Therapeutic potential of LINS01 histamine H3 receptor antagonists as antineoplastic agents for triple negative breast cancer.

The aims of this work were to evaluate the expression of histamine H3 receptor (H3R) in triple negative breast cancer (TNBC) samples and to investigate the antitumoral efficacy and safety of the LINS01 series of H3R antagonists, through in silico, in vitro, and in vivo approaches. Antitumor activity of LINS01009, LINS01010, LINS01022, LINS01023 was assayed in vitro in 4T1 and MDA-MB-231 TNBC cells (0.01-100 µM), and in vivo in 4T1 tumors orthotopically established in BALB/c mice (1 or 20 mg/kg). Additionally, H3R expression was assessed in 50 human TNBC samples. We have described a higher H3R mRNA expression in basal-like/TNBC tumors vs. matched normal tissue using TCGA Pan-Cancer Atlas data, and a higher H3R expression in human tumor samples vs. peritumoral tissue evidenced by immunohistochemistry associated with poorer survival. Furthermore, while all the essayed compounds showed antitumoral properties, LINS01022 and LINS01023 exhibited the most potent antiproliferative effects by: i) inducing cell apoptosis and suppressing cell migration in 4T1 and MDA-MB-231 TNBC cells, and ii) inhibiting cell growth in paclitaxel-resistant 4T1 cells (potentiating the paclitaxel antiproliferative effect). Moreover, 20 mg/kg LINS01022 reduced tumor size in 4T1 tumor-bearing mice, exhibiting a safe toxicological profile and potential for druggability estimated by ADME calculations. We conclude that the H3R is involved in the regulation of TNBC progression, offering promising therapeutic potential for the novel LINS01 series of H3R antagonists.

Nanomicellar Formulations Loaded with Histamine and Paclitaxel as a New Strategy to Improve Chemotherapy for Breast Cancer.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Currently, paclitaxel (PTX) represents the first-line therapy for TNBC; however it presents a hydrophobic behavior and produces severe adverse effects. The aim of this work is to improve the therapeutic index of PTX through the design and characterization of novel nanomicellar polymeric formulations composed of a biocompatible copolymer Soluplus® (S), surface-decorated with glucose (GS), and co-loaded either with histamine (HA, 5 mg/mL) and/or PTX (4 mg/mL). Their micellar size, evaluated by dynamic light scattering, showed a hydrodynamic diameter between 70 and 90 nm for loaded nanoformulations with a unimodal size distribution. Cytotoxicity and apoptosis assays were performed to assess their efficacy in vitro in human MDA-MB-231 and murine 4T1 TNBC cells rendering optimal antitumor efficacy in both cell lines for the nanoformulations with both drugs. In a model of TNBC developed in BALB/c mice with 4T1 cells, we found that all loaded micellar systems reduced tumor volume and that both HA and HA-PTX-loaded SG micelles reduced tumor weight and neovascularization compared with the empty micelles. We conclude that HA-PTX co-loaded micelles in addition to HA-loaded formulations present promising potential as nano-drug delivery systems for cancer chemotherapy.

Genomic and proteomic characterization of two strains of Shigella flexneri 2 isolated from infants' stool samples in Argentina.

BACKGROUND: Shigella specie is a globally important intestinal pathogen disseminated all over the world. In this study we analyzed the genome and the proteomic component of two Shigella flexneri 2a clinical isolates, collected from pediatric patients with gastroenteritis of the Northwest region of Argentina (NWA) in two periods of time, with four years of difference. Our goal was to determine putative changes at molecular levels occurred during these four years, that could explain the presence of this Shigella`s serovar as the prevalent pathogen in the population under study. RESULTS: As previously reported, our findings support the idea of Shigella has a conserved "core" genome, since comparative studies of CI133 and CI172 genomes performed against 80 genomes obtained from the NCBI database, showed that there is a large number of genes shared among all of them. However, we observed that CI133 and CI172 harbors a small number of strain-specific genes, several of them present in mobile genetic elements, supporting the hypothesis that these isolates were established in the population by horizontal acquisition of genes. These differences were also observed at proteomic level, where it was possible to detect the presence of certain secreted proteins in a culture medium that simulates the host environment. CONCLUSION: Great similarities were observed between the CI133 and CI172 strains, confirming the high percentage of genes constituting the "core" genome of S. flexneri 2. However, numerous strain specific genes were also determined. The presence of the here identified molecular elements into other strain of our culture collation, is currently used to develop characteristic markers of local pathogens. In addition, the most outstanding result of this study was the first description of a S. flexneri 2 producing Colicin E, as one of the characteristics that allows S. flexneri 2 to persist in the microbial community. These findings could also contribute to clarify the mechanism and the evolution strategy used by this pathogen to specifically colonize, survive, and cause infection within the NWA population.

