The Prc and CtpA proteases modulate cell-surface signaling activity and virulence in Pseudomonas aeruginosa.

Cell-surface signaling (CSS) is a signal transfer system of Gram-negative bacteria that produces the activation of an extracytoplasmic function σ factor (σECF) in the cytosol in response to an extracellular signal. Activation requires the regulated and sequential proteolysis of the σECF-associated anti-σ factor, and the function of the Prc and RseP proteases. In this work, we have identified another protease that modulates CSS activity, namely the periplasmic carboxyl-terminal processing protease CtpA. CtpA functions upstream of Prc in the proteolytic cascade and seems to prevent the Prc-mediated proteolysis of the CSS anti-σ factor. Importantly, using zebrafish embryos and the A549 lung epithelial cell line as hosts, we show that mutants in the rseP and ctpA proteases of the human pathogen Pseudomonas aeruginosa are considerably attenuated in virulence while the prc mutation increases virulence likely by enhancing the production of membrane vesicles.

The extracytoplasmic function sigma factor σVreI is active during infection and contributes to phosphate starvation-induced virulence of Pseudomonas aeruginosa.

The extracytoplasmic function sigma factor σVreI of the human pathogen Pseudomonas aeruginosa promotes transcription of potential virulence determinants, including secretion systems and secreted proteins. Its activity is modulated by the VreR anti-σ factor that inhibits the binding of σVreI to the RNA polymerase in the absence of a (still unknown) inducing signal. The vreI-vreR genes are expressed under inorganic phosphate (Pi) starvation, a physiological condition often encountered in the host that increases P. aeruginosa pathogenicity. However, whether or not σVreI is active in vivo during infection and contributes to the Pi starvation-induced virulence of this pathogen has not been analyzed yet. Using zebrafish embryos and a human alveolar basal epithelial cell line as P. aeruginosa hosts, we demonstrate in this work that σVreI is active during infection and that lack of σVreI considerably reduces the Pi starvation-induced virulence of this pathogen. Surprisingly, lack of the σVreI inhibitor, the VreR anti-σ factor, also diminishes the virulence of P. aeruginosa. By transcriptomic analyses we show that VreR modulates gene expression not only in a σVreI-dependent but also in a σVreI-independent manner. This includes potential virulence determinants and transcriptional regulators that could be responsible for the reduced virulence of the ΔvreR mutant.

Pseudomonas aeruginosa possesses three distinct systems for sensing and using the host molecule haem.

Pathogens have developed several strategies to obtain iron during infection, including the use of iron-containing molecules from the host. Haem accounts for the vast majority of the iron pool in vertebrates and thus represents an important source of iron for pathogens. Using a proteomic approach, we have identified in this work a previously uncharacterized system, which we name Hxu, that together with the known Has and Phu systems, is used by the human pathogen Pseudomonas aeruginosa to respond to haem. We show that the Has and Hxu systems are functional signal transduction pathways of the cell-surface signalling class and report the mechanism triggering the activation of these signalling systems. Both signalling cascades involve an outer membrane receptor (HasR and HxuA respectively) that upon sensing haem in the extracellular medium produces the activation of an σECF factor in the cytosol. HxuA has a major role in signalling and a minor role in haem acquisition in conditions in which the HasR and PhuR receptors or other sources of iron are present. Remarkably, P. aeruginosa compensates the lack of the HasR receptor by increasing the production of HxuA, which underscores the importance of haem signalling for this pathogen.

Diversity of extracytoplasmic function sigma (σECF ) factor-dependent signaling in Pseudomonas.

Pseudomonas bacteria are widespread and are found in soil and water, as well as pathogens of both plants and animals. The ability of Pseudomonas to colonize many different environments is facilitated by the multiple signaling systems these bacteria contain that allow Pseudomonas to adapt to changing circumstances by generating specific responses. Among others, signaling through extracytoplasmic function σ (σECF ) factors is extensively present in Pseudomonas. σECF factors trigger expression of functions required under particular conditions in response to specific signals. This manuscript reviews the phylogeny and biological roles of σECF factors in Pseudomonas, and highlights the diversity of σECF -signaling pathways of this genus in terms of function and activation. We show that Pseudomonas σECF factors belong to 16 different phylogenetic groups. Most of them are included within the iron starvation group and are mainly involved in iron acquisition. The second most abundant group is formed by RpoE-like σECF factors, which regulate the responses to cell envelope stress. Other groups controlling solvent tolerance, biofilm formation and the response to oxidative stress, among other functions, are present in lower frequency. The role of σECF factors in the virulence of Pseudomonas pathogenic species is described.

The Activity of the Pseudomonas aeruginosa Virulence Regulator σ(VreI) Is Modulated by the Anti-σ Factor VreR and the Transcription Factor PhoB.

Gene regulation in bacteria is primarily controlled at the level of transcription initiation by modifying the affinity of the RNA polymerase (RNAP) for the promoter. This control often occurs through the substitution of the RNAP sigma (σ) subunit. Next to the primary σ factor, most bacteria contain a variable number of alternative σ factors of which the extracytoplasmic function group (σ(ECF)) is predominant. Pseudomonas aeruginosa contains nineteen σ(ECF), including the virulence regulator σ(VreI). σ(VreI) is encoded by the vreAIR operon, which also encodes a receptor-like protein (VreA) and an anti-σ factor (VreR). These three proteins form a signal transduction pathway known as PUMA3, which controls expression of P. aeruginosa virulence functions. Expression of the vreAIR operon occurs under inorganic phosphate (Pi) limitation and requires the PhoB transcription factor. Intriguingly, the genes of the σ(VreI) regulon are also expressed in low Pi despite the fact that the σ(VreI) repressor, the anti-σ factor VreR, is also produced in this condition. Here we show that although σ(VreI) is partially active under Pi starvation, maximal transcription of the σ(VreI) regulon genes requires the removal of VreR. This strongly suggests that an extra signal, probably host-derived, is required in vivo for full σ(VreI) activation. Furthermore, we demonstrate that the activity of σ(VreI) is modulated not only by VreR but also by the transcription factor PhoB. Presence of this regulator is an absolute requirement for σ(VreI) to complex the DNA and initiate transcription of the PUMA3 regulon. The potential DNA binding sites of these two proteins, which include a pho box and -10 and -35 elements, are proposed.

Processing of cell-surface signalling anti-sigma factors prior to signal recognition is a conserved autoproteolytic mechanism that produces two functional domains.

Cell-surface signalling (CSS) enables Gram-negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti-sigma factor in the cytoplasmic membrane, allowing the activation of an extracytoplasmic function (ECF) sigma factor. Recent studies have demonstrated that RseP-mediated proteolysis of the anti-sigma factors is key to σ(ECF) activation. Using the Pseudomonas aeruginosa FoxR anti-sigma factor, we show here that RseP is responsible for the generation of an N-terminal tail that likely contains pro-sigma activity. Furthermore, it has been reported previously that this anti-sigma factor is processed in two separate domains prior to signal recognition. Here, we demonstrate that this process is common in these types of proteins and that the processing event is probably due to autoproteolytic activity. The resulting domains interact and function together to transduce the CSS signal. However, our results also indicate that this processing event is not essential for activity. In fact, we have identified functional CSS anti-sigma factors that are not cleaved prior to signal perception. Together, our results indicate that CSS regulation can occur through both complete and initially processed anti-sigma factors.