PEG10 is associated with treatment-induced neuroendocrine prostate cancer.

Neuroendocrine (NE) differentiation of advanced prostate adenocarcinoma following androgen receptor (AR) axis-directed therapy is becoming increasingly recognized. Several models of this transdifferentiation provide insight into its molecular pathogenesis and have highlighted the placental gene PEG10 for further study. Using our unique model of enzalutamide resistance (ENZR) and NE differentiation, we studied PEG10/AR interplay in enzalutamide treatment-resistant cell lines 42DENZR and 42FENZR compared to LNCaP and castration-resistant 16DCRPC cells. ENZR cell lines with positive terminal NE marker status also displayed higher baseline expression of PEG10 compared to LNCaP and 16DCRPC. Antagonism of AR activity increased PEG10 expression followed by an increase in terminal NE markers. Conversely, stimulating AR activity via androgen supplementation reversed PEG10 and NE marker expression in a time and dose-dependent manner. These results were supported by human data showing that PEG10 expression is highest in NEPC and that AR-dependent gene, PSA, is negatively correlated with PEG10 in adenocarcinoma. Further, ChIP assay confirmed binding of activated AR to the PEG10 enhancer, decreasing PEG10 expression. While PEG10 did not drive NEPC, its knockdown reduced NE markers in our cell lines. Moreover, PEG10 knockdown in vitro- and in vivo-attenuated tumor growth. Overall, these observations indicate that PEG10 is an AR-repressed gene which modulates NE markers in ENZR cells and targeting PEG10 in advanced prostate cancer with NE features is a rational and viable option.

Galiellalactone inhibits the STAT3/AR signaling axis and suppresses Enzalutamide-resistant Prostate Cancer.

Most prostate cancer patients will progress to a castration-resistant state (CRPC) after androgen ablation therapy and despite the development of new potent anti-androgens, like enzalutamide (ENZ), which prolong survival in CRPC, ENZ-resistance (ENZR) rapidly occurs. Re-activation of the androgen receptor (AR) is a major mechanism of resistance. Interrogating our in vivo derived ENZR model, we discovered that transcription factor STAT3 not only displayed increased nuclear localization but also bound to and facilitated AR activity. We observed increased STAT3 S727 phosphorylation in ENZR cells, which has been previously reported to facilitate AR binding. Strikingly, ENZR cells were more sensitive to inhibition with STAT3 DNA-binding inhibitor galiellalactone (GPA500) compared to CRPC cells. Treatment with GPA500 suppressed AR activity and significantly reduced expression of Cyclin D1, thus reducing cell cycle progression into S phase and hindering cell proliferation. In vivo, GPA500 reduced tumor volume and serum PSA in ENZR xenografts. Lastly, the combination of ENZ and GPA500 was additive in the inhibition of AR activity and proliferation in LNCaP and CRPC cells, providing rationale for combination therapy. Overall, these results suggest that STAT3 inhibition is a rational therapeutic approach for ENZR prostate cancer, and could be valuable in CRPC in combination with ENZ.

Effects of a compression garment on sensory feedback transmission in the human upper limb.

Compression apparel is popular in both medical and sport performance settings. Perceived benefits are suggested to include changes in sensory feedback transmission caused by activation of mechanoreceptors. However, little is known about effects of compression apparel on sensorimotor control. Our purpose was to mechanistically examine whether compression apparel modulates sensory feedback transmission and reaching accuracy in the upper limb. Two experiments were completed under CONTROL and COMPRESSION (sleeve applied across the elbow joint) conditions. M-waves and H-reflexes were elicited by stimulating the median nerve and were recorded via surface electromyography (EMG). In experiment 1, H-reflexes and M-H recruitment curves were assessed at REST, during wrist flexion (10% EMGmax), and during a cutaneous conditioning of the superficial radial (SR) or distal median (MED) nerve. Cutaneous reflexes were elicited during 10% wrist flexion via stimulation of SR or MED. In experiment 2, unconditioned H-reflex measures were assessed at rest, during arm cycling, and during a discrete reaching task. Results indicate that compression apparel modulates spinal cord excitability across multiple sensory pathways and movement tasks. Interestingly, there was a significant improvement in reaching accuracy while wearing the compression sleeve. Taken together, the compression sleeve appears to increase precision and sensitivity around the joint where the sleeve is applied. Compression apparel may function as a "filter" of irrelevant mechanoreceptor information allowing for optimal task-related sensory information to enhance proprioception. NEW & NOTEWORTHY Wearing a customized compression sleeve was shown to alter the excitability of multiple pathways within the central nervous system regardless of conditioning input or movement task and was accompanied by improved accuracy of reaching movements and determination of movement end point. Compression apparel may assist as a type of "filter function" of tonic and nonspecific mechanoreceptor information leading to increased precision and movement sensitivity around the joint where compression is applied.

