. Furthermore, there is increasing evidence that the recently discovered asthma airway microbiota may play a critical role in pathophysiological processes associated with the disease. Here, we discuss the current data regarding a possible role for infection in chronic asthma with a particular focus on the role bacteria may play. We discuss recent advances that are beginning to elucidate the complex relations between the microbiota and the immune response in asthma patients. We also highlight the clinical implications of these recent findings in regards to the development of novel therapeutic strategies.

Microglial activation with atypical proinflammatory cytokine expression in a rat model of Parkinson's disease.

Microglial activation has been associated with the pathogenesis of Parkinson's disease (PD). Among the many components of this reaction, cytokines have been proposed as candidates to mediate neurodegenerative or neuroprotective effects. We investigated the interleukin-1 system and tumour necrosis factor-alpha mRNA and protein levels at different time intervals in the subacute intrastriatal 6-hydroxydopamine rat model of PD, in parallel with the inflammatory response. Immunohistochemistry showed that microglial cells were activated from days 6-30 postlesion in the substantia nigra pars compacta. This microglial activation was accompanied by an atypical proinflammatory cytokine production: Interleukin-1alpha and beta mRNAs were found to be elevated 30 days post-6-hydroxydopamine injection (2- and 16-fold, respectively), but no induction for interleukin-1alpha or beta at the protein level was detected by ELISA. As a control, a classical proinflammatory stimulus, namely endotoxin, was capable of inducing these cytokines at similar mRNA levels but also at the protein level. In addition, tumour necrosis factor-alpha mRNA was hardly or not detected in the substantia nigra at any time point studied. Our data point out a tight control of key proinflammatory cytokine production in our model of PD. This work supports the notion that chronic neuronal death per se does not induce secretion of these proinflammatory cytokines but that an additional stimulus is necessary to stimulate proinflammatory cytokine production. The production of proinflammatory cytokines from "primed" microglia may in turn modulate disease progression as has been recently proposed in a model of prion disease.