Ru-ching Hsia

Unconventional Secretion of Tissue Transglutaminase Involves Phospholipid-Dependent Delivery into Recycling Endosomes

Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG). The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.

Chlamydia trachomatis-infected patients display variable antibody profiles against the nine-member polymorphic membrane protein family.

ABSTRACT Genomic analysis of the Chlamydiaceae has revealed a multigene family encoding large, putatively autotransported polymorphic membrane proteins (Pmps) with nine members in the sexually transmitted pathogen Chlamydia trachomatis. While various pathogenesis-related functions are emerging for the Pmps, observed genotypic and phenotypic variation among several chlamydial Pmps in various Chlamydia species has led us to hypothesize that the pmp gene repertoire is the basis of a previously undetected mechanism of antigenic variation. To test this hypothesis, we chose to examine the serologic response of C. trachomatis-infected patients to each Pmp subtype. Immune serum samples were collected from four populations of patients with confirmed C. trachomatis genital infection: 40 women with pelvic inflammatory disease from Pittsburgh, PA; 27 and 34 adolescent/young females from Oakland, CA, and Little Rock, AR, respectively; and 58 adult male patients from Baltimore, MD. The Pmp-specific antibody response was obtained using immunoblot analysis against each of the nine recombinantly expressed Pmps and quantified by densitometry. Our results show that nearly all C. trachomatis-infected patients mount a strong serologic response against individual or multiple Pmp subtypes and that the antibody specificity profile varies between patients. Moreover, our analysis reveals differences in the strengths and specificities of the Pmp subtype-specific antibody reactivity relating to gender and clinical outcome. Overall, our results indicate that the Pmps elicit various serologic responses in C. trachomatis-infected patients and are consistent with the pmp gene family being the basis of a mechanism of antigenic variation.

Variable expression of surface-exposed polymorphic membrane proteins in in vitro-grown Chlamydia trachomatis.

The hypothesized variable expression of polymorphic membrane proteins (PmpA–PmpI) in Chlamydia trachomatis‐infected patients was tested by examination of the expression of each Pmp subtype in in vitro‐grown C. trachomatis. A panel of monospecific polyclonal and monoclonal antibodies was used to demonstrate surface exposure of Pmps of each subtype by differential immunofluorescence (IF) with and without prior detergent permeabilization of paraformaldehyde‐fixed inclusions and for selected Pmps by immunogold labelling. Although specific transcript was detected for each pmp gene late in development, IF experiments with Pmp subtype‐specific antibodies reveal that a number of inclusions in a single infection do not express Pmps of a given subtype. Coexpression experiments suggest that pmp genes are shut off independently from one another in non‐expressing inclusions, i.e. different inclusions are switched off for different Pmps. Overall, these studies establish the existence of an efficient shutoff mechanism independently affecting the expression of each member of the pmp gene family in in vitro‐grown C. trachomatis. Like other paralogous gene families of bacterial pathogens, the pmp gene family of C. trachomatis may serve the critical dual function of a highly adaptable virulence factor also providing antigenic diversity in the face of the host adaptive immune response.

Isolation of a New Chlamydia species from the Feral Sacred Ibis (Threskiornis aethiopicus): Chlamydia ibidis.

Investigations conducted on feral African Sacred Ibises ( Threskiornis aethiopicus ) in western France led to the isolation of a strain with chlamydial genetic determinants. Ultrastructural analysis, comparative sequence analysis of the 16S rRNA gene, ompA, and of a concatenate of 31 highly conserved genes, as well as determination of the whole genome sequence confirmed the relatedness of the new isolate to members of the Chlamydiaceae, while, at the same time demonstrating a unique position outside the currently recognized species of this family. We propose to name this new chlamydial species Chlamydia ibidis .

