Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis

The catalytic adenyl cyclase (AC) domain of the protein CyaA from Bordetella pertussis is activated by interaction with the C terminal lobe of calmodulin (C-CaM). The AC/C-CaM complex displays an elongated shape, but hydrodynamics measurements on the isolated AC domain allowed to characterize the shape of the protein as spherical. Here, we study by molecular dynamics simulations the complexes between AC and the apo and Ca(2+)-loaded C-CaM, as well as the isolated AC, to characterize the features of AC conformational variability and of AC/C-CaM interaction. The removal of calcium ions from C-CaM increases the AC flexibility, but the removal of C-CaM induces a dramatic drift of the AC conformation. Isolated AC conformations show a general tendency to become less elongated, as the two protein extremities (regions SA and CB) tend to get closer. An analysis of the energetic influences between the C-CaM and the AC regions shows a simple influence scheme, in agreement with the high affinity of AC to CaM. In this scheme, a single influence is observed from C-CaM to the region CA of the AC domain. This influence is correlated to the presence of hydrogen bonds involving residues from C-CaM, and from regions CA, C-terminal tail, and catalytic loop of AC. This study reveals a C-CaM/AC interaction picture where C-CaM stabilizes AC by a steric hindrance on the conformational drift of SA, whereas the Ca(2+) ions allow further stabilization by the establishment of a hydrogen bond network extending from C-CaM to the AC catalytic loop.     

The role of proteases in the differentiation of Acanthamoeba castellanii

Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii . Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.     

Pathogen–pathogen interactions: a comparative study of Escherichia coli interactions with the clinical and environmental isolates of Acanthamoeba

In this study, we compared the interactions of invasive and non-invasive strains of E. coli with clinical and environmental isolates of Acanthamoeba. The environmental isolate of Acanthamoeba exhibited significantly higher association with E. coli compared with the clinical isolates of Acanthamoeba. The ratio of E. coli per amoebae was more than 8-fold higher in the environmental isolate compared with the clinical isolates of Acanthamoeba. Interestingly, non-pathogenic environmental Acanthamoeba showed uptake and/or survival of the non-invasive E. coli. In contrast, clinical isolates of Acanthamoeba did not support uptake and/or survival of non-invasive E. coli. Using several mutants derived from K1, we demonstrated that outer membrane protein A (OmpA) and lipopolysaccharide (LPS) are crucial bacterial determinants responsible for E. coli K1 interactions and in the intracellular survival of E. coli in Acanthamoeba. The use of Acanthamoeba as a model to study E. coli K1 pathogenesis and to understand bacterial immune evasion strategies is discussed further.     

Cellulose biosynthesis pathway is a potential target in the improved treatment of <Emphasis Type="Italic">Acanthamoeba</Emphasis> keratitis

Acanthamoeba is an opportunistic protozoan pathogen that can cause blinding keratitis as well as fatal granulomatous encephalitis. One of the distressing aspects in combating Acanthamoeba infections is the prolonged and problematic treatment. For example, current treatment against Acanthamoeba keratitis requires early diagnosis followed by hourly topical application of a mixture of drugs that can last up to a year. The aggressive and prolonged management is due to the ability of Acanthamoeba to rapidly adapt to harsh conditions and switch phenotypes into a resistant cyst form. One possibility of improving the treatment of Acanthamoeba infections is to inhibit the ability of these parasites to switch into the cyst form. The cyst wall is partially made of cellulose. Here, we tested whether a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), can enhance the effects of the antiamoebic drug pentamidine isethionate (PMD). Our findings revealed that DCB can block Acanthamoeba encystment and may improve the antiamoebic effects of PMD. Using in vitro assays, the findings revealed that DCB enhanced the inhibitory effects of PMD on Acanthamoeba binding to and cytotoxicity of the host cells, suggesting the cellulose biosynthesis pathway as a novel target for the improved treatment of Acanthamoeba infections.     

Allosteric Activation of Bordetella pertussis Adenylyl Cyclase by Calmodulin: MOLECULAR DYNAMICS AND MUTAGENESIS STUDIES*

Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg³³⁸, Asn³⁴⁷, and Asp³⁶⁰) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn³⁴⁷; ACm1A), two (Arg³³⁸ and Asp³⁶⁰; ACm2A), or three residues (Arg³³⁸, Asn³⁴⁷, and Asp³⁶⁰; ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3A·C-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.     

Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin

Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, secretes several virulence factors, among which is the adenylate cyclase toxin (CyaA) that plays a crucial role in the early stages of human respiratory tract colonization. CyaA invades target cells by translocating its catalytic domain directly across the plasma membrane and overproduces cAMP, leading to cell death. The molecular process leading to the translocation of the catalytic domain remains largely unknown. We have previously shown that the catalytic domain per se, AC384, encompassing residues 1–384 of CyaA, did not interact with lipid bilayer, whereas a longer polypeptide, AC489, spanning residues 1–489, binds to membranes and permeabilizes vesicles. Moreover, deletion of residues 375–485 within CyaA abrogated the translocation of the catalytic domain into target cells. Here, we further identified within this region a peptidic segment that exhibits membrane interaction properties. A synthetic peptide, P454, corresponding to this sequence (residues 454–485 of CyaA) was characterized by various biophysical approaches. We found that P454 (i) binds to membranes containing anionic lipids, (ii) adopts an α-helical structure oriented in plane with respect to the lipid bilayer, and (iii) permeabilizes vesicles. We propose that the region encompassing the helix 454–485 of CyaA may insert into target cell membrane and induce a local destabilization of the lipid bilayer, thus favoring the translocation of the catalytic domain across the plasma membrane.     

Effects of low concentrations of ampicillin in feed on the intestinal Escherichia coli of chicks

Ampicillin in low concentrations (1.7 and 5 g t^-1) was incorporated in the feed given to 1-d-old chicks for 2 weeks. This was sufficient to select, in the intestinal contents, resistant Escherichia coli strains for which the minimum inhibitory concentration of ampicillin was > 100 μg ml^-1. Different clones of E. coli were identified by their biotype, pattern of resistance to antibacterial agents and plasmid profile (designated P-P types). The experiment was repeated twice and the average proportion of ampicillin-resistant P-P types among 72 isolates of E. coli from chicks given feed containing 0, 1.7 and 5 g ampicillin t^-1 were 10%, 31% and 46% respectively.     

Mutations in C12orf57 Cause a Syndromic Form of Colobomatous Microphthalmia

Microphthalmia is an important developmental eye disorder. Although mutations in several genes have been linked to this condition, they only account for a minority of cases. We performed autozygome analysis and exome sequencing on a multiplex consanguineous family in which colobomatous microphthalmia is associated with profound global developmental delay, intractable seizures, and corpus callosum abnormalities, and we identified a homozygous truncating mutation in C12orf57 [c.1A>G; p.Met1?]. In a simplex case with a similar phenotype, we identified compound heterozygosity for the same mutation and another missense mutation [c.152T>A; p.Leu51Gln]. Little is known about C12orf57 but we show that it is expressed in several mouse tissues, including the eye and brain. Our data strongly implicate mutations in C12orf57 in the pathogenesis of a clinically distinct autosomal-recessive syndromic form of colobomatous microphthalmia.     

Profiling Inflammatory Responses with Microfluidic Immunoblotting

Rapid profiling of signaling pathways has been a long sought after goal in biological sciences and clinical medicine. To understand these signaling pathways, their protein components must be profiled. The protein components of signaling pathways are typically profiled with protein immunoblotting. Protein immunoblotting is a powerful technique but has several limitations including the large sample requirements, high amounts of antibody, and limitations in assay throughput. To overcome some of these limitations, we have designed a microfluidic protein immunoblotting device to profile multiple signaling pathways simultaneously. We show the utility of this approach by profiling inflammatory signaling pathways (NFκB, JAK-STAT, and MAPK) in cell models and human samples. The microfluidic immunoblotting device can profile proteins and protein modifications with 5380-fold less antibody compared to traditional protein immunoblotting. Additionally, this microfluidic device interfaces with commonly available immunoblotting equipment, has the ability to multiplex, and is compatible with several protein detection methodologies. We anticipate that this microfluidic device will complement existing techniques and is well suited for life science applications.