Characterization of dominantly negative mutant ClyA cytotoxin proteins in Escherichia coli

We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.     

Phylogenetic Relationships among Indigenous Sheep Populations in East Asia based on Five Informative Blood Protein and Nonprotein Polymorphisms

The phylogenetic relationships among 23 local sheep breeds and varieties in East Asia were determined based on allele frequency data for five informative and polymorphic blood protein and nonprotein loci (transferrin, arylesterase, hemoglobin-β, X-protein, and potassium transport) using electrophoresis and ion-densitometric techniques. Comparatively striking differences in the frequency distributions, especially of hemoglobin-β and X-protein alleles, were seen between the northern population of the Khalkhas, Bhyanglung, Baruwal, Jakar, Sakten, and five Chinese breeds and the southern population of the Bengal, Kagi, Lampuchhre, Myanmar, and Sipsu breeds. Clustering analyses using UPGMA and NJ methods and principal component analysis, using the data of all populations and loci tested, clearly demonstrated the difference. The two population groups are divided by the boundary of the Himalayas, and each diverged into three subgroups: Mongolian, Tibetan, and Himalayan groups in the north and Indian groups I, II, and III in the south. It was noted that the genetic differentiation of populations was more distinct in the northern group. These findings strongly suggest the existence of at least two large and phylogenetically different gene pools of sheep in East Asia.     

Impact of Community-Based Maternal Health Workers on Coverage of Essential Maternal Health Interventions among Internally Displaced Communities in Eastern Burma: The MOM Project

Background

Access to essential maternal and reproductive health care is poor throughout Burma, but is particularly lacking among internally displaced communities in the eastern border regions. In such settings, innovative strategies for accessing vulnerable populations and delivering basic public health interventions are urgently needed.

Methods

Four ethnic health organizations from the Shan, Mon, Karen, and Karenni regions collaborated on a pilot project between 2005 and 2008 to examine the feasibility of an innovative three-tiered network of community-based providers for delivery of maternal health interventions in the complex emergency setting of eastern Burma. Two-stage cluster-sampling surveys among ever-married women of reproductive age (15–45 y) conducted before and after program implementation enabled evaluation of changes in coverage of essential antenatal care interventions, attendance at birth by those trained to manage complications, postnatal care, and family planning services.

Results

Among 2,889 and 2,442 women of reproductive age in 2006 and 2008, respectively, population characteristics (age, marital status, ethnic distribution, literacy) were similar. Compared to baseline, women whose most recent pregnancy occurred during the implementation period were substantially more likely to receive antenatal care (71.8% versus 39.3%, prevalence rate ratio [PRR] = 1.83 [95% confidence interval (CI) 1.64–2.04]) and specific interventions such as urine testing (42.4% versus 15.7%, PRR = 2.69 [95% CI 2.69–3.54]), malaria screening (55.9% versus 21.9%, PRR = 2.88 [95% CI 2.15–3.85]), and deworming (58.2% versus 4.1%, PRR = 14.18 [95% CI 10.76–18.71]. Postnatal care visits within 7 d doubled. Use of modern methods to avoid pregnancy increased from 23.9% to 45.0% (PRR = 1.88 [95% CI 1.63–2.17]), and unmet need for contraception was reduced from 61.7% to 40.5%, a relative reduction of 35% (95% CI 28%–40%). Attendance at birth by those trained to deliver elements of emergency obstetric care increased almost 10-fold, from 5.1% to 48.7% (PRR = 9.55 [95% CI 7.21–12.64]).

Conclusions

Coverage of maternal health interventions and higher-level care at birth was substantially higher during the project period. The MOM Project''s focus on task-shifting, capacity building, and empowerment at the community level might serve as a model approach for similarly constrained settings. Please see later in the article for the Editors'' Summary     

Biologically active alkylated coumarins from Kayea assamica

Four coumarin derivatives, theraphins A (1), B (2), C (3), and D (4), along with three known xanthones, 2-hydroxyxanthone, 1,7-dihydroxyxanthone, and 5-hydroxy-1-methoxyxanthone, were isolated from the bark of Kayea assamica (Clusiaceae) native to Myanmar. Their structures were determined using spectroscopic and chemical techniques. The absolute configuration of 1 was established by the modified Mosher ester method. Theraphins A (1), B (2), and C (3) exhibited good cytotoxicity against Col2, KB, and LNCaP human cancer cell lines. Theraphin D (4) showed mild activity only against the KB cell line. The coumarins also exhibited mild antimalarial activities.     

Release of the type I secreted alpha-haemolysin via outer membrane vesicles from Escherichia coli

The alpha-haemolysin is an important virulence factor commonly expressed by extraintestinal pathogenic Escherichia coli. The secretion of the alpha-haemolysin is mediated by the type I secretion system and the toxin reaches the extracellular space without the formation of periplasmic intermediates presumably in a soluble form. Surprisingly, we found that a fraction of this type I secreted protein is located within outer membrane vesicles (OMVs) that are released by the bacteria. The alpha-haemolysin appeared very tightly associated with the OMVs as judged by dissociation assays and proteinase susceptibility tests. The alpha-haemolysin in OMVs was cytotoxically active and caused lysis of red blood cells. The OMVs containing the alpha-haemolysin were distinct from the OMVs not containing alpha-haemolysin, showing a lower density. Furthermore, they differed in protein composition and one component of the type I secretion system, the TolC protein, was found in the lower density vesicles. Studies of natural isolates of E. coli demonstrated that the localization of alpha-haemolysin in OMVs is a common feature among haemolytic strains. We propose an alternative pathway for the transport of the type I secreted alpha-haemolysin from the bacteria to the host cells during bacterial infections.     

