The Renin-Angiotensin System Involvement in Cisplatin-Induced Nephrotoxicity: An Overview of Physiological and Pathological Mechanisms-A Systematic Review.

Cisplatin (CDDP) is a highly potent chemotherapy drug. But its nephrotoxicity poses a significant limitation to its use. The renin-angiotensin system (RAS) has been proposed to play a role in drug-induced nephrotoxicity. This systematic review (SR) sought to identify the link between CDDP-induced nephrotoxicity and the RAS pathway. In this SR, relevant keywords were employed to explore databases such as PubMed (MEDLINE), Scopus (Elsevier), and Institute for Scientific Information (ISI) Web of Science up to October 2023. Nine studies were selected based on predefined inclusion/exclusion criteria. The findings support the involvement of the RAS in the CDDP-induced nephrotoxicity model, along with the activation of inflammatory mediators, lipid peroxidation, and changes in markers of kidney tissue damage. Furthermore, physiology and pathology of RAS-related interventions in CDDP-induced nephrotoxicity models have involved the factors such as human organic cation transporter 2 (hOCT2), organic anion transporting polypeptides 1B1 (OATP1B1) and 1B3, kallikrein-kinin system, and bradykinin receptors. CDDP-induced nephrotoxicity has been found to be substantially influenced by both classic and nonclassic RAS axes. Angiotensin II exacerbates renal damage induced by CDDP. Conversely, inhibiting the pressor arm of RAS in males mitigates this damage. However, activation of the renal vasodepressor arm of RAS exacerbates CDDP-induced nephrotoxicity in females. These findings underscore gender differences in renal function and response to RAS-related interventions in the presence of CDDP. This SR provides insights into both beneficial and adverse interventions associated with RAS in the CDDP-induced nephrotoxicity, offering valuable considerations for researchers and clinicians.

Establishment of a 96-well transwell system using primary human gut organoids to capture multiple quantitative pathway readouts.

Disruptions in the gut epithelial barrier can lead to the development of chronic indications such as inflammatory bowel disease (IBD). Historically, barrier function has been assessed in cancer cell lines, which do not contain all human intestinal cell types, leading to poor translatability. To bridge this gap, we adapted human primary gut organoids grown as monolayers to quantify transcription factor phosphorylation, gene expression, cytokine production, and barrier function. In this work we describe and characterize a novel 96-well human gut organoid-derived monolayer system that enables quantitative assessment of candidate therapeutics. Normal human intestine differentiation patterns and barrier function were characterized and confirmed to recapitulate key aspects of in vivo biology. Next, cellular response to TNF-α (a central driver of IBD) was determined using a diverse cadre of quantitative readouts. We showed that TNF-α pathway antagonists rescued damage caused by TNF-α in a dose-dependent manner, indicating that this system is suitable for quantitative assessment of barrier modulating factors. Taken together, we have established a robust primary cell-based 96-well system capable of interrogating questions around mucosal response. This system is well suited to provide pivotal functional data to support translational target and drug discovery efforts.

Importance of gut microbiota metabolites in the development of cardiovascular diseases (CVD).

Cardiovascular disease (CVD) remains the most common cause of death worldwide and has become a public health concern. The proven notable risk factors for CVD are atherosclerosis, hypertension, diabetes, dyslipidemia, inflammation, and some genetic defects. However, research has shown a correlation between metabolic health, gut microbiota, and dietary risk factors. The gut microbiota makes an important contribution to human functional metabolic pathways by contributing enzymes that are not encoded by the human genome, for instance, the breakdown of polysaccharides, polyphenols and vitamins synthesis. TMAO and SCFAs, human gut microbiota compounds, have respective immunomodulatory and pro-inflammatory effects. Choline and l-carnitine are abundant in high-fat diets and are transformed into TMA by gut bacteria. The liver's phase of metabolism then changes TMA into TMAO. In turn, TMAO promotes the activation of macrophages, damages vascular endothelium, and results in CVD-however, dysbiosis decreases SCFAs and bile acids, which raises intestinal permeability. Congestion in the portal vein, a drop in cardiac output, a reduction in intestinal perfusion, and intestinal leakage are all caused by heart failure. These factors induce systemic inflammation by increasing intestinal leakage. By raising CRP and pro-inflammatory reactions, human gut dysbiosis and elevated TMAO levels promote the development of arterial plaque, hasten the beginning of atherosclerosis, and raise the risk of CAD. A healthy symbiosis between the gut microbiota and host is a key factor in shaping the biochemical profile of the diet, therefore which are crucial for maintaining the intestinal epithelial barrier, growing mucosa, reducing inflammation, and controlling blood pressure.

