Université catholique de Louvain (UCL-Bruxelles)
Louvain Drug Research Institute > Cellular and Molecular Pharmacology
Chemotherapy of the intracellular infection
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The efficacy of the chemotherapy of intracellular infection depends on an effective cooperation between antibiotics and the host.

Antibiotics must not only penetrate inside the cells and reach the infected cellular compartments, but also express their activity in the corresponding environment(s).

Using models of uninfected or infected cells (macrophages and non phagocytic cells), we study the intracellular pharmacokinetics of antibiotics, their efficacy against intracellular pathogens localized in different subcellular compartments, and the modulation of their activity by cytokines.

These research programs are closely linked to those exploring the cellular toxicity of antibiotics and the novel antibiotic targets

Team

Collaborations

Main current research programs

General principles

Intracellular bacterial infection remains a medical and economical threat in spite of the availability of a large array of antibiotics potentially active against those organisms in a-cellular systems. Most failures actually stem from the inability of the drugs to reach the offending organism(s), and/or to effectively act in the intracellular environment (see figure). More recently, we also noted the importance of a cooperation with the natural host defenses and their modulators. Our aim is to determine the main pharmacodynamic and pharmacokinetic parameters of antibiotics at the cellular level, and to correlate them with their activities against intracellular organisms in quantitative models (see Figure 1). We therefore study, mostly by biochemical and microbiological techniques, the intracellular fate of antibiotics, and assess their effectiveness against sensitive bacteria of medical or economical importance. These are selected on the basis of their capacity to invade and thrive in distinct subcellular compartments (cytosol, phagosomes,...). We also examine the influence of selected cytokines on these properties.

These studies have allowed to describe in detail the cellular properties of many classes of antibiotics of interest in the context of the intracellular infection (fluoroquinolones, macrolides, synergistins,beta-lactams, oxazolidinones glyco(lipo)peptides, ansamycins, ...) in a series of models of uninfected cells and cells infected with Staphylococcus aureus or epidermidis, Listeria monocytogenes Legionella spp., Pseudomonas aeruginosa, ...).

Pharmacokinetic and Pharmacodynamic parameters
 Figure 1: Cellular pharmacokinetic and pharmacodynamic parameters governing the activity of antibiotics in cells

Pharmacokinetic parameters:
  • influx (transmembrane / endocytosis)
  • accumulation and bioavailability (including subcellular localisation, binding to cellular constituents, metabolic inactivation)
  • efflux (active or passive)
Pharmacodynamic parameters
  • bacterial responsiveness (fast or slowly growing bacteria, metabolic modulation, ...)
  • impact of physicochemical conditions (pH, other intracellular consttuents, ...)
  • cooperation with host defenses
from Carryn et al, 2003.

Intracellular pharmacokinetics

We study the cellular accumulation (including the mechanisms of entry) and the subcellular localization of of antibiotics, including of novel molecules in preclinical and clinical developement, as a basis for further studies examining their intracellular activities in specific compartments.  

The main results of what has been observed over the last 10 years is shown on Table 1.

In a nutshel, beta-lactams penetrate but do not accumulate in cells, and are distributed in the cytosol. Macrolides and fluoroquinolones accumulate rapidly in cells and are distribute primarily in lysosomes and cytosol, respectively. Aminoglycosides and glycopeptides enter cells by endocytosis and accumulate specifically in lysosomes. Of note, the level of accumulation of new lipopeptides like oritavancin can be very high. Linezolid does not accumulate in cells, but some novel derivatives accumulate to much larger extents.

Table 1: Main cellular pharmacokinetic properties of antibiotics. From Van Bambeke et al. 2006

Selected references on cellular pharmacokinetics (by reverse chronological order; for a full reference list, see our publication list)

Intracellular pharmacodynamics

We study the activity of antibiotics in different models of  intracellular infection caused by bacteria sojourning in different subcellular compartments, as illustrated in Figure 3A for L. monocytogenes(cytosol) and Figure 3B for S. aureus (phagolysosomes). We then try to establish their intracellular pharmacodynamic profile (influence of  concentration and of time on activity). 

Intraphagoytic L. monocytogenes (THP1 macrophages)

Figure 3A: Following the intracellular fate of Listeria monocytogenes with electron microcopy:

Listeria monocytogenes hly+ (virulent variant) has been phagocytozed by THP-1 macrophages (A). The bacteria quickly escapes from its cytoplasmic vacuole (B) to multiply in the cytosol while sourrounding it-self with a tick layer of finely granular and filamentous material (actin; C).

In cells pre-treated with interferon-gamma (D, E), Listeria monocytogenes hly+ remain confined within membrane-bound vacuoles.

