Abstract Background Crush syndrome (consisting of hyperkalemia, acidosis, hypocalcemia, and acute kidney injury), is the second-most common cause of death in earthquakes, and a frequent cause of critical illness after burn, blast, and prolonged immobility. No specific treatment exists; supportive treatment is burdensome, contributing to deaths in austere environments, especially disasters and conflicts. There is urgent need for specific treatment which reduces burden of care. Crush syndrome is dependent on the renal megalin-dependent endocytic system. We investigated whether cilastatin sodium, a megalin inhibitor which is US Food and Drug Administration-approved for another purpose, has efficacy as a crush syndrome treatment in a highly translational large animal trauma model. Methods Anesthetized 40 kg female pigs received blunt muscle injury and 48 h protocolized critical care management. Cilastatin sodium or vehicle was administered 30 minutes after injury in randomized, blinded fashion. Renal function and injury were assessed by repeated quantification of iohexol clearance, serial plasma assessment, and histopathologic analysis. Linear mixed models and Kaplan-Meier analysis were used to assess differences. A power estimate for a clinical trial was performed. Results Here we show that cilastatin has efficacy to reduce kidney impairment from crush syndrome, resulting in increased measured glomerular filtration rate and reduced creatinine, histopathologic kidney damage, and need for treatment of hyperkalemia. Animals receiving cilastatin excrete more myoglobin in the urine and are more likely to recover from acute kidney injury. The effect size suggests feasibility of future clinical trials. Conclusions Cilastatin sodium has efficacy to ameliorate crush syndrome in a translational large animal model These results support further efforts to translate this potential therapy.

Background: In the United States over 10% of all neonates are born premature (less than 37 weeks gestational age), and many face complications, such as necrotizing enterocolitis (NEC). NEC is the most deadly gastrointestinal disease in preterm neonates with up to 50% mortality. Survivors face lifelong complications. NEC is a multifactorial disease caused in part by intestinal immaturity, including a poorly developed physical barrier between luminal contents and the rest of the body, leading to irreparable damage to the intestine. Since there is no cure for NEC, prevention is the best strategy. Enhancing our understanding of intestinal epithelial cell [IEC] barrier regulation during early development will provide novel therapeutic targets to prevent NEC. Published evidence indicates roles for the RNA-binding protein insulin-like growth factor 2 mRNA binding protein 1 (IMP1) in intestinal development, barrier function, and intestinal repair. Sequencing data from the inflamed tissue of a NEC patient indicates that IMP1 is down-regulated relative to uninflamed tissue. Notably, roles for IMP1 in NEC are not defined. Goblet cells produce protective mucus in the intestine, and their mature function is dependent on the transcription factor Spdef. Emerging evidence suggests that goblet cell mucus complexity impacts barrier function and inflammation susceptibility. This study aimed to define the role of IMP1 in NEC pathogenesis using a model of NEC-like intestinal injury in mice with IEC-specific Imp1 overexpression. Hypothesis: IMP1 expression is protective in a mouse model of NEC. Methods: All studies are OHSU IACUC-approved. This study used wildtype (WT, control) and Imp1 IEC-overexpression (VillinCre;lslFLAG-Imp1OE; Imp1IEC-OE) mice. At post-natal day 3, mice of both sexes were randomly assigned to control or NEC groups. The control mice remained with the dam. NEC was induced with the well-established experimental NEC-like intestinal injury model. Mice were separated from the dam and maintained in an incubator. Pups were gavage-fed a mixture of infant formula, puppy replacer, bacteria derived from a premature infant with NEC, and 2.5 ug/g body weight of lipopolysaccharide every 3 hours from 7:30 am-10 pm. Twice daily, the pups were exposed to hypoxia (5% oxygen). After 72 hours, tissue was collected for histology, gene, and protein expression analyses. Total RNA sequencing was performed using the NovaSeq S4. Goblet cells were evaluated by immunostaining. Statistical analyses were performed using 2-way ANOVA with Tukey’s test for multiple comparisons. Results: The NEC model induces pneumatosis, histological damage, and a significant up-regulation of inflammatory cytokine gene expression Lcn2 and Il1B, p< 0.05, n=30-31. Pathway analysis of RNA sequencing data revealed a unique and significant enrichment of Spdef transcriptional activity in Imp1IEC-OE during NEC. This included significant upregulation of Spdef target genes such as, Agr2 (in NEC WT: 617.8 +/- 33.56 vs Imp1IEC-OE: 812.9 +/- 111.3, p=0.02) and Fut2 (in NEC WT: 219.4 +/- 34 vs Imp1IEC-OE: 396.6 +/- 62.9, p=0.05). In silico analysis predicted Imp1 binding to Spdef mRNA. Although genotype did not affect goblet cell number, Imp1IEC-OE mice with NEC exhibited significant increases (p< 0.05) in genes responsible for goblet cell function (Spink4, Klk1, Tspg1) mucus glycosylation (Gcnt3, B3gnt7, Qsox1), and mucus secretion (Creb3l4, Ramp1, Chrm1). Conclusion: Our data indicate that during NEC, Imp1 may enhance goblet cell function via Spdef. Ongoing studies will examine mucus glycosylation and secretion, and assess Imp1 binding targets. Funding: NIH K01DK129401, R01HD097367, R03DK142841-01, the Gerber Foundation This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

