The Biochemistry of Sensing: Enteric Pathogens Regulate Type III Secretion in Response to Environmental and Host Cues

Abstract

Enteric pathogens employ sophisticated strategies to colonize and infect mammalian hosts. Gram-negative bacteria, such as Escherichia coli, Salmonella, and Campylobacter jejuni, are among the leading causes of gastrointestinal tract infections worldwide. The virulence strategies of many of these Gram-negative pathogens rely on type III secretion systems (T3SSs), which are macromolecular syringes that translocate bacterial effector proteins directly into the host cytosol. However, synthesis of T3SS proteins comes at a cost to the bacterium in terms of growth rate and fitness, both in the environment and within the host. Therefore, expression of the T3SS must be tightly regulated to occur at the appropriate time and place during infection. Enteric pathogens have thus evolved regulatory mechanisms to control expression of their T3SSs in response to specific environmental and host cues. These regulatory cascades integrate multiple physical and chemical signals through complex transcriptional networks. Although the power of bacterial genetics has allowed elucidation of many of these networks, the biochemical interactions between signal and sensor that initiate the signaling cascade are often poorly understood. Here, we review the physical and chemical signals that Gram-negative enteric pathogens use to regulate T3SS expression during infection. We highlight the recent structural and functional studies that have elucidated the biochemical properties governing both the interaction between sensor and signal and the mechanisms of signal transduction from sensor to downstream transcriptional networks.

Keywords: T3SS; cell signaling; enteric pathogens; environmental cues; nutritional stress; pathogenesis; surface sensing.

FIG 1

Thermosensing by intergenic RNAT and thermolabile protein YmoA in Y. pseudotuberculosis. (A) At moderate temperatures (25°C), the transcription of the yscW-lcrF operon is partially repressed by YmoA homodimers and/or YmoA–H-NS heterodimers. Translation is fully repressed by a two-stem-loop structure in the 5′-UTR of the lcrF mRNA, the RNAT. (B) At 37°C, the thermolabile protein YmoA is rapidly degraded by ClpP and Lon proteases, and this lifts the transcriptional repression of lcrF. High temperatures also melt the inhibitory two-stem-loop structure in the lcrF mRNA, allowing for enhanced translation of LcrF, the transcriptional activator of the pYV-encoded T3SS genes.

FIG 2

Regulation of the LEE-encoded T3SS by hormone and nutritional sensing in EHEC. (A) The histidine sensor kinase QseC binds host hormones Epi and NE as well as the microbiome-produced AI-3. Upon ligand binding, QseC autophosphorylates and transfers phosphate to the RRs QseB, QseF, and KdpE. P-KdpE activates expression of the LEE-encoded T3SS through Ler. P-QseB modulates flagellar operons, while P-QseF induces expression of the non-LEE T3SS effector EspFU and activates the SOS response, which in turn activates stx2 expression. QseF is also phosphorylated by the sensor HK QseE in response to Epi, SO₄²⁻, and PO₄³⁻. (B) Within the intestinal lumen, EHEC senses fucose produced by B. thetaiotaomicron through the HK/RR pair FusKR, which represses T3SS expression when fucose is present. To reach the intestinal epithelium, EHEC produces mucinases that obliterate the mucosal layer and create a gluconeogenic environment. B. thetaiotaomicron then switches to gluconeogenic metabolism and secretes large amounts of succinate, which EHEC senses through the transcriptional regulator Cra. Sensing of succinate by Cra induces T3SS expression and AE lesion formation. The gluconeogenic environment also triggers EHEC’s stringent response, during which the alarmone ppGpp is synthesized. ppGpp directly binds RNA polymerase and modulates its activity, such that LEE expression is upregulated.