Altered developmental expression of polymorphic membrane proteins in penicillin-stressed Chlamydia trachomatis

Late Chlamydia trachomatis inclusions express each member of the surface‐exposed polymorphic membrane protein family (Pmp subtypes A through I) with a reproducible distribution of fully‐on, fully‐off and intermediate phenotypes. This observation is consistent with observed variable Pmp antibody profiles in C. trachomatis‐infected patients and has led to the hypothesis that the pmp gene family forms the basis of a phase variation‐like mechanism of antigenic variation. Here we investigate and compare the developmental expression of each of the nine pmp genes under conditions of optimal in vitro growth with that under conditions that promote prolonged survival of chlamydiae when exposed to penicillin‐induced stress. We demonstrate that the pmp gene family includes distinct transcriptional units that are differentially expressed along development and differentially responsive to stress. In particular, our results indicate that expression of pmpA, pmpD and pmpI is uniquely unaffected by stress, suggesting that the PmpA, PmpD and PmpI proteins play a critical role in the pathogenesis of C. trachomatis.

SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors

SINC, a secreted effector of Chlamydia psittaci, uniquely targets the inner nuclear membrane of infected cells and uninfected neighbors. Candidate partners include ELYS, lamin B1, and four proteins of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, and chromatin organization.

Identification of Bacillus anthracis Spore Component Antigens Conserved across Diverse Bacillus cereus sensu lato Strains

We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.

Analysis of polymorphic membrane protein expression in cultured cells Identifies PmpA and PmpH of Chlamydia psittaci as candidate factors in pathogenesis and immunity to infection

The polymorphic membrane protein (Pmp) paralogous families of Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia abortus are putative targets for Chlamydia vaccine development. To determine whether this is also the case for Pmp family members of C. psittaci, we analyzed transcription levels, protein production and localization of several Pmps of C. psittaci. Pmp expression profiles were characterized using quantitative real-time PCR (RT-qPCR), immunofluorescence (IF) and immuno-electron microscopy (IEM) under normal and stress conditions. We found that PmpA was highly produced in all inclusions as early as 12 hpi in all biological replicates. In addition, PmpA and PmpH appeared to be unusually accessible to antibody as determined by both immunofluorescence and immuno-electron microscopy. Our results suggest an important role for these Pmps in the pathogenesis of C. psittaci, and make them promising candidates in vaccine development.

Temporospatial Relationship of Lipid Droplets and Mitochondria in Cardiac Muscle by Focused Ion Beam Scanning Electron Microscopy

Obesity is a rising health problem in the United States and many developed countries. The World Health Organization estimates that, in 2014, more than 1.9 billion adults were overweight worldwide, and more than 600 millions were obese [1]. Obesity is the result of an imbalance in lipid homeostasis and is evidenced by the excessive accumulation of cytoplasmic lipid droplets (CLDs) in cardiac muscles. The CLD is a dynamic organelle that interacts with various cell organelles such as the endoplasmic reticulum, endosomes and mitochondria. Our previous studies suggested that the LD specific protein, perilipin 5, is involved in promoting association of mitochondria to LDs [2]. Here we investigate the temporospatial relationship of LDs, mitochondria, endoplasmic reticulum (sarcoplasmic reticulum) and muscle fibrils in a 3D volume of mouse cardiac muscle using a focused ion beam scanning electron microscope (FB-SEM).

Immunoelectron Microscopy Reveals Varied Surface Expression of Potential Vaccine Targets of Chlamydia trachomatis

Chlamydia trachomatis, an obligate intracellular bacterium, is a leading cause of sexually transmitted infection in the world. It is estimated that 6.8 % of sexually active young females aged 14-19 in the US may be infected with Chlamydia [1]. Previous studies have demonstrated that a nine-member C. trachomatis protein family, the polymorphic membrane proteins (Pmps), plays a critical role in pathogenesis and may be a potential vaccine target [2]. These proteins share specific structural motifs and are transported to the chlamydial surface via an autotransporter mechanism [2]. We report here on the differential subcellular location of two of these proteins, PmpD and PmpI, at different stages of chlamydial development using immuno electron microscopy.