Evaluating the effectiveness of tranexamic acid administration in reducing bleeding in benign prostate hyperplasia patients underwent open prostatectomy: A double-blind randomized clinical trial.

Background: Blood loss of postoperative after prostate surgery could be related with an increase in urinary fibrinolytic activity. Tranexamic acid (TXA) is both a potent inhibitor of plasminogen and urokinase activators and a low molecular weight substance that is excreted unchanged in the urinary tract and can be administered both orally and intravenously. This study aimed to evaluate the effectiveness TXA administration in reducing bleeding in benign prostatic hyperplasia (BPH) patients who underwent open prostatectomy. Materials and Methods: This double-blind randomized clinical trial was conducted on patients with BPH who underwent open prostatectomy. The first group received TXA (1 gr IV from during surgery to 48 h after surgery, 3 times/day). Twenty-four hours after surgery, the two groups were compared in terms of bleeding rate. Hemoglobin (Hb), hematocrit (HCT), and platelet (Plt) counts were also assessed before and after the intervention. Results: Intervention and control groups were comparable in terms of basic and baseline values of variables at the beginning of the study (P > 0.05). The mean bleeding volume in TXA group was significantly lower than the control group 112.11 ± 53.5 and 190.00 ± 97.5 CC; P ≤ 0.001). Mean hospitalization (3.28±0.46 vs. 4.38 ± 0.95 days P < 0.001) and surgery duration (98.11 ± 37.11 vs. 128.00 ± 39.12 h; P = 0.001) were significantly lower in TXA group compared to control intervention. Conclusion: According to the findings of the current study, the administration of TXA led to reduce bleeding in BPH patients who underwent open prostatectomy. Furthermore, the mean Hb, HCT, levels were significantly affected by TXA. TXA treatment approach also can reduce the surgery and hospitalization time effectively. TXA approach is recommended as effective procedure in BPH patients who underwent open prostatectomy.

Buspirone Ameliorates Colon Inflammation in TNBS-Induced Rat Acute Colitis: The Involvement of TLR4/NF-kB Pathway.

Inflammatory bowel disease (IBD) is an inflammatory situation involving the whole digestive system. This illness includes ulcerative colitis and Crohn's disease. According to scientific research, the immune system plays an essential part in developing this disease. Recently, buspirone has been discovered to have anti-inflammatory properties. As a result, this research aims to see if buspirone provides anti-inflammatory effects in a rat model of TNBS-induced colitis. Control, TNBS, dexamethasone (2 mg/kg), and buspirone (5, 10, and 20 mg/kg) were randomly given to six groups of 36 male Wistar rats. Colitis was induced by intrarectal instillation of TNBS in all research groups except the control group, and rats were meliorated with dexamethasone and buspirone. Macroscopic and microscopic lesions appeared after colitis induction, while therapy with dexamethasone and buspirone significantly improved the lesions. TLR4 and pNF-κB expression were also enhanced during colitis induction. On the other hand, the administration of dexamethasone or buspirone resulted in a considerable reduction in their expression. Tissue TNF-α and MPO activity were enhanced after induction of colitis in terms of biochemical variables; however, administration of dexamethasone or buspirone reduced TNF-α and MPO activity. Eventually, in an animal model of severe colitis, buspirone displayed anti-inflammatory characteristics via lowering the TLR4/NF-ĸB signaling pathway's activity in an animal model of acute colitis.

Assessment of Sumatriptan on Sepsis-Induced Kidney injury in the Cecal Ligation and Puncture Mice Model.