BACKGROUND Hemorrhage accounts for the most preventable deaths after trauma. Resuscitation is guided by studies that demonstrate improved outcomes in patients receiving whole blood or balanced administration of blood products. Platelets present a logistical challenge due to short shelf life and need for refrigeration. Platelet-derived extracellular vesicles (PEVs) are a possible platelet alternative. Platelet-derived extracellular vesicles are secreted from platelets, have hemostatic effects and mitigate inflammation and vascular injury, similar to platelets. This pilot study aimed to elucidate the therapeutic effects of PEVs in a rat model of uncontrolled hemorrhage. METHODS Male rats were anesthetized and femoral vessels cannulated. Vital signs (MAP, HR, and RR) were monitored. Electrolytes, lactate and ABG were obtained at baseline, 1-hour and 3-hours post injury. Laparotomy was performed, 50% of the middle hepatic lobe excised and the abdomen packed with gauze. Rats received 2 mL PEVs or lactated Ringers (LR) over 6 minutes immediately after injury. Peritoneal blood loss was quantified using preweighed gauze at 5 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes. Laparotomy was closed 1-hour postinjury. Animals were monitored for 3 hours postinjury then euthanized. Generalized Linear Mixed Effects models were performed to assess effects of treatment and time on lactate and MAP. RESULTS Twenty-one rats were included (11 LR, 10 PEV). Overall blood loss was between 6 mL and 10 mL and not significantly different between groups. There was a 36% mortality rate in the LR group and 0% mortality in the PEV group ( p = 0.03). The LR group had significantly higher lactates at 1 hour ( p = 0.025). At 15 minutes, 45 minutes, 60 minutes, and 180 minutes, the MAP of the PEV group was significantly higher than the LR group. CONCLUSION Early studies are encouraging regarding the potential use of PEVs in uncontrolled hemorrhagic shock based on improved survival and hemodynamics.

The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix proteins. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods ( synapse.org/LINCS_MCF10A ). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes. Beyond these analyses, this dataset will serve as a resource for the broader scientific community to mine for biological insights, to compare signals carried across distinct molecular modalities, and to develop new computational methods for integrative data analysis.