FIG 3

SP1-1 and SPI-2 T3SS regulation by CAMPs in Salmonella. (A) CAMPs are sensed by the PhoQ HK. When CAMPs are not present, divalent cations (gray spheres) bridge the acidic patch (yellow) of the PhoQ PD and the negatively charged IM phosphate groups, tethering the PhoQ PD to the membrane and locking it in a repressed state. When CAMPs are present, they compete for binding to the PhoQ PD acidic patch and displace the bound divalent cations, which frees the PhoQ PD from its membrane-locked state and induces a conformational change that activates PhoQ HK activity. The activated HK domain of PhoQ then phosphorylates the RR PhoP, which in turn induces SPI-2 expression and represses SPI-1 expression. (B) The crystal structure of the S. Typhimurium PhoQ PD dimer in a divalent cation-bound (gray spheres) state (PDB ID 1YAX). The N- and C-terminal TM helices, HAMP domain, and HK domain are represented as rectangles. Acidic patch residues (yellow sticks) coordinate cations (gray spheres) at the IM outer leaflet interface.

FIG 4

Long- and short-chain fatty acids regulate SPI-1 expression in Salmonella. The SCFAs acetate and formate activate BarA HK activity and phosphotransfer to the SirA RR. The acetate metabolite, acetyl-P, can also directly phosphorylate SirA independently of BarA HK activity. P-SirA induces expression of CsrB and CsrC RNAs, which sequester the CsrA protein, thereby alleviating CsrA-mediated repression of hilD translation. The HilD protein then induces expression of the regulators HilC and RtsA, which through a feed-forward loop further amplify HilD expression. HilD, HilC, and RtsA also activate expression of HilA, which in turn induces expression of SPI-1 T3SS genes. Conversely, both LCFAs and proprionate negatively repress HilD activity. LCFAs bind to HilD and reduce its affinity for target DNA, including the hilA promoter. The propionate metabolite propionyl-CoA destabilizes the HilD protein, thereby inhibiting its transcription factor activity and activation of SPI-1 expression.

FIG 5

Iron sensing and SPI-1 regulation by Fur in Salmonella. (A) Under iron-limiting conditions, apo-Fur cannot bind the Fur boxes upstream of the hilD or hns promoters and does not activate or repress expression of HilD or H-NS, respectively. H-NS then blocks transcription of hilA, the primary transcriptional activator of the SPI-1 T3SS. (B) Under iron-rich conditions (e.g., within the intestinal lumen), Fur binds free iron (Fe²⁺), inducing a conformational change that enables binding to Fur boxes upstream of hilD and hns. DNA-bound Fur then represses H-NS expression and activates expression of HilD, a transcriptional activator of hilA, resulting in HilA expression and downstream SPI-1 T3SS expression. (C) Crystal structures of M. gryphiswaldense MSR-1 apo, holo, and DNA-bound Fur. Binding of divalent cations (Mn²⁺, Fe²⁺) induces hinge-like movement of the DNA-binding domains of each monomer, creating a two-fold rotational axis within the holo-dimer and promoting DNA binding (PDB IDs 4RAY, 4RAZ, and 4RB2).

FIG 6

Bile salt sensing and regulation of the Vp-PAI by VtrABC. The V. parahaemolyticus IM proteins VtrA and VtrC form a complex through interactions between their PDs. As V. parahaemolyticus enters the small intestine, bile salts bind to the VtrA-VtrC complex and activate the DBD of VtrA to induce vtrB transcription. VtrB then directly activates the transcription of T3SS2 and other Vp-PAI genes. (B) The crystal structure of the VtrA (purple) and VtrC (teal) PD complex bound to the bile salt taurodeoxycholate (green sticks) (PDB ID 5KEW). TM helices of both VtrA and VtrC are represented as rectangles, as is the DBD of VtrA. An overlay of the bile salt-bound (teal) and apo (yellow) structures illustrates the conformational change that results from bile salt binding (inset).

FIG 7

Signals that regulate T3S in the mammalian GI tract. Spatial depiction of the signals that govern T3S in the small and large intestine. Certain signals, like bile salts and iron, are known to be constrained to a specific compartment, based on mammalian physiology, while others are depicted in the compartment where they are postulated to be at the greatest concentration and play the largest role in T3S regulation. Pathogens are listed based on their known major site of colonization within mammalian hosts. Although many host signals and enteric pathogens exist, this figure illustrates only the signals and specific pathogens discussed in this review.