Stress Relief during Host Infection: The Phage Shock Protein Response Supports Bacterial Virulence in Various Ways

Inducible extracytoplasmic stress responses (ESRs) help to maintain the integrity and function of the bacterial cell envelope in unfavorable conditions. ESRs can also have highly specialized functions linked to virulence-associated systems directly. One of the most intriguing and yet enigmatic examples is the widely conserved phage shock protein (Psp) response [1]–[4]. This article outlines the significance of envelope stress and the roles of the Psp response in supporting bacterial virulence. This particular ESR might be critical for different reasons in different bacteria, with implications for both extracellular and intracellular pathogenesis, as well as processes that include antibiotic resistance and biofilm formation.


The Phage Shock Protein (Psp) Response
The ''phage shock'' name comes from the founding discovery that filamentous phage f1 infection massively induces the synthesis of an E. coli protein, which was named phage shock protein A (PspA) [15]. In E. coli, PspA is encoded by the pspABCDE operon, which is positively controlled by the transcription factor PspF ( Figure 1). Filamentous phage infection induces pspA operon expression because a phage-encoded outer membrane poreforming ''secretin'' protein (pIV) has a tendency to mislocalize within the E. coli envelope [3]. The Psp response is also induced by environmental shocks that can negatively affect the cell envelope. This, together with various other observations, has led to a hypothesis that the Psp system responds to, and helps mitigate, some aspect of cytoplasmic membrane perturbation [1][2][3][4].
Psp protein homologues, especially PspA, are present in Gramnegative and Gram-positive bacteria, as well as archaea and plants (reviewed in [16]). However, the Psp response has been studied most in the Gram-negative Enterobacteriaceae E. coli, Yersinia enterocolitica, and Salmonella enterica serovar Typhimurium (S. Typhimurium), where only PspF, -A, -B, and -C have been associated with robust phenotypes. One of their well-characterized functions is to autoregulate psp gene expression. This is achieved by PspA changing from an inhibitory interaction with PspF in the cytoplasm of uninduced cells, to a complex with the cytoplasmic membrane proteins PspB and -C of induced cells ( Figure 1) [17,18]. However, once pspA operon expression is induced, PspA, -B, and -C have additional roles in mitigating envelope stress. PspA has been focused upon due to its abundance, as well as experiments suggesting that it maintains the proton motive force (PMF) by preventing proton leakage across a damaged cytoplasmic membrane [19,20].

The Psp Response Supports Virulence by Preventing T3SS-Induced Envelope Stress in the Extracellular Pathogen Yersinia enterocolitica
The Y. enterocolitica Psp system is essential for virulence because of a specific connection to the Ysc-Yop T3SS. A pspC null mutant The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The author has declared that no competing interests exist.
* E-mail: andrew.darwin@med.nyu.edu is avirulent in a mouse model of infection, and grows slower than the wild type in conditions that induce production of the Ysc-Yop system [21,22]. This growth inhibition is caused by the outer membrane pore-forming component of the T3SS, YscC [22]. Like the phage pIV protein that induces the E. coli Psp system, YscC is a secretin. Indeed, when the Y. enterocolitica Psp system is intact, production of the native Ysc-Yop T3SS, or of only YscC, induces pspA operon expression [22]. Therefore, the Psp system is induced by production of the T3SS, and is then essential to mitigate a growth-inhibiting stress that its YscC secretin component can cause. The consequence is that a psp null mutant essentially kills itself during host infection by producing the Ysc-Yop T3SS to Figure 1. The Psp response and its involvement in various virulence-assocaied processes. In uninduced cells, PspA forms an inhibitory complex with the transcription factor PspF in the cytoplasm. An inducing trigger, such as the mislocalization of a pore-forming secretin protein, causes PspA to relocate to the cytoplasmic membrane, perhaps both in complex with PspBC and by making direct membrane contact. PspF induces pspA operon expression, leading to increased concentrations of PspA, -B, and -C that play roles in stress mitigation. PspA is believed to prevent proton (H + ) leakage across the cytoplasmic membrane and maintain the proton motive force. This is thought to support S. Typhimurium virulence in Nramp1-positive mice by ensurng an energy supply for metal ion (Me 2+ ) importers. PspB and -C prevent secretins from causing lethal cytoplasmic membrane permeability, which supports the T3SS-dependent virulence of Y. enterocolitica. The Psp response has also been shown to be required for the formation of biofilms and antibiotic-resistant (Ab R ) persister cells in E. coli, although the Psp protein(s) involved has not yet been reported. doi:10.1371/journal.ppat.1003388.g001 evade the immune response, but being unable to survive the stress that T3SS production causes.
Secretins have the unique ability to insert into either membrane of Gram-negative bacteria [23]. It is their mislocalization into the cytoplasmic membrane that induces psp gene expression and kills a psp null strain. Therefore, the toxicity of native T3SS production to a psp null strain suggests that endogenously produced YscC can mislocalize [22]. The cell death results from severe cytoplasmic membrane permeability, thought to be caused by the multimeric secretin channel (Figure 1) [24]. Remarkably, only the small membrane proteins PspB and PspC are required to prevent this toxicity from occurring, although it is not yet known how they do it [24]. Whatever the mechanism, the relationship between the Psp system and secretin mislocalization is highly specific. Secretin production induces psp gene expression without having much effect on any other genes in Y. enterocolitica, E. coli, or S. Typhimurium [25,26]. It is surprising that PspA, the most abundant Psp protein, linked with maintenance of the PMF in E. coli, is not required to prevent secretin toxicity [24]. However, this does not mean that PspA is without an important role to play, or that it is always dispensable for virulence, as explained below.