Targeting Prostate Cancer Subtype 1 by Forkhead Box M1 Pathway Inhibition.

Purpose: Prostate cancer was recently classified to three clinically relevant subtypes (PCS) demarcated by unique pathway activation and clinical aggressiveness. In this preclinical study, we investigated molecular targets and therapeutics for PCS1, the most aggressive and lethal subtype, with no treatment options available in the clinic.Experimental Design: We utilized the PCS1 gene set and our model of enzalutamide (ENZR) castration-resistant prostate cancer (CRPC) to identify targetable pathways and inhibitors for PCS1. The findings were evaluated in vitro and in the ENZR CRPC xenograft model in vivoResults: The results revealed that ENZR CRPC cells are enriched with PCS1 signature and that Forkhead box M1 (FOXM1) pathway is the central driver of this subtype. Notably, we identified Monensin as a novel FOXM1-binding agent that selectively targets FOXM1 to reverse the PCS1 signature and its associated stem-like features and reduces the growth of ENZR CRPC cells and xenograft tumors.Conclusions: Our preclinical data indicate FOXM1 pathway as a master regulator of PCS1 tumors, namely in ENZR CRPC, and targeting FOXM1 reduces cell growth and stemness in ENZR CRPC in vitro and in vivo These preclinical results may guide clinical evaluation of targeting FOXM1 to eradicate highly aggressive and lethal PCS1 prostate cancer tumors. Clin Cancer Res; 23(22); 6923-33. ©2017 AACR.

The Master Neural Transcription Factor BRN2 Is an Androgen Receptor-Suppressed Driver of Neuroendocrine Differentiation in Prostate Cancer.

Mechanisms controlling the emergence of lethal neuroendocrine prostate cancer (NEPC), especially those that are consequences of treatment-induced suppression of the androgen receptor (AR), remain elusive. Using a unique model of AR pathway inhibitor-resistant prostate cancer, we identified AR-dependent control of the neural transcription factor BRN2 (encoded by POU3F2) as a major driver of NEPC and aggressive tumor growth, both in vitro and in vivo Mechanistic studies showed that AR directly suppresses BRN2 transcription, which is required for NEPC, and BRN2-dependent regulation of the NEPC marker SOX2. Underscoring its inverse correlation with classic AR activity in clinical samples, BRN2 expression was highest in NEPC tumors and was significantly increased in castration-resistant prostate cancer compared with adenocarcinoma, especially in patients with low serum PSA. These data reveal a novel mechanism of AR-dependent control of NEPC and suggest that targeting BRN2 is a strategy to treat or prevent neuroendocrine differentiation in prostate tumors. SIGNIFICANCE: Understanding the contribution of the AR to the emergence of highly lethal, drug-resistant NEPC is critical for better implementation of current standard-of-care therapies and novel drug design. Our first-in-field data underscore the consequences of potent AR inhibition in prostate tumors, revealing a novel mechanism of AR-dependent control of neuroendocrine differentiation, and uncover BRN2 as a potential therapeutic target to prevent emergence of NEPC. Cancer Discov; 7(1); 54-71. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1.

Molecular chaperone Hsp27 regulates the Hippo tumor suppressor pathway in cancer.

Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers.

Regulation of tumor cell plasticity by the androgen receptor in prostate cancer.