Background Resistance to HER2-targeted therapeutics remains a significant clinical problem in HER2+ breast cancer patients with advanced disease. This may be particularly true for HER2+ patients with basal subtype disease, as recent evidence suggests they receive limited benefit from standard of care HER2-targeted therapies. Identification of drivers of resistance and aggressive disease that can be targeted clinically has the potential to impact patient outcomes. Methods We performed siRNA knockdown screens of genes differentially expressed between lapatinib-responsive and -resistant HER2+ breast cancer cells, which corresponded largely to luminal versus basal subtypes. We then validated hits in 2-d and 3-d cell culture systems. Results Knockdown of one of the genes, INHBA, significantly slowed growth and increased sensitivity to lapatinib in multiple basal HER2+ cell lines in both 2-d and 3-d cultures, but had no effect in luminal HER2+ cells. Loss of INHBA altered metabolism, eliciting a shift from glycolytic to oxidative phosphorylative metabolism, which was also associated with a decrease in tumor invasiveness. Analysis of breast cancer datasets showed that patients with HER2+ breast cancer and high levels of INHBA expression had worse outcomes than patients with low levels of INHBA expression. Conclusions Our data suggest that INHBA is associated with aggressiveness of the basal subtype of HER2+ tumors, resulting in poor response to HER2-targeted therapy and an invasive phenotype. We hypothesize that targeting this pathway could be an effective therapeutic strategy to reduce invasiveness of tumor cells and to improve therapeutic response.

SUMMARYThe phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods (synapse.org/LINCS_MCF10A). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes.

Abstract Background HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype. Methods We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. Results Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts. Conclusion Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Representative in vitro model systems that accurately model response to therapy and allow the identification of new targets are important for improving our treatment of prostate cancer. Here we describe molecular characterization and drug testing in a panel of 20 prostate cancer cell lines. The cell lines cluster into distinct subsets based on RNA expression, which is largely driven by functional Androgen Receptor (AR) expression. KLK3 , the AR-responsive gene that encodes prostate specific antigen, shows the greatest variability in expression across the cell line panel. Other common prostate cancer associated genes such as TMPRSS2 and ERG show similar expression patterns. Copy number analysis demonstrates that many of the most commonly gained (including regions containing TERC and MYC ) and lost regions (including regions containing TP53 and PTEN) that were identified in patient samples by the TCGA are mirrored in the prostate cancer cell lines. Assessment of response to the anti-androgen enzalutamide shows a distinct separation of responders and non-responders, predominantly related to status of wild-type AR. Surprisingly, several AR-null lines responded to enzalutamide. These AR-null, enzalutamide-responsive cells were characterized by high levels of expression of glucocorticoid receptor (GR) encoded by NR3C1 . Treatment of these cells with the anti-GR agent mifepristone showed that they were more sensitive to this drug than enzalutamide, as were several of the enzalutamide non-responsive lines. This is consistent with several recent reports that suggest that GR expression is an alternative signaling mechanism that can bypass AR blockade. This study reinforces the utility of large cell line panels for the study of cancer and identifies several cell lines that represent ideal models to study AR-null cells that have upregulated GR to sustain growth.

