Márta Szaszák

Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell

Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.

When oxygen runs short: the microenvironment drives host–pathogen interactions

Pathogens that colonize or infect the human body have to face varying oxygen concentrations within different organs. Inflammation itself promotes oxygen consumption within affected tissues and creates a low oxygen environment. As a consequence, pathogens and the host immune system have to adapt to rapid changes in oxygen availability. Here we summarize recent findings on the adaptation of pathogens, host defense mechanisms and treatment strategies against intracellular pathogens in a low oxygen environment.

Impact of a Low-Oxygen Environment on the Efficacy of Antimicrobials against Intracellular Chlamydia trachomatis

ABSTRACT Emergence of chronic inflammation in the urogenital tract induced by Chlamydia trachomatis infection in females is a long-standing concern. To avoid the severe sequelae of C. trachomatis infection, such as pelvic inflammatory diseases (PID), ectopic pregnancies, and tubal infertility, antibiotic strategies aim to eradicate the pathogen even in asymptomatic and uncomplicated infections. Although first-line antimicrobials have proven successful for the treatment of C. trachomatis infection, treatment failures have been observed in a notable number of cases. Due to the obligate intracellular growth of C. trachomatis, reliable antimicrobial susceptibility assays have to consider environmental conditions and host cell-specific factors. Oxygen concentrations in the female urogenital tract are physiologically low and decrease further during an inflammatory process. We compared MIC testing and time-kill curves (TKC) for doxycycline, azithromycin, rifampin, and moxifloxacin under hypoxia (2% O2) and normoxia (20% O2). While low oxygen availability only moderately decreased the antichlamydial activity of azithromycin in conventional MIC testing (0.08 μg/ml versus 0.04 μg/ml; P < 0.05), TKC analyses revealed profound divergences for antibiotic efficacies between the two conditions. Thus, C. trachomatis was significantly less rapidly killed by doxycycline and azithromycin under hypoxia, whereas the efficacies of moxifloxacin and rifampin remained unaffected using concentrations at therapeutic serum levels. Chemical inhibition of multidrug resistance protein 1 (MDR-1), but not multidrug resistance-associated protein 1 (MRP-1), restored doxycycline activity against intracellular C. trachomatis under hypoxia. We suggest careful consideration of tissue-specific characteristics, including oxygen availability, when testing antimicrobial activities of antibiotics against intracellular bacteria.

Two-photon fluorescence lifetime imaging monitors metabolic changes during wound healing of corneal epithelial cells in vitro.

BackgroundEarly and correct diagnosis of delayed or absent corneal epithelial wound healing is a key factor in the prevention of infection and consecutive destruction of the corneal stroma with impending irreversible visual loss. Two-photon microscopy (TPM) is a novel technology that has potential to depict epithelial cells and to evaluate cellular function by measuring autofluorescence properties such as fluorescence intensity and fluorescence lifetimes of metabolic co-factors such as NAD(P)H.MethodsUsing non-invasive TPM in a tissue-culture scratch model and an organ-culture erosion model, fluorescence intensity and fluorescence lifetimes of NAD(P)H were measured before and during closure of the epithelial wounds. Influence of temperature and selective inhibition of metabolism on intensity and lifetimes were tested additionally.ResultsDecrease of temperature resulted in significant increase of fluorescence lifetimes and decrease of the relative amount of free NAD(P)H due to decreased global metabolism. Increase in temperature and upregulation of glycolysis through blocking the mitochondrial electron transport chain by rotenone resulted in increased intensity, decreased lifetimes and increase in the relative amount of free NAD(P)H. Changes of lifetimes and free:protein-bound NAD(P)H ratios were similar to changes measured during wound healing in both scratch and erosion models.ConclusionsFluorescence lifetime measurements (FLIM) detected enhancement of cellular metabolism following epithelial damage in both models. The prospective detection of cellular autofluorescence in vivo, in particular FLIM of metabolic cofactor NAD(P)H, has the potential to become an indispensible tool in clinical use to differentiate healing from non-healing epithelial cells and to evaluate effects of newly developed substances on cellular metabolism in preclinical and clinical trials.

Imaging of Chlamydia and host cell metabolism.

Chlamydial infections cause a wide range of acute and chronic diseases. Chlamydia trachomatis is the most common sexually transmitted bacterium while Chlamydia pneumoniae causes infections of the upper and lower respiratory tract. Chlamydia are obligate, intracellular bacteria with a biphasic developmental cycle that involves unique metabolic changes. Aside from entering an actively replicating state, Chlamydia may also implement persistent infections depending on different microenvironmental factors. In addition, changes in local oxygen availability and the composition of surrounding host microbiota are suggested to affect chlamydial growth and metabolism. Both bacteria and host cells endure characteristic metabolic changes during infection. Technical developments in recent years enable us to separately characterize chlamydial and host cell metabolism in living cells. This article focuses on novel approaches to analyze chlamydial metabolism such as NAD(P)H fluorescence lifetime imaging by two-photon microscopy. In addition, we provide an overview regarding promising future possibilities to further elucidate host-pathogen metabolic interactions.

