Identification and biochemical characterization of the CHLAMYDIA TRACHOMATIS type III secretion chaperone, SLC1, and its role in the translocation of the invasion-associated effector TARP.

Chlamydia trachomatis is an obligate intracellular pathogen that utilizes a type III secretion system to enter mammalian cells and establish an intracellular niche. TARP, the translocated actin recruitment protein, is a chlamydial invasion protein known to be type III secreted by the metabolically inert elementary body upon docking to the mammalian cell surface. Because immediate secretion of TARP into host cells is necessary for entry, I hypothesized that a chlamydial chaperone binds to TARP and facilitates its translocation through the type III secretion apparatus. Most effector-binding type III secretion chaperones are small (14-18 kDa), have an acidic pI, and share a specific secondary structure of alternating alpha-helices (a) and beta-sheets (~). Typically, type III secretion chaperones dimerize and interact with their effectors as a complex of two molecules of chaperone to one effector molecule. Only 3 Chlamydia trachomatis proteins have been identified in EB\u27 s that are predicted to be putative chlamydial type III effector chaperones. These are CT043, CT663, and CT088, which I have designated as SIc 1, SIc2, and Scc 1, respectively. These chaperones were tested for their interaction with the N-terminal 200 amino acids of TARP (HIS6 TARpl-200) by co immunoprecipitation. HIS6 TARpl-200 interacted specifically with SlcI, but not Scc1 or Slc2. This interaction was enhanced by coexpression of the recombinant proteins. To confirm this interaction and rule out the possibility of Slc 1 heterodimerization enhancing the interaction with TARP, I employed a 2-hybrid system to test for TARP: chaperone and chaperone:chaperone interactions. I confirmed the specific interaction between Cya18- TARpl-200 and Cya25-Slc 1. I was also able to detect SIc1 interaction with itself as well as confirm a few other previously described chaperone-chaperone interactions. Analysis by crosslinking and gel filtration chromatography indicated that Slc 1 forms a stable dimer in solution. Complexes of the Slci chaperone dimer with TARP in a 2:1 stoichiometry were detected following purification from co-expressing bacteria, but not following addition of singly purified species. Expression ofbeta-Iactamase fused to TARl-200 by the heterologous system Yersinia enterocolitica allowed for secretion of TARP into type-III inducing media (low calcium). Furthermore I was able to detect SIc I-dependent translocation of T ARP into HeLa cells via the heterologous type III secretion system of Y enterocolitica, and also by the SPI-2 system of Salmonella enterica serovar typhimurium

Optimization of Tick Attachment and Detection of B. Burgdorferi Transmission into In Vitro Generated Skin Rafts

Borrelia burgdorferi (B. burgdorferi) is a Lyme Disease causing pathogen that is transmitted via tick-bite by blacklegged (deer) ticks (Ixodes scapularis). These ticks tend choose hosts such as rabbits and mice to feed on, sometimes transmitting harmful pathogens to these animals such as B. burgdorferi, which cycles between rodents and large animals to complete its enzootic cycle. Humans are accidental dead-end hosts for B. burgdorferi. Those infected with B. burgdorferi can develop Lyme Disease, which includes symptoms such as neurological, cardiac, and joint-related complications which are exacerbated by increased feeding time on the host1. Due to the many negative affects Lyme Disease can have on humans, there is a need for medicinal options that could prevent the accumulation of the bacteria at the tick bite site or inhibit the ability of ticks to feed on the human host. We rationalize that a human skin model to study tick-human skin interactions and possible therapeutic screening is needed as there are currently no cost-effective or high-throughput options. Our in vitro generated skin rafts will enable us to study tick feeding habits and B. burgdorferi transmission comparable to use of in vivo animal models, but more applicable to human infection. We hypothesize that, as B. burgdorferi-positive ticks feed on the rafts, they will transmit the bacteria into the epidermal layer which can later be extracted and quantified. Once these skin rafts have been optimized for maximal tick feeding and B. burgdorferi transmission, they can then be used in high-throughput experiments to find therapies that could modify the bacteria or tick interactions with the host and prevent development of Lyme Disease.https://digitalcommons.unmc.edu/surp2022/1013/thumbnail.jp

Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion

IntroductionThe obligate intracellular pathogen Chlamydia trachomatis is the causative agent of the most common bacterial sexually transmitted disease worldwide. While the host response to infection by this pathogen has been well characterized, it remains unclear to what extent host gene expression during infection is the product of Chlamydia-directed modulation of host transcription factors.MethodsTo identify transcription factors potentially modulated by Chlamydia during infection, we infected immortalized endocervical epithelial cells (End1/E6E7) with the anogenital C. trachomatis serovar L2, harvesting polyadenylated RNA for bulk RNA-sequencing. Subsequent experiments elucidating the mechanism of infection-mediated YAP activation assayed YAP target gene expression via qRT-PCR, YAP nuclear translocation via quantitative immunofluorescence, and YAP phosphorylation via Western blotting.ResultsRNA sequencing of Chlamydia-infected endocervical epithelial cells revealed gene expression consistent with activity of YAP, a transcriptional coactivator implicated in cell proliferation, wound healing, and fibrosis. After confirming induction of YAP target genes during infection, we observed an infection-dependent increase in YAP nuclear translocation sensitive to inhibition of bacterial protein synthesis. While Hippo-mediated phosphoinhibition of YAP at S127 was unaffected by C. trachomatis infection, Hippo-independent phosphorylation at Y357 was increased. Infection did not enhance nuclear translocation of Y357F mutant YAP, illustrating a requirement for phosphorylation at this residue. Pharmacological inhibition of host Src-family kinase activity attenuated YAP Y357 phosphorylation, but not nuclear translocation – which was instead sensitive to inhibition of Abl.DiscussionOur results define a transcriptome-altering mechanism of pathogen-directed YAP activation that bypasses canonical inhibition by the Hippo kinase cascade, with a potential link to chlamydial fibrosis and other advanced disease sequelae. Additional study is required to determine the specific role of infection-associated Y357 phosphorylation and Abl activity in chlamydial induction of YAP

Possible detection of two giant extrasolar planets orbiting the eclipsing polar UZ Fornacis

We present new high-speed, multi-observatory, multi-instrument photometry of the eclipsing polar UZ For in order to measure precise mid-eclipse times with the aim of detecting any orbital period variations. When combined with published eclipse times and archival data spanning ~27 years, we detect departures from a linear and quadratic trend of ~60 s. The departures are strongly suggestive of two cyclic variations of 16(3) and 5.25(25) years. The two favoured mechanisms to drive the periodicities are either two giant extrasolar planets as companions to the binary (with minimum masses of 6.3(1.5)M(Jupiter) and 7.7(1.2)M(Jupiter)) or a magnetic cycle mechanism (e.g. Applegate's mechanism) of the secondary star. Applegate's mechanism would require the entire radiant energy output of the secondary and would therefore seem to be the least likely of the two, barring any further refinements in the effect of magnetic fieilds (e.g. those of Lanza et al.). The two planet model can provide realistic solutions but it does not quite capture all of the eclipse times measurements. A highly eccentric orbit for the outer planet would fit the data nicely, but we find that such a solution would be unstable. It is also possible that the periodicities are driven by some combination of both mechanisms. Further observations of this system are encouraged.Comment: 10 pages, 4 figures, 2 table

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Manipulation of Focal Adhesion Signaling by Pathogenic Microbes

Focal adhesions (FAs) serve as dynamic signaling hubs within the cell. They connect intracellular actin to the extracellular matrix (ECM) and respond to environmental cues. In doing so, these structures facilitate important processes such as cell–ECM adhesion and migration. Pathogenic microbes often modify the host cell actin cytoskeleton in their pursuit of an ideal replicative niche or during invasion to facilitate uptake. As actin-interfacing structures, FA dynamics are also intimately tied to actin cytoskeletal organization. Indeed, exploitation of FAs is another avenue by which pathogenic microbes ensure their uptake, survival and dissemination. This is often achieved through the secretion of effector proteins which target specific protein components within the FA. Molecular mimicry of the leucine–aspartic acid (LD) motif or vinculin-binding domains (VBDs) commonly found within FA proteins is a common microbial strategy. Other effectors may induce post-translational modifications to FA proteins through the regulation of phosphorylation sites or proteolytic cleavage. In this review, we present an overview of the regulatory mechanisms governing host cell FAs, and provide examples of how pathogenic microbes have evolved to co-opt them to their own advantage. Recent technological advances pose exciting opportunities for delving deeper into the mechanistic details by which pathogenic microbes modify FAs

Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP

Bacterial type III secretion system (T3SSs) chaperones pilot substrates to the export apparatus in a secretion-competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well-characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two-hybrid, co-precipitation, cross-linking, and size exclusion chromatography we show that Slc1 (SycE-like chaperone 1; CT043) specifically interacts with a 200 amino acid residue N-terminal region of TARP (TARP 1–200 ). Slc1 formed homodimers in vitro , as shown in crosslinking and gel filtration experiments. Biochemical analysis of an isolated Slc1-TARP 1–200 complex was consistent with a characteristic 2:1 chaperone-effector stoichiometry. Furthermore, Slc1 was co-immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, co-expression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP

Identification of <i>K</i>. <i>pneumoniae</i> outer membrane proteins by 2DE.

<p>(A) Two-dimensional map of outer membrane proteins from isolate 30660 cultured to stationary phase of growth in LB. Numbers refer to the extracted spots that were processed for identification by LC-MS/MS. The pI and molecular weight markers are shown on the top and right, respectively. (B) Categorical representation by function of predicted outer membrane proteins. (C) Categorical representation by function of predicted outer membrane proteins.</p