Characterization of Escherichia coli DNA lesions generated within J774 macrophages

Abstract

Macrophages are armed with multiple oxygen-dependent and -independent bactericidal properties. However, the respiratory burst, generating reactive oxygen species, is believed to be a major cause of bacterial killing. We exploited the susceptibility of Escherichia coli in macrophages to characterize the effects of the respiratory burst on intracellular bacteria. We show that E. coli strains recovered from J774 macrophages exhibit high rates of mutations. We report that the DNA damage generated inside macrophages includes DNA strand breaks and the modification 8-oxo-2'-deoxyguanosine, which are typical oxidative lesions. Interestingly, we found that under these conditions, early in the infection the majority of E. coli cells are viable but gene expression is inhibited. Our findings demonstrate that macrophages can cause severe DNA damage to intracellular bacteria. Our results also suggest that protection against the macrophage-induced DNA damage is an important component of the bacterial defense mechanism within macrophages.

FIG. 1

(A) Plasmid DNA topology within macrophages. J774 macrophages were activated and infected with E. coli MC4100 or S. enterica serovar Typhimurium SL1344 bearing pKK177-3. Plasmid DNA was extracted by the alkali lysis procedure before infection (control) and from intracellular bacteria 30 min after infection (Macrophages). Equal amounts of DNA, as quantified by dot blot analysis using γ-³²P-end labeled primer 374 corresponding to pKK177-3 (data not shown), were separated on a 1.4% agarose gel containing 10 μg of chloroquine per ml and transferred to a nylon membrane. The membrane was probed with labeled primer 374. (B) Equal amounts of plasmid DNA from the experiment in panel A were used to transform E. coli and S. enterica serovar Typhimurium by electroporation.

FIG. 2

Survival in J774 macrophages. Macrophages were activated, infected with bacterial cultures for 20 min, washed, and lysed, and aliquots were plated to determine the number of viable intracellular bacteria or further incubated for an additional 20, 40, or 70 min in medium containing gentamicin. The results are means of four independent experiments, each carried out with three or four cultures of each strain. Shown are the numbers of viable intracellular bacteria from 20 to 90 min after infection. The number of viable cells at 20 min represents bacterial cells attached to or within macrophages. Since S. enterica serovar Typhimurium actively invades macrophages, the initial number of intracellular S. enterica serovar Typhimurium bacteria is larger than the initial number of intracellular E. coli bacteria.

FIG. 3

PoxyS-gfp induction by H₂O₂ (A) and Ptac-gfp induction by IPTG (B) in LB medium. Exponentially growing S. enterica serovar Typhimurium SL1344 and E. coli MC4100 bearing PoxyS-gfp and Ptac-gfp-lacI were treated with 0.2 mM H₂O₂ and 1 mM IPTG, respectively. The GFP expression of treated (+) and untreated (−) cultures was analyzed by fluorescence-activated cell sorting. A similar pattern of expression was obtained with the PoxyS E. coli clone (PoxySEc-gfp) (see Materials and Methods), although the levels were reduced twofold (data not shown).

FIG. 4

(A) PoxyS-gfp expression within macrophages. J774 cells activated with PMA were infected with S. enterica serovar Typhimurium/PoxyS-gfp or E. coli/PoxyS-gfp. At 30 min after infection, the macrophages were fixed, stained with anti-E. coli or anti-Salmonella antibodies and Cy5-conjugated secondary antibody, and subjected to confocal microscopy. (Bottom) Cy5 conjugate (red); (middle) GFP (green); (top) Cy5 and GFP superimposed (yellow). (B) Ptac-gfp expression within macrophages. J774 cells activated with PMA were infected with S. enterica serovar Typhimurium/Ptac-gfp-lacI or E. coli/Ptac-gfp-lacI. At 30 min after infection, the macrophages were washed and further incubated for 40 min in medium containing gentamicin (50 μg/ml) and IPTG (1 mM). Thereafter, the macrophages were fixed and stained as in panel A and subjected to confocal microscopy. (Bottom) Cy5 conjugate (red); (middle) GFP (green); (top) Cy5 and GFP superimposed (yellow).