Microinjection of specific anti-IMPDH2 antibodies induces disassembly of cytoplasmic rods/rings that are primarily stationary and stable structures

Background: Our laboratory previously reported interesting rods 3-10 mu m long and rings 2-5 mu m diameter (RR) in the cytoplasm of mammalian cells. Experimental evidence show that both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthetase (CTPS) are components of RR structures. Several cell types, including mouse embryonic stem cells, and cell lines, such as mouse 3 T3 and rat NRK, naturally present RR structures, while other cells can present RR when treated with compounds interfering with GTP/CTP biosynthetic pathways. in this study, we aimed to investigate the dynamic behavior of these RR in live cells.Results: RR were detected in > 90% of COS-7 and HeLa cells treated with 1 mM ribavirin or 6-Diazo-5-oxo-L-norleucine (DON) for 24 h, and in 75% of COS-7 cells treated with 1 mM mycophenolic acid (MPA) for the same period of time. Microinjection of affinity-purified anti-IMPDH2 antibodies in live COS-7 cells treated with ribavirin, DON, or MPA showed mature forms of RR presented as stable and stationary structures in 71% of cells. in the remaining 29% of cells, RR acquired erratic movement and progressively disassembled into fragments and disappeared within 10 min. the specific stationary state and antibody-dependent disassembling of RR structures was independently confirmed in COS-7 and HeLa cells transfected with GFP-tagged IMPDH2.Conclusions: This is the first demonstration of disassembly of RR structures upon microinjection of anti-IMPDH2 antibodies that led to the disappearance of the molecular aggregates. the disassembly of RR after microinjection of anti-IMPDH2 antibody further strengthens the notion that IMPDH2 are major building blocks of RR. Using two independent methods, this study demonstrated that the induced RR are primarily stationary structures in live cells and that IMPDH2 is a key component of RR.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Florida, Dept Oral Biol, Gainesville, FL 32610 USAUniversidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilFleury Med & Hlth Labs, Div Immunol, BR-04102050 São Paulo, BrazilUniv Idaho, Dept Biol Sci, Moscow, ID 83844 USAUniversidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilCAPES: 9028-11-0FAPESP: 2011/12448-0Web of Scienc

The Chlamydia effector TarP mimics the mammalian leucine-aspartic acid motif of paxillin to subvert the focal adhesion kinase during invasion

Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34Arc, and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector

Activation of epidermal growth factor receptor is required for Chlamydia trachomatis development

Background Chlamydia trachomatis (C. trachomatis) is a clinically significant human pathogen and one of the leading causative agents of sexually transmitted diseases. As obligate intracellular bacteria, C. trachomatis has evolved strategies to redirect the host’s signaling and resources for its own survival and propagation. Despite the clinical notoriety of Chlamydia infections, the molecular interactions between C. trachomatis and its host cell proteins remain elusive. Results In this study, we focused on the involvement of the host cell epidermal growth factor receptor (EGFR) in C. trachomatis attachment and development. A combination of molecular approaches, pharmacological agents and cell lines were used to demonstrate distinct functional requirements of EGFR in C. trachomatisinfection. We show that C. trachomatis increases the phosphorylation of EGFR and of its downstream effectors PLCγ1, Akt and STAT5. While both EGFR and platelet-derived growth factor receptor-β (PDGFRβ) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRβ that affects the formation of C. trachomatis inclusions in the host cells. Inhibition of EGFR results in small immature inclusions, and prevents C. trachomatis-induced intracellular calcium mobilization and the assembly of the characteristic F-actin ring at the inclusion periphery. By using complementary approaches, we demonstrate that the coordinated regulation of both calcium mobilization and F-actin assembly by EGFR are necessary for maturation of chlamydial inclusion within the host cells. A particularly important finding of this study is the co-localization of EGFR with the F-actin at the periphery of C. trachomatis inclusion where it may function to nucleate the assembly of signaling protein complexes for cytoskeletal remodeling required for C. trachomatisdevelopment. Conclusion Cumulatively, the data reported here connect the function of EGFR to C. trachomatis attachment and development in the host cells, and this could lead to new venues for targeting C. trachomatis infections and associated diseases

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Computational Modeling of the Chlamydial Developmental Cycle Reveals a Potential Role for Asymmetric Division