Histamine H4 Receptor Expression in Triple-negative Breast Cancer: An Exploratory Study.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype. There are neither universally accepted prognostic markers nor molecular targets related to TNBC. The histamine H4 receptor (H4R) has been characterized in TNBC experimental models, demonstrating its critical role in tumor development and progression. In this study, H4R expression was compared in breast cancer subtypes and correlated with clinical features using The Cancer Genome Atlas data (Pan-Cancer Atlas). The H4R status was further evaluated by immunohistochemistry in 30 TNBC human samples in relation to clinicopathological parameters. Results indicate that H4R was downregulated in basal-like/TNBC compared with luminal A and normal breast-like tumors. The higher expression of H4R was associated with improved progression-free and overall survival outcomes in basal-like/TNBC. H4R immunoreactivity was detected in about 70% of tumors, and its expression was positively correlated with the levels in the histologically normal peritumoral tissue. High H4R expression in peritumoral tissue correlated with reduced number of lymph node involvement and unifocal TNBC, while it was associated with increased patient survival. In conclusion, the H4R might represent a potential prognostic biomarker in TNBC. Further studies in large cohorts are needed to better understand the significance of H4R in breast cancer biology.

Histamine H4 Receptor Agonism Induces Antitumor Effects in Human T-Cell Lymphoma.

The discovery of the human histamine H4 receptor (H4R) has contributed to our understanding of the role of histamine in numerous physiological and pathological conditions, including tumor development and progression. The lymph nodes of patients with malignant lymphomas have shown to contain high levels of histamine, however, less is known regarding the expression and function of the H4R in T-cell lymphoma (TCL). In this work we demonstrate the expression of H4R isoforms (mRNA and protein) in three human aggressive TCL (OCI-Ly12, Karpas 299, and HuT78). Histamine and specific H4R agonists (VUF8430 and JNJ28610244) significantly reduced cell viability in a dose-dependent manner (p < 0.05). The combined treatment with the H4R antagonist (JNJ7777120, 10 µM) reversed the effects of the H4R ligands. Importantly, we screened a drug repurposing library of 433 FDA-approved compounds (1 µM) in combination with histamine (10 µM) in Hut78 cells. Histamine produced a favorable antitumor effect with 18 of these compounds, including the histone deacetylase inhibitor panobinostat. Apoptosis, proliferation, and oxidative stress studies confirmed the antitumoral effects of the combination. We conclude that the H4R is expressed in TCL, and it is involved in histamine-mediated responses.

Histamine in cancer immunology and immunotherapy. Current status and new perspectives.

Cancer is the second leading cause of death globally and its incidence and mortality are rapidly increasing worldwide. The dynamic interaction of immune cells and tumor cells determines the clinical outcome of cancer. Immunotherapy comes to the forefront of cancer treatments, resulting in impressive and durable responses but only in a fraction of patients. Thus, understanding the characteristics and profiles of immune cells in the tumor microenvironment (TME) is a necessary step to move forward in the design of new immunomodulatory strategies that can boost the immune system to fight cancer. Histamine produces a complex and fine-tuned regulation of the phenotype and functions of the different immune cells, participating in multiple regulatory responses of the innate and adaptive immunity. Considering the important actions of histamine-producing immune cells in the TME, in this review we first address the most important immunomodulatory roles of histamine and histamine receptors in the context of cancer development and progression. In addition, this review highlights the current progress and foundational developments in the field of cancer immunotherapy in combination with histamine and pharmacological compounds targeting histamine receptors.

Impact of histamine H4 receptor deficiency on the modulation of T cells in a murine breast cancer model.

