Åsa Gylfe

A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis

The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3β to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity.

Reactivation of Borrelia infection in birds

Birds often carry ticks infected with Borrelia burgdorferi sensu lato — the spirochaete that causes Lyme disease — but their role as possible hosts and amplifiers for this illness has long been discounted. We find, however, that migratory birds are able to carry Lyme disease as a latent infection for several months, and that this infection can be reactivated and passed on to ticks as a result of migratory restlessness. Our results indicate that migratory birds may be efficient long-distance carriers of this pathogen.

Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle

Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re‐differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti‐chlamydial drugs.

Socioeconomic status, pathogen burden and cardiovascular disease risk

Objective: Socioeconomic status (SES) is inversely associated with coronary heart disease (CHD) risk. Cumulative pathogen burden may also predict future CHD. The hypothesis was tested that lower SES is associated with a greater pathogen burden, and that pathogen burden accounts in part for SES differences in cardiovascular risk factors. Methods: This was a cross-sectional observational study involving the clinical examination of 451 men and women aged 51–72 without CHD, recruited from the Whitehall II epidemiological cohort. SES was defined by grade of employment, and pathogen burden by summing positive serostatus for Chlamydia pneumoniae, cytomegalovirus and herpes simplex virus 1. Cardiovascular risk factors were also assessed. Results: Pathogen burden averaged 1.94 (SD) 0.93 in the lower grade group, compared with 1.64 (0.97) and 1.64 (0.93) in the intermediate and higher grade groups (p = 0.011). Pathogen burden was associated with a higher body mass index, waist/hip ratio, blood pressure and incidence of diabetes. There were SES differences in waist/hip ratio, high-density lipoprotein-cholesterol, fasting glucose, glycated haemoglobin, lung function, smoking and diabetes. The SES gradient in these cardiovascular risk factors was unchanged when pathogen burden was taken into account statistically. Conclusions: Although serological signs of infection with common pathogens are more frequent in lower SES groups, their distribution across the social gradient does not match the linear increases in CHD risk present across higher, intermediate and lower SES groups. Additionally, pathogen burden does not appear to mediate SES differences in cardiovascular risk profiles.

Statistical molecular design of a focused salicylidene acylhydrazide library and multivariate QSAR of inhibition of type III secretion in the Gram-negative bacterium Yersinia

A combined application of statistical molecular design (SMD), quantitative structure-activity relationship (QSAR) modeling and prediction of new active compounds was effectively used to develop salicylidene acylhydrazides as inhibitors of type III secretion (T3S) in the Gram-negative pathogen Yersinia pseudotuberculosis. SMD and subsequent synthesis furnished 50 salicylidene acylhydrazides in high purity. Based on data from biological evaluation in T3S linked assays 18 compounds were classified as active and 25 compounds showed a dose-dependent inhibition. The 25 compounds were used to compute two multivariate QSAR models and two multivariate discriminant analysis models were computed from both active and inactive compounds. Three of the models were used to predict 4416 virtual compounds in consensus and eight new compounds were selected as an external test set. Synthesis and biological evaluation of the test set in Y. pseudotuberculosis and the intracellular pathogen Chlamydia trachomatis validated the models. Y. pseudotuberculosis and C. trachomatis cell-based infection models showed that compounds identified in this study are selective and non-toxic inhibitors of T3S dependent virulence.

Epidemiology: Reactivation of Borrelia infectionin birds

Birds often carry ticks infected with Borrelia burgdorferi sensu lato — the spirochaete that causes Lyme disease — but their role as possible hosts and amplifiers for this illness has long been discounted. We find, however, that migratory birds are able to carry Lyme disease as a latent infection for several months, and that this infection can be reactivated and passed on to ticks as a result of migratory restlessness. Our results indicate that migratory birds may be efficient long-distance carriers of this pathogen.

Mutations in hemG Mediate Resistance to Salicylidene Acylhydrazides, Demonstrating a Novel Link between Protoporphyrinogen Oxidase (HemG) and Chlamydia trachomatis Infectivity

Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.

Derivatives of 8-hydroxyquinoline—antibacterial agents that target intra- and extracellular Gram-negative pathogens

Small molecule screening identified 5-nitro-7-((4-phenylpiperazine-1-yl-)methyl)quinolin-8-ol INP1750 as a putative inhibitor of type III secretion (T3S) in the Gram-negative pathogen Yersinia pseudotuberculosis. In this study we report structure-activity relationships for inhibition of T3S and show that the most potent compounds target both the extracellular bacterium Y. pseudotuberculosis and the intracellular pathogen Chlamydia trachomatis in cell-based infection models.

Pre-clinical pharmacokinetics and anti-chlamydial activity of salicylidene acylhydrazide inhibitors of bacterial type III secretion

Salicylidene acylhydrazides belong to a class of compounds shown to inhibit bacterial type III secretion (T3S) in pathogenic Gram-negative bacteria. This class of compounds also inhibits growth and replication of Chlamydiae, strict intracellular bacteria that possess a T3S system. In this study a library of 58 salicylidene acylhydrazides was screened to identify inhibitors of Chlamydia growth. Compounds inhibiting growth of both Chlamydia trachomatis and Chlamydophila pneumoniae were tested for cell toxicity and seven compounds were selected for preliminary pharmacokinetic analysis in mice using cassette dosing. Two compounds, ME0177 and ME0192, were further investigated by individual pharmacokinetic analysis. Compound ME0177 had a relatively high peak plasma concentration (Cmax) and area under curve and therefore may be considered for systemic treatment of Chlamydia infections. The other compound, ME0192, had poor pharmacokinetic properties but the highest anti-chlamydial activity in vitro and therefore was tested for topical treatment in a mouse vaginal infection model. ME0192 administered vaginally significantly reduced the infectious burden of C. trachomatis and the number of infected mice.

Thiazolino 2-Pyridone Amide Inhibitors of Chlamydia trachomatis Infectivity

The bacterial pathogen Chlamydia trachomatis is a global health burden currently treated with broad-spectrum antibiotics which disrupt commensal bacteria. We recently identified a compound through phenotypic screening that blocked infectivity of this intracellular pathogen without host cell toxicity (compound 1, KSK 120). Herein, we present the optimization of 1 to a class of thiazolino 2-pyridone amides that are highly efficacious (EC50 ≤ 100 nM) in attenuating infectivity across multiple serovars of C. trachomatis without host cell toxicity. The lead compound 21a exhibits reduced lipophilicity versus 1 and did not affect the growth or viability of representative commensal flora at 50 μM. In microscopy studies, a highly active fluorescent analogue 37 localized inside the parasitiphorous inclusion, indicative of a specific targeting of bacterial components. In summary, we present a class of small molecules to enable the development of specific treatments for C. trachomatis.