ppGpp conjures bacterial virulence

Abstract

Like for all microbes, the goal of every pathogen is to survive and replicate. However, to overcome the formidable defenses of their hosts, pathogens are also endowed with traits commonly associated with virulence, such as surface attachment, cell or tissue invasion, and transmission. Numerous pathogens couple their specific virulence pathways with more general adaptations, like stress resistance, by integrating dedicated regulators with global signaling networks. In particular, many of nature's most dreaded bacteria rely on nucleotide alarmones to cue metabolic disturbances and coordinate survival and virulence programs. Here we discuss how components of the stringent response contribute to the virulence of a wide variety of pathogenic bacteria.

FIG. 1.

Domain structure of the enzymes that modulate bacterial pools of ppGpp. Four functional regions have been identified: the ppGpp synthetase domain, the ppGpp hydrolase domain, and the TGS and ACT regulatory domains. The bifunctional SpoT and RSH (RelA/SpoT homologue) proteins contain both synthetase and hydrolase activities in an N-terminal enzymatic domain. RelA proteins behave as monofunctional synthetases. The RelA and SpoT proteins of E. coli share 31% amino acid identity, and amino acid divergence renders the hydrolase domain inactive (*). The activity of the SpoT/RSH and RelA proteins is controlled through their TGS and ACT domains. RelP, RelQ, and RelV, referred to as small alarmone synthases (SASs), are monofunctional enzymes with little similarity to RelA or each other at the amino acid sequence level. For example, the S. mutans RelP and RelQ proteins share 8% identity with the synthetase domain of E. coli RelA and 29% identity with each other (109).

FIG. 2.

SpoT activity is regulated through ACP interactions. Gammaproteobacteria that encode both RelA and SpoT have evolved a SpoT-dependent stringent response to fatty acid starvation that is mediated by an interaction between SpoT and ACP. ACP transfers fatty acyl chains to enzymes devoted to phospholipid and secondary metabolite biosynthesis. SpoT interacts with functional acyl-bound ACP at a nonenzymatic region known as the TGS domain. During fatty acid starvation, metabolic signals are transduced through an ACP-SpoT interaction, resulting in an increase in cellular ppGpp pools. It remains to be determined whether, in response to fatty acid stress, the ACP-SpoT interaction specifically modulates the synthetase (SD) or hydrolase (HD) activity of SpoT.

FIG. 3.

ppGpp and DksA control transcription directly. In response to stress, gammaproteobacteria use ppGpp and DksA to control RNAP activity at particular promoters. Although DksA is known to bind at the secondary channel of RNAP, a binding site for ppGpp has not been confirmed. In the presence of DksA and elevated ppGpp levels, transcription can be either activated or deactivated. Whether transcription is stimulated or repressed depends upon intrinsic properties of the promoter. Activated targets such as the E. coli promoter for the histidine biosynthetic (his) operon typically have an AT-rich DNA sequence between the −10 hexamer and the +1 transcriptional start site, known as the discriminator region. Conversely, repressed targets such as the P1 promoter of rRNA (rrn) operons typically have a GC-rich discriminator sequence. Promoters controlled directly by ppGpp and DksA generally depend on the housekeeping/vegetative sigma factor σ⁷⁰.

FIG. 4.

ppGpp and DksA control transcription indirectly through “sigma factor competition.” During exponential growth or favorable conditions, ppGpp levels are low in gammaproteobacteria, and transcription from strong σ⁷⁰-dependent promoters such as those of rRNA operons is robust. As bacteria exit the exponential phase, or during high stress, ppGpp levels accumulate. DksA, whose levels remain constant during growth, cooperates with ppGpp to repress the transcription of rRNA operons, liberating RNAP to bind alternative sigma factors (σ^S, σ⁵⁴, σ²⁸, σ³², and σ^E). As a result, transcription from promoters targeted by these sigma factors increases, inducing specialized stress responses. In this manner, ppGpp and DksA contribute indirectly to bacterial adaptation.

FIG. 5.

ppGpp controls dimerization and DNA binding of SlyA. In addition to controlling RNAP activity, ppGpp can directly control transcriptional activators. After phagocytosis, S. enterica serovar Typhimurium encounters an acidic, magnesium-limited environment with high levels of cationic antimicrobial peptides (CAMP), activating the PhoP/PhoQ two-component system. The PhoP response regulator acts with the transcriptional activator SlyA in a feed-forward loop. PhoP itself activates the transcription of the slyA gene; together, the PhoP and SlyA proteins control the expression of SPI2 genes, which are critical for intracellular survival and replication. SPI2 gene expression is intimately linked to ppGpp signaling, as the alarmone facilitates dimerization and DNA binding of SlyA, facilitating the SlyA- and PhoP-dependent activation of several divergent operons that promote the pathogen's intracellular virulence program, including the SPI2 genes pagC and pagD.

FIG. 6.

SpoT governs the Legionella life cycle in macrophages. During its life cycle, the intracellular pathogen L. pneumophila differentiates between two forms, replicative and transmissive. When nutrients become scarce, ppGpp levels increase, coordinating the differentiation of replicative bacteria to the highly resilient, motile, transmissive form. After phagocytosis, transmissive bacteria inhibit fusion with degradative lysosomes (small, dashed, empty vacuole). To convert to the replicative form, L. pneumophila must sense favorable vacuolar conditions that stimulate the bifunctional SpoT enzyme to reduce alarmone pools via ppGpp hydrolysis (HD). In a vacuole derived from the ER, replicative L. pneumophila cells divide exponentially. Gradually, the replication vacuole acidifies and acquires lysosomal markers. Deteriorating vacuolar conditions elicit SpoT synthetase (SD) activity, cueing replicative bacteria to differentiate back to the transmissive form. Transmissive L. pneumophila cells resist lysosomal degradation and migrate to a naïve host cell, primed to establish a new infection.

FIG. 7.

rel_Mtb expression is induced in response to carbon stress. In addition to controlling the activity of ppGpp synthetases, some bacteria regulate the transcription of the gene that encodes this enzyme. In response to carbon limitation, M. smegmatis and M. tuberculosis accumulate polyphosphate. This high-energy molecule donates phosphate to the MprA/MprB two-component system. MprA/MprB and the alternative sigma factor σ^E comprise a positive-feedback loop: MprA/MprB activates the transcription of the sigE gene; in return, the σ^E protein controls the activation of the mprA mprB locus. Together, MprA/MprB and σ^E activate the transcription of rel_Mtb, thereby increasing levels of ppGpp in the cell. This positive-feedback mechanism amplifies the response to carbon limitation, leading to a robust activation of the stringent response.