Prostate cancer (PCa) has become the most common form of cancer in men in the developed world, and it ranks second in cancer-related deaths. Men that succumb to PCa have a disease that is resistant to hormonal therapies that suppress androgen receptor (AR) signaling, which plays a central role in tumor development and progression. Although AR continues to be a clinically relevant therapeutic target in PCa, selection pressures imposed by androgen-deprivation therapies promote the emergence of heterogeneous cell populations within tumors that dictate the severity of disease. This cellular plasticity, which is induced by androgen deprivation, is the focus of this review. More specifically, we address the emergence of cancer stem-like cells, epithelial-mesenchymal or myeloid plasticity, and neuroendocrine transdifferentiation as well as evidence that demonstrates how each is regulated by the AR. Importantly, because all of these cell phenotypes are associated with aggressive PCa, we examine novel therapeutic approaches for targeting therapy-induced cellular plasticity as a way of preventing PCa progression.

Inhibition of the HER2-YB1-AR axis with Lapatinib synergistically enhances Enzalutamide anti-tumor efficacy in castration resistant prostate cancer.

Incurable castration-resistant prostate cancer (CRPC) is driven by androgen receptor (AR) activation. Potent therapies that prevent AR signaling, such as Enzalutamide (ENZ), are mainstay treatments for CRPC; however patients eventually progress with ENZ resistant (ENZR) disease. In this study, we investigated one mechanism of ENZ resistance, and tried to improve therapeutic efficiency of ENZ. We found HER2 expression is increased in ENZR tumors and cell lines, and is induced by ENZ treatment of LNCaP cells. ENZ-induced HER2 overexpression was dependent on AKT-YB1 activation and modulated AR activity. HER2 dependent AR activation in LNCaP and ENZR cells was effectively blocked by treatment with the EGFR/HER2 inhibitor Lapatinib, which reduced cell viability and increased apoptosis. Despite efficacy in vitro, in vivo monotherapy with Lapatinib did not prevent ENZR tumor growth. However, combination treatment of Lapatinib with ENZ most effectively induced cell death in LNCaP cells in vitro and was more effective than ENZ alone in preventing tumor growth in an in vivo model of CRPC. These results suggest that while HER2 overexpression and subsequent AR activation is a targetable mechanism of resistance to ENZ, therapy using Lapatinib is only a rational therapeutic approach when used in combination with ENZ in CRPC.

PD-L1 is highly expressed in Enzalutamide resistant prostate cancer.

Efficacy of Enzalutamide (ENZ) in castration resistant prostate cancer (CRPC) patients is short-lived. Immunotherapy like T cell checkpoint blockade may improve patient survival. However, when and where checkpoint molecules are expressed in CRPC and whether immune evasion is a mechanism of ENZ resistance remains unclear. Thus, we investigated whether clinically relevant immunotherapy targets, specifically PD-L1/2 , PD-1 and CTLA-4, are upregulated in ENZ resistant (ENZR) patients and in a pre-clinical model of ENZ resistance. We show for the first time that patients progressing on ENZ had significantly increased PD-L1/2+ dendritic cells (DC) in blood compared to those naïve or responding to treatment, and a high frequency of PD-1+T cells. These data supported our pre-clinical results, in which we found significantly increased circulating PD-L1/2+ DCs in mice bearing ENZR tumors compared to CRPC, and ENZR tumors expressed significantly increased levels of tumor-intrinsic PD-L1. Importantly, the expression of PD-L1 on ENZR cells, or the ability to modulate PD-L1/2+ DC frequency, was unique to ENZR cell lines and xenografts that did not show classical activation of the androgen receptor. Overall, our results suggest that ENZ resistance is associated with the strong expression of anti-PD-1 therapy targets in circulating immune cells both in patients and in a pre-clinical model that is non-AR driven. Further evaluation of the contribution of tumor vs. immune cell PD-L1 expression in progression of CRPC to anti-androgen resistance and the utility of monitoring circulating cell PD-L1 pathway activity in CRPC patients to predict responsiveness to checkpoint immunotherapy, is warranted.

Hsp27 regulates EGF/ß-catenin mediated epithelial to mesenchymal transition in prostate cancer.