Sepsis is a severe systemic inflammatory response with high mortality rate resulting from different microorganisms. Cytokines activation is essential for the immune response, but in painful conditions like sepsis, cytokines act as a double-edged sword and dysregulate immune response which is life-threatening owing to multiple organ dysfunction. The abnormality in 5-HT function is involved in pathological conditions like irritable bowel syndrome, inflammation, myocardial ischemia, itch and renal injury. Sumatriptan, a 5-HT1B/1D agonist, has anti-inflammatory and anti-oxidative stress effects on animal models. This study was aimed to assess the effects of sumatriptan on kidney injury, the levels of pro-inflammatory cytokines and the percentage of survival in (CLP)-induced sepsis were examined.Cecal ligation and puncture (CLP) model was done on adult C57BL/6 male mice to induce Polymicrobial sepsis. Sumatriptan was injected intraperitoneally 1 h after the sepsis induction by CLP at doses of 0.1, 0.3, and 1 mg/kg in 3 treatment groups. To study the effect of sumatriptan on short-term survival, septic animals were detected 72 h after CLP. Serum levels of TNF-α, IL-1ß, IL-6 and IL-10 were evaluated. To study sepsis-induced acute renal failure, kidney functional biomarkers and histopathological alterations were evaluated.Sumatriptan (0.3 mg/kg) administration significantly enhanced survival rate (P<0.01) compared to the CLP group. The beneficial effects of sumatriptan were related to a significant decrease in the pro-inflammatory cytokines and elevated level of IL-10. Sumatriptan presented protective effects on kidney biomarkers and histopathology assay.Anti-inflammatory effects of sumatriptan lead to decrease mortality rate and inflammatory cytokines in CLP induction sepsis in C57BL/6 mice.

Assessing donor-to-donor variability in human intestinal organoid cultures.

Donor-to-donor variability in primary human organoid cultures has not been well characterized. As these cultures contain multiple cell types, there is greater concern that variability could lead to increased noise. In this work we investigated donor-to-donor variability in human gut adult stem cell (ASC) organoids. We examined intestinal developmental pathways during culture differentiation in ileum- and colon-derived cultures established from multiple donors, showing that differentiation patterns were consistent among cultures. This finding indicates that donor-to-donor variability in this system remains at a manageable level. Intestinal metabolic activity was evaluated by targeted analysis of central carbon metabolites and by analyzing hormone production patterns. Both experiments demonstrated similar metabolic functions among donors. Importantly, this activity reflected intestinal biology, indicating that these ASC organoid cultures are appropriate for studying metabolic processes. This work establishes a framework for generating high-confidence data using human primary cultures through thorough characterization of variability.

Chronically altered NMDAR signaling in epilepsy mediates comorbid depression.

Depression is the most common psychiatric comorbidity of epilepsy. However, the molecular pathways underlying this association remain unclear. The NMDA receptor (NMDAR) may play a role in this association, as its downstream signaling has been shown to undergo long-term changes following excitotoxic neuronal damage. To study this pathway, we used an animal model of fluoxetine-resistant epilepsy-associated depression (EAD). We determined the molecular changes associated with the development of depressive symptoms and examined their response to various combinations of fluoxetine and a selective neuronal nitric oxide synthase inhibitor, 7-nitroindazole (NI). Depressive symptoms were determined using the forced swim test. Furthermore, expression and phosphorylation levels of markers in the ERK/CREB/ELK1/BDNF/cFOS pathway were measured to determine the molecular changes associated with these symptoms. Finally, oxidative stress markers were measured to more clearly determine the individual contributions of each treatment. While chronic fluoxetine (Flxc) and NI were ineffective alone, their combination had a statistically significant synergistic effect in reducing depressive symptoms. The development of depressive symptoms in epileptic rats was associated with the downregulation of ERK2 expression and ELK1 and CREB phosphorylation. These changes were exactly reversed upon Flxc + NI treatment, which led to increased BDNF and cFOS expression as well. Interestingly, ERK1 did not seem to play a role in these experiments. NI seemed to have augmented Flxc's antidepressant activity by reducing oxidative stress. Our findings suggest NMDAR signaling alterations are a major contributor to EAD development and a potential target for treating conditions associated with underlying excitotoxic neuronal damage.

