José Luis Bocco

Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming toxin from V. cholerae

Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg5−/− cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins.

Molecular mechanisms implicated in galectin-1-induced apoptosis: activation of the AP-1 transcription factor and downregulation of Bcl-2

Galectins are emerging as a new class of bioactive molecules with specific immunomodulatory properties. Galectin-1 (Gal-1), a member of this family, has been shown to induce apoptosis of mature T cells and immature thymocytes. To gain insight into the intracellular signals transduced by Gal-1 upon binding to mature T cells, we investigated whether this protein triggered activation of the dimeric AP-1 transcription factor. A marked increase in the binding of nuclear extracts to synthetic oligonucleotides containing the AP-1 consensus sequence, could be detected by an electrophoretic mobility shift assay, when T cells were cultured for 30 min in the presence of Gal-1. This DNA-binding activity was preceded by a rapid increase in the levels of c-Jun mRNA, as determined by Northern blot analysis. Requirement of AP-1 for Gal-1-induced apoptosis was confirmed by the dose-dependent reduction on the level of DNA fragmentation observed when cells were pre-treated with curcumin (an inhibitor of AP-1 activation) before exposure to Gal-1. Finally, evidence is also provided by Western blot analysis, showing that Gal-1 inhibits Concanavalin A (Con A) induction of Bcl-2 protein. Results presented in this study provide the first experimental evidence regarding AP-1 and Bcl-2 as targets of the signal transduction pathway triggered by Gal-1 and set the basis for a more in depth understanding of the molecular mechanisms of T-cell death regulation. Cell Death and Differentiation (2000) 7, 747–753

A new role for the Krüppel-like transcription factor KLF6 as an inhibitor of c-Jun proto-oncoprotein function

Krüppel-like transcription factors (KLFs) represent one of the most diverse set of regulators in vertebrate organisms. KLF family members are involved in cell proliferation and differentiation control in normal as well as in pathological situations. Here, we demonstrate that KLF6 behaves as a functional antagonist of the c-Jun proto-oncoprotein. Thus, KLF6 overexpression downregulated c-Jun-dependent transcription and a physical interaction between c-Jun and KLF6 was detected. Moreover, cell proliferation induced by c-Jun was significantly decreased by KLF6. The inhibition of c-Jun functions correlates directly with c-Jun protein degradation induced by KLF6. We also show that all KLF6 effects on c-Jun were largely dependent on phorbol ester (TPA/ionomycin) extracellular stimulation, which enhanced KLF6 nuclear translocation and transcriptional activity and modified its phosphorylation status. Our data are consistent with a novel mechanism of KLF6s role as an inhibitor of cell proliferation by counteracting the function of the c-Jun proto-oncoprotein involving enhanced c-Jun degradation by the proteasome-dependent pathway, and further reinforces KLF6 as a potential tumor suppressor gene product.

Mucoidy, Quorum Sensing, Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.

Trypanosoma cruzi-induced immunosuppression : B cells undergo spontaneous apoptosis and lipopolysaccharide (LPS) arrests their proliferation during acute infection

Acute infection with Trypanosoma cruzi is characterized by multiple manifestations of immunosuppression of both cellular and humoral responses. B cells isolated at the acute stage of infection have shown marked impairment in their response to polyclonal activators in vitro. The present work aims at studying the B cell compartment in the context of acute T. cruzi infection to provide evidence for B cell activation, spontaneous apoptosis and arrest of the cell cycle upon mitogenic stimulation as a mechanism underlying B cell hyporesponse. We found that B cells from acutely infected mice, which fail to respond to the mitogen LPS, showed spontaneous proliferation and production of IgM, indicating a high level of B cell activation. Furthermore, these activated B cells also exhibited an increase in Fas expression and apoptosis in cultures without an exogenous stimulus. On the other hand, B cells from early acute and chronic infected mice did not present activation or apoptosis, and were able to respond properly to the mitogen. Upon in vitro stimulation with LPS, B cells from hyporesponder mice failed to progress through the cell cycle (G0/G1 arrest), nor did they increase the levels of apoptosis. These results indicate that B cell apoptosis and cell cycle arrest could be the mechanisms that control intense B cell expansion, but at the same time could be delaying the emergence of a specific immune response against the parasite.

