Macrophage environment turns otherwise MccJ25-resistant Salmonella into sensitive

Abstract

BackgroundMicrocin J25 (MccJ25) is a plasmid-encoded antibiotic peptide produced by Escherichia coli (E. coli). MccJ25 enters into the sensitive E. coli strains by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD and SbmA. The resistance of Salmonella enterica serovar Typhimurium (S. Typhimurium) to MccJ25 is attributed to the inability of its FhuA protein to incorporate the antibiotic into the cell.ResultsIn this work we demonstrate that S. Typhimurium becomes notably susceptible to MccJ25 when replicating within macrophages. In order to determine the possible cause of this phenomenon, we studied the sensitivity of S. Typhimurium to MccJ25 at conditions resembling those of the internal macrophage environment, such as low pH, low magnesium and iron deprivation. We observed that the strain was only sensitive to the antibiotic at low pH, leading us to attribute the bacterial sensitization to this condition. A MccJ25-resistant E. coli strain in which fhuA is deleted was also inhibited by the antibiotic at low pH. Then, we could assume that the MccJ25 sensitivity change observed in both E. coli fhuA and S. Typhimurium is mediated by a MccJ25 uptake independent of the FhuA receptor. Moreover, low pH incubation also sensitized S. Typhimurium to the hydrophobic antibiotic novobiocin, which does not affect enteric bacteria viability because it is unable to penetrate the bacterial outer membrane. This observation supports our hypothesis about low pH producing a modification in the bacterial membrane permeability that allows an unspecific MccJ25 uptake. On the other hand, MccJ25 inhibited S. Typhimurium when cells were preincubated in acidic pH medium and then treated at neutral pH with the antibiotic.ConclusionsOur results suggest that acidic condition does not alter MccJ25 hydrophobicity but irreversibly modifies bacterial membrane permeability. This would allow an unspecific antibiotic uptake into the cell.From our data it is possible to infer that intracellular pathogenic strains, which are in vitro resistant to MccJ25, could become susceptible ones in vivo. Therefore, the MccJ25 action spectrum would be broader than what in vitro experiments indicate.

DNA supercoiling differences in bacteria result from disparate DNA gyrase activation by polyamines

“…A mildly acidic pH was previously proposed to play a role based on an in vivo synergistic effect of the DNA gyrase inhibitor novobiocin and acidic pH on DNA relaxation [ 50 ]. However, this observation can be explained by a mildly acidic pH increasing permeability to novobiocin [ 51 ]. Changes in the abundance of putrescine and spermidine, or in a metabolite yet to be identified, may contribute to the reported decrease in global DNA supercoiling.…”

Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress

“…Novobiocin at 10 μg ml −1 resulted in greater DNA relaxation at pH 4 than at pH 7. This was consistent with the known ability of acid treatment to enhance the uptake of novobiocin by the bacterium (Ofek et al ., ; Pomares et al ., ; see Discussion ).…”

“…In considering our results, we took into account previous data showing that growth at acid pH enhances novobiocin uptake by Salmonella (Ofek et al ., ; Pomares et al ., ). In a pH 7 culture, the effect of 10 μg ml −1 novobiocin was similar to the degree of relaxation seen due to acid treatment alone in the drug‐free pH 4 culture (Fig.…”

Engineering and characterization of human β-defensin-3 and its analogues and microcin J25 peptides against Mannheimia haemolytica and bovine neutrophils

“…There are some data on the characterization of outer membrane proteins of M. haemolytica [ 36 , 37 ] but we don’t know whether specific membrane proteins exist to facilitate transport of MccJ25. Furthermore, MccJ25 is not effective against many bacteria of the family Enterobacteriaceae that are not related to the MccJ25 producing members [ 38 ]. It could be argued that MccJ25 shows a narrow action spectrum activity in vitro but could be active in vivo against resistant species [ 38 , 39 ].…”

“…Furthermore, MccJ25 is not effective against many bacteria of the family Enterobacteriaceae that are not related to the MccJ25 producing members [ 38 ]. It could be argued that MccJ25 shows a narrow action spectrum activity in vitro but could be active in vivo against resistant species [ 38 , 39 ]. It has been shown that resistant bacteria become susceptible to antibiotics upon their entry into the low pH environment in the phagolysosomes of macrophages likely due to the nonspecific MccJ25 uptake into the bacterial cell through altered bacterial membrane permeability.…”