Objectives To test vaccines, formulated with novel antigens, to protect mice against Chlamydia infections. Methods To determine the ability of polymorphic membrane proteins (Pmps) to induce cross-species protective immune responses, recombinant fragments from all nine C. trachomatis serovar E Pmps were used to vaccinate BALB/c mice utilizing CpG-1826 and Montanide ISA 720 as adjuvants. C. muridarum recombinant MOMP and PBS, formulated with the same adjuvants, were used as positive and negative controls, respectively. Mice were challenged intranasally with 104 inclusion-forming units (IFU) of C. muridarum. Animals were weighed daily and at 10 days post-challenge, they were euthanized, their lungs harvested, weighed and the number of chlamydial IFU counted. Results Following vaccination the nine Pmps elicited immune responses. Based on body weight changes, or number of IFU recovered from lungs, mice vaccinated with Pmp C, G or H were the best protected. For example, over the 10-day period, the negative control group vaccinated with PBS lost significantly more body weight than mice immunized with PmpC or G (P < 0.05). C. muridarum MOMP vaccinated mice were better protected against body weight losses than any group immunized with Pmps. Also, the median number of IFU recovered from the lungs of mice vaccinated with PmpC (72 x 106) or PmpH (61 x 106) was significantly less than from mice immunized with PBS (620 x 106; P < 0.05). As determined by the number of IFU, all Pmps elicited less protection than C. muridarum MOMP (0.078 x 106 IFU; P < 0.05). Conclusions This is the first time PmpC has been shown to elicit cross-protection against a respiratory challenge. Additional work with Pmps C, G and H is recommended to determine their ability to protect animal models against genital and ocular challenges. Keywords: C. trachomatis, polymorphic membrane proteins (Pmps), vaccine, C. muridarum, PmpC,

Uptake of obligate intracellular bacterial pathogens into mammalian epithelial cells is critically dependent on modulation of the host’s endocytic machinery. It is an open question how the invading pathogens generate a membrane-bound vesicle appropriate to their size. This requires extensive deformation of the host plasma membrane itself by pathogen-derived membrane-binding proteins, accompanied by substantial F-actin-based forces to further expand and finally pinch off the vesicle. Here we show that upon adhesion to the host cell, the human pathogenic bacterium Chlamydia pneumoniae secretes the scaffolding effector protein CPn0677, which binds to the inner leaflet of the invaginating host’s PM, induces inwardly directed, negative membrane curvature, and forms a recruiting platform for the membrane-deforming BAR-domain containing proteins Pacsin and SNX9. In addition, while bound to the membrane, CPn0677 recruits monomeric G-actin, and its C-terminal region binds and activates N-WASP, which initiates branching actin polymerization via the Arp2/3 complex. Together, these membrane-bound processes enable the developing endocytic vesicle to engulf the infectious elementary body, while the associated actin network generates the forces required to reshape and detach the nascent vesicle from the PM. Thus, Cpn0677 (now renamed SemD) acts as recruiting platform for central components of the endocytic machinery during uptake of chlamydia.

Integral membrane proteins are critical for many cellular functions. Roughly 25% of all human genes code for membrane proteins, and about 70% of all approved drugs target them. Despite their importance, laborious and harsh purification conditions often hinder their characterization. Traditionally, they are removed from the membrane using detergents, thereby taking the proteins out of their native environment, affecting their function. Recently, a variety of synthetic polymers have been introduced, which can extract membrane proteins together with their native lipids into a so-called native nanodisc. However, they usually show lesser solubilization capacity than detergents, and their general applicability for membrane protein biochemistry is poorly understood. Here, we used Hek293 cell membrane extracts and LC-MS-based proteomics to compare the ability of nanodisc-forming polymers against state-of-the-art detergents to solubilize the membrane proteome. Our data demonstrates the general ability of synthetic co-polymers to extract membrane proteins, rivalling the efficacy of commonly used detergents. Interestingly, each class of solubilization agent presents specific solubilization profiles. Importantly, we found no correlation between efficiency and number of transmembrane domains, isoelectric point, or GRAVY score for any compound. Combined, our data show that these polymers are a versatile alternative to detergents for the biochemical and structural study of membrane proteins, functional proteomics or as components of a native lysis/solubilization buffers. Our work here represents the first attempt at a proteome-scale comparison of the efficacy of nanodisc-forming polymers. These data should serve as starting reference to researchers looking to purify membrane proteins in near native conditions.

We study the extension of non-monotonic disjunctive logic programs with terms that represent sets of constants, called DLP(S), under the stable model semantics. This strictly increases expressive power, but keeps reasoning decidable, though cautious entailment is coNEXPTIME^NP-complete, even for data complexity. We present two new reasoning methods for DLP(S): a semantics-preserving translation of DLP(S) to logic programming with function symbols, which can take advantage of lazy grounding techniques, and a ground-and-solve approach that uses non-monotonic existential rules in the grounding stage. Our evaluation considers problems of ontological reasoning that are not in scope for traditional ASP (unless EXPTIME =ΠP2 ), and we find that our new existential-rule grounding performs well in comparison with native implementations of set terms in ASP.