Abstract
Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine.
Highlights
Chemokines are small (8- to 12-kDa) proteins originally recognized for their ability to act as cellular messengers that promote cell differentiation, activation, and migration, such as routing leukocytes to areas of inflammation as part of the innate immune response [1]
To what we found with B. anthracis, specific bacterial targets necessary for CXCL10-mediated killing would be present in other bacteria that were susceptible to the interferon-inducible ELRϪ CXC chemokines
Microscopic examination of untreated CAV1036 revealed that this clinical isolate formed long chains of bacteria, but exposure to lower, sublethal concentrations of CXCL10 resulted in separation of the long chains into multiple, shorter chains of bacteria as well as individual bacteria that exhibited a tendency to form aggregates in the wells
Summary
Chemokines are small (8- to 12-kDa) proteins originally recognized for their ability to act as cellular messengers that promote cell differentiation, activation, and migration, such as routing leukocytes to areas of inflammation as part of the innate immune response [1]. PDHc in CXCL10-Mediated Killing of E. coli with CXCR3 primarily via the N-terminal region of the chemokine and amino acid residues in two loop regions [15] and, through this interaction with CXCR3, recruit additional immune cells to their location Their positively charged C-terminal end contains an amphipathic alpha-helical moiety that is structurally similar to those of CAMPs [13]. Studies investigating the mechanism of action of other antimicrobial peptides, including human defensins, support the possibility that the specific targets and mechanisms of antimicrobial action may differ among the diverse range of microorganisms affected by a given antimicrobial peptide [17, 18] The identification of such bacterial targets has the potential to open up new avenues of inquiry into the mechanisms by which chemokinemediated antimicrobial effects occur. Such information has important implications for the development of novel therapeutic agents, including those with activity against multidrug-resistant pathogens
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