Mycobacterium smegmatis Resists the Bactericidal Activity of Hypochlorous Acid Produced in Neutrophil Phagosomes

Abstract

Neutrophils are often the major leukocyte at sites of mycobacterial infection, yet little is known about their ability to kill mycobacteria. In this study we have investigated whether the potent antibacterial oxidant hypochlorous acid (HOCl) contributes to killing of Mycobacterium smegmatis when this bacterium is phagocytosed by human neutrophils. We found that M. smegmatis were ingested by neutrophils into intracellular phagosomes but were killed slowly. We measured a t 1/2 of 30 min for the survival of M. smegmatis inside neutrophils, which is 5 times longer than that reported for Staphylococcus aureus and 15 times longer than Escherichia coli. Live-cell imaging indicated that neutrophils generated HOCl in phagosomes containing M. smegmatis; however, inhibition of HOCl production did not alter the rate of bacterial killing. Also, the doses of HOCl that are likely to be produced inside phagosomes failed to kill isolated bacteria. Lethal doses of reagent HOCl caused oxidation of mycothiol, the main low-m.w. thiol in this bacterium. In contrast, phagocytosed M. smegmatis maintained their original level of reduced mycothiol. Collectively, these findings suggest that M. smegmatis can cope with the HOCl that is produced inside neutrophil phagosomes. A mycothioldeficient mutant was killed by neutrophils at the same rate as wild-type bacteria, indicating that mycothiol itself is not the main driver of M. smegmatis resistance. Understanding how M. smegmatis avoids killing by phagosomal HOCl could provide new opportunities to sensitize pathogenic mycobacteria to destruction by the innate immune system. The Journal of Immunology,

Antimicrobial Activity of Neutrophils Against Mycobacteria

“…Phagocytosis of M. abscessus was also found to be slower than that of S. aureus when examined by counting the number of neutrophils containing fluorescently labelled bacteria ( 129 ). In another study, approximately half of a population of M. fortuitum was phagocytosed within 30 min ( 130 ), only slightly faster than the 43 min we measured for M. smegmatis , and still considerably slower than the 9 min for S. aureus and 12 min for E. coli ( 127 , 131 ). As far as we are aware no in vitro studies have directly compared phagocytosis of different mycobacteria by the same neutrophils; however, neutrophils were found to ingest M. abscessus more frequently than either M. tuberculosis or M. avium in an ex vivo infection of human lung tissue ( 99 ).…”

“…Recently we measured the rate of phagocytosis of M. smegmatis and found that it was five times slower than the phagocytosis of Staphylococcus aureus and 3.5 times slower than that for Escherichia coli ( 127 , 128 ). Phagocytosis of M. abscessus was also found to be slower than that of S. aureus when examined by counting the number of neutrophils containing fluorescently labelled bacteria ( 129 ).…”

“…While Hartman et al reported near complete killing of M. avium by neutrophils in two hours ( 120 ), only around 40% of populations of M. abscessus and M. fortuitum were killed after two hours incubation with neutrophils ( 146 ). We have previously reported half-lives for E. coli and S. aureus inside the neutrophil phagosome of 2 min and 6 min, respectively ( 128 ), and recently measured a half-life for M. smegmatis of 30 min inside the neutrophil phagosome, which indicates that even though killing occurs, it is slow ( 127 ).…”

“…The purified MPO/H 2 O 2 /Cl - system is capable of killing M. tuberculosis and M. leprae ( 149 , 150 ); however, it has been reported that the complete system did not augment killing of M. tuberculosis over that observed by H 2 O 2 alone ( 141 ). Reagent HOCl killed M. smegmatis , but approximately seven times more HOCl was required to kill M. smegmatis than S. aureus suggesting mycobacteria may be innately more resistant to HOCl ( 127 ). Further studies are required to determine if other mycobacteria are similarly resistant to HOCl.…”

“…Additionally, the neutrophil phagosome contains many proteins and amines that can react with phagosomal HOCl before it reaches the bacterium ( 151 , 152 ). Using a fluorescent probe we observed HOCl production in the phagosome and that MPO inhibition abrogated HOCl production ( 127 ). By modelling the data obtained in our study we estimated that it would take 30-40 minutes at full MPO capacity for sufficient HOCl to be produced to kill a single ingested M. smegmatis .…”