An Experimental Pipeline for Initial Characterization of Bacterial Type III Secretion System Inhibitor Mode of Action Using Enteropathogenic Yersinia.

Dozens of Gram negative pathogens use one or more type III secretion systems (T3SS) to disarm host defenses or occupy a beneficial niche during infection of a host organism. While the T3SS represents an attractive drug target and dozens of compounds with T3SS inhibitory activity have been identified, few T3SS inhibitors have been validated and mode of action determined. One issue is the lack of standardized orthogonal assays following high throughput screening. Using a training set of commercially available compounds previously shown to possess T3SS inhibitory activity, we demonstrate the utility of an experiment pipeline comprised of six distinct assays to assess the stages of type III secretion impacted: T3SS gene copy number, T3SS gene expression, T3SS basal body and needle assembly, secretion of cargo through the T3SS, and translocation of T3SS effector proteins into host cells. We used enteropathogenic Yersinia as the workhorse T3SS-expressing model organisms for this experimental pipeline, as Yersinia is sensitive to all T3SS inhibitors we tested, including those active against other T3SS-expressing pathogens. We find that this experimental pipeline is capable of rapidly distinguishing between T3SS inhibitors that interrupt the process of type III secretion at different points in T3SS assembly and function. For example, our data suggests that Compound 3, a malic diamide, blocks either activity of the assembled T3SS or alters the structure of the T3SS in a way that blocks T3SS cargo secretion but not antibody recognition of the T3SS needle. In contrast, our data predicts that Compound 4, a haloid-containing sulfonamidobenzamide, disrupts T3SS needle subunit secretion or assembly. Furthermore, we suggest that misregulation of copy number control of the pYV virulence plasmid, which encodes the Yersinia T3SS, should be considered as a possible mode of action for compounds with T3SS inhibitory activity against Yersinia.

mTORC1 signaling suppresses Wnt/ß-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level.

Wnt/ß-catenin signaling plays pivotal roles in cell proliferation and tissue homeostasis by maintaining somatic stem cell functions. The mammalian target of rapamycin (mTOR) signaling functions as an integrative rheostat that orchestrates various cellular and metabolic activities that shape tissue homeostasis. Whether these two fundamental signaling pathways couple to exert physiological functions still remains mysterious. Using a genome-wide CRISPR-Cas9 screening, we discover that mTOR complex 1 (mTORC1) signaling suppresses canonical Wnt/ß-catenin signaling. Deficiency in tuberous sclerosis complex 1/2 (TSC1/2), core negative regulators of mTORC1 activity, represses Wnt/ß-catenin target gene expression, which can be rescued by RAD001. Mechanistically, mTORC1 signaling regulates the cell surface level of Wnt receptor Frizzled (FZD) in a Dishevelled (DVL)-dependent manner by influencing the association of DVL and clathrin AP-2 adaptor. Sustained mTORC1 activation impairs Wnt/ß-catenin signaling and causes loss of stemness in intestinal organoids ex vivo and primitive intestinal progenitors in vivo. Wnt/ß-catenin-dependent liver metabolic zonation gene expression program is also down-regulated by mTORC1 activation. Our study provides a paradigm that mTORC1 signaling cell autonomously regulates Wnt/ß-catenin pathway to influence stem cell maintenance.

ATP2B1 rs2681472 and STK39 rs35929607 polymorphisms and risk of Hypertension in Iranian Population.

Background: ATP2B1 and STK39 have been introduced as essential hypertension candidate genes. The association of these genes' variations have not been studied in Iranian population yet. Here we aimed to investigate the association of ATP2B1 rs2681472 and STK39 rs35929607 polymorphisms with the risk of hypertension in an Iranian population. Methods: We included 400 individuals in our case-control study: 200 cases with essential hypertension and 200 healthy sex and age matched controls. All subjects were genotyped for rs2681472 and rs35929607 using a PCR-RFLP method. Genotype and allele frequencies were compared between the two groups using chi-squared test. The association was further assessed under log-additive, dominant and recessive genetic models. Results: There was no association between rs2681472 and rs35929607 polymorphisms and risk of essential hypertension in our population (p>0.05). There was also no association between the studied polymorphisms and hypertension under different genetic models. Conclusion: Our study indicated that rs2681472 of ATP2B1 and rs35929607 of STK39 may not have a significant effect on the risk of essential hypertension in Iranian population. More studies are still needed to validate our results.