Evolution and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Epidemic and Sporadic Clones in Cordoba, Argentina

ABSTRACT Since 1999, a new, epidemic, methicillin-resistant Staphylococcus aureus (MRSA) strain, named the “Cordobes clone,” has emerged in Argentina and coexists with the pandemic Brazilian clone. The purpose of this study was to determine the stability over time of the new clone and to investigate its evolutionary relationship with epidemic international MRSA lineages and with other MRSA and methicillin-susceptible S. aureus (MSSA) major clones distributed in this region. One hundred three MRSA isolates recovered in 2001 from Cordoba, Argentina, hospitals and 31 MSSA strains collected from 1999 to 2002 were analyzed by their antibiotic resistance patterns, phage typing, and pulsed-field gel electrophoresis. Additionally, representative members of most MRSA defined genotypes (A, B, C, E, K, and I) were characterized by multilocus sequence typing (MLST) and spaA and SCCmec typing. The most prevalent MSSA pulsotypes were also analyzed by MLST. Our results support the displacement of the Brazilian clone (sequence type [ST] 239, spaA type WGKAOMQ, SCCmec type IIIA) by the Cordobes clone (ST5, spaA type TIMEMDMGMGMK, SCCmec type I) in the hospital environment. MRSA and MSSA isolates shared only ST5. The data support the origin of the Cordobes clone as a member of a lineage that includes the pediatric and New York/Japan international clones and that is genetically related to the British EMRSA-3 strain. Interestingly, the pediatric clone, isolated from most community-acquired infections in Cordoba, was characterized by ST100, a single-locus variant of ST5 and a new variant of SCCmec type related to SCCmec type IVc.

Co-localization of KLF6 and KLF4 with pregnancy-specific glycoproteins during human placenta development

Pregnancy-specific glycoproteins (PSGs) are major placental proteins essential for the maintenance of normal gestation. However, little is known about their gene expression regulation during placentation. It was previously demonstrated that the human core promoter binding protein recently renamed Krüppel-like factor (KLF) 6 binds to a highly conserved sequence within the PSG promoters and is mainly expressed in human term placenta. Here, we determined the expression pattern of the 13 other KLFs during human placental development. We demonstrate that eight KLFs exhibit specific expression patterns in human placental tissues and membranes, in favor of a functional cooperation of specific KLFs during placentation. In addition, we demonstrate that KLF6, KLF4 and PSG proteins are co-expressed in same cell types of placental villi and membranes. This experimental evidence further strengthens the potential cross talk of both transcription factors for PSG gene regulation in vivo.

Effects of microcystin-LR on the expression of P-glycoprotein in Jenynsia multidentata.

The multixenobiotic resistance phenomenon (MXR) related to the P-glycoprotein multidrug transporter protein (P-gp) has been identified and characterized in several aquatic organisms. In the present work, we prove the presence of a P-gp in liver, gills and brain of Jenynsia multidentata by Western Blot and RT-PCR. A 170 kDa protein has been found in liver and gills while in brain a approximately 80 kDa protein has been detected. The partial nucleotide sequence obtained in this autochthonous fish showed high similarity ranging from 83% to 92% with other fishes. In addition, P-gp expression in this fish was evaluated after time and dose-dependent exposures to the cyanotoxin microcystin-LR. Individuals were exposed to MC-LR at concentrations of 2, 5 and 10 microg L(-1) for 24h and for 6, 12 and 24h at 2 microg L(-1) MC-LR. Changes in P-gp expression were observed in liver, gills and brain. However, this response was tissue specific. Only in gills of J. multidentata P-gp expression, measured either by real-time RT-PCR or Western Blot, was significantly higher compared to controls at most tested times and doses. A 3-fold increase with respect to controls was found at 12h by RT-PCR and after 24h by Western Blot. In dose-dependent experiments the maximum P-gp expression was observed at 2 microg L(-1) MC-LR, measured by both RT-PCR and Western Blot. In the liver, P-gp protein levels were significantly increased after 24h of exposure, at every toxin dose tested. Thus, probably longer exposures would show also significant increases in this tissue. Considering these results we can propose that P-gp belongs to the defence system involved in the response to MC-LR in J. multidentata.

