The Ras-ERK (extracellular signal–regulated kinase) pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors for treating cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends upon multifunctional-autoprocessing repeats-in-toxin (MARTX), a toxin that releases bacterial effector domains into host cells. One of the effector domains delivered by this toxin is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance (NMR) analysis and nucleotide exchange assays, we determined that RRSP processing did not release any fragments of KRAS or cause KRAS to dissociate from its bound nucleotide, but instead only locally impacted the structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor (GEF)-mediated nucleotide exchange and RAF binding. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance to its target.

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are pore-forming bacterial toxins that translocate multiple functionally independent effector domains into a target eukaryotic cell. Vibrio cholerae colonizes intestinal epithelial cells (IECs) and uses a MARTX toxin with three effector domains—an actin cross-linking domain (ACD), a Rho inactivation domain (RID), and an α/β hydrolase domain (ABH)—to suppress innate immunity and enhance colonization. We investigated whether these multiple catalytic enzymes delivered from a single toxin functioned in a coordinated manner to suppress intestinal innate immunity. Using cultured human IECs, we demonstrated that ACD-induced cytoskeletal collapse activated extracellular signal–regulated kinase, p38, and c-Jun amino-terminal kinase mitogen-activated protein kinase (MAPK) signaling to elicit a robust proinflammatory response characterized by the secretion of interleukin-8 (IL-8; also called CXCL8) and the expression of CXCL8, tumor necrosis factor (TNF), and other proinflammatory genes. However, RID and ABH, which are naturally delivered together with ACD, blocked MAPK activation through Rac1 and thus prevented ACD-induced inflammation. RID also abolished IL-8 secretion induced by heat-killed bacteria, TNF, or latrunculin A. Thus, MARTX toxins use enzymatic multifunctionality to silence the host response to bacterial factors and to the damage caused by the toxins. Furthermore, these data show how V. cholerae MARTX toxin suppresses intestinal inflammation and contributes to cholera being classically defined as a noninflammatory diarrheal disease.

Bacteria often coordinate virulence factors to fine-tune the host response during infection. These coordinated events can include toxins counteracting or amplifying effects of another toxin or though regulating the stability of virulence factors to remove their function once it is no longer needed. Multifunctional autoprocessing repeats-in toxin (MARTX) toxins are effector delivery toxins that form a pore into the plasma membrane of a eukaryotic cell to deliver multiple effector proteins into the cytosol of the target cell. The function of these proteins includes manipulating actin cytoskeletal dynamics, regulating signal transduction pathways and inhibiting host secretory pathways. Investigations into the molecular mechanisms of these effector domains are providing insight into how the function of some effectors overlap and regulate one another during infection. Coordinated crosstalk of effector function suggests that MARTX toxins are not simply a sum of all their parts. Instead, modulation of cell function by effector domains may depend on which other effector domain are co-delivered. Future studies will elucidate how these effectors interact with each other to modulate the bacterial host interaction.

The etiology of chronic pelvic pain syndromes remains unknown. In a murine urinary tract infection (UTI) model, lipopolysaccharide of uropathogenic E. coli and its receptor TLR4 are required for post-UTI chronic pain development. However, downstream mechanisms of post-UTI chronic pelvic pain remain unclear. Because the TRPV1 and MCP-1/CCR2 pathways are implicated in chronic neuropathic pain, we explored their role in post-UTI chronic pain. Mice were infected with the E. coli strain SΦ874, known to produce chronic allodynia, and treated with the TRPV1 antagonist capsazepine. Mice treated with capsazepine at the time of SΦ874 infection failed to develop chronic allodynia, whereas capsazepine treatment of mice at two weeks following SΦ874 infection did not reduce chronic allodynia. TRPV1-deficient mice did not develop chronic allodynia either. Similar results were found using novelty-suppressed feeding (NSF) to assess depressive behavior associated with neuropathic pain. Imaging of reporter mice also revealed induction of MCP-1 and CCR2 expression in sacral dorsal root ganglia following SΦ874 infection. Treatment with a CCR2 receptor antagonist at two weeks post-infection reduced chronic allodynia. Taken together, these results suggest that TRPV1 has a role in the establishment of post-UTI chronic pain, and CCR2 has a role in maintenance of post-UTI chronic pain.

The host immune response is highly effective to detect and clear infecting bacterial pathogens. Given the elaborate surveillance systems of the host, it is evident that in order to productively infect a host, the bacteria often coordinate virulence factors to fine-tune the host response during infection. These coordinated events can include either suppressing or activating the signaling pathways that control the immune response and thereby promote bacterial colonization and infection. This review will cover the surveillance and signaling systems for detection of bacteria in the intestine and a sample of the toxins and effectors that have been characterized that cirumvent these signaling pathways. These factors that promote infection and disease progression have also been redirected as tools or therapeutics. Thus, these toxins are enemies deployed to enhance infection, but can also be redeployed as allies to enable research and protect against infection.

Vibrio vulnificus is an opportunistic pathogen that causes gastroenteritis and septicemia in humans. The V. vulnificus multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin is a pore-forming toxin that translocates multiple functionally independent effector domains into target cells and an essential virulence factor for fatal disease.

Interorganelle communication regulates cellular homeostasis through the formation of tightly-associated membrane contact sites 1,3. Prior work has identified several ways that intracellular pathogens alter contacts between eukaryotic membranes 4,6, but there is no existing evidence for contact sites spanning eukaryotic and prokaryotic membranes. Here, using a combination of live-cell microscopy and transmission and focused-ion-beam scanning electron microscopy, we demonstrate that the intracellular bacterial pathogenRickettsia parkeriforms a direct membrane contact site between its bacterial outer membrane and the rough endoplasmic reticulum (ER), with tethers that are approximately 55 nm apart. Depletion of the ER-specific tethers VAPA and VAPB reduced the frequency of rickettsia-ER contacts, suggesting these interactions mimic organelle-ER contacts. Overall, our findings illuminate a direct, interkingdom membrane contact site uniquely mediated by rickettsia that seems to mimic traditional host MCSs.

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are pore-forming toxins that translocate multiple functionally independent effector domains into a target eukaryotic cell. Vibrio cholerae colonizes intestinal epithelial cells (IECs) and utilizes a MARTX toxin with three effector domains -an actin cross-linking domain (ACD), a Rho inactivation domain (RID), and an a/b hydrolase domain (ABH) -to suppress innate immunity and enhance colonization. We investigated whether these multiple catalytic enzymes delivered from a single toxin function in a coordinated manner to regulate intestinal innate immunity. Using cultured IECs, we demonstrate that ACD-induced cytoskeletal collapse activated ERK, p38, and JNK mitogen-activated protein kinase (MAPK) signaling to elicit a robust proinflammatory response characterized by production of interleukin-8 (IL-8) and expression of CXCL8, TNF, and other proinflammatory genes. However, RID and ABH, which are naturally delivered along with ACD, blocked MAPK activation via Rac1 and thus prevented the ACD-induced inflammation. RID also abolished IL-8 secretion induced by heat-killed bacteria, tumor necrosis factor, and latrunculin A.Thus, MARTX toxins utilize enzymatic multifunctionality to silence the host response to bacterial factors and to the damage it causes. Further, these data show how V. cholerae MARTX toxin suppresses 2 intestinal inflammation and contributes to cholera being classically defined as non-inflammatory diarrheal disease.