How Vibrio cholerae survive during starvation

Vibrio cholerae, a Gram-negative, motile, aquatic bacterium, is the causal agent of the diarrheal disease cholera. Cholera is a serious epidemic disease that has killed millions of people and continues to be a major health problem world-wide. The hypothesis that V. cholerae occupies an ecological niche in the estuarine environment requires that this organism is able to survive the dynamics of physiochemical stresses, including nutrient starvation. As a result of these stresses, bacteria in nature often exist in non-growth or very slow growth states with a low metabolic activity. Because microorganisms have little ability to control their environment, environmental changes have led to changes in cell function and structure. Such cellular responses can originate in one of two ways: by changes in genetic constitution or by phenotypic adaptation. In this review, we will focus on the phenotypic responses of V. cholerae of a given genotype to starvation stress.     

Vibrio cholerae O1 Strain TSI-4 Produces the Exopolysaccharide Materials That Determine Colony Morphology,Stress Resistance,and Biofilm Formation

Vibrio cholerae O1 strain TSI-4 (El Tor, Ogawa) can shift to a rugose colony morphology from its normal translucent colony morphology in response to nutrient starvation. We have investigated differences between the rugose and translucent forms of V. cholerae O1 strain TSI-4. Electron microscopic examination of the rugose form of TSI-4 (TSI-4/R) revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the ferritin-stained material was absent around the translucent form of TSI-4 (TSI-4/T). The exopolysaccharide produced by V. cholerae TSI-4/R was found to have a composition of N-acetyl-d-glucosamine, d-mannose, 6-deoxy-d-galactose, and d-galactose (7.4:10.2:2.4:3.0). The expression of an amorphous exopolysaccharide promotes biofilm development under static culture conditions. Biofilm formation by the rugose strain was determined by scanning electron microscopy, and most of the surface of the film was colonized by actively dividing rod cells. The corresponding rugose and translucent strains were compared for stress resistance. By having exopolysaccharide materials, the rugose strains acquired resistance to osmotic and oxidative stress. Our data indicated that an exopolysaccharide material on the surface of the rugose strain promoted biofilm formation and resistance to the effects of two stressing agents.Cholera is a serious epidemic disease that has killed millions of people and continues to be a major health problem worldwide. Vibrio cholerae, the bacterium that causes cholera, is a motile, gram-negative, curved rod with a single polar flagellum. The hypothesis that V. cholerae occupies an ecological niche in the estuarine environment requires that this organism be able to survive the dynamics of various physiochemical changes, including variations in nutrient concentrations. As a response to nutrient depletion, copiotrophic (31, 42), heterotrophic bacteria may undergo considerable morphological, physiological, and chemical changes (13, 22, 23, 26–28). In fact, to survive energy- and nutrient-deprived conditions, non-spore-forming, heterotrophic bacteria are known to undergo an active adaptation program (28). Brown and Williams have provided detailed experimental evidence that the molecular composition of the bacterial cell walls is essentially plastic and is remarkably responsive to the cell’s growth environment (5). Rice et al. (33) discovered that V. cholerae O1 from the Peru epidemic was able to shift to a phenotype having a wrinkled or rugose colony morphology. They also suggested that the V. cholerae rugose phenotype represents a fully virulent survival form of the organism that can persist in the presence of free chlorine. Morris et al. (29) reported that V. cholerae can shift to a rugose colony morphology associated with the expression of an amorphous exopolysaccharide (EPS) that promotes cell aggregation, and they also confirmed that rugose strains displayed resistance to killing by chlorine and complement-mediated serum bactericidal activity. They also indicated that these rugose strains cause human disease. However, the phenotypic characteristics associated with rugose morphology, relationships between these characteristics, and their relative importance in pathogenicity still remained to be identified.A large variety of EPSs are synthesized by gram-negative bacteria. While some have been implicated in the pathogenicity of plant and mammalian hosts, others have not been assigned a function, but many serve a structural role, benefiting the bacterium by enabling attachment to surfaces, improving nutrient acquisition, or providing protection from environmental stresses and host defenses (36). The EPSs cover the surfaces of many gram-negative and gram-positive bacteria. They may form a capsule composed of a high-molecular-weight polysaccharide attached to the cell surface, or they may produce slime either loosely attached to the cell surface or released to the culture fluid. Bacterial cells initiate the process of irreversible adhesion by binding to the surface by using EPS glycocalyx polymers and the development of microcolonies. The eventual production of a continuous biofilm on the colonized surface is a function of cell division within microcolonies and recruitment of bacteria from the planktonic phase. The biofilm concept has drawn attention to the bacterium’s ecological and biotechnological importance (8–11). We must now accept the unequivocal evidence that bacteria respond to changes in their environment by profound phenotypic variations in enzymatic activity, cell wall composition (34), and surface structure (2).In this study, we have isolated the rugose variants of V. cholerae O1 strain TSI-4 from starvation medium and determined EPS expression on the cell surface of the rugose strain by polycationic ferritin-labeled thin-section electron microscopy. While examining the morphological characteristics of these rugose strains, we found that they produced a continuous biofilm on the colonized surface and culture tube walls. Directly sampled, intact biofilms were subjected to electron microscopic analysis. We have also studied the role of the slime polysaccharide of V. cholerae TSI-4 in the bacterium’s resistance to osmotic and oxidative stress.