Identification of a Novel Methicillin-Resistant Staphylococcus aureus Epidemic Clone in Córdoba, Argentina, Involved in Nosocomial Infections

ABSTRACT Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are increasingly a main health concern worldwide for hospitalized patients. In addition, the prevalence of community-acquired infection has risen continuously during the last few years. Some MRSA clones spread easier than others within the hospital environment and therefore are frequently implicated in outbreaks. Thus, the spread of a unique epidemic multiresistant clone, the so-called South American clone, is the main cause of nosocomial infections produced by this bacterium in Brazil and in some regions of Argentina, Chile, and Uruguay. In the present work we describe the identification of a novel clone of MRSA that is involved in nosocomial infections and that shows a prevalence as high as that for the South American clone. A total of 53 consecutive single-patient MRSA isolates were recovered during a 3-month period (May to July 1999) from six different hospitals (955 beds) in Córdoba. The isolates were initially typed according the antibiotic resistance and phage susceptibility patterns, followed by genotyping using pulsed-field gel electrophoresis (PFGE). PFGE analysis of the 53 MRSA isolates revealed six major types (A to F) and 25 subtypes. The B-type DNA pattern was indistinguishable from that of the South American epidemic clone observed in 34% of the isolates. A novel highly prevalent clone, showing the A-type DNA pattern and representing 38% of the isolates, was also identified. Moreover, the most frequent subtype of the A clonal family triggered an outbreak in a hospital 2 months later, further confirming its epidemic feature.

Spread of Epidemic MRSA-ST5-IV Clone Encoding PVL as a Major Cause of Community Onset Staphylococcal Infections in Argentinean Children

Background Community-associated methicillin-resistant Staphylococcus aureus-(CA-MRSA) strains have emerged in Argentina. We investigated the clinical and molecular evolution of community-onset MRSA infections (CO-MRSA) in children of Córdoba, Argentina, 2005–2008. Additionally, data from 2007 were compared with the epidemiology of these infections in other regions of the country. Methodology/Principal Findings Two datasets were used: i) lab-based prospective surveillance of CA-MRSA isolates from 3 Córdoba pediatric hospitals-(CBAH1-H3) in 2007–2008 (compared to previously published data of 2005) and ii) a sampling of CO-MRSA from a study involving both, healthcare-associated community-onset-(HACO) infections in children with risk-factors for healthcare-associated infections-(HRFs), and CA-MRSA infections in patients without HRFs detected in multiple centers of Argentina in 2007. Molecular typing was performed on the CA-MRSA-(n: 99) isolates from the CBAH1-H3-dataset and on the HACO-MRSA-(n: 51) and CA-MRSA-(n: 213) isolates from other regions. Between 2005–2008, the annual proportion of CA-MRSA/CA-S. aureus in Córdoba hospitals increased from 25% to 49%, P<0.01. Total CA-MRSA infections increased 3.6 fold-(5.1 to 18.6 cases/100,000 annual-visits, P<0.0001), associated with an important increase of invasive CA-MRSA infections-(8.5 fold). In all regions analyzed, a single genotype prevailed in both CA-MRSA (82%) and HACO-MRSA(57%), which showed pulsed-field-gel electrophoresis-(PFGE)-type-“I”, sequence-type-5-(ST5), SCCmec-type-IVa, spa-t311, and was positive for PVL. The second clone, pulsotype-N/ST30/CC30/SCCmecIVc/t019/PVL+, accounted for 11.5% of total CA-MRSA infections. Importantly, the first 4 isolates of Argentina belonging to South American-USA300 clone-(USA300/ST8/CC8/SCCmecIVc/t008/PVL+/ACME−) were detected. We also demonstrated that a HA-MRSA clone-(pulsotype-C/ST100/CC5) caused 2% and 10% of CA-MRSA and HACO-MRSA infections respectively and was associated with a SCCmec type closely related to SCCmecIV(2B&5). Conclusions/Significance The dissemination of epidemic MRSA clone, ST5-IV-PVL+ was the main cause of increasing staphylococcal community-onset infections in Argentinean children (2003–2008), conversely to other countries. The predominance of this clone, which has capacity to express the h-VISA phenotype, in healthcare-associated community-onset cases suggests that it has infiltrated into hospital-settings.