BACKGROUND: The histamine H4 receptor (H4R) is preferentially expressed in immune cells and is a potential therapeutic target for inflammatory and autoimmune diseases. This study aimed at further exploring the role of H4R in the immunobiology of breast cancer. METHODS: We used wild type (WT) and H4R deficient mice (KO) to evaluate whether H4R genotypes show a different distribution of T cell subsets in spleens, tumours and tumour draining lymph nodes (TDLN) in a syngeneic ErbB2-positive breast cancer model developed orthotopically with LM3 cells and its impact on tumour growth. RESULTS: The presence of tumours had a differential impact on the distribution of T cells in TDLN from KO mice compared to WT ones. At day 21 post-inoculation (p.i.) of cells, despite no significant changes in the tumour weight, TDLN from KO mice showed a significantly increased proportion of CD8+ T cells compared to WT mice. At day 38 p.i. of cells a reduced tumour weight was evident in KO mice. This was accompanied by a decreased proportion of CD4+CD25+FoxP3+ regulatory T cells in TDLN of KO compared to WT mice. Tumour-bearing KO mice showed a better survival compared to WT mice. CONCLUSIONS: H4R-mediated mechanisms may modulate the immune tumour microenvironment, promoting an immunosuppressive milieu. Results suggest that H4R could be explored as an immunotherapeutic target with potential benefit in combination with immunotherapy. Further preclinical and clinical studies are necessary to confirm this hypothesis.

Histamine H4 receptor agonists induce epithelial-mesenchymal transition events and enhance mammosphere formation via Src and TGF-ß signaling in breast cancer cells.

Epithelial-mesenchymal transition (EMT) contributes to cell invasion and metastasis during the progression of epithelial cancers. Though preclinical evidence suggests a role for histamine H4 receptor (H4R) in breast cancer growth, its function in the EMT is less known. In this study we proposed to investigate the effects of H4R ligands on EMT and mammosphere formation as a surrogate assay for cancer stem cells in breast cancer cells with different invasive phenotype. We also investigated the participation of Src and TGF-ß signaling in these events. Breast cancer cells were treated with the H4R agonists Clobenpropit, VUF8430 and JNJ28610244 and the H4R antagonist JNJ7777120. Immunodetection studies showed cytoplasmic E-cadherin, cytoplasmic and nuclear beta-catenin, nuclear Slug and an increase in vimentin and α-smooth muscle actin expression. There was also an enhancement in cell migration and invasion assessed by transwell units. All these effects were prevented by JNJ7777120. Moreover, H4R agonists induced an increase in phospho-Src levels detected by Western blot. Results revealed the involvement of phospho-Src in EMT events. Upon treatment with H4R agonists there was an increase in phospho-ERK1/2 and TGF-ß1 levels by Western blot, in Smad2/3 positive nuclei by indirect immunofluorescence, and in tumor spheres formation by the mammosphere assay. Notably, the selective TGF-ß1 kinase/activin receptor-like kinase inhibitor A83-01 blocked these effects. Moreover, cells derived from mammospheres exhibited higher Slug expression and enhanced migratory behavior. Collectively, findings support the interaction between H4R and TGF-ß receptor signaling in the enhancement of EMT features and mammosphere formation and point out intracellular TGF-ß1 as a potential mediator of these events.

Stability of the Salmonella Typhimurium rcsC11 mutant under different stress conditions.

The virulence genes of Salmonella are modulated during infection by several regulatory systems, and the RcsCDB system is one of the most important of these. The S. Typhimurium EG14873 (rcsC11) strain harbours the rcsC11 point mutation, displaying a constitutive activation of this system, which is characterized by mucoid colonies and attenuated virulence phenotypes. In this work, the stability of the rcsC11 mutation was analysed under stress conditions. Under acid and anaerobic stresses, we observed the appearance of small and non-mucoid colonies of the rcsC11 strain. The sequencing of the rcsC gene from these colonies showed that the mutation is conserved. Moreover, we found that small colonies were also generated when the wild-type strain grew in acid and anaerobic conditions. It is worth noting that the transition from normal to atypical colonies of both strains only took place after several days of incubation and was not observed during eukaryotic cell infection. Therefore, the appearance of these atypical colonies is a characteristic feature of S. Typhimurium strains under stressful situations and does not involve a reversion of the rcsC11 allele and nor does it imply any risk to mammalian cells. Therefore, we propose that the S. Typhimurium rcsC11 strain is a good candidate for the development of attenuated vaccines.

Study of the antitumour effects and the modulation of immune response by histamine in breast cancer.