Increased expression of the molecular chaperone Hsp27 is associated with the progression of prostate cancer (PCa) to castration-resistant disease, which is lethal due to metastatic spread of the prostate tumor. Metastasis requires epithelial to mesenchymal transition (EMT), which endows cancer cells with the ability to disseminate from the primary tumor and colonize new tissue sites. A wide variety of secreted factors promote EMT, and while overexpression and constitutive activation of epidermal growth factor (EGF) signaling is associated with poor prognosis of PCa, a precise role of EGF in PCa progression to metastasis remains unclear. Here, we show that Hsp27 is required for EGF-induced cell migration, invasion and MMPs activity as well as the expression of EMT markers including Fibronectin, Vimentin and Slug with concomitant decrease of E-cadherin. Mechanistically, we found that Hsp27 is required for EGF-induced AKT and GSK3ß phosphorylation and ß-catenin nuclear translocation. Moreover, silencing Hsp27 decreases EGF dependent phosphorylation of ß-catenin on tyrosine 142 and 654, enhances ß-catenin ubiquitination and degradation, prevents ß-catenin nuclear translocation and binding to the Slug promoter. These data suggest that Hsp27 is required for EGF-mediated EMT via modulation of the ß-catenin/Slug signaling pathway. Together, our findings underscore the importance of Hsp27 in EGF induced EMT in PCa and highlight the use of Hsp27 knockdown as a useful strategy for patients with advanced disease.

Beyond immune checkpoint blockade: new approaches to targeting host-tumor interactions in prostate cancer: report from the 2014 Coffey-Holden prostate cancer academy meeting.

INTRODUCTION: The 2014 Coffey-Holden Prostate Cancer Academy Meeting, held in La Jolla, CA from June 26 to 29, 2014, was themed: "Beyond Immune Checkpoint Blockade: New Approaches to Targeting Host-Tumor Interactions in Prostate Cancer." METHODS: Sponsored by the Prostate Cancer Foundation (PCF), this annual, invitation-only meeting is structured as an action-tank, and brought together 72 investigators with diverse academic backgrounds to discuss the most relevant topics in the fields of prostate cancer immunotherapy and the tumor microenvironment. RESULTS: The questions addressed at the meeting included: mechanisms underlying the successes and failures of prostate cancer immunotherapies, how to trigger an effective immune response against prostate cancer, the tumor microenvironment and its role in therapy resistance and tumor metastasis, clinically relevant prostate cancer mouse models, how host-tumor interactions affect current therapies and tumor phenotypes, application of principles of precision medicine to prostate cancer immunotherapy, optimizing immunotherapy clinical trial design, and complex multi-system interactions that affect prostate cancer and immune responses including the effects of obesity and the potential role of the host microbiome. DISCUSSION: This article highlights the most significant recent progress and unmet needs that were discussed at the meeting toward the goal of speeding the development of optimal immunotherapies for the treatment of prostate cancer.

Lyn deficiency leads to increased microbiota-dependent intestinal inflammation and susceptibility to enteric pathogens.

The Lyn tyrosine kinase governs the development and function of various immune cells, and its dysregulation has been linked to malignancy and autoimmunity. Using models of chemically induced colitis and enteric infection, we show that Lyn plays a critical role in regulating the intestinal microbiota and inflammatory responses as well as protection from enteric pathogens. Lyn(-/-) mice were highly susceptible to dextran sulfate sodium (DSS) colitis, characterized by significant wasting, rectal bleeding, colonic pathology, and enhanced barrier permeability. Increased DSS susceptibility in Lyn(-/-) mice required the presence of T but not B cells and correlated with dysbiosis and increased IFN-γ(+) and/or IL-17(+) colonic T cells. This dysbiosis was characterized by an expansion of segmented filamentous bacteria, associated with altered intestinal production of IL-22 and IgA, and was transmissible to wild-type mice, resulting in increased susceptibility to DSS. Lyn deficiency also resulted in an inability to control infection by the enteric pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Lyn(-/-) mice exhibited profound cecal inflammation, bacterial dissemination, and morbidity following S. Typhimurium challenge and greater colonic inflammation throughout the course of C. rodentium infection. These results identify Lyn as a key regulator of the mucosal immune system, governing pathophysiology in multiple models of intestinal disease.