Triple negative breast cancer (TNBC) lacks expression of hormone receptors (ER and PR) and HER2 and is characterized by aggressive disease with poor outcomes. Recent work suggests that TNBC also has a high degree of intratumoral heterogeneity, as measured by lineage differentiation status. This heterogeneity may impact therapeutic response, as it has been shown that treatment with PI3K/mTOR (BEZ235) or MEK (trametinib) inhibitors can drive TNBC cells into more homogeneous states, but that the surviving cells are resistant to the targeted therapy. In this study, we sought to understand how the microenvironment impacts differentiation state heterogeneity and response to targeted therapeutics in HCC1143 cells using our microenvironment microarray (MEMA) platform. Under low serum growth conditions, we found that several ligands could drive the growth of HCC1143, particularly EGF family ligands like AREG and EGF. With respect to differentiation state and heterogeneity, EGF and TGFB1 drove HCC1143 cells into a more mesenchymal like state, with increased expression of VIM and decreased expression of KRT14. In contrast, BMP2 led to higher levels of KRT14 and lower levels of VIM, leading to a more basal-like state. We also grew HCC1143 on MEMA with trametinib treatment. Here we found that combinations of collagen-based substrates and NRG1, HGF, and EGF ligands all led to higher cell counts and EdU incorporation rates compared to PBS-control treated cells. However, the levels of resistance conferred by the microenvironment was less than we had previously seen in HER2 positive MEMA, as the GR50 values (dose required to inhibit growth by 50%) only increased modestly (18 nM for untreated cells, 40 nM for NRG1, 45 nM for HGF). Interestingly, in that HER2 positive MEMA study, we identified HGF and NRG1 as potent resistance factors to lapatinib, but that they functioned in a subtype specific manner. HGF was effective in basal subtype cells and NRG1 in luminal, but not vice versa. We postulated that the modest resistance we observed was due to ligands acting on subsets of cells. We thus treated cells with a combination of NRG1 plus HGF, and found that this resulted in increased resistance (GR50= 91 nM). Imaging showed that trametinib drove HCC1143 cells to a homogenous KRT14 positive state, but surprisingly, addition of ligands reverted the cells to a more heterogeneous state that was resistant to trametinib. These data demonstrate that the microenvironment can impact the differentiation state of TNBC cells and is also capable of conferring resistance within subsets of the heterogeneous cell populations. Citation Format: Rebecca Smith, Kaylyn Devlin, Moqing Liu, Tiera Liby, David Kilburn, Elmar Bucher, Damir Sudar, Guillaume Thibault, Mark Dane, Joe Gray, Laura Heiser, James E. Korkola. The impact of the microenvironment on heterogeneity and trametinib response in HCC1143 triple negative breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1870.

Extrinsic signals are implicated in breast cancer resistance to HER2-targeted tyrosine kinase inhibitors (TKIs). To examine how microenvironmental signals influence resistance, we monitored TKI-treated breast cancer cell lines grown on microenvironment microarrays composed of printed extracellular matrix proteins supplemented with soluble proteins. We tested ∼2,500 combinations of 56 soluble and 46 matrix microenvironmental proteins on basal-like HER2+ (HER2E) or luminal-like HER2+ (L-HER2+) cells treated with the TKIs lapatinib or neratinib. In HER2E cells, hepatocyte growth factor, a ligand for MET, induced resistance that could be reversed with crizotinib, an inhibitor of MET. In L-HER2+ cells, neuregulin1-β1 (NRG1β), a ligand for HER3, induced resistance that could be reversed with pertuzumab, an inhibitor of HER2-HER3 heterodimerization. The subtype-specific responses were also observed in 3D cultures and murine xenografts. These results, along with bioinformatic pathway analysis and siRNA knockdown experiments, suggest different mechanisms of resistance specific to each HER2+ subtype: MET signaling for HER2E and HER2-HER3 heterodimerization for L-HER2+ cells.

Treatment options remain limited for men with castration resistant prostate cancer, with current chemotherapy offering mainly palliative benefits. Thus, there is an urgent need to identify new targets for therapeutic intervention in men who have progressed on anti-androgen therapy. We have compiled a panel of 20 prostate cancer cell lines that has been extensively characterized at the molecular level that enables us to perform high throughput drug and siRNA screens to identify novel therapeutic targets. We performed an siRNA screen targeting kinases using eight representative lines, both in the presence and absence of the anti-androgen MDV3100. Knockdown of NTRK1 (TRKA) significantly inhibited growth in 4/8 of the cell lines tested. To confirm this, we screened the entire panel of cell lines with the inhibitor CEP-701, which targets FLT3, JAK2, and TRK-A/B/C. CEP-701 showed GI50 values (dose required to inhibit growth by 50%) less than 400 nM in 8/20 cell lines. Since this drug had already been tested in clinical trials for prostate cancer and the trials were discontinued due to lack of PSA decline, we looked for other drugs that also inhibit TRKA. Both BIBF-1120 and GSK-1363089 are also known to inhibit TRKA, but neither have been tested in prostate cancer expressing TRKA. We tested GSK-136089 in a subset of 9 prostate cell lines, and found that 4/9 lines reached GI50 at 1 uM or less, including AR mutant or null lines such as 22rv1 and PC3 that are non-responsive to MDV3100. Additional tests with BIBF-1120 and GSK-1363089 are underway. These data suggest that NTRK1 may be a potential target for patients with castration resistant prostate cancer. Citation Format: James E. Korkola, Moqing Liu, Rebecca Smith, Tiera Liby, Joe W. Gray. NTRK1/TRKA as a therapeutic target in castration resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1347.