BACKGROUND: The aim of this work was to improve the knowledge of the role of histamine in breast cancer by assessing the therapeutic efficacy of histamine and histamine H4 receptor (H4R) ligands in a triple-negative breast cancer (TNBC) model developed in immunocompetent hosts. By using publicly available genomic data, we further investigated whether histidine decarboxylase (HDC) could be a potential biomarker. METHODS: Tumours of 4T1 TNBC cells were orthotopically established in BALB/c mice. Treatments employed (mg kg-1): histamine (1 and 5), JNJ28610244 (H4R agonist, 1 and 5) and JNJ7777120 (H4R antagonist, 10). RESULTS: Increased HDC gene expression is associated with better relapse-free and overall survival in breast cancer patients. Histamine treatment (5 mg kg-1) of 4T1 tumour-bearing mice reduced tumour growth and increased apoptosis. Although no immunomodulatory effects were observed in wild-type mice, significant correlations between tumour weight and cytotoxic lymphocyte infiltration were detected in H4R knockout mice. H4R agonist or antagonist differentially modulated tumour growth and immunity in 4T1 tumour-bearing mice. CONCLUSIONS: Histamine plays a complex role and stands out as a promising drug for TNBC treatment, which deserves to be tested in clinical settings. HDC expression level is associated with clinicopathological characteristics, suggesting a prognostic value in breast cancer.

RcsB-dependent effects on nar operon regulation during the aerobic growth of Salmonella Typhimurium.

The intracellular pathogen Salmonella is an important cause of human foodborne diseases worldwide. Salmonella takes advantage of the phosphorelay regulatory systems to survive in the hostile environment of the host's gastrointestinal tract. It has been reported that the nitrate reductase Z (NR-Z), encoded by the narUZYV operon, is required during Salmonella transition to anaerobic environments and is constitutively produced at low levels, but little is known about the regulatory mechanism involved in the operon gene expression. In this work, we found that the RcsCDB system is activated by high concentrations of specific sugars as a carbon source. In this activation state, the RcsCDB system participates in the negative control of narUZYWV expression. This control strategy occurs during exponential growth when the carbon source is high, allowing for normal aerobic respiration. The RcsCDB system's participation in aerobic respiration is necessary to ensure efficient metabolism and optimal energy consumption when the bacteria are growing exponentially.

Transcriptional regulation of the Salmonella enterica std fimbrial operon by the RcsCDB system.

In Salmonella enterica serovar Typhimurium, the RcsCDB regulatory system controls the expression of genes involved in synthesis of colanic acid, formation of flagella and virulence. Here, we show that activation of the RcsCDB system downregulates expression of std, an operon that encodes fimbriae involved in Salmonella attachment to the mucus layer in the large intestine. Bioinformatic analysis predicts the existence of an RcsB-binding site located 180 bp upstream to the +1 transcription start site of the std promoter, and electrophoretic mobility shift assays confirm that RcsB binds the std promoter region in vitro. This study adds RcsB to the list of regulators of std transcription and provides an example of modulation of fimbriae synthesis by a signal transduction system.

Cross-talk between the RcsCDB and RstAB systems to control STM1485 gene expression in Salmonella Typhimurium during acid-resistance response.

Bacterial survive and respond to adverse changes in the environment by regulating gene transcription through two-component regulatory systems. In Salmonella Typhimurium the STM1485 gene expression is induced under low pH (4.5) during replication inside the epithelial host cell, but it is not involved in sensing or resisting to this condition. Since the RcsCDB system is activated under acidic conditions, we investigated whether this system is able to modulate STM1485 expression. We demonstrated that acid-induced activation of the RcsB represses STM1485 transcription by directly binding to the promoter. Under the same condition, the RstA regulator activates the expression of this gene. Physiologically, we observed that RcsB-dependent repression is required for the survival of bacteria when they are exposed to pancreatic fluids. We hypothesized that STM1485 plays an important role in Salmonella adaptation to pH changes, during transition in the gastrointestinal tract. We suggest that bacteria surviving the gastrointestinal environment invade the epithelial cells, where they can remain in vacuoles. In this new environment, acidity and magnesium starvation activate the expression of the RstA regulator in a PhoPQ-dependent manner, which in turn induces STM1485 expression. These levels of STM1485 allow increased bacterial replication within vacuoles to continue the course of infection.

Regulatory Effect of SlyA on rcsB Expression in Salmonella enterica Serovar Typhimurium.

