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Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin.

ABSTRACT: Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness.

SUBMITTER: Olatunji S

PROVIDER: S-EPMC6952399 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin.

Olatunji Samir S Yu Xiaoxiao X Bailey Jonathan J Huang Chia-Ying CY Zapotoczna Marta M Bowen Katherine K Remškar Maja M Müller Rolf R Scanlan Eoin M EM Geoghegan Joan A JA Olieric Vincent V Caffrey Martin M

Nature communications 20200109 1

Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed w ...[more]

PMID: 31919415

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Project description:Methicillin-resistant Staphylococcus aureus (MRSA) resists nearly all β-lactam antibiotics that have a bactericidal activity. However, whether the empirically used β-lactams enhance MRSA pathogenicity in vivo remains unclear. In this study, we showed that a cluster of lipoprotein-like genes (lpl, sa2275 to sa2273 [sa2275-sa2273]) was upregulated in MRSA in response to subinhibitory concentrations of β-lactam induction. The increasing expression of lpl by β-lactams was directly controlled by the global regulator SarA. The β-lactam-induced Lpls stimulated the production of interleukin-6 and tumor necrosis factor alpha in RAW 264.7 macrophages. The lpl deletion mutants (N315Δlpl and USA300Δlpl) decreased the proinflammatory cytokine levels in vitro and in vivo Purified lipidated SA2275-his proteins could trigger a Toll-like-receptor-2 (TLR2)-dependent immune response in primary mouse bone marrow-derived macrophages and C57BL/6 mice. The bacterial loads of N315Δlpl in the mouse kidney were lower than those of the wild-type N315. The β-lactam-treated MRSA exacerbated cutaneous infections in both BALB/c and C57BL/6 mice, presenting increased lesion size; destroyed skin structure; and easily promoted abscess formation compared with those of the untreated MRSA. However, the size of abscesses caused by the β-lactam-treated N315 was negligibly different from those caused by the untreated N315Δlpl in C57BL/6 TLR2-/- mice. Our findings suggest that β-lactams must be used carefully because they might aggravate the outcome of MRSA infection compared to inaction in treatment.IMPORTANCE β-Lactam antibiotics are widely applied to treat infectious diseases. However, certain poor disease outcomes caused by β-lactams remain poorly understood. In this study, we have identified a cluster of lipoprotein-like genes (lpl, sa2275-sa2273) that is upregulated in the major clinically prevalent MRSA clones in response to subinhibitory concentrations of β-lactam induction. The major highlight of this work is that β-lactams stimulate the expression of SarA, which directly binds to the lpl cluster promoter region and upregulates lpl expression in MRSA. Deletion of lpl significantly decreases proinflammatory cytokine levels in vitro and in vivo The β-lactam-induced Lpls enhance host inflammatory responses by triggering the Toll-like-receptor-2-mediated expressions of interleukin-6 and tumor necrosis factor alpha. The β-lactam-induced Lpls are important virulence factors that enhance MRSA pathogenicity. These data elucidate that subinhibitory concentrations of β-lactams can exacerbate the outcomes of MRSA infection through induction of lpl controlled by the global regulator SarA.

Project description:Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use.IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.

Dataset Information

Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin.

Publications

Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin.

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