Efficacy of Indolicidin,Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48 h of incubation in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24 h and 48 h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24 h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

     

Ecology of Listeria monocytogenes and Listeria species in India: the occurrence,resistance to biocides,genomic landscape and biocontrol

Listeria monocytogenes, the causative agent of listeriosis, has been implicated in increasing foodborne outbreaks worldwide. The disease is manifested in various forms ranging from severe sepsis in immune-compromised individuals, febrile gastroenteritis, still birth, abortions and meningoencephalitis. In India, data from studies on the detection and molecular epidemiological analysis of L. monocytogenes are only recently emerging. The presence of Listeria in different ecological niches has been recorded from India, including foods, soil, vegetables, mangrove swamps, seafood, freshwater fishes, clinical cases, and also insects. The organism has also been isolated from women with spontaneous abortions, miscarriage or recurrent obstetric history, aborted foetuses, animal clinical cases and wildlife samples. A novel species of Listeria has also been characterized. Listeria monocytogenes strains isolated from clinical, environmental, and foods showed biofilm-forming abilities. Listeria monocytogenes serotype 4b isolates of ST328, a predominant and unique ST observed in India, was repeatedly isolated from different sources, times, and geographical locations. Here, we reviewed the occurrence of Listeria in different sources in India, its resistance to biocides, and provide epidemiological analysis on its genomic landscape.     

Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution. However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric. To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range. We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 +/- 50 nM at 4 degrees C. To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed. These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant. The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments. These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form.     

Actinomyces israelii endocarditis misidentified as “Diptheroids”

Actinomyces is a rare cause of endocarditis, however misidentification as Cornyebacteria often hampers the diagnosis.     

A high-throughput differential filtration assay to screen and select detergents for membrane proteins

Structural studies on integral membrane proteins are routinely performed on protein-detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and “protein-expensive” process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours.     

The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (enterokinase, factor Xa, human rhinovirus 3C protease, SUMOstar, tobacco etch virus protease, and thrombin) by use of a panel of 94 individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and factor Xa were only affected by a small number of detergents, making them good choices as well.     

CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi) /CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo) /CD16(pos) monocytes in acute liver failure

Changes in monocytes and their subsets (CD14(hi) /CD16(neg) , CD14(hi) /CD16(pos) and CD14(lo) /CD16(pos) ) have been described in several diseases. The combination of CD14, CD16 and HLA-DR has been suggested to discriminate monocytes from the CD16(pos) /HLA-DR(neg) NK-cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA-DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66b(pos) neutrophils, CD56(pos) NKcells, CD19(pos) B-cells, and CD3(pos) T-cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA-DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA-DR expression]. Bland-Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14(hi) /CD16(pos) monocytes was higher (7% vs 4%) whilst the percentage of CD14(lo) /CD16(pos) was lower (1.9% vs. 7%) (P ≤ 0.001); HLA-DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA-DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody-fluorochrome "backbone" targeting HLA-DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody-fluorochrome "backbone" of HLA-DR, CD14, and CD16. CD16(pos) monocytes continue to constitutively express HLA-DR even in conditions where HLA-DR is pathologically reduced on CD14(hi) /CD16(neg) monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico-pathological diseases may allow the development of novel biomarkers or therapeutic strategies. ? 2012 International Society for Advancement of Cytometry.     

Antibacterial efficacy of in-house designed cell-penetrating peptide against multi-drug resistant strains of Salmonella Enteritidis and Salmonella Typhimurium

The in vitro antibacterial efficacy of an in-house designed cell-penetrating peptide (CPP) variant of Cecropin A (1–7)-Melittin (CAMA) (CAMA-CPP) against the characterized multi-drug resistant (MDR) field strains of Salmonella Enteritidis and Salmonella Typhimurium were evaluated and compared with two identified CPPs namely, P7 and APP, keeping CAMA as control. Initially, the minimum inhibitory concentration (MIC) (μg ml⁻¹) of in-house designed CAMA-CPP, APP and CAMA was determined to be 3.91, whereas that of P7 was 7.81; however, the minimum bactericidal concentration (MBC) of all the peptides were twice the MIC. CAMA-CPP and CAMA were found to be stable under different conditions (high-end temperatures, proteinase-K, cationic salts, pH and serum) when compared to the other CPPs. Moreover, CAMA-CPP exhibited negligible cytotoxicity in HEp-2 and RAW 264.7 cell lines as well as haemolysis in the sheep and human erythrocytes with no adverse effects against the commensal gut lactobacilli. In vitro time-kill assay revealed that the MBC levels of CAMA-CPP and APP could eliminate the intracellular MDR-Salmonella infections from mammalian cell lines; however, CAMA and P7 peptides were ineffective. CAMA-CPP appears to be a promising antimicrobial candidate and opens up further avenues for its in vivo clinical translation.     

Catalytic mechanism of the cyclohydrolase activity of human aminoimidazole carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Using recently determined crystal structures of ATIC as a guide, four candidate residues, Lys66, Tyr104, Asp125, and Lys137, were identified for site-directed mutagenesis to study the cyclohydrolase activity of this bifunctional enzyme. Steady-state kinetic experiments on these mutants have shown that none of these residues are absolutely required for catalytic activity; however, they strongly influence the efficiency of the reaction. Since the FAICAR binding site is made up mostly of backbone interactions with highly conserved residues, we postulate that these conserved interactions orient FAICAR in the active site to favor the intramolecular ring closure reaction and that this reaction may be catalyzed by an orbital steering mechanism. Furthermore, it was shown that Lys137 is responsible for the increase in cyclohydrolase activity for dimeric ATIC, which was reported previously by our laboratory. From the experiments presented here, a catalytic mechanism for the cyclohydrolase activity is postulated.