Abstract Background. Pertuzumab and T-DM1 are two recently approved monoclonal antibody based therapies targeting HER2+ breast cancer. Pertuzumab interferes with dimerization of HER family members, while T-DM1 binds to HER2 and interferes with its oncogenic function while also specifically delivering a cytotoxic agent (emtansine). One arm of the I-SPY 2 clinical trial is to investigate the efficacy of a combination Pertuzumab plus T-DM1 in HER2+ breast cancer patients. Methods. We performed pre-clinical screening of response to each agent alone and in combination in a set of 21 HER2+ breast cancer cell lines, with an end goal of identifying markers of response to the therapies. There were five treatment regimens employed in the initial screen: i) pertuzumab alone for 72 h; ii) T-DM1 alone for 72h; iii) pertuzumab plus T-DM1 concurrently for 72h; iv) pertuzumab for 24h followed by addition of T-DM1 for 48h more; and iv) T-DM1 for 24h followed by addition of pertuzumab for 48h more. Response was assessed using the Cell Titer Glo assay as a measure of cell viability. To assess the effects of drug combinations, we used a stringent measure of synergy and antagonism employing the median effect method of Chou and Talalay that included 95% confidence intervals to determine significance. Results. Initial screens showed that concurrent treatment of cells with pertuzumab plus T-DM1 gave significant synergistic interactions in 15/21 cell lines as measured by the median effect method, with combination indices (CI) less than 0.5 (and 95% upper confidence levels less than 1.0) for at least one drug concentration. However, 24h pretreatment with pertuzumab followed by T-DM1 significantly diminished the response of cells to T-DM1, resulting in significant antagonism in 17/21 cell lines test (CI>1.5, lower confidence level greater than 1). Since this could be due to a shorter exposure time to T-DM1, and since patients are scheduled to be treated with pertuzumab first followed by T-DM1 one hour later, we repeated the experiment with one hour between pertuzumab and T-DM1 rather than 24h. While the inhibitory effect was diminished, this treatment regimen still resulted in significant antagonism when T-DM1 was given 1 hour after pertuzumab in 5/5 cell lines tested, in contrast to concurrent pertuzumab plus T-DM1 treatment, which showed synergy. Conclusions. Pertuzumab plus T-DM1 appears to be beneficial when given concurrently, but pretreatment with pertuzumab appears to blunt the efficacy of T-DM1. This has important potential ramifications for patient treatment, and may further elucidate mechanisms of action for both compounds. Further testing will be necessary to determine whether these timing effects are operational in vivo and whether immune effects mitigate the antagonism. Citation Format: James E Korkola, Moqing Liu, Tiera Liby, Laura Heiser, Heidi Feiler, Joe W Gray. Detrimental effects of sequential compared to concurrent treatment of pertuzumab plus T-DM1 in HER2+ breast cancer cell lines [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S6-07.

ORF40 (named fatE) in the Vibrio anguillarum pJM1 plasmid-encoding anguibactin iron transport systems is a homolog of ATPase genes involved in ferric-siderophore transport. Mutation of fatE did not affect ferric-anguibactin transport, indicating that there must be other ATPase gene(s) in addition to fatE. By searching the genomic sequence of V.anguillarum 775(pJM1), we identified a homolog of fatE named fvtE on chromosome 2. It is of interest that in this locus, we also identified homologs of fatB, fatC, and fatD that we named fvtB, fvtC and fvtD, respectively. The fvtE mutant still showed ferric-anguibactin transport, while the double fatE and fvtE mutation completely abolished the ferric-anguibactin transport indicating that fatE and fvtE are functional ATPase homologs for ferric-anguibactin transport. Furthermore, we demonstrate that fvtB, fvtC, fvtD, and fvtE are essential for ferric-vanchrobactin and ferric-enterobactin transport.