The Multifaceted Roles of STAT3 Signaling in the Progression of Prostate Cancer.

The signal transducer and activator of transcription (STAT)3 governs essential functions of epithelial and hematopoietic cells that are often dysregulated in cancer. While the role for STAT3 in promoting the progression of many solid and hematopoietic malignancies is well established, this review will focus on the importance of STAT3 in prostate cancer progression to the incurable metastatic castration-resistant prostate cancer (mCRPC). Indeed, STAT3 integrates different signaling pathways involved in the reactivation of androgen receptor pathway, stem like cells and the epithelial to mesenchymal transition that drive progression to mCRPC. As equally important, STAT3 regulates interactions between tumor cells and the microenvironment as well as immune cell activation. This makes it a major factor in facilitating prostate cancer escape from detection of the immune response, promoting an immunosuppressive environment that allows growth and metastasis. Based on the multifaceted nature of STAT3 signaling in the progression to mCRPC, the promise of STAT3 as a therapeutic target to prevent prostate cancer progression and the variety of STAT3 inhibitors used in cancer therapies is discussed.

Hsp27 regulates epithelial mesenchymal transition, metastasis, and circulating tumor cells in prostate cancer.

Defining the mechanisms underlying metastatic progression of prostate cancer may lead to insights into how to decrease morbidity and mortality in this disease. An important determinant of metastasis is epithelial-to-mesenchymal transition (EMT), and the mechanisms that control the process of EMT in cancer cells are still emerging. Here, we report that the molecular chaperone Hsp27 (HSPB1) drives EMT in prostate cancer, whereas its attenuation reverses EMT and decreases cell migration, invasion, and matrix metalloproteinase activity. Mechanistically, silencing Hsp27 decreased IL-6-dependent STAT3 phosphorylation, nuclear translocation, and STAT3 binding to the Twist promoter, suggesting that Hsp27 is required for IL-6-mediated EMT via modulation of STAT3/Twist signaling. We observed a correlation between Hsp27 and Twist in patients with prostate cancer, with Hsp27 and Twist expression each elevated in high-grade prostate cancer tumors. Hsp27 inhibition by OGX-427, an antisense therapy currently in phase II trials, reduced tumor metastasis in a murine model of prostate cancer. More importantly, OGX-427 treatment decreased the number of circulating tumor cells in patients with metastatic castration-resistant prostate cancer in a phase I clinical trial. Overall, this study defines Hsp27 as a critical regulator of IL-6-dependent and IL-6-independent EMT, validating this chaperone as a therapeutic target to treat metastatic prostate cancer.

Glenoid inclination: in vivo measures in rotator cuff tear patients and associations with superior glenohumeral joint translation.

Glenoid inclination has been associated with rotator cuff tears and superior humeral translation, but the relationship between glenoid inclination and superior humeral translation has not been assessed in vivo. This study compared glenoid inclination between repaired and contralateral shoulders in 21 unilateral rotator cuff repair patients. As a secondary analysis, we assessed the relationship between glenoid inclination and in vivo superior humeral translation. Glenoid inclination was measured from patient-specific, computed tomography-based bone models. Glenohumeral joint motion was measured from biplane radiographs collected during coronal-plane abductions. Glenoid inclination was significantly lower for the rotator cuff tear shoulders (90.7 degrees ) than the asymptomatic, contralateral shoulders (92.3 degrees , P = .04). No significant correlation existed between increased glenoid inclination and superior-inferior translation of the uninjured shoulder (P > .30). This study failed to support the theory that glenoid inclination is responsible for superior humeral translation and the development of subacromial impingement.

Mechanisms of traumatic rupture of the aorta and associated peri-isthmic motion and deformation.

