High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil

Carbapenem resistance amongst Acinetobacter spp. has been increasing in the last decade. This study evaluated the outer membrane protein (OMP) profile and production of carbapenemases in 50 carbapenem-resistant Acinetobacter spp. isolates from bloodstream infections. Isolates were identified by API20NE. Minimum inhibitory concentrations (MICs) for carbapenems were determined by broth microdilution. Carbapenemases were studied by phenotypic tests, detection of their encoding gene by polymerase chain reaction (PCR) amplification, and imipenem hydrolysis. Nucleotide sequencing confirming the enzyme gene type was performed using MegaBACE 1000. The presence of OMPs was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and PCR. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). All isolates were resistant to carbapenems. Moreover, 98% of the isolates were positive for the gene encoding the enzyme OXA-51-like, 18% were positive for OXA-23-like (only one isolate did not show the presence of the insertion sequence ISAba1 adjacent to this gene) and 76% were positive for OXA-143 enzyme. Five isolates (10%) showed the presence of the IMP-1 gene. Imipenem hydrolysing activity was detected in only three strains containing carbapenemase genes, comprising two isolates containing the bla(IMP) gene and one containing the bla(OXA-51/OXA-23-like) gene. The OMP of 43 kDa was altered in 17 of 25 strains studied, and this alteration was associated with a high meropenem MIC (256 mu g/mL) in 5 of 7 strains without 43 kDa OMP. On the other hand, decreased OMP 33-36 kDa was found in five strains. The high prevalence of OXA-143 and alteration of OMPs might have been associated with a high level of carbapenem resistance. (C) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Immune receptors and adhesion molecules in human pulmonary leptospirosis

Pulmonary involvement in leptospirosis has been increasingly reported in the last 20 years, being related to the severity and mortality of the disease. The pathogenesis of pulmonary hemorrhage in leptospirosis is not understood. Lung endothelial cells have been proposed as targets in the pathogenesis of lung involvement in leptospirosis through the activation of Toll-like receptor 2 or the complement system, which stimulates the release of cytokines that lead to the activation of adhesion molecules. The aim of this study was to investigate the involvement of immune pathways and of the intercellular and vascular cell adhesion molecules (intercellular adhesion molecule and vascular cell adhesion molecule, respectively) in the lungs of patients with pulmonary involvement of leptospirosis. We studied the lungs of 18 patients who died of leptospirosis and compared them with 2 groups of controls: normal and noninfectious hemorrhagic lungs. Using immunohistochemistry and image analysis, we quantified the expression of the C3a anaphylatoxin receptor, intercellular adhesion molecule, vascular cell adhesion molecule, and Toll-like receptor 2 in small pulmonary vessels and in the alveolar septa. There was an increased expression of intercellular adhesion molecule (P <.03) and C3a anaphylatoxin receptor (P <.008) in alveolar septa in the leptospirosis group compared with the normal and hemorrhagic controls. In the vessels of the leptospirosis group, there was an increased expression of intercellular adhesion molecule (P=.004), vascular cell adhesion molecule (P=.030), and Toll-like receptor 2 (P=.042) compared with the normal group. Vascular cell adhesion molecule expression in vessels was higher in the leptospirosis group compared with the hemorrhagic group (P=.015). Our results indicate that immune receptors and adhesion molecules participate in the phenomena leading to pulmonary hemorrhage in leptospirosis. (C) 2012 Elsevier Inc. All rights reserved.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Differential expression of toll-like receptor mRNAs in recurrent aphthous ulceration

BACKGROUND: Toll-like receptors (TLR) are membrane proteins that recognize conserved molecules derived from bacterial, viral, fungal or host tissues. They are responsible for promoting the production of cytokines and chemokines, increasing the expression of costimulatory molecules and influencing the T Helper response (Th) toward either a Th1 or Th2 profile, thereby modulating the regulatory T cell response and controlling the integrity of the epithelial barrier. The key factors responsible for increased susceptibility to recurrent aphthous ulceration (RAU) are unclear, and because TLRs are involved in both immune regulation and control of the epithelial barrier, a deficiency in TLR activity is likely to cause increased susceptibility. METHODS: We investigated the gene expression of TLRs one through 10 in tissue samples and peripheral blood mononuclear cells (PBMC) of RAU patients in comparison to healthy controls using real-time quantitative reverse transcription PCR. RESULTS: The analysis of mRNA expression levels in oral lesion showed significant (P < 0.01) overexpression of the TLR2(similar to 6-fold) gene and decreased expression of the TLR3 (similar to 5-fold) and TLR5 (similar to 6-fold) genes in comparison with healthy oral mucosa. The analysis of mRNA expression in PBMC indicated a down-regulation of TLR5 gene expression in the cells from RAU patients (P < 0.05; similar to 2-fold). CONCLUSION: Our results support the hypothesis that a subset of RAU patients has fewer TLR expression that have been tentatively implicated in antiinflammatory effects. This derangement of TLR gene expression may cause an overlay exuberant inflammation reaction in situations where normal individuals are resistant. J Oral Pathol Med (2012) 41: 8085

OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical beta-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 +/- 871.03 nM and 1,239.23 +/- 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 +/- 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coil BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K-D) of 292 +/- 24 nM and 157 +/- 35 nM, respectively. Moreover, the Lsa30 is a plasminogen (PLC) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. (C) 2012 Elsevier Ltd. All rights reserved.

Proteomic profiling of the rat hypothalamus

Background: The hypothalamus plays a pivotal role in numerous mechanisms highly relevant to the maintenance of body homeostasis, such as the control of food intake and energy expenditure. Impairment of these mechanisms has been associated with the metabolic disturbances involved in the pathogenesis of obesity. Since rodent species constitute important models for metabolism studies and the rat hypothalamus is poorly characterized by proteomic strategies, we performed experiments aimed at constructing a two-dimensional gel electrophoresis (2-DE) profile of rat hypothalamus proteins. Results: As a first step, we established the best conditions for tissue collection and protein extraction, quantification and separation. The extraction buffer composition selected for proteome characterization of rat hypothalamus was urea 7 M, thiourea 2 M, CHAPS 4%, Triton X-100 0.5%, followed by a precipitation step with chloroform/methanol. Two-dimensional (2-D) gels of hypothalamic extracts from four-month-old rats were analyzed; the protein spots were digested and identified by using tandem mass spectrometry and database query using the protein search engine MASCOT. Eighty-six hypothalamic proteins were identified, the majority of which were classified as participating in metabolic processes, consistent with the finding of a large number of proteins with catalytic activity. Genes encoding proteins identified in this study have been related to obesity development. Conclusion: The present results indicate that the 2-DE technique will be useful for nutritional studies focusing on hypothalamic proteins. The data presented herein will serve as a reference database for studies testing the effects of dietary manipulations on hypothalamic proteome. We trust that these experiments will lead to important knowledge on protein targets of nutritional variables potentially able to affect the complex central nervous system control of energy homeostasis.

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5 gamma 2 chain as well as alpha 3, beta 1 subunits of alpha 3 beta 1 heterodimer and beta 4 subunit of integrin alpha 6 beta 4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of beta 1, beta 4 and alpha 3 integrins, and SISCCs lacked beta 1, beta 4 and alpha 3 integrins in the invasive front. AC cases were negative for the Ln-5 gamma 2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5 gamma 2 chain expression in the invasive front of the tumor. Integrin beta 1, beta 4 and alpha 3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5 gamma 2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype. (C) 2012 Elsevier GmbH. All rights reserved.

Tumour cells incorporate exosomes derived from dendritic cells through a mechanism involving the tetraspanin CD9

Exosomes (Exos) are secreted nanovesicles that contain membrane proteins and genetic material, which can be transferred between cells and contribute to their communication in the body. We show that Exos, obtained from mature human dendritic cells (DCs), are incorporated by tumour cells, which after Exos treatment, acquire the expression of HLA‐class I, HLA‐class II, CD86, CD11c, CD54 and CD18. This incorporation reaches its peak eight hours after treatment, can be observed in different cell tumour lines (SK‐BR‐3, U87 and K562) and could be a means to transform non‐immunogenic into immunogenic tumour cells. Interestingly, tetraspanins, which are expressed by the tumour cells, have their surface level decreased after Exo treatment. Furthermore, the intensity of Exo incorporation by the different tumour cell lines was proportional to their CD9 expression levels and pretreatment of Exos with anti‐CD9 decreased their incorporation (by SK‐BR‐3 cells). This modification of tumour cells by DC‐derived Exos may allow their use in new immunotherapeutic approaches to cancer. Furthermore, by showing the involvement of CD9 in this incorporation, we provide a possible selection criterion for tumours to be addressed by this strategy