Listeria monocytogenes hly- (non-virulent variant) remains constantly in vacuoles in control (F, G) as well as in interferon-gamma-treated cells.

A = 1 h post infection;
B, C, D, F, G = 3 h post infection
E, H = 5 h post infection

Bars = 1 µ except for inset of A, where bar is 0.1 µ)
 

From: Ouadrhiri et al.,1999
Intraphagocytic S. aureus (J774 macrophages)

Figure 3B: Examining the subcellular localization of phagocytized S. aureus

Electron microscopy of J774 macrophages fixed 1 h (A and B) or 24 h (C and D) after phagocytosis of opsonized S. aureus. In both cases, incubation was carried out in the presence of 0.5 mg/L of gentamicin (to avoid extracellular multiplication of bacteria released from died cells and subsequent acidification of the medium).

A,B: after 1 h of phagocytosis, bacteria appear isolated (A) or sometimes in clusters (B) without evidence of damage but with no sign of division.

C,D: after 24 h of phagocytosis, most of the bacteria are in the active process of division.

In both cases, all bacteria were seen in membrane-bounded structures with no evidence of transfer to cytosol. Bars are 0.3 µm (A, B, and D) and 1 µm (C)

from Seral et al, 2003

The model allows to determine 3 key pharmacological descriptors of activity, namely

These descriptors allow to quantitavely assessd the the activity of a given drug against different intracellular bacteria or different strains with various resistance phenotypes, and to also to compare difefrent antibiotics.

At the present time, and using this approach, we are studying antibiotic intracellular activity in models of phagocytic (monocytes and macrophages) or non-phagocytic (endothelial cells, keratinocytes, fibroblasts, bronchial epithelial cells) infected by S. aureus or S. epidermidis, L. monocytogenes, L. pneumophila, or P. aeruginosa, using both collection strains and clinical isolates coming from patients with recurrent or persistent infections.

These studies are leading to largely unanticipated conclusions.  Thus, beta-lactams, which are not accumulated by eucaryotic cells, are actually more active against the intracellular than the extracellular forms of L.monocytogenes (Figure 4A).  Quinolones, which do accumulate in cells, show a similar level of maximal activity (Emax) intracellularly and extracellularly (Figure 4B).  Macrolides, which accumulate to a very large extent in cells, are only bacteriostatic. Some new glycopeptides, which also accumulate to very large extents but are located exclusively in the lysosomal compartment, do not act on intracellular L. monocytogenes but are highly bactericidal against intracellular S. aureus.

The reasons for these discrepancies are now examined by studying the influence that the intracellular medium can exert on antibiotic activity (influence of pH or of redox status) or on bacterial metabolism (using proteomic approaches), to try explaining the modifications of susceptibility to antibiotics observed intracellularly.

Figure 4A

Illustration of dose-effect relationships in intracellular models of infection. The left panel shows the influence of time and concentration on the intracellular activity of ampicillin against Listeria monocytogenes in infected THP1macrophages exposed for 5 or 24 h to increasing concentrations of the drug, expressed as the log of multiples of its minimum inhibitory
concentration (MIC). The right panel provides a comparison of the dose-effect relationship of the activity of ampicillin and moxifloxacin against Staphylococcus aureus in infected THP1 macrophages exposed for over 24 h to increasing multiples of their MIC.

from Van Bambeke et al, 2006

 Pharmacodynamics of antibiotics against Listeria
 Figure 4B

Illustration of some paradoxical observations made in the model of intracellular infection by L. moncytogenes.  In the left panel, one sees that the quinolone moxifloxacin shows the same activity against intracellular and extracellular bacteria despite the fact it is accumulated about 8-fold in the macrophages.  In the right panel, one sees that two beta-lactams, which enter the cells but do not accumulate (cellular concentration lower than the extracellular one) are essentially  bacteriostatic against L. monocytogenes in broth but become bactericidal intracellularly after 24 h of exposure of the bacteria to the drugs

from Carryn et al., 2003

Selected References on cellular pharmacodynamics (by reverse chronological order; for a full reference list, see our publication list)

Modulation of resistance mechanisms in the intracellular environment

In the course of our studies comparing the intracellular activity of antibiotics against extracellular and intracellular forms of bacteria harboring typical resistance mechanisms, we made the unanticipated observation that beta-lactams regain activity against MRSA when intracellular. This was explained by the fact that the acidic pH prevailing in the lysosomes where S. aureus sojourns inside the cells modifies the conformation of PBP2a, allowing for the binding of beta-lactams (see Figure 5).

We have also shown that most antibiotics are only poorly and slowly efficient against SCV (Small Colony variants) of S. aureus, probably in relation with the slow intracellular multiplication rate of these organisms.