Indoleacrylic Acid Produced by Commensal Peptostreptococcus Species Suppresses Inflammation.

Host factors in the intestine help select for bacteria that promote health. Certain commensals can utilize mucins as an energy source, thus promoting their colonization. However, health conditions such as inflammatory bowel disease (IBD) are associated with a reduced mucus layer, potentially leading to dysbiosis associated with this disease. We characterize the capability of commensal species to cleave and transport mucin-associated monosaccharides and identify several Clostridiales members that utilize intestinal mucins. One such mucin utilizer, Peptostreptococcus russellii, reduces susceptibility to epithelial injury in mice. Several Peptostreptococcus species contain a gene cluster enabling production of the tryptophan metabolite indoleacrylic acid (IA), which promotes intestinal epithelial barrier function and mitigates inflammatory responses. Furthermore, metagenomic analysis of human stool samples reveals that the genetic capability of microbes to utilize mucins and metabolize tryptophan is diminished in IBD patients. Our data suggest that stimulating IA production could promote anti-inflammatory responses and have therapeutic benefits.

Quince seed mucilage magnetic nanocomposites as novel bioadsorbents for efficient removal of cationic dyes from aqueous solutions.

This study investigated the potential use of quince seed mucilage (QSM) as alternative bioadsorbents for methylene blue (MB) dye from aqueous solutions. This novel magnetic nanocomposite adsorbent (MNCA) based on QSM was synthesized by in situ formation of magnetic iron oxide nanoparticles into QSM solution. The MNCAs were characterized using FTIR, SEM, TEM, XRD, and VSM. Removal of MB was investigated by batch adsorption technique. The thermodynamic parameters suggest that the dye adsorption process is spontaneous and exothermic in nature. Moreover, the adsorbents showed high selectivity for the adsorption of cationic dyes with regenerated properties. The pseudo-second-order kinetics and Langmuir adsorption isotherm models also provide the best correlation of the experimental data for MB adsorption. The results indicate that the MNCAs can be employed as efficient low cost adsorbents with excellent dye adsorption performance in wastewater treatment process.

Community behavior and spatial regulation within a bacterial microcolony in deep tissue sites serves to protect against host attack.

Bacterial pathogens express virulence-specific transcriptional programs that allow tissue colonization. Although phenotypic variation has been noted in the context of antibiotic exposure, no direct evidence exists for heterogeneity in virulence-specific transcriptional programs within tissues. In a mouse model of Yersinia pseudotuberculosis infection, we show that at least three subpopulations of bacteria develop within a single tissue site in response to distinct host signals. Bacteria growing on the exterior of spleen microcolonies responded to soluble signals and induced the nitric oxide (NO)-detoxifying gene, hmp. Hmp effectively eliminated NO diffusion and protected the interior bacterial population from exposure to NO-derived inducing signals. A third subpopulation, constituting the most peripherally localized bacteria, directly contacted neutrophils and transcriptionally upregulated a virulence factor. These studies demonstrate that growth within tissues results in transcriptional specialization within a single focus of microbial replication, facilitating directed pathogen counterattack against the host response.

Cdc42 interacts with the exocyst complex to promote phagocytosis.

