Penicillin Kills Chlamydia following the Fusion of Bacteria with Lysosomes and Prevents Genital Inflammatory Lesions in C. muridarum-Infected Mice

The obligate intracellular bacterium Chlamydia exists as two distinct forms. Elementary bodies (EBs) are infectious and extra-cellular, whereas reticulate bodies (RBs) replicate within a specialized intracellular compartment termed an ‘inclusion’. Alternative persistent intra-cellular forms can be induced in culture by diverse stimuli such as IFNγ or adenosine/EHNA. They do not grow or divide but revive upon withdrawal of the stimulus and are implicated in several widespread human diseases through ill-defined in vivo mechanisms. β-lactam antibiotics have also been claimed to induce persistence in vitro. The present report shows that upon penicillin G (pG) treatment, inclusions grow as fast as those in infected control cells. After removal of pG, Chlamydia do not revert to RBs. These effects are independent of host cell type, serovar, biovar and species of Chlamydia. Time-course experiments demonstrated that only RBs were susceptible to pG. pG-treated bacteria lost their control over host cell apoptotic pathways and no longer expressed pre-16S rRNA, in contrast to persistent bacteria induced with adenosine/EHNA. Confocal and live-video microscopy showed that bacteria within the inclusion fused with lysosomal compartments in pG-treated cells. That leads to recruitment of cathepsin D as early as 3 h post pG treatment, an event preceding bacterial death by several hours. These data demonstrate that pG treatment of cultured cells infected with Chlamydia results in the degradation of the bacteria. In addition we show that pG is significantly more efficient than doxycycline at preventing genital inflammatory lesions in C. muridarum-C57Bl/6 infected mice. These in vivo results support the physiological relevance of our findings and their potential therapeutic applications.     

A Cell-surface Phylome for African Trypanosomes

The cell surface of Trypanosoma brucei, like many protistan blood parasites, is crucial for mediating host-parasite interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the related trypanosomatid parasites T. cruzi and Leishmania major suggest that the cell-surface proteome of T. brucei is largely taxon-specific. Here we compare genes predicted to encode cell surface proteins of T. brucei with those from two related African trypanosomes, T. congolense and T. vivax. We created a cell surface phylome (CSP) by estimating phylogenies for 79 gene families with putative surface functions to understand the more recent evolution of African trypanosome surface architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in T. brucei are conserved in T. congolense but absent from T. vivax and include an expanded gene family of insect stage-specific surface glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and innovations and confirm that these include most expression site-associated genes (ESAGs) in T. brucei, which are absent from T. congolense and T. vivax. The CSP presents the first global picture of the origins and dynamics of cell surface architecture in African trypanosomes, representing the principal differences in genomic repertoire between African trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface architectures. All data can be accessed at: http://www.genedb.org/Page/trypanosoma_surface_phylome.     

Analysis of the Airway Microbiota of Healthy Individuals and Patients with Chronic Obstructive Pulmonary Disease by T-RFLP and Clone Sequencing

Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways. Objective: The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.     

Characteristics and Functional Relevance of Apolipoprotein-A1 and Cholesterol Binding in Mammary Gland Tissues and Epithelial Cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of ¹²⁵I-apoA-I and ³H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular ³H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell^® plates. The amounts of isolated EPM and the maximal binding capacity of ¹²⁵I-apoA-I to EPM differed depending on the MG’s physiological state, while the kinetics of ³H-cholesterol and ¹²⁵I-apoA-I binding were similar. ³H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of ¹²⁵I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (K_D) of ¹²⁵I-apoA-I ranged between 40–74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and ¹²⁵I-apoA-I binding. The ABCA1 inhibitor Probucol displaced ¹²⁵I-apoA-I binding to EPM and reduced ³H-cholesterol efflux in MeBo. Time-dependent ³H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell^® plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of ³H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.     

Tetrathionate stimulated growth of Campylobacter jejuni identifies a new type of bi‐functional tetrathionate reductase (TsdA) that is widely distributed in bacteria

Tetrathionate (S₄O₆^2?) is used by some bacteria as an electron acceptor and can be produced in the vertebrate intestinal mucosa from the oxidation of thiosulphate (S₂O₃^2?) by reactive oxygen species during inflammation. Surprisingly, growth of the microaerophilic mucosal pathogen Campylobacter jejuni under oxygen‐limited conditions was stimulated by tetrathionate, although it does not possess any known type of tetrathionate reductase. Here, we identify a dihaem cytochrome c (C8j_0815; TsdA) as the enzyme responsible. Kinetic studies with purified recombinant C. jejuni TsdA showed it to be a bifunctional tetrathionate reductase/thiosulphate dehydrogenase with a high affinity for tetrathionate. A tsdA null mutant still slowly reduced, but could not grow on, tetrathionate under oxygen limitation, lacked thiosulphate‐dependent respiration and failed to convert thiosulphate to tetrathionate microaerobically. A TsdA paralogue (C8j_0040), lacking the unusual His–Cys haem ligation of TsdA, had low thiosulphate dehydrogenase and tetrathionate reductase activities. Our data highlight a hitherto unrecognized capacity of C. jejuni to use tetrathionate and thiosulphate in its energy metabolism, which may promote growth in the host. Moreover, as TsdA represents a new class of tetrathionate reductase that is widely distributed among bacteria, we predict that energy conserving tetrathionate respiration is far more common than currently appreciated.     

The activation of RhoC in vascular endothelial cells is required for the S1P receptor type 2-induced inhibition of angiogenesis

Sphingosine-1-phosphate (S1P) is a multifunctional phospholipid inducing a variety of cellular responses in endothelial cells (EC). S1P responses are mediated by five G protein coupled receptors of which three types (S1P₁R–S1P₃R) have been described to be of importance in vascular endothelial cells (EC). Whereas the S1P₁R regulates endothelial barrier function by coupling to Gα_i and the monomeric GTPase Rac1, the signaling pathways involved in the S1P-induced regulation of angiogenesis are ill defined. We therefore studied the sprouting of human umbilical vein EC (HUVEC) in vitro and analyzed the activation of the RhoGTPases RhoA and RhoC. Physiological relevant concentrations of S1P (100–300 nM) induce a moderate activation of RhoA and RhoC. Inhibition or siRNA-mediated depletion of the S1P₂R preferentially decreased the activation of RhoC. Both manipulations caused an increase of sprouting in a spheroid based in vitro sprouting assay. Interestingly, a similar increase in sprouting was detected after effective siRNA-mediated knockdown of RhoC. In contrast, the depletion of RhoA had no influence on sprouting. Furthermore, suppression of the activity of G proteins of the Gα_12/13 subfamily by adenoviral overexpression of the regulator of G protein signaling domain of LSC as well as siRNA-mediated knockdown of the Rho specific guanine nucleotide exchange factor leukemia associated RhoGEF (LARG) inhibited the S1P-induced activation of RhoC and concomitantly increased sprouting of HUVEC with similar efficacy. We conclude that the angiogenic sprouting of EC is suppressed via the S1P₂R subtype. Thus, the increase in basal sprouting can be attributed to blocking of the inhibitory action of autocrine S1P stimulating the S1P₂R. This inhibitory pathway involves the activation of RhoC via Gα_12/13 and LARG, while the simultaneously occurring activation of RhoA is apparently dispensable here.