ABSTRACT Chlamydia trachomatis is an obligate intracellular bacterium that progresses through an essential multicell form developmental cycle. Infection of the host is initiated by the elementary body (EB). Once in the host, the EB cell differentiates into the noninfectious, but replication-competent, reticulate body, or RB. After multiple rounds of replication, RBs undergo secondary differentiation, eventually producing newly infectious EBs. Here, we generated paired cell-type promoter reporter constructs and determined the kinetics of the activities of the euo, hctA, and hctB promoters. The paired constructs revealed that the developmental cycle produces at least three phenotypically distinct cell types, the RB (euoprom+), intermediate body (IB; hctAprom+), and EB (hctBprom+). The kinetic data from the three dual-promoter constructs were used to generate two computational agent-based models to reproduce the chlamydial developmental cycle. Both models simulated EB germination, RB amplification, IB formation, and EB production but differed in the mechanism that generated the IB. The direct conversion and the asymmetric production models predicted different behaviors for the RB population, which were experimentally testable. In agreement with the asymmetric production model, RBs acted as stem cells after the initial amplification stage, producing one IB and self-renewing after every division. We also demonstrated that IBs are a transient cell population, maturing directly into EBs after formation without the need for cell division. The culmination of these results suggests that the developmental cycle can be described by a four-stage model, EB germination, RB amplification/maturation, IB production, and EB formation. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for both ocular and sexually transmitted infections. All Chlamydiae are reliant on a complex developmental cycle, consisting of both infectious and noninfectious cell forms. The EB cell form initiates infection, whereas the RB cell replicates. The infectious cycle requires both cell types, as RB replication increases the cell population while EB formation disseminates the infection to new hosts. The mechanisms of RB-to-EB development are largely unknown. Here, we developed unique dual promoter reporters and used live-cell imaging and confocal microscopy to visualize the cycle at the single-cell and kinetic levels. These data were used to develop and test two agent-based models, simulating either direct conversion of RBs to EBs or production of EBs via asymmetric RB division. Our results suggest that RBs mature into a stem cell-like population producing intermediate cell forms through asymmetric division, followed by maturation of the intermediate cell type into the infectious EB. Ultimately, a more complete mechanistic understanding of the developmental cycle will lead to novel therapeutics targeting cell type development to eliminate chlamydial dissemination

IhtA sequence is conserved across species.

<p>The sequence of <i>ihtA</i> of <i>C. trachomatis</i> serovar D, <i>C. muridarum, C. caviae</i>, and <i>C. pneumoniae</i> compared to <i>C. trachomatis</i> serovar L2. The TSS of serovars L2 and D and <i>C. pneumoniae</i> ihtA (indicated in bold) has been experimentally proven while the TSS of the other <i>Chlamydia</i> is predicted.</p

Translation Inhibition of the Developmental Cycle Protein HctA by the Small RNA IhtA Is Conserved across <em>Chlamydia</em>

<div><p>The developmental cycle of the obligate intracellular pathogen <em>Chlamydia trachomatis</em> serovar L2 is controlled in part by the small non-coding RNA (sRNA), IhtA. All <em>Chlamydia</em> alternate in a regulated fashion between the infectious elementary body (EB) and the replicative reticulate body (RB) which asynchronously re-differentiates back to the terminal EB form at the end of the cycle. The histone like protein HctA is central to RB:EB differentiation late in the cycle as it binds to and occludes the genome, thereby repressing transcription and translation. The sRNA IhtA is a critical component of this regulatory loop as it represses translation of <em>hctA</em> until late in infection at which point IhtA transcription decreases, allowing HctA expression to occur and RB to EB differentiation to proceed. It has been reported that IhtA is expressed during infection by the human pathogens <em>C. trachomatis</em> serovars L2, D and L2b and <em>C. pneumoniae</em>. We show in this work that IhtA is also expressed by the animal pathogens <em>C. caviae</em> and <em>C. muridarum</em>. Expression of HctA in <em>E. coli</em> is lethal and co-expression of IhtA relieves this phenotype. To determine if regulation of HctA by IhtA is a conserved mechanism across pathogenic chlamydial species, we cloned <em>hctA</em> and <em>ihtA</em> from <em>C. trachomatis</em> serovar D, <em>C. muridarum, C. caviae</em> and <em>C. pneumoniae</em> and assayed for rescue of growth repression in <em>E. coli</em> co-expression studies. In each case, co-expression of <em>ihtA</em> with the cognate <em>hctA</em> resulted in relief of growth repression. In addition, expression of each chlamydial species IhtA rescued the lethal phenotype of <em>C. trachomatis</em> serovar L2 HctA expression. As biolayer interferometry studies indicate that IhtA interacts directly with <em>hctA</em> message for all species tested, we predict that conserved sequences of IhtA are necessary for function and/or binding.</p> </div

Identification of the Base-Pairing Requirements for Repression of <i>hctA</i> Translation by the Small RNA IhtA Leads to the Discovery of a New mRNA Target in <i>Chlamydia trachomatis</i>

<div><p>The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen <i>Chlamydia trachomatis</i> modulates the translation of HctA, a key protein involved in replicative to infectious cell type differentiation. Using a combination of bioinformatics and mutagenesis we sought to identify the base pairing requirement for functional repression of HctA protein expression, with an eye to applying our findings towards the identification of additional targets. IhtA is predicted to fold into a three stem:loop structure. We found that loop 1 occludes the initiation codon of <i>hctA</i>, while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of <i>hctA</i>. Two additional genes in the chlamydial genome, <i>CTL0322</i> and <i>CTL0097</i>, contained some elements of the <i>hctA</i>:IhtA recognition sequence. The mRNA of both <i>CTL0322</i>and <i>CTL0097</i> interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of <i>CTL0322</i>. The proposed IhtA recognition site in the <i>CTL0322 </i>message contains significant G/C base pairing on either side of the initiation codon while <i>CTL0097</i> only contains G/C base pairing 3’ to the AUG initiation codon. These data suggest that as the functional interacting region is only 6-7nt in length that full translation repression is dependent on the degree of G/C base pairing. Additionally our results indicate that IhtA may regulate multiple mRNAs involved in the chlamydial infectious cycle.</p></div