Active efflux is a general mechanism of resistance, with efflux pumps for antibiotics being expressed at the surface of both eucaryotic cells and bacteria (see also antibiotic efflux and permeability resistance mechanisms). We study the cooperation between efflux pumps expressed by eucaryotic cells and bacteria to reduce the intracellular activity of antibiotics, as clearly demonstrated for fluoroquinolones and intracellular Listeria.


Figure 5: Restauration of activity of beta-lactams against intracellular MRSA, in relation with the change of conformation of PBP2a at acidic pH

Top: Concentration killing effects of meropenem and cloxacillin toward MSSA strain ATCC 25923 and MRSA strain ATCC 33591 after phagocytosis by THP-1 macrophages. Cells were incubated with the antibiotics for 24 h at the concentrations (total drug) indicated on the abscissa. The arrows along the abscissa point to the MIC of the organisms determined in broth at pH 7.4 (open arrows, MSSA strain ATCC 25923; closed arrows, MRSA ATCC 33591).

from Lemaire et al. 2007.

Bottom: Circular dichroic spectra of PBP2a at pH 7.0 (left) and pH 5.5 (right) in the absence (open symbols) and in the presence (closed symbols) of oxacillin (30 µM) for 30 min at 25°C. The thin-dotted lines in each graph represent minima of PBP2a molar ellipticity for each condition.

from Lemaire et al.,2008.

Selected References on expression of resistance mechanisms (by reverse chronological order; for a full reference list, see our publication list)

Assessment of cytokines-antibiotic cooperation and antivirulence strategies

Host defenses can modify the intracellular fate of bacteria and the intracellular activity of antibiotics. We have studied the influence of gamma-interferon and other cytokines on antibiotic action against intracellular Listeria monocytogenes, a typical exemple of food-borne intracellular infection. 

In the absence of a sufficiently powerful cellular immune response, Listeria quiclky speads through its host. These virulent bacteria gains access to cells by endoyctosis but escape destruction by egressing from the phagocytic vacuole to reach and multiply in the cytosol. Interferon-gamma, one of the key cytokine involved in the immune response against Listeria infection, prevents this evasion from the phagosome (see Figure 6). Interestingly, Listeria constrained in the phagosomes become unable to multiply.

The protection afforded by the cytokines appears mediated by overproduction of oxygen- and nitrogen-reactive species. We showed an overexpression of the inducible NO synthase by Interferon-gamma and IL-6 causes in Caco-2 and by GM-SCF in THP-1 cells, making a direct link between the immune response and the effective control of Listeria infection.
   

Figure 6:
Following the intracellular fate of Listeria monocytogenes with confocal microcopy.

Listeria, stained in green with fluorescein-isothiocyanate (FITC) has been phagocyozed by THP-1 macrophages.  The cell actin has been stained in red with rhodamine-phalloidin.  In control cells,  the virulent variant (Listeria monocytogenes hly +), is quickly surrounded by actin and appears red/yellow, most likely because bacteria have reached the cytosol. The a-virulant strain (hly - ) remains stained in green.  In the presence of interferon-gamma, bacteria from both strains remain stained in green and presumably in vacuoles. 

From Ouadrhiri et al. ,1999

As antibiotic activity is limited against intracellular infections, we have started to examine other strategies to control intracellular infection.

A first example is that of dehydrosqualene synthase inhibitors that inhibit the synthesis of staphyloxanthin is S. aureus, a pigment required for protecting S. aureus against oxydative stress. Strains producing high amounts of this pigment (like the CA-MRSA US300) show a high capacity to multiply within the cells, which returns to low values in the presence of dehydrosqualene synthase inhibitors (Figure 7).

 
Figure 7:

Effects of the dehydrosqualene synthase inhibitor BPH-652 on staphyloxantin production (top) and on intracellular growth of different strains of S. aureus (bottom)

A: Bacteria were grown for 2 days in the absence (CONTR) or in the presence of the inhibitor (100 µM) before being pelleted for photography (8325-4 strain subjected to the same treatments is also shown, to demonstrate the near absence of pigmentation under all conditions).

B: Intracellular growth of SH1000 in HUVEC cells and of SH1000, USA300, and three clinical isolates in THP-1 macrophages in the absence (control) or in the presence of BPH-652. The dotted lines show the response observed for S. aureus 8325-4 rsbU - (non pigmented strain).

from Olivier et al., 2009


Selected references on cytokine-antibiotic cooperations (by reverse chronological order; for a full reference list, see the publication list)

Expertise

Additional information:  <tulkens@facm.ucl.ac.be>
Last significant update: June 2014