Assignment of Putative Functions to Membrane "Hypothetical Proteins" from the Trypanosoma cruzi Genome

Protozoan parasites cause thousands of deaths each year in developing countries. The genome projects of these parasites opened a new era in the identification of therapeutic targets. However, the putative function could be predicted for fewer than half of the protein-coding genes. In this work, all Trypanosoma cruzi proteins containing predicted transmembrane spans were processed through an automated computational routine and further analyzed in order to assign the most probable function. The analysis consisted of dissecting the whole predicted protein in different regions. More than 5,000 sequences were processed, and the predicted biological functions were grouped into 19 categories according to the hits obtained after analysis. One focus of interest, due to the scarce information available on trypanosomatids, is the proteins involved in signal-transduction processes. In the present work, we identified 54 proteins belonging to this group, which were individually analyzed. The results show that by means of a simple pipeline it was possible to attribute probable functions to sequences annotated as coding for "hypothetical proteins.'' Also, we successfully identified the majority of candidates participating in the signal-transduction pathways in T. cruzi.

Mitochondrial Swelling and Incipient Outer Membrane Rupture in Preapoptotic and Apoptotic Cells

Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM rupture is not caused by a membrane stretching promoted by a markedly swollen matrix. It is shown that the widths of the ruptured regions of the OMM vary from 6 to 250 nm. Independent of the perforation size, herniation of the mitochondrial matrix appeared to have resulted in pushing the IMM through the perforation. A large, long focal herniation of the mitochondrial matrix, covered with the IMM, was associated with a rupture of the OMM that was as small as 6 nm. Contextually, the collapse of the selective permeability of the IMM may precede or follow the release of the mitochondrial proteins of the intermembrane space into the cytoplasm. When the MPT is a late event, exit of the intermembrane space proteins to the cytoplasm is unimpeded and occurs through channels that transverse the outer membrane, because so far, the inner membrane is impermeable. No channel within the outer membrane can expose to the cytoplasm a permeable inner membrane, because it would serve as a conduit for local herniation of the mitochondrial matrix. Anat Rec, 2012. (c) 2012 Wiley Periodicals, Inc.

Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring a-fetoprotein, alpha-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-alpha-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-alpha-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions

Background: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (K-D) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a K-D of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca2+. Recent crystal structures have been obtained for the protein in the apo-and Ca2+-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca2+ and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca2+ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca2+ affinity as the wild-type protein. We then evaluated if Ca2+ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca2+ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.

Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clusters within proteobacteria

Abstract Background Heavy metal Resistance-Nodulation-Division (HME-RND) efflux systems help Gram-negative bacteria to keep the intracellular homeostasis under high metal concentrations. These proteins constitute the cytoplasmic membrane channel of the tripartite RND transport systems. Caulobacter crescentus NA1000 possess two HME-RND proteins, and the aim of this work was to determine their involvement in the response to cadmium, zinc, cobalt and nickel, and to analyze the phylogenetic distribution and characteristic signatures of orthologs of these two proteins. Results Expression assays of the czrCBA operon showed significant induction in the presence of cadmium and zinc, and moderate induction by cobalt and nickel. The nczCBA operon is highly induced in the presence of nickel and cobalt, moderately induced by zinc and not induced by cadmium. Analysis of the resistance phenotype of mutant strains showed that the ΔczrA strain is highly sensitive to cadmium, zinc and cobalt, but resistant to nickel. The ΔnczA strain and the double mutant strain showed reduced growth in the presence of all metals tested. Phylogenetic analysis of the C. crescentus HME-RND proteins showed that CzrA-like proteins, in contrast to those similar to NczA, are almost exclusively found in the Alphaproteobacteria group, and the characteristic protein signatures of each group were highlighted. Conclusions The czrCBA efflux system is involved mainly in response to cadmium and zinc with a secondary role in response to cobalt. The nczCBA efflux system is involved mainly in response to nickel and cobalt, with a secondary role in response to cadmium and zinc. CzrA belongs to the HME2 subfamily, which is almost exclusively found in the Alphaproteobacteria group, as shown by phylogenetic analysis. NczA belongs to the HME1 subfamily which is more widespread among diverse Proteobacteria groups. Each of these subfamilies present distinctive amino acid signatures.