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VERAPAMIL AND RESERPIN PROMOTE THE KILLING OF INTRACELLULAR BACTERIA
Abstract
The killing activity of macrophages has been shown to be due to the availability of K+ as needed for the activation of hydrolases present in the lysosome that fuses with the phagosome containing the phagocytosed bacterium (1). We have shown that the calcium channel blockers chlorpromazine and thioridazine kill
intracellularpathogenic bacteria (2, 3). We now show that the calcium channel blockers verapamil and reserpin, that have no antibacterial activity, promote the killing of methicillin resistant bacteria after such bacteria are phagocytosed by monocyte-derived human macrophages that have little killing activity of their own. Because verapamil and reserpin are known to be common inhibitors of ABC type efflux pumps (4), it is hypothesised that the killing activity noted is the result of their ABC type efflux pumps, which originate from the plasma membrane component that forms the phagosome. These pumps, that normally function at the plasma membrane site and are responsible for the import of K+, are now found in the phagosome complex to be oriented in an inverted manner and would export the ion to the outside of the phagosome. Inhibition of these pumps would mean that K+ effux would be inhibited and, therefore, the availability of this ion for the activation of hydrolases present in the lysosome that fuses with the phagosome, would be insured, hence killing would
take place. The similar employment of ouabain, an inhibitor of the sodium-potassium pump, supports the aforementioned hypothesis.
References
1 Reeves EP, Lu H, Jacobs HL, Messina CG, Bolsover S,
Gabella G, Potma EO, Warley A, Roes J and Segal AW:
Killing activity of neutrophils is mediated through activation
of proteases by K+ flux. Nature 416: 291-7, 2002.
2 Kristiansen MM, Leandro C, Ordway D, Martins M,
Viveiros M, Pacheco T, Kristiansen JE and Amaral L:
Phenothiazines alter resistance of methicillin-resistant
strains of Staphylococcus aureus (MRSA) to oxacillin in
vitro. Int J Antimicrob Agents 22: 250-3, 2003.
3 Ordway D, Viveiros M, Leandro C, Bettencourt R, Almeida
J, Martins M, Kristiansen JE, Molnar J and Amaral L:
Clinical concentrations of thioridazine kill intracellular
multidrug-resistant Mycobacterium tuberculosis. Antimicrob
Agents Chemother 47: 917-22, 2003.
4 Viveiros M, Leandro C and Amaral L: Mycobacterial
efflux pumps and chemotherapeutic implications. Int J
Antimicrob Agents 22: 274-8, 2003.
The killing activity of macrophages has been shown to be
due to the availability of K+ as needed for the activation
of hydrolases present in the lysosome that fuses with the
phagosome containing the phagocytosed bacterium (1).
We have shown that the calcium channel blockers
chlorpromazine and thioridazine kill intracellular
pathogenic bacteria (2, 3). We now show that the calcium
channel blockers verapamil and reserpin, that have no
antibacterial activity, promote the killing of methicillinresistant
bacteria after such bacteria are phagocytosed by
monocyte-derived human macrophages that have little
killing activity of their own. Because verapamil and
reserpin are known to be common inhibitors of ABC type
efflux pumps (4), it is hypothesised that the killing activity
noted is the result of their ABC type efflux pumps, which
originate from the plasma membrane component that
forms the phagosome. These pumps, that normally
function at the plasma membrane site and are responsible
for the import of K+, are now found in the phagosome
complex to be oriented in an inverted manner and would
export the ion to the outside of the phagosome. Inhibition
of these pumps would mean that K+ effux would be
inhibited and, therefore, the availability of this ion for the
activation of hydrolases present in the lysosome that fuses
with the phagosome, would be insured, hence killing would
take place. The similar employment of ouabain, an inhibitor
of the sodium-potassium pump, supports the aforementioned
hypothesis.
1 Reeves EP, Lu H, Jacobs HL, Messina CG, Bolsover S,
Gabella G, Potma EO, Warley A, Roes J and Segal AW:
Killing activity of neutrophils is mediated through activation
of proteases by K+ flux. Nature 416: 291-7, 2002.
2 Kristiansen MM, Leandro C, Ordway D, Martins M,
Viveiros M, Pacheco T, Kristiansen JE and Amaral L:
Phenothiazines alter resistance of methicillin-resistant
strains of Staphylococcus aureus (MRSA) to oxacillin in
vitro. Int J Antimicrob Agents 22: 250-3, 2003.
3 Ordway D, Viveiros M, Leandro C, Bettencourt R, Almeida
J, Martins M, Kristiansen JE, Molnar J and Amaral L:
Clinical concentrations of thioridazine kill intracellular
multidrug-resistant Mycobacterium tuberculosis. Antimicrob
Agents Chemother 47: 917-22, 2003.
4 Viveiros M, Leandro C and Amaral L: Mycobacterial
efflux pumps and chemotherapeutic implications. Int J
Antimicrob Agents 22: 274-8, 2003.
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According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.
The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant Mycobacterium tuberculosis. These compounds have not been used as anti-M. tuberculosis agents because their in vitro activities take place at concentrations which are beyond those that are clinically achievable. In addition, chronic administration of CPZ produces frequent severe side effects. Because CPZ has been shown to enhance the killing of intracellular M. tuberculosis at concentrations in the medium that are clinically relevant, we have investigated whether TZ, a phenothiazine whose negative side effects are less frequent and serious than those associated with CPZ, kills M. tuberculosis organisms that have been phagocytosed by human macrophages, which have nominal killing activities against these bacteria. Both CPZ and TZ killed intracellular antibiotic-sensitive and -resistant M. tuberculosis organisms when they were used at concentrations in the medium well below those present in the plasma of patients treated with these agents. These concentrations in vitro were not toxic to the macrophage, nor did they affect in vitro cellular immune processes. TZ thus appears to be a serious candidate for the management of a freshly diagnosed infection of pulmonary tuberculosis or as an adjunct to conventional antituberculosis therapy if the patient originates from an area known to have a high prevalence of multidrug-resistant M. tuberculosis isolates. Nevertheless, we must await the outcomes of clinical trials to determine whether TZ itself may be safely and effectively used as an antituberculosis agent.
The demonstration of the existence of active efflux pumps in mycobacteria raises the question of whether or not these can increase in number and activity rendering wild-type mycobacteria increasingly resistant to a given antibiotic. This could be a mechanism by which mutated resistant strains become better fit to the selective environment. Mycobacterium tuberculosis genome analysis reveals several genes encoding putative drug efflux pumps. During the course of tuberculosis chemotherapy many of these pumps might play a role in the survival of the mycobacterial populations. Compounds capable of inactivating these pumps could improve anti-tuberculous therapeutics.
Mechanisms of antibiotic resistance of bacteria include efflux pumps which extrude the antibiotic prior to reaching its target. Phenothiazines inhibit the activity of some efflux pumps thereby altering the susceptibility of bacteria. This study demonstrated that chlorpromazine and thioridazine reduce the susceptibility of methicillin-resistant strains (MRSA) but not that of methicillin-susceptible Staphylococcus aureus (MSSA) strains to oxacillin (MIC of oxacillin reduced from >500 to 10 mg/l). Reserpine, an inhibitor of antibiotic efflux pumps also reduced the resistance of MRSA strains to oxacillin suggesting the presence of an efflux pump that contributes to antibiotic resistance of MRSA strains.