Host metabolism promotes growth of Chlamydia pneumoniae in a low oxygen environment.

Chlamydia pneumoniae infections of the respiratory tract are common and are associated with acute and chronic diseases such as community-acquired pneumonia (CAP) and chronic obstructive pulmonary disease (COPD). Recent studies have shown that reduced environmental oxygen availability promotes chlamydial growth in infected host cells. The underlying mechanisms remain unclear. We performed a targeted siRNA screen coupled with an automated high-throughput microscopic analysis to identify key host cell genes that play a role in promoting the hypoxic growth of C. pneumoniae. A total of 294 siRNAs - targeting 98 selected genes including central mediators of metabolic, trafficking and signaling pathways - were tested on chlamydial inclusion formation in C. pneumoniae infected A549 cells under normoxic (20% O2) and hypoxic (2% O2) conditions 48 h post infection. Evaluation of the different functional clusters of genes revealed that under hypoxic conditions, enhanced growth of C. pneumoniae was centrally mediated by the host cell glycolytic pathway. Inhibition of the phosphofructokinase (PFK), lactate dehydrogenase (LDH), glycerol-3-phosphate dehydrogenase (GPD2) and the forkheadbox O3 (FOXO3) gene-expression by siRNAs abrogated chlamydial progeny. The pivotal role of host cell glycolysis in chlamydial development under hypoxia was further confirmed by pharmacological inhibition of the pathway by 2-fluoro-deoxy-glucose. The results indicate that the microenvironment of the host cell determines the fate of C. pneumoniae by controlling pathogen-induced metabolic pathways.

Characterizing the intracellular distribution of metabolites in intact Chlamydia-infected cells by Raman and two-photon microscopy

Chlamydia species are obligate intracellular pathogens that proliferate only within infected cells. Currently, there are no known techniques or systems that can probe the spatial distribution of metabolites of interest within intact Chlamydia-infected cells. Here we investigate the ability of Raman microscopy to probe the chemical composition of different compartments (nucleus, inclusion, and cytoplasm) of Chlamydia trachomatis-infected epithelial cells. The overall intensity of the Raman spectrum is greatest in the inclusions and lowest in the cytoplasm in fixed cells. Difference spectra generated by normalizing to the intensity of the strong 1004 cm(-1) phenylalanine line show distinct differences among the three compartments. Most notably, the concentrations of adenine are greater in both the inclusions and the nucleus than in the cytoplasm, as seen by Raman microscopy. The source of the adenine was explored through a complementary approach, using two-photon microscopy imaging. Autofluorescence measurements of living, infected cells show that the adenine-containing molecules, NAD(P)H and FAD, are present mainly in the cytoplasm, suggesting that these molecules are not the source of the additional adenine signal in the nucleus and inclusions. Experiments of infected cells stained with a DNA-binding dye, Hoechst 33258, reveal that most of the DNA is present in the nucleus and the inclusions, suggesting that DNA/RNA is the main source of the additional Raman adenine signal in the nucleus and inclusions. Thus, Raman and two-photon microscopy are among the few non-invasive methods available to investigate cells infected with Chlamydia and, together, should also be useful for studying infection by other intracellular pathogens that survive within intracellular vacuoles.

Non-Invasive Multi-Dimensional Two-Photon Microscopy enables optical fingerprinting (TPOF) of immune cells

Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non-invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two-photon microscopy to non-invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence-based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients. Two photon based autofluorescence features of immune cells enables non-invasive differentiation.

Growth of Chlamydia pneumoniae is enhanced in cells with impaired mitochondrial function

Effective growth and replication of obligate intracellular pathogens depend on host cell metabolism. How this is connected to host cell mitochondrial function has not been studied so far. Recent studies suggest that growth of intracellular bacteria such as Chlamydia pneumoniae is enhanced in a low oxygen environment, arguing for a particular mechanistic role of the mitochondrial respiration in controlling intracellular progeny. Metabolic changes in C. pneumoniae infected epithelial cells were analyzed under normoxic (O2 ≈ 20%) and hypoxic conditions (O2 < 3%). We observed that infection of epithelial cells with C. pneumoniae under normoxia impaired mitochondrial function characterized by an enhanced mitochondrial membrane potential and ROS generation. Knockdown and mutation of the host cell ATP synthase resulted in an increased chlamydial replication already under normoxic conditions. As expected, mitochondrial hyperpolarization was observed in non-infected control cells cultured under hypoxic conditions, which was beneficial for C. pneumoniae growth. Taken together, functional and genetically encoded mitochondrial dysfunction strongly promotes intracellular growth of C. pneumoniae.