The Salmonella enterica serovar Typhimurium RcsCDB system regulates the synthesis of colanic acid and the flagellum as well as the expression of virulence genes. We previously demonstrated that the rcsC11 mutant, which constitutively activates the RcsB regulator, attenuates Salmonella virulence in an animal model. This attenuated phenotype was also produced by deletion of the slyA gene. In this work, we investigated if this antagonistic behavior is produced by modulating the expression of both regulator-encoding genes. We demonstrated that SlyA overproduction negatively regulates rcsB transcription. A bioinformatics analysis enabled us to identify putative SlyA binding sites on both promoters, P rcsDB and P rcsB , which control rcsB transcriptional levels. We also determined that SlyA is able to recognize and bind to these predicted sites to modulate the activity of both rcsB promoters. According to these results, SlyA represses rcsB transcription by direct binding to specific sites located on the rcsB promoters, thus accounting for the attenuated/virulence antagonistic behaviors. Moreover, we showed that the opposite effect between both regulators also physiologically affects the Salmonella motility phenotype. In this sense, we observed that under SlyA overproduction, P rcsB is repressed, and consequently, bacterial motility is increased. On the basis of these results, we suggest that during infection, the different RcsB levels produced act as a switch between the virulent and attenuated forms of Salmonella Thereby, we propose that higher concentrations of RcsB tilt the balance toward the attenuated form, while absence or low concentrations resulting from SlyA overproduction tilt the balance toward the virulent form.IMPORTANCE The antagonistic behavior of RcsB and SlyA on virulence gene expression led us to hypothesize that there is interplay between both regulators in a regulatory network and these could be considered coordinators of this process. Here, we report that the SlyA virulence factor influences motility behavior by controlling rcsB transcription from the P rcsB promoter. We also demonstrate that SlyA negatively affects the expression of the rcsB gene by direct binding to P rcsDB and P rcsB promoters. We suggest that different levels of RcsB act as a switch between the virulent and attenuated forms of Salmonella, where high concentrations of the regulator tend to tilt the balance toward the attenuated form and low concentrations or its absence tilt it toward the virulent form.

Immunomodulatory role of histamine H4 receptor in breast cancer.

BACKGROUND: Although the role of histamine H4 receptor (H4R) in immune cells is being extensively investigated, its immunomodulatory function in cancer is completely unknown. This study aimed to investigate the role of H4R in antitumour immunity in a model of triple-negative breast cancer. METHODS: We evaluated growth parameters, histological characteristics and the composition of tumour, splenic and tumour draining lymph node (TDLN) immune subsets, in a syngeneic model, developed orthotopically with 4T1 cells in H4R knockout (H4R-KO) and wild-type mice. RESULTS: Mice lacking H4R show reduced tumour size and weight, decreased number of lung metastases and percentage of CD4+ tumour-infiltrating T cells, while exhibiting increased infiltration of NK cells and CD19+ lymphocytes. Likewise, TDLN of H4R-KO mice show decreased CD4+ T cells and T regulatory cells (CD4+CD25+FoxP3+), and increased percentages of NK cells. Finally, H4R-deficient mice show decreased Tregs in spleens and non-draining lymph nodes, and a negative correlation between tumour weight and the percentages of CD4+, CD19+ and NK splenic cells, suggesting that H4R also regulates antitumour immunity at a systemic level. CONCLUSIONS: This is the first report that demonstrates the participation of H4R in antitumour immunity, suggesting that H4R could be a target for cancer treatment.

Histamine prevents radiation-induced mesenchymal changes in breast cancer cells.

Radiotherapy is a prime option for treatment of solid tumors including breast cancer though side effects are usually present. Experimental evidence shows an increase in invasiveness of several neoplastic cell types through conventional tumor irradiation. The induction of epithelial to mesenchymal transition is proposed as an underlying cause of metastasis triggered by gamma irradiation. Experiments were conducted to investigate the role of histamine on the ionizing radiation-induced epithelial to mesenchymal transition events in breast cancer cells with different invasive phenotype. We also evaluated the potential involvement of Src phosphorylation in the migratory capability of irradiated cells upon histamine treatment. MCF-7 and MDA-MB-231 mammary tumor cells were exposed to a single dose of 2Gy of gamma radiation and five days after irradiation mesenchymal-like phenotypic changes were observed by optical microscope. The expression and subcellular localization of E-cadherin, ß-catenin, vimentin and Slug were determined by immunoblot and indirect immunofluorescence. There was a decrease in the epithelial marker E-cadherin expression and an increase in the mesenchymal marker vimentin after irradiation. E-cadherin and ß-catenin were mainly localized in cytoplasm. Slug positive nuclei, matrix metalloproteinase-2 activity and cell migration and invasion were significantly increased. In addition, a significant enhancement in Src phosphorylation/activation could be determined by immunoblot in irradiated cells. MCF-7 and MDA-MB-231 cells also received 1 or 20µM histamine during 24h previous to be irradiated. Notably, pre-treatment of breast cancer cells with 20µM histamine prevented the mesenchymal changes induced by ionizing radiation and also reduced the migratory behavior of irradiated cells decreasing phospho-Src levels. Collectively, our results suggest that histamine may block events related to epithelial to mesenchymal transition in irradiated mammary cancer cells and open a perspective for the potential use of histamine to improve radiotherapy efficacy.