This study investigated the mechanisms of traumatic rupture of the aorta (TRA). Eight unembalmed human cadavers were tested using various dynamic blunt loading modes. Impacts were conducted using a 32-kg impactor with a 152-mm face, and high-speed seatbelt pretensioners. High-speed biplane x-ray was used to visualize aortic motion within the mediastinum, and to measure deformation of the aorta. An axillary thoracotomy approach was used to access the peri-isthmic region to place radiopaque markers on the aorta. The cadavers were inverted for testing. Clinically relevant TRA was observed in seven of the tests. Peak average longitudinal Lagrange strain was 0.644, with the average peak for all tests being 0.208 +/- 0.216. Peak intraluminal pressure of 165 kPa was recorded. Longitudinal stretch of the aorta was found to be a principal component of injury causation. Stretch of the aorta was generated by thoracic deformation, which is required for injury to occur. The presence of atherosclerosis was demonstrated to promote injury. The isthmus of the aorta moved dorsocranially during frontal impact and submarining loading modes. The aortic isthmus moved medially and anteriorly during impact to the left side. The results of this study provide a better understanding of the mechanisms associated with TRA, and can be used for the validation of finite element models developed for the examination and prediction of TRA.

The inositol phosphatase SHIP controls Salmonella enterica serovar Typhimurium infection in vivo.

The SH2 domain-containing inositol 5'-phosphatase, SHIP, negatively regulates various hematopoietic cell functions and is critical for maintaining immune homeostasis. However, whether SHIP plays a role in controlling bacterial infections in vivo remains unknown. Salmonella enterica causes human salmonellosis, a disease that ranges in severity from mild gastroenteritis to severe systemic illness, resulting in significant morbidity and mortality worldwide. The susceptibility of ship(+/+) and ship(-/-) mice and bone marrow-derived macrophages to S. enterica serovar Typhimurium infection was compared. ship(-/-) mice displayed an increased susceptibility to both oral and intraperitoneal serovar Typhimurium infection and had significantly higher bacterial loads in intestinal and systemic sites than ship(+/+) mice, indicating a role for SHIP in the gut-associated and systemic pathogenesis of serovar Typhimurium in vivo. Cytokine analysis of serum from orally infected mice showed that ship(-/-) mice produce lower levels of Th1 cytokines than do ship(+/+) animals at 2 days postinfection, and in vitro analysis of supernatants taken from infected bone marrow-derived macrophages derived to mimic the in vivo ship(-/-) alternatively activated (M2) macrophage phenotype correlated with these data. M2 macrophages were the predominant population in vivo in both oral and intraperitoneal infections, since tissue macrophages within the small intestine and peritoneal macrophages from ship(-/-) mice showed elevated levels of the M2 macrophage markers Ym1 and Arginase 1 compared to ship(+/+) cells. Based on these data, we propose that M2 macrophage skewing in ship(-/-) mice contributes to ineffective clearance of Salmonella in vivo.

SseL is a salmonella-specific translocated effector integrated into the SsrB-controlled salmonella pathogenicity island 2 type III secretion system.

Bacterial pathogens use horizontal gene transfer to acquire virulence factors that influence host colonization, alter virulence traits, and ultimately shape the outcome of disease following infection. One hallmark of the host-pathogen interaction is the prokaryotic type III secretion system that translocates virulence factors into host cells during infection. Salmonella enterica possesses two type III secretion systems that are utilized during host colonization and intracellular replication. Salmonella pathogenicity island 2 (SPI2) is a genomic island containing approximately 30 contiguous genes required to assemble a functional secretion system including the two-component regulatory system called SsrA-SsrB that positively regulates transcription of the secretion apparatus. We used transcriptional profiling with DNA microarrays to search for genes that coregulate with the SPI2 type III secretion machinery in an SsrB-dependent manner. Here we report the identification of a Salmonella-specific translocated effector called SseL that is required for full virulence during murine typhoid-like disease. Analysis of infected macrophages using fluorescence-activated cell sorting revealed that sseL is induced inside cells and requires SsrB for expression. SseL is retained predominantly in the cytoplasm of infected cells following translocation by the type III system encoded in SPI2. Animal infection experiments with sseL mutant bacteria indicate that integration of SseL into the SsrB response regulatory system contributes to systemic virulence of this pathogen.