The process of phagocytosis in multicellular organisms is required for homeostasis, clearance of foreign particles, and establishment of long-term immunity, yet the molecular determinants of uptake are not well characterized. Cdc42, a Rho guanosine triphosphatase, is thought to orchestrate critical actin remodeling events needed for internalization. In this paper, we show that Cdc42 controls exocytic events during phagosome formation. Cdc42 inactivation led to a selective defect in large particle phagocytosis as well as a general decrease in the rate of membrane flow to the cell surface. Supporting the connection between Cdc42 and exocytic function, we found that the overproduction of a regulator of exocytosis, Rab11, rescued the large particle uptake defect in the absence of Cdc42. Additionally, we demonstrated a temporal interaction between Cdc42 and the exocyst complex during large particle uptake. Furthermore, disruption of exocyst function through Exo70 depletion led to a defect in large particle internalization, thereby establishing a functional role for the exocyst complex during phagocytosis.

Identification of MrtAB, an ABC transporter specifically required for Yersinia pseudotuberculosis to colonize the mesenteric lymph nodes.

A highly conserved virulence plasmid encoding a type III secretion system is shared by the three Yersinia species most pathogenic for mammals. Although factors encoded on this plasmid enhance the ability of Yersinia to thrive in their mammalian hosts, the loss of this virulence plasmid does not eliminate growth or survival in host organs. Most notably, yields of viable plasmid-deficient Yersinia pseudotuberculosis (Yptb) are indistinguishable from wild-type Yptb within mesenteric lymph nodes. To identify chromosomal virulence factors that allow for plasmid-independent survival during systemic infection of mice, we generated transposon insertions in plasmid-deficient Yptb, and screened a library having over 20,000 sequence-identified insertions. Among the previously uncharacterized loci, insertions in mrtAB, an operon encoding an ABC family transporter, had the most profound phenotype in a plasmid-deficient background. The absence of MrtAB, however, had no effect on growth in the liver and spleen of a wild type strain having an intact virulence plasmid, but caused a severe defect in colonization of the mesenteric lymph nodes. Although this result is consistent with lack of expression of the type III secretion system by Wt Yptb in the mesenteric lymph nodes, a reporter for YopE indicated that expression of the system was robust. We demonstrate that the ATPase activity of MrtB is required for growth in mice, indicating that transport activity is required for virulence. Indeed, MrtAB appears to function as an efflux pump, as the ATPase activity enhances resistance to ethidium bromide while increasing sensitivity to pyocyanin, consistent with export across the inner membrane.

Legionella pneumophila LidA affects nucleotide binding and activity of the host GTPase Rab1.

Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease, intercepts material from host cell membrane transport pathways to create a specialized vacuolar compartment that supports bacterial replication. Delivery of bacterial effector proteins into the host cell requires the Dot/Icm type IV secretion system. Several effectors, including SidM, SidD, and LepB, were shown to target the early secretory pathway by manipulating the activity of the host GTPase Rab1. While the function of these effectors has been well characterized, the role of another Rab1-interacting protein from L. pneumophila, the effector protein LidA, is poorly understood. Here, we show that LidA binding to Rab1 stabilized the Rab1-guanosine nucleotide complex, protecting it from inactivation by GTPase-activating proteins (GAPs) and from nucleotide extraction. The protective effect of LidA on the Rab1-guanine nucleotide complex was concentration dependent, consistent with a 1:1 stoichiometry of the LidA-Rab1 complex. The central coiled-coil region of LidA was sufficient for Rab1 binding and to prevent GAP-mediated inactivation or nucleotide extraction from Rab1. In addition, the central region mediated binding to phosphatidylinositol 3-phosphate and other phosphoinositides. When bound to Rab1, LidA interfered with the covalent modification of Rab1 by phosphocholination or AMPylation, and it also blocked de-AMPylation of Rab1 by SidD and dephosphocholination by Lem3. Based on these findings, we propose a role for LidA in bridging the membrane of the Legionella-containing vacuole (LCV) with that of secretory transport vesicles surrounding the LCV.

Yersinia pseudotuberculosis virulence determinants invasin, YopE, and YopT modulate RhoG activity and localization.