The RcsCDB regulatory system plays a crucial role in the protection of Salmonella enterica serovar Typhimurium against oxidative stress.

Dps, the most abundant protein during the stationary growth phase, in Salmonella enterica is required for resistance to reactive oxygen species produced by the host during infection. It has been reported that in Salmonella dps expression is controlled by RpoS and Fur proteins. However, the regulation and function of Dps remain to be resolved. In the present work we demonstrate that activation of the complex RcsCDB regulatory system increases dps expression during exponential growth of Salmonella. In addition, we show that such dps upregulation produces high levels of H2O2 resistance. This phenotype allows the bacteria to avoid reactive oxygen species killing at early stages of growth, thus protecting its genetic material.

A novel insight on signal transduction mechanism of RcsCDB system in Salmonella enterica serovar typhimurium.

The RcsCDB system of Salmonella enterica serovar Typhimurium is implicated in the control of capsule and flagella synthesis. The hybrid sensor RcsC, the phosphotransferase RcsD and the RcsB regulator, constitute the main components of the RcsCDB system. The proposed Rcs signaling cascade involves the autophosphorylation of RcsC and the transfer of the phosphate group to RcsB, mediated by RcsD. We previously reported that the overexpression of rcsB repress the transcription of rcsD by an autoregulation mechanism. Moreover, we demonstrated that during the rcsD repression, the RcsB-dependent flagellar modulation remained active. These results suggest that the Rcs phosphorelay mechanism occurs even in the absence of RcsD. In this work, we established the existence of two alternative phosphorelay pathways driving activation of this system. We demonstrated that RcsC and RcsD can act as histidine kinase proteins which, after autophosphorylated, are able to independently transfer the phosphate to RcsB. Our results suggest that these pathways could be activated by different environmental signals, leading different levels of RcsB-phosphorylated to produce a differential gene modulation. These findings contribute to a better understanding of the complexity and importance of the Rcs system activation, where more than one phosphate flow pathway increases the possibilities to exert gene regulation for a quick environmental changes response.

Macrophage environment turns otherwise MccJ25-resistant Salmonella into sensitive.

BACKGROUND: Microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide produced by Escherichia coli (E. coli). MccJ25 enters into the sensitive E. coli strains by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD and SbmA. The resistance of Salmonella enterica serovar Typhimurium (S. Typhimurium) to MccJ25 is attributed to the inability of its FhuA protein to incorporate the antibiotic into the cell. RESULTS: In this work we demonstrate that S. Typhimurium becomes notably susceptible to MccJ25 when replicating within macrophages. In order to determine the possible cause of this phenomenon, we studied the sensitivity of S. Typhimurium to MccJ25 at conditions resembling those of the internal macrophage environment, such as low pH, low magnesium and iron deprivation. We observed that the strain was only sensitive to the antibiotic at low pH, leading us to attribute the bacterial sensitization to this condition. A MccJ25-resistant E. coli strain in which fhuA is deleted was also inhibited by the antibiotic at low pH. Then, we could assume that the MccJ25 sensitivity change observed in both E. coli fhuA and S. Typhimurium is mediated by a MccJ25 uptake independent of the FhuA receptor. Moreover, low pH incubation also sensitized S. Typhimurium to the hydrophobic antibiotic novobiocin, which does not affect enteric bacteria viability because it is unable to penetrate the bacterial outer membrane. This observation supports our hypothesis about low pH producing a modification in the bacterial membrane permeability that allows an unspecific MccJ25 uptake. On the other hand, MccJ25 inhibited S. Typhimurium when cells were preincubated in acidic pH medium and then treated at neutral pH with the antibiotic. CONCLUSIONS: Our results suggest that acidic condition does not alter MccJ25 hydrophobicity but irreversibly modifies bacterial membrane permeability. This would allow an unspecific antibiotic uptake into the cell.From our data it is possible to infer that intracellular pathogenic strains, which are in vitro resistant to MccJ25, could become susceptible ones in vivo. Therefore, the MccJ25 action spectrum would be broader than what in vitro experiments indicate.