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Loss of the ClpXP Protease Leads to Decreased Resistance to Cell-Envelope Targeting Antimicrobials in Bacillus anthracis Sterne.

ABSTRACT: The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.

SUBMITTER: Zou L

PROVIDER: S-EPMC8419472 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Susceptibility to penicillin and other beta-lactam-containing compounds is a common trait of Bacillus anthracis. Beta-lactam agents, particularly penicillin, have been used worldwide to treat anthrax in humans. Nonetheless, surveys of clinical and soil-derived strains reveal penicillin G resistance in 2 to 16% of isolates tested. Bacterial resistance to beta-lactam agents is often mediated by production of one or more types of beta-lactamases that hydrolyze the beta-lactam ring, inactivating the antimicrobial agent. Here, we report the presence of two beta-lactamase (bla) genes in the penicillin-susceptible Sterne strain of B. anthracis. We identified bla1 by functional cloning with Escherichia coli. bla1 is a 927-nucleotide (nt) gene predicted to encode a protein with 93.8% identity to the type I beta-lactamase gene of Bacillus cereus. A second gene, bla2, was identified by searching the unfinished B. anthracis chromosome sequence database of The Institute for Genome Research for open reading frames (ORFs) predicted to encode beta-lactamases. We found a partial ORF predicted to encode a protein with significant similarity to the carboxy-terminal end of the type II beta-lactamase of B. cereus. DNA adjacent to the 5' end of the partial ORF was cloned using inverse PCR. bla2 is a 768-nt gene predicted to encode a protein with 92% identity to the B. cereus type II enzyme. The bla1 and bla2 genes confer ampicillin resistance to E. coli and Bacillus subtilis when cloned individually in these species. The MICs of various antimicrobial agents for the E. coli clones indicate that the two beta-lactamase genes confer different susceptibility profiles to E. coli; bla1 is a penicillinase, while bla2 appears to be a cephalosporinase. The beta-galactosidase activities of B. cereus group species harboring bla promoter-lacZ transcriptional fusions indicate that bla1 is poorly transcribed in B. anthracis, B. cereus, and B. thuringiensis. The bla2 gene is strongly expressed in B. cereus and B. thuringiensis and weakly expressed in B. anthracis. Taken together, these data indicate that the bla1 and bla2 genes of the B. anthracis Sterne strain encode functional beta-lactamases of different types, but gene expression is usually not sufficient to confer resistance to beta-lactam agents.

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Loss of the ClpXP Protease Leads to Decreased Resistance to Cell-Envelope Targeting Antimicrobials in Bacillus anthracis Sterne.

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