The Yersinia pseudotuberculosis surface protein invasin binds to multiple beta1 integrins with high affinity, leading to misregulation of Rac1 activity. Upon host cell binding, alteration of Rho GTPase activity results from the action of several Yersinia outer proteins (Yops) that are translocated into the cytoplasm. We report here that three virulence determinants encoded by Y. pseudotuberculosis manipulate the Rho GTPase RhoG. Y. pseudotuberculosis binding to cells caused robust recruitment of RhoG to the site of attachment, which required high-affinity invasin-beta1 integrin association. Furthermore, inactivation of RhoG significantly reduced the efficiency of invasin-mediated bacterial internalization. To investigate the activation state of RhoG, a fluorescence resonance energy transfer-based activation biosensor was developed and used to show distinct spatial activation of RhoG at the site of bacterial attachment. The biosensor was also used to show efficient RhoG inactivation by Y. pseudotuberculosis YopE, a potent Rho GTPase activating protein. Additionally, RhoG mislocalization by the prenylcysteine endoprotease YopT was demonstrated by two independent assays. Functional bacterial uptake experiments demonstrated that RhoG activation can bypass a deficit in Rac1 activity. Interestingly, increasing the size of the particle gave results more consistent with a linear pathway, in which RhoG acts as an upstream activator of Rac1, indicating that increased surface area introduces constraints on the signaling pathways required for efficient internalization. Taken together, these data demonstrate the misregulation of RhoG by multiple Y. pseudotuberculosis virulence determinants. Since RhoG is imperative for proper neutrophil function, this misregulation may represent a unique mechanism by which Yersinia species dampen the immune response.

The polybasic region of Rac1 modulates bacterial uptake independently of self-association and membrane targeting.

The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase member, is required for Rac1 self-association, membrane localization, nuclear translocation, and interaction with downstream effectors. We previously demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the effectors that requires the polybasic region for interaction, is necessary for efficient invasin-promoted uptake of Yersinia pseudotuberculosis by nonphagocytic cells. Here we further examined the role of this region in invasin-promoted uptake. Using fluorescence resonance energy transfer experiments (FRET), we determined that engagement of integrin receptors by invasin caused elevated levels of Rac1 self-association at the site of bacterial adhesion in a PBR-dependent fashion. Self-association could be disrupted using several strategies: translocation of the Yersinia YopT prenylcysteine protease into host cells, inactivation of the Rac1 isoprenylation signal that is required for membrane localization, and elimination of the PBR. Disruption in each case impaired invasin-promoted uptake. To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail. The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association. This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.

Disruption of RhoGDI and RhoA regulation by a Rac1 specificity switch mutant.

Rho family GTPases are important regulators of the actin cytoskeleton. Activation of these proteins can be promoted by guanine nucleotide exchange factors containing Dbl and Pleckstrin homology domains resulting in membrane insertion of a Rho family member, whereas the inactive GDP-bound form is sequestered primarily in the cytoplasm, bound to the guanosine dissociation inhibitor RhoGDI. Dominant interfering variants of Rac1, but not Cdc42, inhibit beta1 integrin-promoted uptake of Yersinia pseudotuberculosis. Unexpectedly, we found that the Rac1(W56F) guanine nucleotide exchange factors specificity switch mutant blocked invasin-promoted uptake as well as Cdc42-dependent uptake of enteropathogenic Escherichia coli. Fluorescence resonance energy transfer experiments demonstrated that Rac1(W56F) retained the ability to be loaded with GTP, bind a downstream effector, and interact with RhoGDI. Mutational analyses of intragenic suppressors and coexpression studies demonstrated that binding of the Rac1(W56F) mutant to RhoGDI appeared to play a role in the inhibition of uptake. As RhoGDI inhibits RhoA, overactivation of RhoA may account for the uptake interference caused by Rac1(W56F). Consistent with this model, a dominant interfering form of RhoA restored significant uptake in the presence of the Rac1(W56F) mutant but had no effect on another interfering Rac1 form. Furthermore, the cellular GTP-RhoA level was elevated by the presence of Rac1(W56F) mutant protein. These data are consistent with the proposition that Rac1(W56F) blocks invasin-promoted uptake by preventing RhoGDI from inactivating RhoA. We conclude that RhoGDI allows cross-talk between Rho family members that promote potentially antagonistic processes, and disruption of this cross-talk can interfere with invasin-promoted uptake.