Vibrio anguillarum is a marine pathogen that causes vibriosis, a hemorrhagic septicemia in aquatic invertebrate as well as vertebrate animals. The siderophore anguibactin system is one of the most important virulence factors of this bacterium. Most of the anguibactin biosynthesis and transport genes are located in the 65-kb pJM1 virulence plasmid although some of them are found in the chromosome of this fish pathogen. Over 30 years of research unveiled the role numerous chromosomal and pJM1 genes play in the synthesis of anguibactin and the transport of cognate ferric complexes into the bacterial cell. Furthermore, these studies showed that pJM1-carrying strains might be originated from pJM1-less strains producing the chromosome-mediated siderophore vanchrobactin. Additionally, we recently identified a chromosome-mediated anguibactin system in V. harveyi suggesting the possible evolutional origin of the V. anguillarum anguibactin system. In this review, we present our current understanding of the mechanisms and evolution hypothesis of the anguibactin system that might have occurred in these pathogenic vibrios.

HlyU is a master regulator that plays an essential role in the virulence of the human pathogen Vibrio vulnificus. One of the most noteworthy characteristics of HlyU regulation in this organism is its positive control of the expression of the repeat-in-toxin (RtxA1) gene, one of the most important virulence factors accounting for the fulminating and damaging nature of V. vulnificus infections. In this work, we reviewed the latest studies of RtxA1 in this bacterium and highlight the mechanism of gene regulation of rtxA1 expression by HlyU under a broader gene regulatory network.

ABSTRACT The virulence gene rtxA1 , encoding the repeat-in-toxin protein, plays an essential role in the pathogenesis of Vibrio vulnificus infections. Expression of this gene is controlled by the HlyU regulator by direct contact of the DNA upstream of the rtxA1 toxin operon acting as a derepressor of the H-NS protein. The crystal structure suggests that HlyU forms a homodimer in vitro . However, knowledge of the biological implications of these findings in vivo is limited. In this work, we endeavored to dissect, using genetic and biochemical approaches, the domains of this protein that are essential for homodimer formation and the interaction of HlyU with the target DNA. We identified that residues L18, N22, R25, S54, Q55, L57, W59, R61, K70, and Y77 are essential for the HlyU protein binding to the DNA and that amino acids L17 and L91 are important for HlyU dimerization. We also determined that HlyU homodimer formation is an essential requirement for binding to the upstream region of the rtxA1 operon and is the key feature in relieving the H-NS repression of rtxA1 transcription.

In Vibrio vulnificus, HlyU upregulates the expression of the large RTX toxin gene. In this work we identified the binding site of HlyU to -417 to -376 bp of the rtxA1 operon transcription start site. lacZ fusions for a series of progressive deletions from the rtxA1 operon promoter showed that transcriptional activity increased independently of HlyU when its binding site was absent. Thus HlyU must regulate the rtxA1 operon expression by antagonizing a negative regulator. Concomitantly we found that an hns mutant resulted in an increase in the expression of the rtxA1 operon genes. Multiple copies of HlyU can increase the promoter activity only in the presence of H-NS underscoring the hypothesis that HlyU must alleviate the repression by this protein. H-NS binds to a region that extends upstream and downstream of the rtxA1 operon promoter. In the upstream region it binds to five AT-rich sites of which two overlap the HlyU binding site. Competitive footprinting and gel shift data demonstrate HlyU's higher affinity as compared with H-NS resulting in the de-repression and a corresponding increased expression of the rtxA1 operon.