The Psp Response Supports Virulence by Maintaining the Proton Motive Force in the Intracellular Pathogen S. Typhimurium
Attention was drawn to the S. Typhimurium Psp system when it was found to be induced upon inactivation of RpoE [27]. Experiments suggested that PspA was compensating for the absence of the RpoE ESR by preventing a large drop in the PMF. The Psp system is also essential for S. Typhimurium virulence, but the explanation suggests a very different role from that in Y. enterocolitica [28]. S. Typhimurium is an intracellular pathogen that survives inside macrophages within a modified phagosome or Salmonella-containing vacuole (SCV; Figure 1). As a defense mechanism, the host uses the Nramp1 transporter to deplete phagosomes of divalent cations needed by bacteria. However, S. Typhimurium has energy-dependent metal ion importers to counter Nramp1. In Nramp1-positive mice an S. Typhimurium DpspA in-frame deletion mutant is severely attenuated [28]. The proposed explanation lies in the role of PspA in maintaining the PMF and so ensuring the energy to drive the metal ion importers. Consistent with this, in Nramp1-negative mice the DpspA mutation does not affect virulence [28]. Similarly, a DpspA in-frame deletion mutation has only a small effect on virulence of the extracellular pathogen Y. enterocolitica [22].
If PspA maintains the PMF of S. Typhimurium inside macrophages it could also be important for reasons beyond supplying energy to cation pumps. Lee and Groisman showed recently that the acidic pH inside the SCV drives increased ATP synthesis and elevates cytosolic ATP levels in S. Typhimurium [29]. The leader mRNA of the mgtCBR operon senses this elevated ATP concentration, inducing its expression and the production of proteins required for survival inside macrophages. If the absence of PspA decreases S. Typhimurium PMF inside macrophages it might also reduce PMF-driven ATP synthesis. This raises the intriguing but untested possibility that PspA might be important to ensure the induction of virulence genes such as mgtCBR.

Other Links between the Psp Response and Virulence-Associated Processes
Observations suggest additional medically relevant roles for the Psp response ( Figure 1). First, the Shigella flexneri pspA operon is highly induced during macrophage infection [30]. This suggests the possibility of a role for the Psp system in other intracellular pathogens, even though S. flexneri behaves differently from S. Typhimurium by escaping into the host cell cytoplasm. Second, the Psp system is important for biofilm formation by E. coli K-12 [31]. If this extends to pathogens there could be a connection between the Psp system and biofilmmediated disease. Third, the Psp system has been linked to persisters in E. coli [32]. Persisters are dormant antibiotic-resistant cells implicated in chronic and recurrent infections. E. coli persisters can be induced by indole, a molecule produced upon entry into stationary phase. Indole also induces pspA operon expression, and a DpspBC mutant is defective for indole-induced persister formation [32]. The mechanism has not yet been investigated. Nevertheless, this exciting finding reveals a role for the Psp system in a phenomenon thought to have broad clinical significance.
It is becoming apparent that the Psp response has various fingers in various pies related to bacterial virulence. It seems likely that this extends beyond the examples highlighted here. Thus, the Psp system might represent an Achilles' heel for many pathogens. The challenge is to elucidate the molecular mechanisms underlying Psp protein functions and so understand how they affect the function and regulation of so many diverse virulence-associated phenomena.